888 Citations
Respiratory syncytial virus RSV causes lower respiratory tract infections with significant morbidity and mortality at the extremes of age Vaccines based on the viral fusion protein are approved for adults over but infant protection relies on passive immunity via antibody transfer or maternal vaccination An infant vaccine that rapidly elicits protective antibodies would fulfill a critical unmet need Antibodies arising from the VH - VL - gene pairing can neutralize RSV without the need for affinity maturation making them attractive to target through vaccination Here we develop an anti-idiotypic monoclonal antibody ai-mAb immunogen that is specific for unmutated VH - ... More
Respiratory syncytial virus (RSV) causes lower respiratory tract infections with significant morbidity and mortality at the extremes of age. Vaccines based on the viral fusion protein are approved for adults over 60, but infant protection relies on passive immunity via antibody transfer or maternal vaccination. An infant vaccine that rapidly elicits protective antibodies would fulfill a critical unmet need. Antibodies arising from the VH3-21/VL1-40 gene pairing can neutralize RSV without the need for affinity maturation, making them attractive to target through vaccination. Here, we develop an anti-idiotypic monoclonal antibody (ai-mAb) immunogen that is specific for unmutated VH3-21/VL1-40 B cell receptors (BCRs). The ai-mAb efficiently engages B cells with bona fide target BCRs and does not activate off-target non-neutralizing B cells, unlike recombinant pre-fusion (preF) protein used in current RSV vaccines. These results establish proof of concept for using an ai-mAb-derived vaccine to target B cells hardwired to produce RSV-neutralizing antibodies. Less
The opioid overdose epidemic is a growing and evolving public health crisis fueled by the widespread presence of fentanyl and fentanyl analogues F FAs in both street mixtures and counterfeit pills To expand current treatment options drug-targeting monoclonal antibodies mAbs offer a viable therapeutic for both pre- and postexposure clinical scenarios This study reports the isolation in vitro characterization and in vivo efficacy of two murine mAb families targeting fentanyl carfentanil or both Because humanization of the mAbs by CDR grafting negatively impacted affinity for both fentanyl and carfentanil crystal structures of mAbs in complex with fentanyl or carfentanil were ... More
The opioid overdose epidemic is a growing and evolving public health crisis fueled by the widespread presence of fentanyl and fentanyl analogues (F/FAs) in both street mixtures and counterfeit pills. To expand current treatment options, drug-targeting monoclonal antibodies (mAbs) offer a viable therapeutic for both pre- and postexposure clinical scenarios. This study reports the isolation, in vitro characterization, and in vivo efficacy of two murine mAb families targeting fentanyl, carfentanil, or both. Because humanization of the mAbs by CDR grafting negatively impacted affinity for both fentanyl and carfentanil, crystal structures of mAbs in complex with fentanyl or carfentanil were analyzed to identify key residues involved in ligand binding in murine versus humanized structures, and site-directed mutagenesis was used to verify their functional importance. The structural analysis identified a framework residue, Tyr36, present in the murine germline sequence of two mAbs, which was critical for binding to fentanyl and carfentanil. These studies emphasize the importance of structural considerations in mAb engineering to optimize mAbs targeting small molecules including opioids and other drugs of public health interest. Less
-Glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharo lyticus Bgl has been denoted as having an attractive catalytic profile for various industrial applications Bgl catalyses the final step of in the decomposition of cellulose an unbranched glucose polymer that has attracted the attention of researchers in recent years as it is the most abundant renewable source of reduced carbon in the biosphere With the aim of enhancing the thermostability of Bgl for a broad spectrum of biotechnological processes it has been subjected to structural studies Crystal structures of Bgl and its complex with glucose were determined at and resolution respectively Bgl ... More
β-Glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharolyticus (Bgl1) has been denoted as having an attractive catalytic profile for various industrial applications. Bgl1 catalyses the final step of in the decomposition of cellulose, an unbranched glucose polymer that has attracted the attention of researchers in recent years as it is the most abundant renewable source of reduced carbon in the biosphere. With the aim of enhancing the thermostability of Bgl1 for a broad spectrum of biotechnological processes, it has been subjected to structural studies. Crystal structures of Bgl1 and its complex with glucose were determined at 1.47 and 1.95 Å resolution, respectively. Bgl1 is a member of glycosyl hydrolase family 1 (GH1 superfamily, EC 3.2.1.21) and the results showed that the 3D structure of Bgl1 follows the overall architecture of the GH1 family, with a classical (β/α)8 TIM-barrel fold. Comparisons of Bgl1 with sequence or structural homologues of β-glucosidase reveal quite similar structures but also unique structural features in Bgl1 with plausible functional roles. Less
A group of three deep learning tools referred to collectively as CHiMP Crystal Hits in My Plate were created for analysis of micrographs of protein crystallisation experiments at the Diamond Light Source DLS synchrotron UK The first tool a classification network assigns images into categories relating to experimental outcomes The other two tools are networks that perform both object detection and instance segmentation resulting in masks of individual crystals in the first case and masks of crystallisation droplets in addition to crystals in the second case allowing positions and sizes of these entities to be recorded Creation of these tools ... More
A group of three deep learning tools, referred to collectively as CHiMP (Crystal Hits in My Plate) were created for analysis of micrographs of protein crystallisation experiments at the Diamond Light Source (DLS) synchrotron, UK. The first tool, a classification network, assigns images into categories relating to experimental outcomes. The other two tools are networks that perform both object detection and instance segmentation, resulting in masks of individual crystals in the first case, and masks of crystallisation droplets in addition to crystals in the second case, allowing positions and sizes of these entities to be recorded. Creation of these tools used transfer learning, where weights from a pre-trained deep learning network were used as a starting point and re-purposed by further training on a relatively small set of data. Two of the tools are now integrated at the VMXi macromolecular crystallography beamline at DLS where they absolve the need for any user input both for monitoring crystallisation experiments and for triggering in situ data collections. The third is being integrated into the XChem fragment-based drug discovery screening platform, also at DLS, to allow automatic targeting of acoustic compound dispensing into crystallisation droplets. Less
Human - exonuclease PLD a member of the phospholipase D family of enzymes has been validated as a therapeutic target for treating Alzheimer's disease Here we have determined the crystal structure of the luminal domain of the enzyme at resolution revealing a bilobal structure with a catalytic site located between the lobes We then compared the structure with published crystal structures of other human PLD family members which revealed that a number of catalytic and lipid recognition residues previously shown to be key for phospholipase activity are not conserved or are absent This led us to test whether the enzyme ... More
Human 5′-3′ exonuclease PLD3, a member of the phospholipase D family of enzymes, has been validated as a therapeutic target for treating Alzheimer's disease. Here, we have determined the crystal structure of the luminal domain of the enzyme at 2.3 Å resolution, revealing a bilobal structure with a catalytic site located between the lobes. We then compared the structure with published crystal structures of other human PLD family members which revealed that a number of catalytic and lipid recognition residues, previously shown to be key for phospholipase activity, are not conserved or, are absent. This led us to test whether the enzyme is actually a phospholipase. We could not measure any phospholipase activity but the enzyme shows robust nuclease activity. Finally, we have mapped key single nucleotide polymorphisms onto the structure which reveals plausible reasons as to why they have an impact on Alzheimer's disease. Less
This thesis focusses on developing novel data driven oral drug formulation analysis methods by employing technologies such as Fourier transform analysis and generative adversarial learning Data driven measurements have been addressing challenges in advanced manufacturing and analysis for pharmaceutical development for the last two decade Data science combined with analytical chemistry holds the future to solving key problems in the next wave of industrial research and development Data acquisition is expensive in the realm of pharmaceutical development and how to leverage the capability of data science to extract information in data deprived circumstances is a key aspect for improving such ... More
This thesis focusses on developing novel data driven oral drug formulation analysis methods by employing technologies such as Fourier transform analysis and generative adversarial learning. Data driven measurements have been addressing challenges in advanced manufacturing and analysis for pharmaceutical development for the last two decade. Data science combined with analytical chemistry holds the future to solving key problems in the next wave of industrial research and development. Data acquisition is expensive in the realm of pharmaceutical development, and how to leverage the capability of data science to extract information in data deprived circumstances is a key aspect for improving such data driven measurements. Among multiple measurement techniques, chemical imaging is an informative tool for analyzing oral drug formulations. However, chemical imaging can often fall into data deprived situations, where data could be limited from the time-consuming sample preparation or related chemical synthesis. An integrated imaging approach, which folds data science techniques into chemical measurements, could lead to a future of informative and cost-effective data driven measurements. In this thesis, the development of data driven chemical imaging techniques for the analysis of oral drug formulations via Fourier transformation and generative adversarial learning are elaborated. Chapter 1 begins with a brief introduction of current techniques commonly implemented within the pharmaceutical industry, their limitations, and how the limitations are being addressed. Chapter 2 discusses how Fourier transform fluorescence recovery after photobleaching (FT-FRAP) technique can be used for monitoring the phase separated drug-polymer aggregation. Chapter 3 follows the innovation presented in Chapter 1 and illustrates how analysis can be improved by incorporating diffractive optical elements in the patterned illumination. While previous chapters discuss dynamic analysis aspects of drug product formulation, Chapter 4 elaborates on the innovation in composition analysis of oral drug products via use of novel generative adversarial learning methods for linear analyses. Less
Photosensory receptors essential molecular entities across all domains of life enable organisms to detect and respond to light stimuli underpinning their critical involvement in regulating biological processes such as phototropism circadian rhythms photomorphogenesis and photosynthesis Among the myriad types of photosensory receptors blue light sensing proteins such as Blue Light Using Flavin BLUF photoreceptors distinguish themselves through their ability to utilize blue light for signalling Characterized by the conserved structure of their sensor domain BLUF photoreceptors are found in a wide array of organisms from bacteria and algae to plants and certain fungi Known for their capacity to bind flavin ... More
Photosensory receptors, essential molecular entities across all domains of life, enable organisms to
detect and respond to light stimuli, underpinning their critical involvement in regulating biological
processes such as phototropism, circadian rhythms, photomorphogenesis, and photosynthesis.
Among the myriad types of photosensory receptors, blue light sensing proteins such as Blue Light
Using Flavin (BLUF) photoreceptors distinguish themselves through their ability to utilize blue
light for signalling. Characterized by the conserved structure of their sensor domain, BLUF
photoreceptors are found in a wide array of organisms, from bacteria and algae to plants and certain
fungi. Known for their capacity to bind flavin chromophores, typically flavin adenine dinucleotide
(FAD), they undergo conformational changes upon blue photon absorption, leading to downstream
signalling events, highlighting their pivotal role in the adaptive responses of various organisms to
light. This dissertation provides a comprehensive exploration of the BLUF photoreceptors,
particularly focusing on the Photoactivated Adenylate Cyclase protein from Oscillatoria acuminata
(OaPAC), which comprises a BLUF sensor domain linked to an Adenylate Cyclase (AC) effector
domain, catalysing the conversion of ATP into cAMP. This study aims to elucidate the
photoactivation mechanism of OaPAC and the ensuing signal transduction pathway, employing an
integrative approach that leverages time-resolved crystallography, small angle X-ray scattering,
spectroscopy, and biochemical characterization techniques. Special emphasis is placed on the TyrGln-Met triad in the BLUF domain, which plays a crucial role in the initial light-induced
rearrangements. Additionally, significant attention is given to the less understood aspects of BLUF
photoreceptors, particularly the transduction of the initial light signal to more distal parts of the
protein, which ultimately leads to biological activity. This research identifies a Metout/Trpin
transition as a crucial element in conveying the signal to the α-helix linker region. Finally, structural
models of OaPAC with ATP bound in the active site, along with complementary FTIR
investigations, provide a thorough understanding of ATP binding and allosteric communication. As
a result, the research presented in this dissertation not only expands the fundamental understanding
of BLUF photoreceptor biology, but also provides a framework for future studies aimed at
deciphering complete signal transduction pathways in multi-domain BLUF photoreceptors and
towards the development of optogenetic tools Less
detect and respond to light stimuli, underpinning their critical involvement in regulating biological
processes such as phototropism, circadian rhythms, photomorphogenesis, and photosynthesis.
Among the myriad types of photosensory receptors, blue light sensing proteins such as Blue Light
Using Flavin (BLUF) photoreceptors distinguish themselves through their ability to utilize blue
light for signalling. Characterized by the conserved structure of their sensor domain, BLUF
photoreceptors are found in a wide array of organisms, from bacteria and algae to plants and certain
fungi. Known for their capacity to bind flavin chromophores, typically flavin adenine dinucleotide
(FAD), they undergo conformational changes upon blue photon absorption, leading to downstream
signalling events, highlighting their pivotal role in the adaptive responses of various organisms to
light. This dissertation provides a comprehensive exploration of the BLUF photoreceptors,
particularly focusing on the Photoactivated Adenylate Cyclase protein from Oscillatoria acuminata
(OaPAC), which comprises a BLUF sensor domain linked to an Adenylate Cyclase (AC) effector
domain, catalysing the conversion of ATP into cAMP. This study aims to elucidate the
photoactivation mechanism of OaPAC and the ensuing signal transduction pathway, employing an
integrative approach that leverages time-resolved crystallography, small angle X-ray scattering,
spectroscopy, and biochemical characterization techniques. Special emphasis is placed on the TyrGln-Met triad in the BLUF domain, which plays a crucial role in the initial light-induced
rearrangements. Additionally, significant attention is given to the less understood aspects of BLUF
photoreceptors, particularly the transduction of the initial light signal to more distal parts of the
protein, which ultimately leads to biological activity. This research identifies a Metout/Trpin
transition as a crucial element in conveying the signal to the α-helix linker region. Finally, structural
models of OaPAC with ATP bound in the active site, along with complementary FTIR
investigations, provide a thorough understanding of ATP binding and allosteric communication. As
a result, the research presented in this dissertation not only expands the fundamental understanding
of BLUF photoreceptor biology, but also provides a framework for future studies aimed at
deciphering complete signal transduction pathways in multi-domain BLUF photoreceptors and
towards the development of optogenetic tools Less
Neurodegenerative diseases NDDs characterized by progressive neuronal death and misfolded protein aggregation pose significant clinical social and personal challenges Parkinson's Disease PD the second most common neurological disorder is notably associated with the aggregation of alpha-synuclein aSyn Despite its prominence the transition of monomeric aSyn to aggregates remains inadequately understood Recent studies suggest that Liquid-Liquid Phase Separation LLPS and disease related metal ions involve the transition in the molecular pathogenesis of PD LLPS involves the separation of biomolecules into distinct phases without a membrane potentially facilitating aSyn aggregation through dynamic condensates that eventually form solid deposits I aim to investigate ... More
Neurodegenerative diseases (NDDs), characterized by progressive neuronal death and misfolded protein aggregation, pose significant clinical, social, and personal challenges. Parkinson's Disease (PD), the second most common neurological disorder, is notably associated with the aggregation of alpha-synuclein (aSyn). Despite its prominence, the transition of monomeric aSyn to aggregates remains inadequately understood. Recent studies suggest that Liquid-Liquid Phase Separation (LLPS) and disease related metal ions involve the transition in the molecular pathogenesis of PD. LLPS involves the separation of biomolecules into distinct phases without a membrane, potentially facilitating aSyn aggregation through dynamic condensates that eventually form solid deposits. I aim to investigate LLPS of aSyn and macroscopic dynamics of its formed droplets over time, and examine how PD related metal ions, affect the dynamic process of LLPS and modulate its toxicity to neuroblastoma cells. These metal ions, prevalent in the brain and specifically interacting with aSyn are presumably modulating LLPS, toxicity and aggregation of aSyn, making it crucial to understand their roles in the molecular pathogenesis of PD I expressed α-synuclein (aSyn) proteins in E. coli and studied the biophysical properties and toxicities of aSyn-metal ion coacervates using various techniques, including a protein crystallization robotic dispenser and confocal microscopy. In the presence of metal ions such as CuCl₂, MnCl₂, ZnCl₂, and FeCl₃, the number of droplets significantly decreased. I found that CuCl₂ ions immobilize aSyn condensates and increase their toxicity. In contrast, MnCl₂, ZnCl₂, and FeCl₃ help maintain a longer metastable state of the condensates, reducing their toxicity. This project highlights the crucial role of metal ions in modulating aSyn phase behavior, condensate toxicity and their potential involvement in the progression of PD. Less
VRC -class broadly neutralizing antibodies bnAbs have been isolated from people with HIV- but they have not yet been elicited by vaccination They are extensively somatically mutated and sometimes accumulate CDRL deletions Such indels may allow VRC -class antibodies to accommodate the glycans expressed on a conserved N N-linked glycosylation site in loop D of the gp subunit These glycans constitute a major obstacle in the development of VRC -class antibodies as unmutated antibody forms are unable to accommodate them Although immunizations of knock-in mice expressing human VRC -class B-cell receptors BCRs with specifically designed Env-derived immunogens lead to the ... More
VRC01-class broadly neutralizing antibodies (bnAbs) have been isolated from people with HIV-1, but they have not yet been elicited by vaccination. They are extensively somatically mutated and sometimes accumulate CDRL1 deletions. Such indels may allow VRC01-class antibodies to accommodate the glycans expressed on a conserved N276 N-linked glycosylation site in loop D of the gp120 subunit. These glycans constitute a major obstacle in the development of VRC01-class antibodies, as unmutated antibody forms are unable to accommodate them. Although immunizations of knock-in mice expressing human VRC01-class B-cell receptors (BCRs) with specifically designed Env-derived immunogens lead to the accumulation of somatic mutations in VRC01-class BCRs, CDRL1 deletions are rarely observed, and the elicited antibodies display narrow neutralizing activities. The lack of broad neutralizing potential could be due to the absence of deletions, the lack of appropriate somatic mutations, or both. To address this point, we modified our previously determined prime-boost immunization with a germline-targeting immunogen nanoparticle (426c.Mod.Core), followed by a heterologous core nanoparticle (HxB2.WT.Core), by adding a final boost with a cocktail of various stabilized soluble Env trimers. We isolated VRC01-like antibodies with extensive somatic mutations and, in one case, a seven-amino acid CDRL1 deletion. We generated chimeric antibodies that combine the vaccine-elicited somatic mutations with CDRL1 deletions present in human mature VRC01 bnAbs. We observed that CDRL1 indels did not improve the neutralizing antibody activities. Our study indicates that CDRL1 length by itself is not sufficient for the broadly neutralizing phenotype of this class of antibodies. Less
Regulation of Ras GTPases by GTPase activating proteins GAP is essential for their normal signaling Nine of the ten GAPs for Ras contain a C domain immediately proximal to their canonical GAP domain and in RasGAP p GAP p RasGAP RASA mutation of this domain is associated with vascular malformations in humans Here we show that the C domain of RasGAP is required for full catalytic activity towards Ras Analysis of the RasGAP C -GAP crystal structure AlphaFold models and sequence conservation reveal direct C domain interaction with the Ras allosteric lobe This is achieved by an evolutionarily conserved surface ... More
Regulation of Ras GTPases by GTPase activating proteins (GAP) is essential for their normal signaling. Nine of the ten GAPs for Ras contain a C2 domain immediately proximal to their canonical GAP domain, and in RasGAP (p120GAP, p120RasGAP; RASA1) mutation of this domain is associated with vascular malformations in humans. Here, we show that the C2 domain of RasGAP is required for full catalytic activity towards Ras. Analysis of the RasGAP C2-GAP crystal structure, AlphaFold models, and sequence conservation reveal direct C2 domain interaction with the Ras allosteric lobe. This is achieved by an evolutionarily conserved surface centered around RasGAP residue R707, point mutation of which impairs the catalytic advantage conferred by the C2 domain in vitro. In mice, R707C mutation phenocopies the vascular and signaling defects resulting from constitutive disruption of the RASA1 gene. In SynGAP, mutation of the equivalent conserved C2 domain surface impairs catalytic activity. Our results indicate that the C2 domain is required to achieve full catalytic activity of Ras GTPase activating proteins. Less
Bacillus circulans xylanase BcX from the glycoside hydrolase family degrades xylan through a retaining double-displacement mechanism The enzyme is thought to hydrolyze glycosidic bonds in a processive manner and has a large active site cleft with six subsites allowing the binding of six xylose units Such an active site architecture suggests that oligomeric xylose substrates can bind in multiple ways In the crystal structure of the catalytically inactive variant BcX E Q the substrate xylotriose is observed in the active site as well as bound to the known secondary binding site and a third site on the protein surface Nuclear ... More
Bacillus circulans xylanase (BcX) from the glycoside hydrolase family 11 degrades xylan through a retaining, double-displacement mechanism. The enzyme is thought to hydrolyze glycosidic bonds in a processive manner and has a large, active site cleft, with six subsites allowing the binding of six xylose units. Such an active site architecture suggests that oligomeric xylose substrates can bind in multiple ways. In the crystal structure of the catalytically inactive variant BcX E78Q, the substrate xylotriose is observed in the active site, as well as bound to the known secondary binding site and a third site on the protein surface. Nuclear magnetic resonance (NMR) titrations with xylose oligomers of different lengths yield nonlinear chemical shift trajectories for active site nuclei resonances, indicative of multiple binding orientations for these substrates for which binding and dissociation are in fast exchange on the NMR timescale, exchanging on the micro- to millisecond timescale. Active site binding can be modeled with a 2 : 1 model with dissociation constants in the low and high millimolar range. Extensive mutagenesis of active site residues indicates that tight binding occurs in the glycon binding site and is stabilized by Trp9 and the thumb region. Mutations F125A and W71A lead to large structural rearrangements. Binding at the glycon site is sensed throughout the active site, whereas the weak binding mostly affects the aglycon site. The interactions with the two active site locations are largely independent of each other and of binding at the secondary binding site. Less
A strategy for pandemic preparedness is the development of antivirals against a wide set of viral targets with complementary mechanisms of action SARS-CoV- nsp -mac is a viral macrodomain with ADP-ribosylhydrolase activity which counteracts host immune response Targeting the virus' immunomodulatory functionality offers a differentiated strategy to inhibit SARS-CoV- compared to approved therapeutics which target viral replication directly Here we report a fragment-based lead generation campaign guided by computational approaches We discover tool compounds which inhibit nsp -mac activity at low nanomolar concentrations and with responsive structure-activity relationships high selectivity and drug-like properties Using our inhibitors we show that inhibition ... More
A strategy for pandemic preparedness is the development of antivirals against a wide set of viral targets with complementary mechanisms of action. SARS-CoV-2 nsp3-mac1 is a viral macrodomain with ADP-ribosylhydrolase activity, which counteracts host immune response. Targeting the virus' immunomodulatory functionality offers a differentiated strategy to inhibit SARS-CoV-2 compared to approved therapeutics, which target viral replication directly. Here we report a fragment-based lead generation campaign guided by computational approaches. We discover tool compounds which inhibit nsp3-mac1 activity at low nanomolar concentrations, and with responsive structure-activity relationships, high selectivity, and drug-like properties. Using our inhibitors, we show that inhibition of nsp3-mac1 increases ADP-ribosylation, but surprisingly does not translate to demonstrable antiviral activity in cell culture and iPSC-derived pneumocyte models. Further, no synergistic activity is observed in combination with interferon gamma, a main protease inhibitor, nor a papain-like protease inhibitor. Our results question the extent to which targeting modulation of innate immunitydriven ADP-ribosylation can influence SARS-CoV-2 replication. Moreover, these findings suggest that nsp3-mac1 might not be a suitable target for antiviral therapeutics development. Less
The phenazine pyocyanin is an important virulence factor of the pathogen Pseudomonas aeruginosa which is on the WHO list of antibiotic resistant priority pathogens In this study the isomerase PhzF a key bacterial enzyme of the pyocyanin biosynthetic pathway was investigated as a pathoblocker target The aim of the pathoblocker strategy is to reduce the virulence of the pathogen without killing it thus preventing the rapid development of resistance Based on crystal structures of PhzF derivatives of the inhibitor hydroxyanthranilic acid were designed Co-crystal structures of the synthesized derivatives with PhzF revealed spacial limitations of the binding pocket of PhzF ... More
The phenazine pyocyanin is an important virulence factor of the pathogen Pseudomonas aeruginosa, which is on the WHO list of antibiotic resistant “priority pathogens”. In this study the isomerase PhzF, a key bacterial enzyme of the pyocyanin biosynthetic pathway, was investigated as a pathoblocker target. The aim of the pathoblocker strategy is to reduce the virulence of the pathogen without killing it, thus preventing the rapid development of resistance. Based on crystal structures of PhzF, derivatives of the inhibitor 3–hydroxyanthranilic acid were designed. Co-crystal structures of the synthesized derivatives with PhzF revealed spacial limitations of the binding pocket of PhzF in the closed conformation. In contrast, ligands aligned to the open conformation of PhzF provided more room for structural modifications. The intrinsic fluorescence of small 3–hydroxyanthranilic acid derivatives enabled direct affinity determinations using FRET assays. The analysis of structure-activity relationships showed that the carboxylic acid moiety is essential for binding to the target enzyme. The results of this study provide fundamental structural insights that will be useful for the design of PhzF-inhibitors. Less
The COVID- pandemic has demonstrated the need for novel therapeutic interventions and improved pandemic preparedness strategies against severe acute respiratory syndrome coronavirus SARS-CoV- This protocol details an optimized crystallization method for the SARS-CoV- nsp macrodomain a potential drug target Using sitting drop vapor diffusion we describe specific buffer conditions and procedures to consistently produce high-quality crystals suitable for XChem fragment screening The method yields crystals that diffract to an average resolution of enabling high-resolution structural studies All structures solved during the development of tool compounds for the SARS-CoV- nsp macrodomain are deposited on the PDB Group deposition G
The COVID- pandemic has demonstrated the need for novel therapeutic interventions and improved pandemic preparedness strategies against severe acute respiratory syndrome coronavirus SARS-CoV- This protocol details an optimized crystallization method for the SARS-CoV- nsp macrodomain a potential drug target Using sitting drop vapor diffusion with seeding we describe specific buffer conditions and procedures to consistently produce high-quality crystals suitable for XChem fragment screening The method yields crystals that diffract to an average resolution of enabling high-resolution structural studies and can also be used for compound development through co-crystallization experiments All structures solved during the development of tool compounds for the ... More
The COVID-19 pandemic has demonstrated the need for novel therapeutic interventions and improved pandemic preparedness strategies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This protocol details an optimized crystallization method for the SARS-CoV-2 nsp3 macrodomain, a potential drug target. Using sitting drop vapor diffusion with seeding, we describe specific buffer conditions and procedures to consistently produce high-quality crystals suitable for XChem fragment screening. The method yields crystals that diffract to an average resolution of 1.5 Å, enabling high-resolution structural studies and can also be used for compound development through co-crystallization experiments.
All structures solved during the development of tool compounds for the SARS-CoV-2 nsp3 macrodomain are deposited on the PDB (Group deposition: G_1002283). Less
All structures solved during the development of tool compounds for the SARS-CoV-2 nsp3 macrodomain are deposited on the PDB (Group deposition: G_1002283). Less
In diderm bacteria the Lol pathway canonically mediates the periplasmic transport of lipoproteins from the inner membrane IM to the outer membrane OM and therefore plays an essential role in bacterial envelope homeostasis After extrusion of modified lipoproteins from the IM via the LolCDE complex the periplasmic chaperone LolA carries lipoproteins through the periplasm and transfers them to the OM lipoprotein insertase LolB itself a lipoprotein with a LolA-like fold Yet LolB homologs appear restricted to -proteobacteria and are missing from spirochetes like the tick-borne Lyme disease pathogen Borrelia burgdorferi suggesting a different hand-off mechanism at the OM Here we ... More
In diderm bacteria, the Lol pathway canonically mediates the periplasmic transport of lipoproteins from the inner membrane (IM) to the outer membrane (OM) and therefore plays an essential role in bacterial envelope homeostasis. After extrusion of modified lipoproteins from the IM via the LolCDE complex, the periplasmic chaperone LolA carries lipoproteins through the periplasm and transfers them to the OM lipoprotein insertase LolB, itself a lipoprotein with a LolA-like fold. Yet, LolB homologs appear restricted to ψ-proteobacteria and are missing from spirochetes like the tick-borne Lyme disease pathogen Borrelia burgdorferi, suggesting a different hand-off mechanism at the OM. Here, we solved the crystal structure of the B. burgdorferi LolA homolog BB0346 (LolABb) at 1.9 Å resolution. We identified multiple structural deviations in comparative analyses to other solved LolA structures, particularly a unique LolB-like protruding loop domain. LolABb failed to complement an Escherichia coli lolA knockout, even after codon optimization, signal I peptide adaptation, and a C-terminal chimerization which had allowed for complementation with an α-proteobacterial LolA. Analysis of a conditional B. burgdorferi lolA knockout strain indicated that LolABb was essential for growth. Intriguingly, protein localization assays indicated that initial depletion of LolABb led to an emerging mislocalization of both IM and periplasmic OM lipoproteins, but not surface lipoproteins. Together, these findings further support the presence of two separate primary secretion pathways for periplasmic and surface OM lipoproteins in B. burgdorferi and suggest that the distinct structural features of LolABb allow it to function in a unique LolB-deficient lipoprotein sorting system. Less
Iron storage proteins e g vertebrate ferritin and the ferritin-like bacterioferritin Bfr and bacterial ferritin Ftn are spherical hollow proteins that catalyze the oxidation of Fe at binuclear iron ferroxidase centers FOC and store the Fe in their interior thus protecting cells from unwanted Fe Fe redox cycling and storing iron at concentrations far above the solubility of Fe Vertebrate ferritins are heteropolymers of H and L subunits with only the H subunits having FOC Bfr and Ftn were thought to coexist in bacteria as homopolymers but recent evidence indicates these molecules are heteropolymers assembled from Bfr and Ftn subunits ... More
Iron storage proteins, e.g., vertebrate ferritin, and the ferritin-like bacterioferritin (Bfr) and bacterial ferritin (Ftn), are spherical, hollow proteins that catalyze the oxidation of Fe2+ at binuclear iron ferroxidase centers (FOC) and store the Fe3+ in their interior, thus protecting cells from unwanted Fe3+/Fe2+ redox cycling and storing iron at concentrations far above the solubility of Fe3+. Vertebrate ferritins are heteropolymers of H and L subunits with only the H subunits having FOC. Bfr and Ftn were thought to coexist in bacteria as homopolymers, but recent evidence indicates these molecules are heteropolymers assembled from Bfr and Ftn subunits. Despite the heteropolymeric nature of vertebrate and bacterial ferritins, structures have been determined only for recombinant proteins constituted by a single subunit type. Herein we report the structure of Acinetobacter baumannii bacterioferritin, the first structural example of a heteropolymeric ferritin or ferritin-like molecule, assembled from completely overlapping Ftn homodimers harboring FOC and Bfr homodimers devoid of FOC but binding heme. The Ftn homodimers function by catalyzing the oxidation of Fe2+ to Fe3+, while the Bfr homodimers bind a cognate ferredoxin (Bfd) which reduces the stored Fe3+ by transferring electrons via the heme, enabling Fe2+ mobilization to the cytosol for incorporation in metabolism. Less
Inhibition of the proteolytic processing of hepatocyte growth factor HGF and macrophage stimulating protein MSP is an attractive approach for the drug discovery of novel anticancer therapeutics which prevent tumor progression and metastasis Here we utilized an improved and expanded version of positional scanning of substrate combinatorial libraries PS-SCL technique called HyCoSuL to optimize peptidomimetic inhibitors of the HGF MSP activating serine proteases HGFA matriptase and hepsin These inhibitors have an electrophilic ketone serine trapping warhead and thus form a reversible covalent bond to the protease We demonstrate that by varying the P P and P positions of the inhibitor ... More
Inhibition of the proteolytic processing of hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP) is an attractive approach for the drug discovery of novel anticancer therapeutics which prevent tumor progression and metastasis. Here, we utilized an improved and expanded version of positional scanning of substrate combinatorial libraries (PS-SCL) technique called HyCoSuL to optimize peptidomimetic inhibitors of the HGF/MSP activating serine proteases, HGFA, matriptase, and hepsin. These inhibitors have an electrophilic ketone serine trapping warhead and thus form a reversible covalent bond to the protease. We demonstrate that by varying the P2, P3, and P4 positions of the inhibitor with unnatural amino acids based on the protease substrate preferences learned from HyCoSuL, we can predictably modify the potency and selectivity of the inhibitor. We identified the tetrapeptide JH-1144 (8) as a single digit nM inhibitor of HGFA, matriptase and hepsin with excellent selectivity over Factor Xa and thrombin. These unnatural peptides have increased metabolic stability relative to natural peptides of similar structure. The tripeptide inhibitor PK-1-89 (2) has excellent pharmacokinetics in mice with good compound exposure out to 24 h. In addition, we obtained an X-ray structure of the inhibitor MM1132 (15) bound to matriptase revealing an interesting binding conformation useful for future inhibitor design. Less
Nontuberculous mycobacteria NTM are emerging human pathogens linked to severe pulmonary diseases Current treatments involve the prolonged use of multiple drugs and are often ineffective Bacterial dihydrofolate reductase DHFR is a key enzyme targeted by antibiotics in Gram-negative bacterial infections However existing DHFR inhibitors designed for Gram-negative bacteria often fail against mycobacterial DHFRs Here we detail the rational design of NTM DHFR inhibitors based on P a malarial DHFR inhibitor We identified a -diaminopyrimidine exhibiting improved pharmacological properties and activity against purified DHFR and whole cell cultures of two predominant NTM species Mycobacterium avium and Mycobacterium abscessus This study underscores ... More
Nontuberculous mycobacteria (NTM) are emerging human pathogens linked to severe pulmonary diseases. Current treatments involve the prolonged use of multiple drugs and are often ineffective. Bacterial dihydrofolate reductase (DHFR) is a key enzyme targeted by antibiotics in Gram-negative bacterial infections. However, existing DHFR inhibitors designed for Gram-negative bacteria often fail against mycobacterial DHFRs. Here, we detail the rational design of NTM DHFR inhibitors based on P218, a malarial DHFR inhibitor. We identified 8, a 2,4-diaminopyrimidine exhibiting improved pharmacological properties and activity against purified DHFR and whole cell cultures of two predominant NTM species: Mycobacterium avium and Mycobacterium abscessus. This study underscores the potential of 8 as a promising candidate for the in vivo validation of DHFR as an effective treatment against NTM infections. Less
In cyanobacteria Elongation factor Tu EF-Tu plays a crucial role in the repair of photosystem II PSII which is highly susceptible to oxidative stress induced by light exposure and regulated by reactive oxygen species ROS However the specific molecular mechanism governing the functional regulation of EF-Tu by ROS remains unclear Previous research has shown that a mutated EF-Tu where C is substituted with a Ser residue can alleviate photoinhibition highlighting the important role of C in EF-Tu photosensitivity In this study we elucidated how ROS deactivate EF-Tu by examining the crystal structures of EF-Tu in both wild-type and mutated form ... More
In cyanobacteria, Elongation factor Tu (EF-Tu) plays a crucial role in the repair of photosystem II (PSII), which is highly susceptible to oxidative stress induced by light exposure and regulated by reactive oxygen species (ROS). However, the specific molecular mechanism governing the functional regulation of EF-Tu by ROS remains unclear. Previous research has shown that a mutated EF-Tu, where C82 is substituted with a Ser residue, can alleviate photoinhibition, highlighting the important role of C82 in EF-Tu photosensitivity. In this study, we elucidated how ROS deactivate EF-Tu by examining the crystal structures of EF-Tu in both wild-type and mutated form (C82S) individually at resolutions of 1.7 Å and 2.0 Å in Synechococcus elongatus PCC 7942 complexed with GDP. Specifically, the GDP-bound form of EF-Tu adopts an open conformation with C82 located internally, making it resistant to oxidation. Coordinated conformational changes in switches I and II create a tunnel that positions C82 for ROS interaction, revealing the vulnerability of the closed conformation of EF-Tu to oxidation. An analysis of these two structures reveals that the precise spatial arrangement of C82 plays a crucial role in modulating EF-Tu's response to ROS, serving as a regulatory element that governs photosynthetic biosynthesis. Less
Crystalline suspensions of monoclonal antibodies mAbs have great potential to improve drug substance isolation and purification on a large scale and to be used for drug delivery via high-concentration formulations Crystalline mAb suspensions are expected to have enhanced chemical and physical properties relative to mAb solutions delivered intravenously making them attractive candidates for subcutaneous delivery In contrast to small molecules the development of protein crystalline suspensions is not a widely used approach in the pharmaceutical industry This is mainly due to the challenges in finding crystalline hits and the suboptimal physical properties of the resulting crystallites when hits are found ... More
Crystalline suspensions of monoclonal antibodies (mAbs) have great potential to improve drug substance isolation
and purification on a large scale and to be used for drug delivery via high-concentration formulations. Crystalline mAb suspensions
are expected to have enhanced chemical and physical properties relative to mAb solutions delivered intravenously, making them
attractive candidates for subcutaneous delivery. In contrast to small molecules, the development of protein crystalline suspensions is
not a widely used approach in the pharmaceutical industry. This is mainly due to the challenges in finding crystalline hits and the
suboptimal physical properties of the resulting crystallites when hits are found. Modern advances in instrumentation and increased
knowledge of mAb crystallization have, however, resulted in higher probabilities of discovering crystal forms and improving their
particle properties and characterization. In this regard, physical, analytical characterization plays a central role in the initial steps of
understanding and later optimizing the crystallization of mAbs and requires careful selection of the appropriate tools. This
contribution describes a novel crystal structure of the antibody pembrolizumab and demonstrates the usefulness of small-angle X-ray
scattering (SAXS) for characterizing its crystalline suspensions. It illustrates the advantages of SAXS when used to (i) confirm
crystallinity and crystal phase of crystallites produced in batch mode; (ii) confirm crystallinity under various conditions and detect
variations in crystal phases, enabling fine-tuning of the crystallizations for phase control across multiple batches; (iii) monitor the
physical response and stability of the crystallites in suspension with regard to filtration and washing; and (iv) monitor the physical
stability of the crystallites upon drying. Overall, this work highlights how SAXS is an essential tool for mAb crystallization
characterization. Less
and purification on a large scale and to be used for drug delivery via high-concentration formulations. Crystalline mAb suspensions
are expected to have enhanced chemical and physical properties relative to mAb solutions delivered intravenously, making them
attractive candidates for subcutaneous delivery. In contrast to small molecules, the development of protein crystalline suspensions is
not a widely used approach in the pharmaceutical industry. This is mainly due to the challenges in finding crystalline hits and the
suboptimal physical properties of the resulting crystallites when hits are found. Modern advances in instrumentation and increased
knowledge of mAb crystallization have, however, resulted in higher probabilities of discovering crystal forms and improving their
particle properties and characterization. In this regard, physical, analytical characterization plays a central role in the initial steps of
understanding and later optimizing the crystallization of mAbs and requires careful selection of the appropriate tools. This
contribution describes a novel crystal structure of the antibody pembrolizumab and demonstrates the usefulness of small-angle X-ray
scattering (SAXS) for characterizing its crystalline suspensions. It illustrates the advantages of SAXS when used to (i) confirm
crystallinity and crystal phase of crystallites produced in batch mode; (ii) confirm crystallinity under various conditions and detect
variations in crystal phases, enabling fine-tuning of the crystallizations for phase control across multiple batches; (iii) monitor the
physical response and stability of the crystallites in suspension with regard to filtration and washing; and (iv) monitor the physical
stability of the crystallites upon drying. Overall, this work highlights how SAXS is an essential tool for mAb crystallization
characterization. Less
Ubiquitination is an important post-translational modification that maintains cellular homeostasis by regulating various biological processes Deubiquitinases DUBs are enzymes that reverse the ubiquitination process by catalyzing the removal of ubiquitin from a substrate Abnormal expression or function of DUBs is often associated with the onset and progression of various diseases including cancer Ubiquitin specific proteases USPs which constitute the largest family of DUBs in humans have become the center of interest as potential targets in cancer therapy as many of them display increased activity or are overexpressed in a range of malignant tumors or the tumor microenvironment Two related members ... More
Ubiquitination is an important post-translational modification that maintains cellular homeostasis by regulating various biological processes. Deubiquitinases (DUBs) are enzymes that reverse the ubiquitination process by catalyzing the removal of ubiquitin from a substrate. Abnormal expression or function of DUBs is often associated with the onset and progression of various diseases, including cancer. Ubiquitin specific proteases (USPs), which constitute the largest family of DUBs in humans, have become the center of interest as potential targets in cancer therapy as many of them display increased activity or are overexpressed in a range of malignant tumors or the tumor microenvironment. Two related members of the USP family, USP28 and USP25, share high sequence identities but play diverse biological roles. USP28 regulates cell proliferation, oncogenesis, DNA damage repair and apoptosis, whereas USP25 is involved in the anti-viral response, innate immunity and ER-associated degradation in addition to carcinogenesis. USP28 and USP25 also exhibit different oligomeric states – while USP28 is a constitutively active dimer, USP25 assumes an auto-inhibited tetrameric structure. The catalytic domains of both USP28 and USP25 comprise the canonical, globular USP-domain but contain an additional, extended insertion site called USP25/28 catalytic domain inserted domain (UCID) that mediates oligomerization of the proteins. Disruption of the USP25 tetramer leads to the formation of an activated dimeric protein. However, it is still not clear what triggers its activation. Due to their role in maintaining and stabilizing numerous oncoproteins, USP28 and USP25 have emerged as interesting candidates for anti-cancer therapy. Recent advances in smallmolecular inhibitor development have led to the discovery of relatively potent inhibitors of USP28 and USP25. This thesis focuses on the structural elucidation of USP28 and the biochemical characterization of USP28/USP25, both in complex with representatives of three out of the eight compound classes reported as USP28/USP25-specific inhibitors. The crystal structures of USP28 in complex with the AZ compounds, Vismodegib and FT206 reveal that all three inhibitor classes bind into the same allosteric pocket distant from the catalytic center, located between the palm and the thumb subdomains (the S1-site). Intriguingly, this binding pocket is identical to the UCID-tip binding interface in the USP25 tetramer, rendering the protein in a locked, inactive conformation. Formation of the binding pocket in USP28 requires a shift in the helix α5, which induces conformational changes and local distortion of the binding channel that typically accommodates the C-terminal tail of I Ubiquitin, thus preventing catalysis and abrogating USP28 activity. The key residues of the USP28-inhibitor binding pocket are highly conserved in USP25. Mutagenesis studies of these residues accompanied by biochemical and biophysical assays confirm the proposed mechanism of inhibition and similar binding to USP25. This work provides valuable insights into the inhibition mechanism of the small molecule compounds specifically for the DUBs USP28 and USP25. The USP28-inhibitor complex structures offer a framework to develop more specific and potent inhibitors. Less
Chikungunya virus CHIKV causes severe fever rash and debilitating joint pain that can last for months or even years Millions of people have been infected with CHIKV mostly in low- and middle-income countries and the virus continues to spread into new areas due to the geographical expansion of its mosquito hosts The crystallization protocol and buffer conditions used to obtain reproducible Chikungunya Virus nsP macrodomain crystals suitable for XChem fragment screening
Zoonotic coronaviruses are known to produce severe infections in humans and have been the cause of significant morbidity and mortality worldwide SARS-CoV- was the largest and latest contributor of fatal cases even though MERS-CoV has the highest case-fatality ratio among zoonotic coronaviruses These infections pose a high risk to public health worldwide warranting efforts for the expeditious discovery of antivirals Hence we hereby describe a novel series of inhibitors of coronavirus CLpro embodying an N-substituted -pyrrolidone scaffold envisaged to exploit favorable interactions with the S S subsites and connected to an invariant Leu-Gln P P recognition element Several inhibitors showed ... More
Zoonotic coronaviruses are known to produce
severe infections in humans and have been the cause of significant
morbidity and mortality worldwide. SARS-CoV-2 was the largest
and latest contributor of fatal cases, even though MERS-CoV has
the highest case-fatality ratio among zoonotic coronaviruses. These
infections pose a high risk to public health worldwide warranting
efforts for the expeditious discovery of antivirals. Hence, we hereby
describe a novel series of inhibitors of coronavirus 3CLpro
embodying an N-substituted 2-pyrrolidone scaffold envisaged to
exploit favorable interactions with the S3−S4 subsites and
connected to an invariant Leu-Gln P2−P1 recognition element.
Several inhibitors showed nanomolar antiviral activity in enzyme and cell-based assays, with no significant cytotoxicity. Highresolution
crystal structures of inhibitors bound to the 3CLpro were determined to probe and identify the molecular determinants
associated with binding, to inform the structure-guided optimization of the inhibitors, and to confirm the mechanism of action of the
inhibitors. Less
severe infections in humans and have been the cause of significant
morbidity and mortality worldwide. SARS-CoV-2 was the largest
and latest contributor of fatal cases, even though MERS-CoV has
the highest case-fatality ratio among zoonotic coronaviruses. These
infections pose a high risk to public health worldwide warranting
efforts for the expeditious discovery of antivirals. Hence, we hereby
describe a novel series of inhibitors of coronavirus 3CLpro
embodying an N-substituted 2-pyrrolidone scaffold envisaged to
exploit favorable interactions with the S3−S4 subsites and
connected to an invariant Leu-Gln P2−P1 recognition element.
Several inhibitors showed nanomolar antiviral activity in enzyme and cell-based assays, with no significant cytotoxicity. Highresolution
crystal structures of inhibitors bound to the 3CLpro were determined to probe and identify the molecular determinants
associated with binding, to inform the structure-guided optimization of the inhibitors, and to confirm the mechanism of action of the
inhibitors. Less
Lactate dehydrogenase-A LDHA is the major isoform of lactate dehydrogenases LDH that is overexpressed and linked to poor survival in pancreatic ductal adenocarcinoma PDAC Despite some progress current LDH inhibitors have poor structural and physicochemical properties or exhibit unfavorable pharmacokinetics that have hampered their development The present study reports the synthesis and biological evaluation of a novel class of LDHA inhibitors comprising a succinic acid monoamide motif Compounds and are structurally related analogs that demonstrated potent inhibition of LDHA with IC s of nM and nM respectively We solved cocrystal structures of compound -bound to LDHA that showed that the ... More
Lactate dehydrogenase-A (LDHA) is the major isoform of lactate dehydrogenases (LDH) that is overexpressed and linked to poor survival in pancreatic ductal adenocarcinoma (PDAC). Despite some progress, current LDH inhibitors have poor structural and physicochemical properties or exhibit unfavorable pharmacokinetics that have hampered their development. The present study reports the synthesis and biological evaluation of a novel class of LDHA inhibitors comprising a succinic acid monoamide motif. Compounds 6 and 21 are structurally related analogs that demonstrated potent inhibition of LDHA with IC50s of 46 nM and 72 nM, respectively. We solved cocrystal structures of compound 21-bound to LDHA that showed that the compound binds to a distinct allosteric site between the two subunits of the LDHA tetramer. Inhibition of LDHA correlated with reduced lactate production and reduction of glycolysis in MIA PaCa-2 pancreatic cancer cells. The lead compounds inhibit the proliferation of human pancreatic cancer cell lines and patient-derived 3D organoids and exhibit a synergistic cytotoxic effect with the OXPHOS inhibitor phenformin. Unlike current LDHA inhibitors, 6 and 21 have appropriate pharmacokinetics and ligand efficiency metrics, exhibit up to 73% oral bioavailability, and a cumulative half-life greater than 4 h in mice. Less
The middle MID domain of eukaryotic Argonaute Ago proteins and archaeal and bacterial homologues mediates the interaction with the -terminal nucleotide of miRNA and siRNA guide strands The MID domain of human Ago hAgo is comprised of amino acids with a molecular weight of kDa MID adopts a Rossman-like beta -alpha -beta -alpha -beta -alpha -beta -alpha fold with a nucleotide specificity loop between beta and alpha Multiple crystal structures of nucleotides bound to hAgo MID have been reported whereby complexes were obtained by soaking ligands into crystals of MID domain alone This protocol describes a simplified one-step approach to ... More
The middle (MID) domain of eukaryotic Argonaute (Ago) proteins and archaeal and bacterial homologues mediates the interaction with the 5′-terminal nucleotide of miRNA and siRNA guide strands. The MID domain of human Ago2 (hAgo2) is comprised of 139 amino acids with a molecular weight of 15.56 kDa. MID adopts a Rossman-like beta1-alpha1-beta2-alpha2-beta3-alpha3-beta4-alpha4 fold with a nucleotide specificity loop between beta3 and alpha3. Multiple crystal structures of nucleotides bound to hAgo2 MID have been reported, whereby complexes were obtained by soaking ligands into crystals of MID domain alone. This protocol describes a simplified one-step approach to grow well-diffracting crystals of hAgo2 MID-nucleotide complexes by mixing purified His6-SUMO-MID fusion protein, Ulp1 protease, and excess nucleotide in the presence of buffer and precipitant. The crystal structures of MID complexes with UMP, UTP and 2′-3′ linked α-L-threofuranosyl thymidine-3′-triphosphate (tTTP) are presented. This article also describes fluorescence-based assays to measure dissociation constants (Kd) of MID-nucleotide interactions for nucleoside 5′-monophosphates and nucleoside 3′,5′-bisphosphates. Less
The impact of exchanging the light and heavy chains on the structures of bovine ultralong antibodies
The third complementary-determining regions of the heavy-chain CDR H variable regions VH of some cattle antibodies are highly extended consisting of or more residues These ultralong CDR Hs form -ribbon stalks that protrude from the surface of the antibody with a disulfide cross-linked knob region at their apex that dominates antigen interactions over the other CDR loops The structure of the Fab fragment of a naturally paired bovine ultralong antibody D identified by single B-cell sequencing has been determined to A resolution By swapping the D native light chain with that of an unrelated antigen-unknown ultralong antibody it is shown ... More
The third complementary-determining regions of the heavy-chain (CDR3H) variable regions (VH) of some cattle antibodies are highly extended, consisting of 48 or more residues. These ‘ultralong’ CDR3Hs form �-ribbon stalks that protrude from the surface of the antibody with a disulfide cross-linked knob region at their apex that dominates antigen interactions over the other CDR loops. The structure of the Fab fragment of a naturally paired bovine ultralong antibody (D08), identified by single B-cell sequencing, has been determined to 1.6 A ˚ resolution. By swapping the D08 native light chain with that of an unrelated antigen-unknown ultralong antibody, it is shown that interactions between the CDR3s of the variable domains potentially affect the fine positioning of the ultralong CDR3H; however, comparison with other crystallographic structures shows that crystalline packing is also a major contributor. It is concluded that, on balance, the exact positioning of ultralong CDR3H loops is most likely to be due to the constraints of crystal packing. Less
Protein crystallography is an established method to study the atomic structures of macromolecules and their complexes A prerequisite for successful structure determination is diffraction-quality crystals which may require extensive optimization of both the protein and the conditions and hence projects can stretch over an extended period with multiple users being involved The workflow from crystallization and crystal treatment to deposition and publication is well defined and therefore an electronic laboratory information management system LIMS is well suited to management of the data Completion of the project requires key information on all the steps being available and this information should also ... More
Protein crystallography is an established method to study the atomic structures of macromolecules and their complexes. A prerequisite for successful structure determination is diffraction-quality crystals, which may require extensive optimization of both the protein and the conditions, and hence projects can stretch over an extended period, with multiple users being involved. The workflow from crystallization and crystal treatment to deposition and publication is well defined, and therefore an electronic laboratory information management system (LIMS) is well suited to management of the data. Completion of the project requires key information on all the steps being available and this information should also be made available according to the FAIR principles. As crystallized samples are typically shipped between facilities, a key feature to be captured in the LIMS is the exchange of metadata between the crystallization facility of the home laboratory and, for example, synchrotron facilities. On completion, structures are deposited in the Protein Data Bank (PDB) and the LIMS can include the PDB code in its database, completing the chain of custody from crystallization to structure deposition and publication. A LIMS designed for macromolecular crystallography, IceBear, is available as a standalone installation and as a hosted service, and the implementation of key features for the capture of metadata in IceBear is discussed as an example. Less
We report the biochemical structural and functional characterization of the protein coded by gene PA in the P aeruginosa PAO genome The PA gene had been annotated as coding a probable bacterioferritin Our structural work shows that the product of gene PA is a protein that adopts the Dps subunit fold which oligomerizes into a -mer quaternary structure Unlike Dps however the ferroxidase di-iron centers and iron coordinating ligands are buried within each subunit in a manner identical to that observed in the ferroxidase center of P aeruginosa bacterioferritin Since these structural characteristics correspond to Dps-like proteins we term the ... More
We report the biochemical, structural, and functional characterization of the protein coded by gene PA4880 in the P. aeruginosa PAO1 genome. The PA4880 gene had been annotated as coding a probable bacterioferritin. Our structural work shows that the product of gene PA4880 is a protein that adopts the Dps subunit fold, which oligomerizes into a 12-mer quaternary structure. Unlike Dps, however, the ferroxidase di-iron centers and iron coordinating ligands are buried within each subunit, in a manner identical to that observed in the ferroxidase center of P. aeruginosa bacterioferritin. Since these structural characteristics correspond to Dps-like proteins, we term the protein as P. aeruginosa Dps-like, or Pa DpsL. The ferroxidase centers in Pa DpsL catalyze the oxidation of Fe2+ utilizing O2 or H2O2 as oxidant, and the resultant Fe3+ is compartmentalized in the interior cavity. Interestingly, incubating Pa DpsL with plasmid DNA results in efficient nicking of the DNA and at higher concentrations of Pa DpsL the DNA is linearized and eventually degraded. The nickase and endonuclease activities suggest that Pa DpsL, in addition to participating in the defense of P. aeruginosa cells against iron-induced toxicity, may also participate in the innate immune mechanisms consisting of restriction endonucleases and cognate methyl transferases. Less
Talin herein referring collectively to talin and couples the actomyosin cytoskeleton to integrins and transmits tension to the extracellular matrix Talin also interacts with numerous additional proteins capable of modulating the actin-integrin linkage and thus downstream mechanosignaling cascades Here we demonstrate that the scaffold protein Caskin interacts directly with the R domain of talin through its C-terminal LD motif Caskin also associates with the WAVE regulatory complex to promote cell migration in an Abi -dependent manner Furthermore we demonstrate that the Caskin Abi interaction is regulated by growth factor-induced phosphorylation of Caskin on serine In MCF and UACC cells which ... More
Talin (herein referring collectively to talin 1 and 2) couples the actomyosin cytoskeleton to integrins and transmits tension to the extracellular matrix. Talin also interacts with numerous additional proteins capable of modulating the actin-integrin linkage and thus downstream mechanosignaling cascades. Here, we demonstrate that the scaffold protein Caskin2 interacts directly with the R8 domain of talin through its C-terminal LD motif. Caskin2 also associates with the WAVE regulatory complex to promote cell migration in an Abi1-dependent manner. Furthermore, we demonstrate that the Caskin2–Abi1 interaction is regulated by growth factor-induced phosphorylation of Caskin2 on serine 878. In MCF7 and UACC893 cells, which contain an amplification of CASKIN2, Caskin2 localizes in plasma membrane-associated plaques and around focal adhesions in cortical microtubule stabilization complexes. Taken together, our results identify Caskin2 as a novel talin-binding protein that might not only connect integrin-mediated adhesion to actin polymerization but could also play a role in crosstalk between integrins and microtubules. Less
Given the crucial role of the main protease Mpro in the replication cycle of SARS-CoV- this viral cysteine protease constitutes a high-profile drug target We investigated peptidomimetic azapeptide nitriles as auspicious irreversibly acting inhibitors of Mpro Our systematic approach combined an Mpro active-site scanning by combinatorially assembled azanitriles with structure-based design Encouraged by the bioactive conformation of open-chain inhibitors we conceptualized the novel chemotype of macrocyclic azanitriles whose binding mode was elucidated by cocrystallization This strategy provided a favorable entropic contribution to target binding and resulted in the development of the extraordinarily potent Mpro inhibitor with an IC value of ... More
Given the crucial role of the main protease (Mpro) in the replication cycle of SARS-CoV-2, this viral cysteine protease constitutes a high-profile drug target. We investigated peptidomimetic azapeptide nitriles as auspicious, irreversibly acting inhibitors of Mpro. Our systematic approach combined an Mpro active-site scanning by combinatorially assembled azanitriles with structure-based design. Encouraged by the bioactive conformation of open-chain inhibitors, we conceptualized the novel chemotype of macrocyclic azanitriles whose binding mode was elucidated by cocrystallization. This strategy provided a favorable entropic contribution to target binding and resulted in the development of the extraordinarily potent Mpro inhibitor 84 with an IC50 value of 3.23 nM and a second-order rate constant of inactivation, kinac/Ki, of 448,000 M–1s–1. The open-chain Mpro inhibitor 58, along with the macrocyclic compounds 83 and 84, a broad-spectrum anticoronaviral agent, demonstrated the highest antiviral activity with EC50 values in the single-digit micromolar range. Our findings are expected to promote the future development of peptidomimetic Mpro inhibitors as anti-SARS-CoV-2 agents. Less
Protein crystallization as opposed to well-established chromatography processes has the benefits to reduce production costs while reaching a comparable high purity However monitoring crystallization processes remains a challenge as the produced crystals may interfere with analytical measurements Especially for capturing proteins from complex feedstock containing various impurities establishing reliable process analytical technology PAT to monitor protein crystallization processes can be complicated In heterogeneous mixtures important product characteristics can be found by multivariate analysis and chemometrics thus contributing to the development of a thorough process understanding In this project an analytical set-up is established combining offline analytics on-line ultraviolet visible light ... More
Protein crystallization as opposed to well-established chromatography processes has the benefits to reduce production costs while reaching a comparable high purity. However, monitoring crystallization processes remains a challenge as the produced crystals may interfere with analytical measurements. Especially for capturing proteins from complex feedstock containing various impurities, establishing reliable process analytical technology (PAT) to monitor protein crystallization processes can be complicated. In heterogeneous mixtures, important product characteristics can be found by multivariate analysis and chemometrics, thus contributing to the development of a thorough process understanding. In this project, an analytical set-up is established combining offline analytics, on-line ultraviolet visible light (UV/Vis) spectroscopy, and in-line Raman spectroscopy to monitor a stirred-batch crystallization process with multiple phases and species being present. As an example process, the enzyme Lactobacillus kefir alcohol dehydrogenase (LkADH) was crystallized from clarified Escherichia coli (E. coli) lysate on a 300 mL scale in five distinct experiments, with the experimental conditions changing in terms of the initial lysate solution preparation method and precipitant concentration. Since UV/Vis spectroscopy is sensitive to particles, a cross-flow filtration (cross-flow filtration)-based bypass enabled the on-line analysis of the liquid phase providing information on the lysate composition regarding the nucleic acid to protein ratio. A principal component analysis (PCA) of in situ Raman spectra supported the identification of spectra and wavenumber ranges associated with productspecific information and revealed that the experiments followed a comparable, spectral trend when crystals were present. Based on preprocessed Raman spectra, a partial least squares (PLS) regression model was optimized to monitor the target molecule concentration in real-time. The off-line sample analysis provided information on the crystal number and crystal geometry by automated image analysis as well as the concentration of LkADH and host cell proteins (HCPs) In spite of a complex lysate suspension containing scattering crystals and various impurities, it was possible to monitor the target molecule concentration in a heterogeneous, multi-phase process using spectroscopic methods. With the presented analytical set-up of off-line, particle-sensitive on-line, and in-line analyzers, a crystallization capture process can be characterized better in terms of the geometry, yield, and purity of the crystals. Less
Cyanobacterial CO concentrating mechanisms CCMs sequester a globally consequential proportion of carbon into the biosphere Proteinaceous microcompartments called carboxysomes play a critical role in CCM function housing two enzymes to enhance CO fixation carbonic anhydrase CA and Rubisco Despite its importance our current understanding of the carboxysomal CAs found in -cyanobacteria CsoSCA remains limited particularly regarding the regulation of its activity Here we present a structural and biochemical study of CsoSCA from the cyanobacterium Cyanobium sp PCC Our results show that the Cyanobium CsoSCA is allosterically activated by the Rubisco substrate ribulose- -bisphosphate and forms a hexameric trimer of dimers ... More
Cyanobacterial CO2 concentrating mechanisms (CCMs) sequester a globally consequential proportion of carbon into the biosphere. Proteinaceous microcompartments, called carboxysomes, play a critical role in CCM function, housing two enzymes to enhance CO2 fixation: carbonic anhydrase (CA) and Rubisco. Despite its importance, our current understanding of the carboxysomal CAs found in α-cyanobacteria, CsoSCA, remains limited, particularly regarding the regulation of its activity. Here, we present a structural and biochemical study of CsoSCA from the cyanobacterium Cyanobium sp. PCC7001. Our results show that the Cyanobium CsoSCA is allosterically activated by the Rubisco substrate ribulose-1,5-bisphosphate and forms a hexameric trimer of dimers. Comprehensive phylogenetic and mutational analyses are consistent with this regulation appearing exclusively in cyanobacterial α-carboxysome CAs. These findings clarify the biologically relevant oligomeric state of α-carboxysomal CAs and advance our understanding of the regulation of photosynthesis in this globally dominant lineage. Less
Effector proteins are central to the success of plant pathogens while immunity in hostplants is driven by receptor-mediated recognition of these effectors Understanding the mole-cular details of effector receptor interactions is key for the engineering of novel immunereceptors Here we experimentally determined the crystal structure of the Puccinia graminis f sp tri-tici Pgt effector AvrSr which was not accurately predicted using AlphaFold We charac-terised the role of the conserved cysteine residues in AvrSr using in vitro biochemical assaysand examined Sr -mediated recognition using transient expression in Nicotiana spp andwheat protoplasts The AvrSr structure contains a novel b-strand rich modular fold ... More
Effector proteins are central to the success of plant pathogens, while immunity in hostplants is driven by receptor-mediated recognition of these effectors. Understanding the mole-cular details of effector–receptor interactions is key for the engineering of novel immunereceptors. Here, we experimentally determined the crystal structure of the Puccinia graminis f. sp. tri-tici (Pgt) effector AvrSr27, which was not accurately predicted using AlphaFold2. We charac-terised the role of the conserved cysteine residues in AvrSr27 using in vitro biochemical assaysand examined Sr27-mediated recognition using transient expression in Nicotiana spp. andwheat protoplasts. The AvrSr27 structure contains a novel b-strand rich modular fold consisting of two structu-rally similar domains that bind to Zn2+ ions. The N-terminal domain of AvrSr27 is sufficient forinteraction with Sr27 and triggering cell death. We identified two Pgt proteins structurallyrelated to AvrSr27 but with low sequence identity that can also associate with Sr27, albeitmore weakly. Though only the full-length proteins, trigger Sr27-dependent cell death in tran-sient expression systems. Collectively, our findings have important implications for utilising protein prediction plat-forms for effector proteins, and those embarking on bespoke engineering of immunity recep-tors as solutions to plant disease Less
Eukaryotic innate immune systems use pattern recognition receptors PRRs to sense infection by detecting pathogen-associated molecular patterns which then triggers an immune response Bacteria have similarly evolved immunity proteins that sense certain components of their viral predators known as bacteriophages Although different immunity proteins can recognize different phage-encoded triggers individual bacterial immunity proteins have only been found to sense a single trigger during infection suggesting a one-to-one relationship between bacterial PRRs and their ligands Here we demonstrate that the anti-phage defense protein CapRelSJ in Escherichia coli can directly bind and sense two completely unrelated and structurally different proteins using the ... More
Eukaryotic innate immune systems use pattern recognition receptors (PRRs) to sense infection by detecting pathogen-associated molecular patterns, which then triggers an immune response. Bacteria have similarly evolved immunity proteins that sense certain components of their viral predators known as bacteriophages1–6. Although different immunity proteins can recognize different phage-encoded triggers, individual bacterial immunity proteins have only been found to sense a single trigger during infection, suggesting a one-to-one relationship between bacterial PRRs and their ligands7–11. Here, we demonstrate that the anti-phage defense protein CapRelSJ46 in Escherichia coli can directly bind and sense two completely unrelated and structurally different proteins using the same sensory domain, with overlapping but distinct interfaces. Our results highlight the remarkable versatility of an immune sensory domain, which may be a common property of anti-phage defense systems and enable them to keep pace with their rapidly evolving viral predators. We found that Bas11 phages harbor both trigger proteins that are sensed by CapRelSJ46 during infection, and we demonstrate that such phage can only fully evade CapRelSJ46 defense when both triggers are mutated. Our work reveals how a bacterial immune system that senses more than one trigger can help prevent phages from easily escaping detection, and it may allow detection of a broader range of phages. More generally, our findings illustrate unexpected multifactorial sensing by bacterial defense systems and complex coevolutionary relationships between them and their phage-encoded triggers. Less
Pyruvate kinase PK deficiency is a rare genetic disorder that affects this critical enzyme within the glycolysis pathway In recent years Mitapivat MTPV AG- has emerged as a notable allosteric activator for treating PK deficiency However the allosteric regulatory effects exerted on PK by MTPV are yet to be comprehensively elucidated To shed light on the molecular mechanisms of the allosteric effects we employed crystallography and biophysical methods Our efforts yielded a high-resolution crystal structure of the PK tetramer complexed with MTPV at resolution Isothermal titration calorimetry measurements revealed that MTPV binds to human PK with an affinity of M ... More
Pyruvate kinase (PK) deficiency is a rare genetic disorder that affects this critical enzyme within the glycolysis pathway. In recent years, Mitapivat (MTPV, AG-348) has emerged as a notable allosteric activator for treating PK deficiency. However, the allosteric regulatory effects exerted on PK by MTPV are yet to be comprehensively elucidated. To shed light on the molecular mechanisms of the allosteric effects, we employed crystallography and biophysical methods. Our efforts yielded a high-resolution crystal structure of the PK tetramer complexed with MTPV at 2.1 Å resolution. Isothermal titration calorimetry measurements revealed that MTPV binds to human PK with an affinity of 1 μM. The enhanced structural details now allow for unambiguous analysis of the MTPV-filled cavity intricately embedded within the enzyme. Finally, the structure suggests that MTPV binding induces an allosteric effect on the B-domain situated proximal to the active site. In summary, our study provides valuable insights into the allosteric regulation of PK by MTPV and paves the way for further structure-based drug optimization for therapeutic interventions in PK deficiency. Less
Enteroviruses are the causative agents of paediatric hand-foot-and-mouth disease and a target for pandemic preparedness due to the risk of higher order complications in a large-scale outbreak The A protease of these viruses is responsible for the self-cleavage of the poly protein allowing for correct folding and assembly of capsid proteins in the final stages of viral replication These A proteases are highly conserved between Enterovirus species such as Enterovirus A and Coxsackievirus A Inhibition of the A protease deranges capsid folding and assembly preventing formation of mature virions in host cells and making the protease a valuable target for ... More
Enteroviruses are the causative agents of paediatric hand-foot-and-mouth disease, and a target for pandemic preparedness due to the risk of higher order complications in a large-scale outbreak. The 2A protease of these viruses is responsible for the self-cleavage of the poly protein, allowing for correct folding and assembly of capsid proteins in the final stages of viral replication. These 2A proteases are highly conserved between Enterovirus species, such as Enterovirus A71 and Coxsackievirus A16. Inhibition of the 2A protease deranges capsid folding and assembly, preventing formation of mature virions in host cells and making the protease a valuable target for antiviral activity. Herein, we describe a crystallographic fragment screening campaign that identified 75 fragments which bind to the 2A protease including 38 unique compounds shown to bind within the active site. These fragments reveal a path for the development of non-peptidomimetic inhibitors of the 2A protease with broad-spectrum anti-enteroviral activity. Less
The adenosine A A receptor A AAR belongs to the rhodopsin-like G protein-coupled receptor GPCR family which constitutes the largest class of GPCRs Partial agonists show reduced efficacy as compared to physiological agonists and can even act as antagonists in the presence of a full agonist Here we determined an X-ray crystal structure of the partial A AAR agonist -amino- - H-imidazol- -ylmethyl sulfanyl - -p-hydroxyphenyl- -pyridinedicarbonitrile LUF in complex with the A AAR construct A A-PSB -bRIL stabilized in its inactive conformation and being devoid of any mutations in the ligand binding pocket The determined high-resolution structure resolved water ... More
The adenosine A2A receptor (A2AAR) belongs to the rhodopsin-like G protein-coupled receptor (GPCR) family, which constitutes the largest class of GPCRs. Partial agonists show reduced efficacy as compared to physiological agonists and can even act as antagonists in the presence of a full agonist. Here, we determined an X-ray crystal structure of the partial A2AAR agonist 2-amino-6-[(1H-imidazol-2-ylmethyl)sulfanyl]-4-p-hydroxyphenyl-3,5-pyridinedicarbonitrile (LUF5834) in complex with the A2AAR construct A2A-PSB2-bRIL, stabilized in its inactive conformation and being devoid of any mutations in the ligand binding pocket. The determined high-resolution structure (2.43 Å) resolved water networks and crucial binding pocket interactions. A direct hydrogen bond of the p-hydroxy group of LUF5834 with T883.36 was observed, an amino acid that was mutated to alanine in the most frequently used A2AAR crystallization constructs thus preventing the discovery of its interactions in most of the previous A2AAR co-crystal structures. G protein dissociation studies confirmed partial agonistic activity of LUF5834 as compared to that of the full agonist N-ethylcarboxamidoadenosine (NECA). In contrast to NECA, the partial agonist was still able to bind to the receptor construct locked in its inactive conformation by an S913.39K mutation, although with an affinity lower than that at the native receptor. This could explain the compound’s partial agonistic activity: while full A2AAR agonists bind exclusively to the active conformation, likely following conformational selection, partial agonists bind to active as well as inactive conformations, showing higher affinity for the active conformation. This might be a general mechanism of partial agonism also applicable to other GPCRs. Less
The crystallization protocol and buffer conditions used to obtain reproducible SARS C V- Nucelocapsid crystals suitable for XChem fragment screening
The development of effective broad-spectrum antivirals forms an important part of preparing for future pandemics A cause for concern is the currently emerging pathogen Enterovirus D EV-D which primarily spreads through respiratory routes causing mostly mild to severe respiratory illness but in severe cases acute flaccid myelitis The C protease of EV-D is a potential target for the development of antiviral drugs due to its essential role in the viral life cycle and high sequence conservation This protocol was used to grow D C ProB crystals that were applied high-throughput crystallographic follow up compound screening on D C
The crystallization protocol and buffer conditions used to obtain Zika NS helicase crystals suitable for XChem fragment screening The Zika virus ZIKV discovered in Africa in swiftly spread across continents causing significant concern due to its recent association with microcephaly in newborns and Guillain-Barr syndrome in adults Despite a decrease in prevalence the potential for a resurgence remains necessitating urgent therapeutic interventions Like other flaviviruses ZIKV presents promising drug targets within its replication machinery notably the NS helicase NS Hel protein which plays critical roles in viral replication However a lack of structural information impedes the development of specific inhibitors ... More
The crystallization protocol and buffer conditions used to obtain Zika NS3 helicase crystals suitable for XChem fragment screening. The Zika virus (ZIKV), discovered in Africa in 1947, swiftly spread across continents, causing significant concern due to its recent association with microcephaly in newborns and Guillain-Barré syndrome in adults. Despite a decrease in prevalence, the potential for a resurgence remains, necessitating urgent therapeutic interventions. Like other flaviviruses, ZIKV presents promising drug targets within its replication machinery, notably the NS3 helicase (NS3Hel) protein, which plays critical roles in viral replication. However, a lack of structural information impedes the development of specific inhibitors targeting NS3Hel. This protocol was used to grow Zika NS3 crystals that were applied high-throughput crystallographic fragment screening on ZIKV NS3 Helicase. Less
This research delves into the early nucleation stages of phycocyanin a protein pivotal for its fluorescent properties and crystalline stability and holding considerable potential for biotechnological applications The paper contrasts traditional crystallization methods with the innovative Langmuir Blodgett nanotemplate approach aiming to enhance molecular assembly and nucleation processes The study employs Langmuir Blodgett nanotemplates alongside second-order nonlinear imaging of chiral crystal SONICC spectroscopy This combination is designed to orderly organize phycocyanin molecules and provide a sensitive visualization of early-stage crystal formation capturing the intricate dynamics of protein crystallization The experiments were conducted under controlled conditions where surface pressure was maintained ... More
This research delves into the early nucleation stages of phycocyanin, a protein pivotal for its fluorescent properties and crystalline stability and holding considerable potential for biotechnological applications. The paper contrasts traditional crystallization methods with the innovative Langmuir–Blodgett nanotemplate approach, aiming to enhance molecular assembly and nucleation processes. The study employs Langmuir–Blodgett nanotemplates alongside second-order nonlinear imaging of chiral crystal (SONICC) spectroscopy. This combination is designed to orderly organize phycocyanin molecules and provide a sensitive visualization of early-stage crystal formation, capturing the intricate dynamics of protein crystallization. The experiments were conducted under controlled conditions, where surface pressure was maintained at 26 mN/m and barrier speed at 70 cm/min to optimize the monolayer formation at the air–water interface. The Langmuir–Blodgett method, compared to traditional vapor diffusion techniques, shows improvements in the uniformity and efficiency of nucleation. The sensitivity of SONICC spectroscopy significantly enhances the visualization of the nucleation process, revealing a more structured and uniform crystalline assembly in the early stages of formation. This method demonstrates a substantial improvement in nucleation dynamics, leading to a more orderly growth process and potentially larger, well-ordered crystals. Integrating Langmuir–Blodgett nanotemplates with SONICC spectroscopy offers a significant step in understanding protein crystallization processes with insights into the nucleation and growth of protein crystals and broad implications for refining crystallography methodologies of protein-based biomaterials, contributing to the advancement of structural biology and materials science. Less
Crimean Congo hemorrhagic fever virus CCHFV is a tick-borne virus that can cause severe disease in humans with case fatality rates of Although structures of CCHFV glycoproteins GP and Gc have provided insights into viral entry and defined epitopes of neutralizing and protective antibodies the structure of glycoprotein Gn and its interactions with GP and Gc have remained elusive Here we used structure-guided protein engineering to produce a stabilized GP -Gn-Gc heterotrimeric glycoprotein complex GP -GnH-DS-Gc A cryo-EM structure of this complex provides the molecular basis for GP s association on the viral surface reveals the structure of Gn and ... More
Crimean–Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus that can cause severe disease in humans with case fatality rates of 10–40%. Although structures of CCHFV glycoproteins GP38 and Gc have provided insights into viral entry and defined epitopes of neutralizing and protective antibodies, the structure of glycoprotein Gn and its interactions with GP38 and Gc have remained elusive. Here, we used structure-guided protein engineering to produce a stabilized GP38-Gn-Gc heterotrimeric glycoprotein complex (GP38-GnH-DS-Gc). A cryo-EM structure of this complex provides the molecular basis for GP38’s association on the viral surface, reveals the structure of Gn, and demonstrates that GP38-Gn restrains the Gc fusion loops in the prefusion conformation, facilitated by an N-linked glycan attached to Gn. Immunization with GP38-GnH-DS-Gc conferred 40% protection against lethal IbAr10200 challenge in mice. These data define the architecture of a GP38-Gn-Gc protomer and provide a template for structure-guided vaccine antigen development. Less
Rhomboid intramembrane serine proteases have been implicated in several pathologies and emerge as attractive pharmacological target candidates The most potent and selective rhomboid inhibitors available to date are peptidyl -ketoamides but their selectivity for diverse rhomboid proteases and strategies to modulate it in relevant contexts are poorly understood This gap together with the lack of suitable in vitro models hinders ketoamide development for relevant eukaryotic rhomboid enzymes Here we explore the structure-activity relationship principles of rhomboid inhibiting ketoamides by medicinal chemistry and enzymatic in vitro and in-cell assays with recombinant rhomboid proteases GlpG human mitochondrial rhomboid PARL and human RHBDL ... More
Rhomboid intramembrane serine proteases have been implicated in several pathologies, and emerge as attractive pharmacological target candidates. The most potent and selective rhomboid inhibitors available to date are peptidyl α-ketoamides, but their selectivity for diverse rhomboid proteases and strategies to modulate it in relevant contexts are poorly understood. This gap, together with the lack of suitable in vitro models, hinders ketoamide development for relevant eukaryotic rhomboid enzymes. Here we explore the structure-activity relationship principles of rhomboid inhibiting ketoamides by medicinal chemistry and enzymatic in vitro and in-cell assays with recombinant rhomboid proteases GlpG, human mitochondrial rhomboid PARL and human RHBDL2. We use X-ray crystallography in lipid cubic phase to understand the binding mode of one of the best ketoamide inhibitors synthesised here containing branched terminal substituent bound to GlpG. In addition, to extend the interpretation of the co-crystal structure, we use quantum mechanical calculations and quantify the relative importance of interactions along the inhibitor molecule. These combined experimental analyses implicates that more extensive exploration of chemical space at the prime side is unexpectedly powerful for the selectivity of rhomboid inhibiting ketoamides. Together with variations in the peptide sequence at the non-prime side, or its non-peptidic alternatives, this strategy enables targeted tailoring of potent and selective ketoamides towards diverse rhomboid proteases including disease-relevant ones such as PARL and RHBDL2. Less
Vitamin B cobalamin or Cbl functions as a cofactor in two important enzymatic processes in human cells and life is not sustainable without it B is obtained from food and travels from the stomach through the intestine and into the bloodstream by three B -transporting proteins salivary haptocorrin HC gastric intrinsic factor IF and transcobalamin TC which all bind B with high affinity and require proteolytic degradation to liberate Cbl After intracellular delivery of dietary B Cbl in the aquo hydroxo-Cbl HOCbl form can coordinate various nucleophiles e g glutathione GSH giving rise to glutathionylcobalamin GSCbl a naturally-occurring form of ... More
Vitamin B12 (cobalamin or Cbl) functions as a cofactor in two important enzymatic processes in human cells, and life is not sustainable without it. B12 is obtained from food and travels from the stomach, through the intestine and into the bloodstream by three B12-transporting proteins: salivary haptocorrin (HC), gastric intrinsic factor (IF) and transcobalamin (TC), which all bind B12 with high affinity and require proteolytic degradation to liberate Cbl. After intracellular delivery of dietary B12, Cbl in the aquo/hydroxo-Cbl (HOCbl) form can coordinate various nucleophiles, e.g., glutathione (GSH), giving rise to glutathionylcobalamin (GSCbl), a naturally-occurring form of vitamin B12. Currently there is no data showing whether GSCbl is recognized and transported in the human body. Our crystallographic data shows for the first time the complex between a vitamin B12-transporter and GSCbl, which compared to HOCbl, binds TC equally well. Furthermore, sequence analysis and structural comparisons show that TC recognizes and transports GSCbl and that the residues involved are conserved among TCs from different organisms. Interestingly, HC and IF are not structurally tailored to bind GSCbl. This study provides new insights into the interactions between TC and Cbl. Less
The Swiss Light Source facilitates fragment-based drug-discovery campaigns for academic and industrial users through the Fast Fragment and Compound Screening FFCS software suite This framework is further enriched by the option to utilize the Smart Digital User SDU software for automated data collection across the PXI PXII and PXIII beamlines In this work the newly developed HEIDI webpage https heidi psi ch is introduced a platform crafted using state-of-the-art software architecture and web technologies for sample management of rotational data experiments The HEIDI webpage features a data-review tab for enhanced result visualization and provides programmatic access through a representational state ... More
The Swiss Light Source facilitates fragment-based drug-discovery campaigns for academic and industrial users through the Fast Fragment and Compound Screening (FFCS) software suite. This framework is further enriched by the option to utilize the Smart Digital User (SDU) software for automated data collection across the PXI, PXII and PXIII beamlines. In this work, the newly developed HEIDI webpage (https://heidi.psi.ch) is introduced: a platform crafted using state-of-the-art software architecture and web technologies for sample management of rotational data experiments. The HEIDI webpage features a data-review tab for enhanced result visualization and provides programmatic access through a representational state transfer application programming interface (REST API). The migration of the local FFCS MongoDB instance to the cloud is highlighted and detailed. This transition ensures secure, encrypted and consistently accessible data through a robust and reliable REST API tailored for the FFCS software suite. Collectively, these advancements not only significantly elevate the user experience, but also pave the way for future expansions and improvements in the capabilities of the system. Less
Production of value-added compounds and sustainable materials from agro-industrial residues is essential for better waste management and building of circular economy This includes valorization of hemicellulosic fraction of plant biomass the second most abundant biopolymer from plant cell walls aiming to produce prebiotic oligosaccharides widely explored in food and feed industries In this work we conducted biochemical and biophysical characterization of a prokaryotic two-domain R champanellensis xylanase from glycoside hydrolase GH family RcXyn A and evaluated its applicability for XOS production from glucuronoxylan in combination with two endo-xylanases from GH and GH families and a GH xylobiohydrolase RcXyn A liberates ... More
Production of value-added compounds and sustainable materials from agro-industrial residues is essential for better waste management and building of circular economy. This includes valorization of hemicellulosic fraction of plant biomass, the second most abundant biopolymer from plant cell walls, aiming to produce prebiotic oligosaccharides, widely explored in food and feed industries. In this work, we conducted biochemical and biophysical characterization of a prokaryotic two-domain R. champanellensis xylanase from glycoside hydrolase (GH) family 30 (RcXyn30A), and evaluated its applicability for XOS production from glucuronoxylan in combination with two endo-xylanases from GH10 and GH11 families and a GH11 xylobiohydrolase. RcXyn30A liberates mainly long monoglucuronylated xylooligosaccharides and is inefficient in cleaving unbranched oligosaccharides. Crystallographic structure of RcXyn30A catalytic domain was solved and refined to 1.37 Å resolution. Structural analysis of the catalytic domain releveled that its high affinity for glucuronic acid substituted xylan is due to the coordination of the substrate decoration by several hydrogen bonds and ionic interactions in the subsite −2. Furthermore, the protein has a larger β5-α5 loop as compared to other GH30 xylanases, which might be crucial for creating an additional aglycone subsite (+3) of the catalytic site. Finally, RcXyn30A activity is synergic to that of GH11 xylobiohydrolase. Less
With the rapid advancements in sequencing technologies the identification of single nucleotide mutations has surged surpassing our capacity for functional characterization Remarkably approximately of these disease-linked point mutations are situated within protein regions devoid of a well-defined D structure known as intrinsically disordered regions IDRs These IDRs are recognized for their pivotal roles in the regulation signaling and control of biological processes They can harbor short linear motifs SLiMs that act as mediators in protein-protein interactions PPIs often subject to regulation through post-translational modifications such as phosphorylation Investigating the impact of these IDR mutations on protein-protein interactions is essential for ... More
With the rapid advancements in sequencing technologies, the identification of
single nucleotide mutations has surged, surpassing our capacity for functional
characterization. Remarkably, approximately 20% of these disease-linked point
mutations are situated within protein regions devoid of a well-defined 3D structure,
known as intrinsically disordered regions (IDRs). These IDRs are recognized for
their pivotal roles in the regulation, signaling, and control of biological processes.
They can harbor short linear motifs (SLiMs) that act as mediators in protein-protein
interactions (PPIs), often subject to regulation through post-translational
modifications such as phosphorylation. Investigating the impact of these IDR
mutations on protein-protein interactions is essential for comprehending the
molecular mechanisms underlying human diseases.
In this doctoral thesis, I present a comprehensive exploration of a peptide-based
proteomics screen, employed to scrutinize 36 disease-associated mutations that
impair phosphorylation sites within IDRs. This approach entailed the immobilization
of synthetic peptides, corresponding to the mutated regions, onto a cellulose
membrane. These peptides were then utilized to capture interacting proteins from
cellular extracts. This method facilitated the simultaneous comparison of
interaction partners among wild-type, phosphorylated, and mutated peptide forms,
enabling the functional assessment of individual mutations. Our analysis
uncovered significant disparities
between the interactomes of phosphorylated and non-phosphorylated peptides,
changes frequently attributed to the disruption of phosphorylation-dependent
SLiMs.
Building on our findings, we placed particular emphasis on the S102P mutation
within the transcription factor GATAD1, a mutation associated with dilated
cardiomyopathy. Our screening demonstrated that this mutation disrupts a crucial
phosphorylation site responsible for 14-3-3 protein binding. To delve deeper into
this interaction, we conducted a thorough investigation, employing techniques such
as isothermal titration calorimetry, X-ray crystallography, and alanine scanning
coupled with mass spectrometry. Our meticulous analyses hinted at the regulatory
role of 14-3-3 binding in GATAD1's nucleocytoplasmic transport, achieved by
masking its nuclear localization signal.
In conclusion, this doctoral thesis focuses on the profound impact of pathogenic
mutations within human phosphorylation sites on protein-protein interactions. The
insights from our research shed fresh light on potential molecular mechanisms
underpinning the development of various human diseases, offering a promising
avenue for further investigation and therapeutic exploration. Less
single nucleotide mutations has surged, surpassing our capacity for functional
characterization. Remarkably, approximately 20% of these disease-linked point
mutations are situated within protein regions devoid of a well-defined 3D structure,
known as intrinsically disordered regions (IDRs). These IDRs are recognized for
their pivotal roles in the regulation, signaling, and control of biological processes.
They can harbor short linear motifs (SLiMs) that act as mediators in protein-protein
interactions (PPIs), often subject to regulation through post-translational
modifications such as phosphorylation. Investigating the impact of these IDR
mutations on protein-protein interactions is essential for comprehending the
molecular mechanisms underlying human diseases.
In this doctoral thesis, I present a comprehensive exploration of a peptide-based
proteomics screen, employed to scrutinize 36 disease-associated mutations that
impair phosphorylation sites within IDRs. This approach entailed the immobilization
of synthetic peptides, corresponding to the mutated regions, onto a cellulose
membrane. These peptides were then utilized to capture interacting proteins from
cellular extracts. This method facilitated the simultaneous comparison of
interaction partners among wild-type, phosphorylated, and mutated peptide forms,
enabling the functional assessment of individual mutations. Our analysis
uncovered significant disparities
between the interactomes of phosphorylated and non-phosphorylated peptides,
changes frequently attributed to the disruption of phosphorylation-dependent
SLiMs.
Building on our findings, we placed particular emphasis on the S102P mutation
within the transcription factor GATAD1, a mutation associated with dilated
cardiomyopathy. Our screening demonstrated that this mutation disrupts a crucial
phosphorylation site responsible for 14-3-3 protein binding. To delve deeper into
this interaction, we conducted a thorough investigation, employing techniques such
as isothermal titration calorimetry, X-ray crystallography, and alanine scanning
coupled with mass spectrometry. Our meticulous analyses hinted at the regulatory
role of 14-3-3 binding in GATAD1's nucleocytoplasmic transport, achieved by
masking its nuclear localization signal.
In conclusion, this doctoral thesis focuses on the profound impact of pathogenic
mutations within human phosphorylation sites on protein-protein interactions. The
insights from our research shed fresh light on potential molecular mechanisms
underpinning the development of various human diseases, offering a promising
avenue for further investigation and therapeutic exploration. Less
Despite their lack of a defined D structure intrinsically disordered regions IDRs of proteins play important biological roles Many IDRs contain short linear motifs SLiMs that mediate protein-protein interactions PPIs which can be regulated by post-translational modifications like phosphorylation of pathogenic missense mutations are found in IDRs and understanding how such mutations affect PPIs is essential for unraveling disease mechanisms Here we employ peptide-based interaction proteomics to investigate disease-associated mutations affecting phosphorylation sites Our results unveil significant differences in interactomes between phosphorylated and non-phosphorylated peptides often due to disrupted phosphorylation-dependent SLiMs We focused on a mutation of a serine phosphorylation ... More
Despite their lack of a defined 3D structure, intrinsically disordered regions (IDRs) of proteins play important biological roles. Many IDRs contain short linear motifs (SLiMs) that mediate protein-protein interactions (PPIs), which can be regulated by post-translational modifications like phosphorylation. 20% of pathogenic missense mutations are found in IDRs, and understanding how such mutations affect PPIs is essential for unraveling disease mechanisms. Here, we employ peptide-based interaction proteomics to investigate 36 disease-associated mutations affecting phosphorylation sites. Our results unveil significant differences in interactomes between phosphorylated and non-phosphorylated peptides, often due to disrupted phosphorylation-dependent SLiMs. We focused on a mutation of a serine phosphorylation site in the transcription factor GATAD1, which causes dilated cardiomyopathy. We find that this phosphorylation site mediates interaction with 14-3-3 family proteins. Follow-up experiments reveal the structural basis of this interaction and suggest that 14-3-3 binding affects GATAD1 nucleocytoplasmic transport by masking a nuclear localisation signal. Our results demonstrate that pathogenic mutations of human phosphorylation sites can significantly impact protein-protein interactions, offering insights into potential molecular mechanisms underlying pathogenesis. Less
Light-driven sodium pumps NaRs are unique ion-transporting microbial rhodopsins The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region essential for sodium transport Here we identify a subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base We thoroughly characterize a member of this subgroup namely the protein ErNaR from Erythrobacter sp HL- and show that the additional glutamic acid results in almost complete loss of pH sensitivity for sodium-pumping activity which is in contrast to previously studied NaRs ... More
Light-driven sodium pumps (NaRs) are unique ion-transporting microbial rhodopsins. The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region essential for sodium transport. Here we identify a subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base. We thoroughly characterize a member of this subgroup, namely the protein ErNaR from Erythrobacter sp. HL-111 and show that the additional glutamic acid results in almost complete loss of pH sensitivity for sodium-pumping activity, which is in contrast to previously studied NaRs. ErNaR is capable of transporting sodium efficiently even at acidic pH levels. X-ray crystallography and single particle cryo-electron microscopy reveal that the additional glutamic acid residue mediates the connection between the other two Schiff base counterions and strongly interacts with the aspartic acid of the characteristic NDQ motif. Hence, it reduces its pKa. Our findings shed light on a subgroup of NaRs and might serve as a basis for their rational optimization for optogenetics. Less