434 Citations
CYP C from Nocardia farcinica is a P monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the -position Using protein engineering and substrate modifications based on the crystal structure of CYP C an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved Thus conversion of progesterone by mutant CYP C F A resulted in formation of the corresponding -hydroxylated product -deoxycorticosterone in addition to -hydroxylation Using MD simulation this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F A MD simulation ... More
CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that the entrance of water to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5α-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure–function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation. Less
Mutations in the calcium-binding protein calsequestrin cause a highly lethal familial arrhythmia catecholaminergic polymorphic ventricular tachycardia CPVT In vivo calsequestrin multimerizes into filaments but a compelling atomic-resolution structure of a calsequestrin filament is lacking We report a crystal structure of a cardiac calsequestrin filament with supporting mutation analysis provided by an in vitro fomentation assay We also report and characterize a novel disease-associated calsequestrin mutation S I which localizes to the filament-forming interface In addition we show that a previously reported dominant disease mutation K R maps to the same multimerization surface Both mutations disrupt filamentation suggesting that dominant disease ... More
Mutations in the calcium-binding protein calsequestrin cause a highly lethal familial arrhythmia, catecholaminergic polymorphic ventricular tachycardia (CPVT). In vivo, calsequestrin multimerizes into filaments, but a compelling atomic-resolution structure of a calsequestrin filament is lacking. We report a crystal structure of a cardiac calsequestrin filament with supporting mutation analysis provided by an in vitro fomentation assay. We also report and characterize a novel disease-associated calsequestrin mutation, S173I, which localizes to the filament-forming interface. In addition, we show that a previously reported dominant disease mutation, K180R, maps to the same multimerization surface. Both mutations disrupt filamentation, suggesting that dominant disease arises from defects in multimer formation. A ytterbium-derivatized structure pinpoints multiple credible calcium sites at filament-forming interfaces, explaining the atomic basis of calsequestrin filamentation in the presence of calcium. This work advances our understanding of calsequestrin biochemistry and provides a unifying structure-function molecular mechanism by which dominant-acting calsequestrin mutations provoke lethal arrhythmias. Less
Fungal aryl-alcohol oxidases AAOx are extracellular flavoenzymes that belong to glucose-methanol-choline oxidoreductase family and are responsible for the selective conversion of primary aromatic alcohols into aldehydes and aromatic aldehydes to their corresponding acids with concomitant production of hydrogen peroxide H O as by-product The H O can be provided to lignin degradation pathway a biotechnological property explored in biofuel production In the thermophilic fungus Thermothelomyces thermophilus formerly Myceliophthora thermophila just one AAOx was identified in the exo-proteome
Using coevolution-network interference based on the comparison of two phylogenetically distantly related isolates one from the main group M and the other from the minor group O of HIV- we identify in the C-terminal domain CTD of integrase a new functional motif constituted by four non-contiguous amino acids N K N K Mutating the lysines abolishes integration through decreased -processing and inefficient nuclear import of reverse transcribed genomes Solution of the crystal structures of wt and mutated CTDs shows that the motif generates a positive surface potential that is important for integration The number of charges in the motif appears ... More
Using coevolution-network interference based on the comparison of two phylogenetically distantly related isolates, one from the main group M and the other from the minor group O of HIV-1, we identify, in the C-terminal domain (CTD) of integrase, a new functional motif constituted by four non-contiguous amino acids (N222K240N254K273). Mutating the lysines abolishes integration through decreased 3’-processing and inefficient nuclear import of reverse transcribed genomes. Solution of the crystal structures of wt and mutated CTDs shows that the motif generates a positive surface potential that is important for integration. The number of charges in the motif appears more crucial than their position within the motif. Indeed, the positions of the K could be permutated or additional K could be inserted in the motif, generally without affecting integration per se. Despite this potential genetic flexibility, the NKNK arrangement is strictly conserved in natural sequences, indicative of an effective purifying selection exerted at steps other than integration. Accordingly, reverse transcription was reduced even in the mutants that retained wt integration levels, indicating that specifically the wt sequence is optimal for carrying out the multiple functions integrase exerts. We propose that the existence of several amino acids arrangements within the motif, with comparable efficiencies of integration per se, might have constituted an asset for the acquisition of additional functions during viral evolution. Less
Immuno-oncology approaches that utilize T cell receptors TCRs are becoming highly attractive because of their potential to target virtually all cellular proteins including cancer-specific epitopes via the recognition of peptide-human leukocyte antigen pHLA complexes presented at the cell surface However because natural TCRs generally recognize cancer-derived pHLAs with very weak affinities efforts have been made to enhance their binding strength in some cases by several million-fold In this study we investigated the mechanisms underpinning human TCR affinity enhancement by comparing the crystal structures of engineered enhanced affinity TCRs with those of their wild-type progenitors Additionally we performed molecular dynamics simulations ... More
Immuno-oncology approaches that utilize T cell receptors (TCRs) are becoming highly attractive because of their potential to target virtually all cellular proteins, including cancer-specific epitopes, via the recognition of peptide-human leukocyte antigen (pHLA) complexes presented at the cell surface. However, because natural TCRs generally recognize cancer-derived pHLAs with very weak affinities, efforts have been made to enhance their binding strength, in some cases by several million-fold. In this study, we investigated the mechanisms underpinning human TCR affinity enhancement by comparing the crystal structures of engineered enhanced affinity TCRs with those of their wild-type progenitors. Additionally, we performed molecular dynamics simulations to better understand the energetic mechanisms driving the affinity enhancements. These data demonstrate that supra-physiological binding affinities can be achieved without altering native TCR-pHLA binding modes via relatively subtle modifications to the interface contacts, often driven through the addition of buried hydrophobic residues. Individual energetic components of the TCR-pHLA interaction governing affinity enhancements were distinct and highly variable for each TCR, often resulting from additive, or knock-on, effects beyond the mutated residues. This comprehensive analysis of affinity-enhanced TCRs has important implications for the future rational design of engineered TCRs as efficacious and safe drugs for cancer treatment. Less
The genus Streptomyces is characterized by the production of a wide variety of secondary metabolites with remarkable biological activities and broad antibiotic capabilities The presence of an unprecedented number of genes encoding hydrolytic enzymes with industrial appeal such as epoxide hydrolases EHs reveals its resourceful microscopic machinery The whole-genome sequence of Streptomyces sp CBMAI an endophytic actinobacterium isolated from Citrus sinensis branches was explored by genome mining and a putative -epoxide hydrolase named B EPH and encoded by amino acids was selected for functional and structural studies The crystal structure of B EPH was obtained at a resolution of and ... More
The genus Streptomyces is characterized by the production of a wide variety of secondary metabolites with remarkable biological activities and broad antibiotic capabilities. The presence of an unprecedented number of genes encoding hydrolytic enzymes with industrial appeal such as epoxide hydrolases (EHs) reveals its resourceful microscopic machinery. The whole-genome sequence of Streptomyces sp. CBMAI 2042, an endophytic actinobacterium isolated from Citrus sinensis branches, was explored by genome mining, and a putative α/β-epoxide hydrolase named B1EPH2 and encoded by 344 amino acids was selected for functional and structural studies. The crystal structure of B1EPH2 was obtained at a resolution of 2.2 Å and it was found to have a similar fold to other EHs, despite its hexameric quaternary structure, which contrasts with previously solved dimeric and monomeric EH structures. While B1EPH2 has a high sequence similarity to EHB from Mycobacterium tuberculosis, its cavity is similar to that of human EH. A group of 12 aromatic and aliphatic racemic epoxides were assayed to determine the activity of B1EPH2; remarkably, this enzyme was able to hydrolyse all the epoxides to the respective 1,2-diols, indicating a wide-range substrate scope acceptance. Moreover, the (R)- and (S)-enantiomers of styrene oxide, epichlorohydrin and 1,2-epoxybutane were used to monitor enantiopreference. Taken together, the functional and structural analyses indicate that this enzyme is an attractive biocatalyst for future biotechnological applications. Less
T cell-mediated immunity is governed primarily by T cell receptor TCR recognition of peptide-human leukocyte antigen pHLA complexes and is essential for immunosurveillance and disease control This interaction is generally stabilized by interactions between the HLA surface and TCR germline-encoded complementarity-determining region CDR loops and whereas peptide selectivity is guided by direct interactions with the TCR CDR loops Here we solved the structure of a newly identified TCR in complex with a clinically relevant peptide derived from the cancer testis antigen melanoma antigen-A MAGE-A The TCR bound pHLA in a position shifted toward the peptide's N terminus This enabled the ... More
T cell-mediated immunity is governed primarily by T cell receptor (TCR) recognition of peptide-human leukocyte antigen (pHLA) complexes and is essential for immunosurveillance and disease control. This interaction is generally stabilized by interactions between the HLA surface and TCR germline-encoded complementarity-determining region (CDR) loops 1 and 2, whereas peptide selectivity is guided by direct interactions with the TCR CDR3 loops. Here, we solved the structure of a newly identified TCR in complex with a clinically relevant peptide derived from the cancer testis antigen melanoma antigen-A4 (MAGE-A4). The TCR bound pHLA in a position shifted toward the peptide's N terminus. This enabled the TCR to achieve peptide selectivity via an indirect mechanism, whereby the TCR sensed the first residue of the peptide through HLA residue Trp-167, which acted as a tunable gateway. Amino acid substitutions at peptide position 1 predicted to alter the HLA Trp-167 side-chain conformation abrogated TCR binding, indicating that this indirect binding mechanism is essential for peptide recognition. These findings extend our understanding of the molecular rules that underpin antigen recognition by TCRs and have important implications for the development of TCR-based therapies. Less
Knowledge of both apo and holo states of riboswitches aid in elucidating the various mechanisms of ligand-induced conformational switching that underpin their gene-regulating capabilities Previous structural studies on the flavin mononucleotide FMN -binding aptamer of the FMN riboswitch however have revealed minimal conformational changes associated with ligand binding that do not adequately explain the basis for the switching behavior We have determined a - resolution crystal structure of the ligand-free FMN riboswitch aptamer that is distinct from previously reported structures particularly in the conformation and orientation of the P and P helices The nearly symmetrical tertiary structure provides a mechanism ... More
Knowledge of both apo and holo states of riboswitches aid in elucidating the various mechanisms of ligand-induced conformational “switching” that underpin their gene-regulating capabilities. Previous structural studies on the flavin mononucleotide (FMN)-binding aptamer of the FMN riboswitch, however, have revealed minimal conformational changes associated with ligand binding that do not adequately explain the basis for the switching behavior. We have determined a 2.7-Å resolution crystal structure of the ligand-free FMN riboswitch aptamer that is distinct from previously reported structures, particularly in the conformation and orientation of the P1 and P4 helices. The nearly symmetrical tertiary structure provides a mechanism by which one of two pairs of adjacent helices (P3/P4 or P1/P6) undergo collinear stacking in a mutually exclusive manner, in the absence or presence of ligand, respectively. Comparison of these structures suggests the stem-loop that includes P4 and L4 is important for maintaining a global conformational state that, in the absence of ligand, disfavors formation of the P1 regulatory helix. Together, these results provide further insight to the structural basis for conformational switching of the FMN riboswitch. Less
Complete genome sequencing of the kinetoplastid protozoans Trypanosoma cruzi Trypanosoma brucei and Leishmania major Tritryp published in opened up new perspectives for drug development targeting Chagas disease African sleeping sickness and Leishmaniasis neglected diseases affecting millions of most economically disadvantaged people Still half of the Tritryp genes code for proteins of unknown function Moreover almost of conserved eukaryotic protein domains are missing in the Tritryp genomes This suggests that functional and structural characterization of proteins of unknown function could reveal novel protein folds used by the trypanosomes for common cellular processes Furthermore proteins without homologous counterparts in humans may provide ... More
Complete genome sequencing of the kinetoplastid protozoans Trypanosoma cruzi, Trypanosoma brucei and Leishmania major (Tritryp), published in 2005, opened up new perspectives for drug development targeting Chagas disease, African sleeping sickness and Leishmaniasis, neglected diseases affecting millions of most economically disadvantaged people. Still, half of the Tritryp genes code for proteins of unknown function. Moreover, almost 50% of conserved eukaryotic protein domains are missing in the Tritryp genomes. This suggests that functional and structural characterization of proteins of unknown function could reveal novel protein folds used by the trypanosomes for common cellular processes. Furthermore, proteins without homologous counterparts in humans may provide potential targets for therapeutic intervention. Here we describe the crystal structure of the T. cruzi protein Q4D6Q6, a conserved and kinetoplastid-specific protein essential for cell viability. Q4D6Q6 is a representative of a family of 20 orthologs, all annotated as proteins of unknown function. Q4D6Q6 monomers adopt a ββαββαββ topology and form a propeller-like tetramer. Oligomerization was verified in solution using NMR, SAXS, analytical ultra-centrifugation and gel filtration chromatography. A rigorous search for similar structures using the DALI server revealed similarities with propeller-like structures of several different functions. Although a Q4D6Q6 function could not be inferred from such structural comparisons, the presence of an oxidized cysteine at position 69, part of a cluster with phosphorylated serines and hydrophobic residues, identifies a highly reactive site and suggests a role of this cysteine as a nucleophile in a post-translational modification reaction. Less
NKp is one of the main human natural killer NK cell activating receptors used in directed immunotherapy The oligomerization of the NKp ligand binding domain depends on the length of the C-terminal stalk region but our structural knowledge of NKp oligomerization and its role in signal transduction remains limited Moreover ligand binding of NKp is affected by the presence and type of N-glycosylation In this study we assessed whether NKp oligomerization depends on its N-glycosylation Our results show that NKp forms oligomers when expressed in HEK S GnTI cell lines with simple N-glycans However NKp was detected only as monomers ... More
NKp30 is one of the main human natural killer (NK) cell activating receptors used in directed immunotherapy. The oligomerization of the NKp30 ligand binding domain depends on the length of the C-terminal stalk region, but our structural knowledge of NKp30 oligomerization and its role in signal transduction remains limited. Moreover, ligand binding of NKp30 is affected by the presence and type of N-glycosylation. In this study, we assessed whether NKp30 oligomerization depends on its N-glycosylation. Our results show that NKp30 forms oligomers when expressed in HEK293S GnTI− cell lines with simple N-glycans. However, NKp30 was detected only as monomers after enzymatic deglycosylation. Furthermore, we characterized the interaction between NKp30 and its best-studied cognate ligand, B7-H6, with respect to glycosylation and oligomerization, and we solved the crystal structure of this complex with glycosylated NKp30, revealing a new glycosylation-induced mode of NKp30 dimerization. Overall, this study provides new insights into the structural basis of NKp30 oligomerization and explains how the stalk region and glycosylation of NKp30 affect its ligand affinity. This furthers our understanding of the molecular mechanisms involved in NK cell activation, which is crucial for the successful design of novel NK cell-based targeted immunotherapeutics. Less
T cell recognition of peptides presented by human leukocyte antigens HLAs is mediated by the highly variable T cell receptor TCR Despite this built-in TCR variability individuals can mount immune responses against viral epitopes by using identical or highly related TCRs expressed on CD T cells Characterization of these TCRs has extended our understanding of the molecular mechanisms that govern the recognition of peptide-HLA However few examples exist for CD T cells Here we investigate CD T cell responses to the internal proteins of the influenza A virus that correlate with protective immunity We identify five internal epitopes that are ... More
T cell recognition of peptides presented by human leukocyte antigens (HLAs) is mediated by the highly variable T cell receptor (TCR). Despite this built-in TCR variability, individuals can mount immune responses against viral epitopes by using identical or highly related TCRs expressed on CD8+ T cells. Characterization of these TCRs has extended our understanding of the molecular mechanisms that govern the recognition of peptide-HLA. However, few examples exist for CD4+ T cells. Here, we investigate CD4+ T cell responses to the internal proteins of the influenza A virus that correlate with protective immunity. We identify five internal epitopes that are commonly recognized by CD4+ T cells in five HLA-DR1+ subjects and show conservation across viral strains and zoonotic reservoirs. TCR repertoire analysis demonstrates several shared gene usage biases underpinned by complementary biochemical features evident in a structural comparison. These epitopes are attractive targets for vaccination and other T cell therapies. Less
Ketol-acid reductoisomerase KARI the second enzyme in the branched-chain amino acid biosynthesis pathway is a potential drug target for bacterial infections including Mycobacterium tuberculosis Here we have screened the Medicines for Malaria Venture Pathogen Box against purified M tuberculosis Mt KARI and identified two compounds that have Ki values below nm In Mt cell susceptibility assays one of these compounds exhibited an IC value of m Co-crystallization of this compound - methylsulfonyl methyl - H-benzo b oxazin- -one MMV in complex with Staphylococcus aureus KARI which has identity with Mt KARI NADPH and Mg yielded a structure to resolution However ... More
Ketol-acid reductoisomerase (KARI), the second enzyme in the branched-chain amino acid biosynthesis pathway, is a potential drug target for bacterial infections including Mycobacterium tuberculosis. Here, we have screened the Medicines for Malaria Venture Pathogen Box against purified M. tuberculosis (Mt) KARI and identified two compounds that have Ki values below 200 nm. In Mt cell susceptibility assays one of these compounds exhibited an IC50 value of 0.8 μm. Co-crystallization of this compound, 3-((methylsulfonyl)methyl)-2H-benzo[b][1,4]oxazin-2-one (MMV553002), in complex with Staphylococcus aureus KARI, which has 56 % identity with Mt KARI, NADPH and Mg2+ yielded a structure to 1.72 Å resolution. However, only a hydrolyzed product of the inhibitor (i.e. 3-(methylsulfonyl)-2-oxopropanic acid, missing the 2-aminophenol attachment) is observed in the active site. Surprisingly, Mt cell susceptibility assays showed that the 2-aminophenol product is largely responsible for the anti-TB activity of the parent compound. Thus, 3-(methylsulfonyl)-2-oxopropanic acid was identified as a potent KARI inhibitor that could be further explored as a potential biocidal agent and we have shown 2-aminophenol, as an anti-TB drug lead, especially given it has low toxicity against human cells. The study highlights that careful analysis of broad screening assays is required to correctly interpret cell-based activity data. Less
Outbreaks of human epidemic nonbacterial gastroenteritis are mainly caused by noroviruses Viral replication requires a C-like cysteine protease CLpro which processes the kDa viral polyprotein into six functional proteins The CLpro has attracted much interest due to its potential as a target for antiviral drugs A system for growing high-quality crystals of native Southampton norovirus CLpro SV CP has been established allowing the ligand-free crystal structure to be determined to in a tetrameric state This also allowed crystal-based fragment screening to be performed with various compound libraries ultimately to guide drug discovery for SV CP A total of fragments were ... More
Outbreaks of human epidemic nonbacterial gastroenteritis are mainly caused by noroviruses. Viral replication requires a 3C-like cysteine protease (3CLpro) which processes the 200 kDa viral polyprotein into six functional proteins. The 3CLpro has attracted much interest due to its potential as a target for antiviral drugs. A system for growing high-quality crystals of native Southampton norovirus 3CLpro (SV3CP) has been established, allowing the ligand-free crystal structure to be determined to 1.3 Å in a tetrameric state. This also allowed crystal-based fragment screening to be performed with various compound libraries, ultimately to guide drug discovery for SV3CP. A total of 19 fragments were found to bind to the protease out of the 844 which were screened. Two of the hits were located at the active site of SV3CP and showed good inhibitory activity in kinetic assays. Another 5 were found at the enzyme’s putative RNA-binding site and a further 11 were located in the symmetric central cavity of the tetramer. Less
The major bottlenecks in structure elucidation of nucleic acids are crystallization and phasing Co-crystallization with proteins is a straight forward approach to overcome these challenges The human RNA-binding protein U A has previously been established as crystallization module however the absence of UV-active residues and the predetermined architecture in the asymmetric unit constitute clear limitations of the U A system Here we report three crystal structures of tryptophan-containing U A variants which expand the crystallization toolbox for nucleic acids Analysis of the structures complemented by SAXS NMR spectroscopy and optical spectroscopy allow for insights into the potential of the U ... More
The major bottlenecks in structure elucidation of nucleic acids are crystallization and phasing. Co-crystallization with proteins is a straight forward approach to overcome these challenges. The human RNA-binding protein U1A has previously been established as crystallization module, however, the absence of UV-active residues and the predetermined architecture in the asymmetric unit constitute clear limitations of the U1A system. Here, we report three crystal structures of tryptophan-containing U1A variants, which expand the crystallization toolbox for nucleic acids. Analysis of the structures complemented by SAXS, NMR spectroscopy, and optical spectroscopy allow for insights into the potential of the U1A variants to serve as crystallization modules for nucleic acids. In addition, we report a fast and efficient protocol for crystallization of RNA by soaking and present a fluorescence-based approach for detecting RNA-binding in crystallo. Our results provide a new tool set for the crystallization of RNA and RNA:DNA complexes. Less
Tumor-associated peptide human leukocyte antigen complexes pHLAs represent the largest pool of cell surface expressed cancer-specific epitopes making them attractive targets for cancer therapies Soluble bispecific molecules that incorporate an anti-CD effector function are being developed to redirect T cells against these targets using different approaches The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs e g immune-mobilizing monoclonal T cell receptors against cancer ImmTAC molecules whereas the second harnesses an antibody-based format TCR-mimic antibodies For both classes of reagent target specificity is vital considering the vast universe of potential pHLA molecules that can be presented on healthy ... More
Tumor-associated peptide–human leukocyte antigen complexes (pHLAs) represent the largest pool of cell surface–expressed cancer-specific epitopes, making them attractive targets for cancer therapies. Soluble bispecific molecules that incorporate an anti-CD3 effector function are being developed to redirect T cells against these targets using 2 different approaches. The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs (e.g., immune-mobilizing monoclonal T cell receptors against cancer [ImmTAC] molecules), whereas the second harnesses an antibody-based format (TCR-mimic antibodies). For both classes of reagent, target specificity is vital, considering the vast universe of potential pHLA molecules that can be presented on healthy cells. Here, we made use of structural, biochemical, and computational approaches to investigate the molecular rules underpinning the reactivity patterns of pHLA-targeting bispecifics. We demonstrate that affinity-enhanced TCRs engage pHLA using a comparatively broad and balanced energetic footprint, with interactions distributed over several HLA and peptide side chains. As ImmTAC molecules, these TCRs also retained a greater degree of pHLA selectivity, with less off-target activity in cellular assays. Conversely, TCR-mimic antibodies tended to exhibit binding modes focused more toward hot spots on the HLA surface and exhibited a greater degree of crossreactivity. Our findings extend our understanding of the basic principles that underpin pHLA selectivity and exemplify a number of molecular approaches that can be used to probe the specificity of pHLA-targeting molecules, aiding the development of future reagents. Less
Ruthenium-catalysed azide alkyne cycloaddition RuAAC provides access to -disubstituted -triazole motifs in peptide engineering applications However investigation of this motif as a disulfide mimetic in cyclic peptides has been limited and the structural consequences remain to be studied We report synthetic strategies to install various triazole linkages into cyclic peptides through backbone cyclisation and RuAAC cross-linking reactions These linkages were evaluated in four serine protease inhibitors based on sunflower trypsin inhibitor- NMR and X-ray crystallography revealed exceptional consensus of bridging distance and backbone conformations RMSD of the triazole linkages compared to the parent disulfide molecules The triazole-bridged peptides also displayed ... More
Ruthenium-catalysed azide–alkyne cycloaddition (RuAAC) provides access to 1,5-disubstituted 1,2,3-triazole motifs in peptide engineering applications. However, investigation of this motif as a disulfide mimetic in cyclic peptides has been limited, and the structural consequences remain to be studied. We report synthetic strategies to install various triazole linkages into cyclic peptides through backbone cyclisation and RuAAC cross-linking reactions. These linkages were evaluated in four serine protease inhibitors based on sunflower trypsin inhibitor-1. NMR and X-ray crystallography revealed exceptional consensus of bridging distance and backbone conformations (RMSD<0.5 Å) of the triazole linkages compared to the parent disulfide molecules. The triazole-bridged peptides also displayed superior half-lives in liver S9 stability assays compared to disulfide-bridged peptides. This work establishes a foundation for the application of 1,5-disubstituted 1,2,3-triazoles as disulfide mimetics. Less
Cellular signaling via binding of the cytokines IL- and along with binding of the accessory protein IL- RAcP to their cognate receptor IL- R is believed to play a major role in epithelial and immune cell-mediated inflammation responses Antagonizing the signaling cascade that results from these binding events via a directed monoclonal antibody provides an opportunity to suppress such immune responses We report here the molecular structure of a complex between an extracellular portion of human IL- R and a Fab derived from a high affinity anti-IL- R neutralizing monoclonal antibody at resolution This structure the first of IL- R ... More
Cellular signaling via binding of the cytokines IL-36α, β, and γ along with binding of the accessory protein IL-36RAcP, to their cognate receptor IL-36R is believed to play a major role in epithelial and immune cell-mediated inflammation responses. Antagonizing the signaling cascade that results from these binding events via a directed monoclonal antibody provides an opportunity to suppress such immune responses. We report here the molecular structure of a complex between an extracellular portion of human IL-36R and a Fab derived from a high affinity anti-IL-36R neutralizing monoclonal antibody at 2.3 Å resolution. This structure, the first of IL-36R, reveals similarities with other structurally characterized IL-1R family members and elucidates the molecular determinants leading to the high affinity binding of the monoclonal antibody. The structure of the complex reveals that the epitope recognized by the Fab is remote from both the putative ligand and accessory protein binding interfaces on IL-36R, suggesting that the functional activity of the antibody is noncompetitive for these binding events. Less
The molecular rules driving TCR cross-reactivity are poorly understood and consequently it is unclear the extent to which TCRs targeting the same Ag recognize the same off-target peptides We determined TCR peptide HLA crystal structures and using a single-chain peptide HLA phage library we generated peptide specificity profiles for three newly identified human TCRs specific for the cancer testis Ag NY-ESO- HLA-A Two TCRs engaged the same central peptide feature although were more permissive at peripheral peptide positions and accordingly possessed partially overlapping peptide specificity profiles The third TCR engaged a flipped peptide conformation leading to the recognition of off-target ... More
The molecular rules driving TCR cross-reactivity are poorly understood and, consequently, it is unclear the extent to which TCRs targeting the same Ag recognize the same off-target peptides. We determined TCR–peptide–HLA crystal structures and, using a single-chain peptide–HLA phage library, we generated peptide specificity profiles for three newly identified human TCRs specific for the cancer testis Ag NY-ESO-1157–165–HLA-A2. Two TCRs engaged the same central peptide feature, although were more permissive at peripheral peptide positions and, accordingly, possessed partially overlapping peptide specificity profiles. The third TCR engaged a flipped peptide conformation, leading to the recognition of off-target peptides sharing little similarity with the cognate peptide. These data show that TCRs specific for a cognate peptide recognize discrete peptide repertoires and reconciles how an individual’s limited TCR repertoire following negative selection in the thymus is able to recognize a vastly larger antigenic pool. Less
Formulation conditions have a significant influence on the degree of freeze thaw FT stress-induced protein instabilities Adding cryoprotectants might stabilize the induced FT stress instabilities However a simple preservation of protein stability might be insufficient and further methods are necessary This study aims to evaluate the addition of a heat cycle following FT application as a function of different cryoprotectants with lysozyme as exemplary protein Sucrose and glycerol were shown to be the most effective cryoprotectants when compared to PEG and Tween In terms of heat-induced reversibility of aggregates glycerol showed the best performance followed by sucrose NaCl and Tween ... More
Formulation conditions have a significant influence on the degree of freeze/thaw (FT) stress-induced protein instabilities. Adding cryoprotectants might stabilize the induced FT stress instabilities. However, a simple preservation of protein stability might be insufficient and further methods are necessary. This study aims to evaluate the addition of a heat cycle following FT application as a function of different cryoprotectants with lysozyme as exemplary protein. Sucrose and glycerol were shown to be the most effective cryoprotectants when compared to PEG200 and Tween20. In terms of heat-induced reversibility of aggregates, glycerol showed the best performance followed by sucrose, NaCl and Tween20 systems. The analysis was performed using a novel approach to visualize complex interplays by a clustering and data reduction scheme. In addition, solubility and structural integrity were measured and confirmed the obtained results. Less
The glycyl radical enzyme GRE superfamily utilizes a glycyl radical cofactor to catalyze difficult chemical reactions in a variety of anaerobic microbial metabolic pathways Recently a GRE trans- -hydroxy-L-proline Hyp dehydratase HypD was discovered that catalyzes the dehydration of Hyp to S - -pyrroline- -carboxylic acid P C This enzyme is abundant in the human gut microbiome and also present in prominent bacterial pathogens However we lack an understanding of how HypD performs its unusual chemistry Here we have solved the crystal structure of HypD from the pathogen Clostridioides difficile with Hyp bound in the active site Biochemical studies have ... More
The glycyl radical enzyme (GRE) superfamily utilizes a glycyl radical cofactor to catalyze difficult chemical reactions in a variety of anaerobic microbial metabolic pathways. Recently, a GRE, trans-4-hydroxy-L-proline (Hyp) dehydratase (HypD), was discovered that catalyzes the dehydration of Hyp to (S)-Δ1-pyrroline-5-carboxylic acid (P5C). This enzyme is abundant in the human gut microbiome and also present in prominent bacterial pathogens. However, we lack an understanding of how HypD performs its unusual chemistry. Here, we have solved the crystal structure of HypD from the pathogen Clostridioides difficile with Hyp bound in the active site. Biochemical studies have led to the identification of key catalytic residues and have provided insight into the radical mechanism of Hyp dehydration. Less
The human formyl peptide receptor FPR plays a crucial role in host defense and inflammation and has been considered as a drug target for chronic inflammatory diseases A variety of peptides with different structures and origins have been characterized as FPR ligands However the ligand-binding modes of FPR remain elusive thereby limiting the development of potential drugs Here we report the crystal structure of FPR bound to the potent peptide agonist WKYMVm at resolution The structure adopts an active conformation and exhibits a deep ligand-binding pocket Combined with mutagenesis ligand binding and signaling studies key interactions between the agonist and ... More
The human formyl peptide receptor 2 (FPR2) plays a crucial role in host defense and inflammation, and has been considered as a drug target for chronic inflammatory diseases. A variety of peptides with different structures and origins have been characterized as FPR2 ligands. However, the ligand-binding modes of FPR2 remain elusive, thereby limiting the development of potential drugs. Here we report the crystal structure of FPR2 bound to the potent peptide agonist WKYMVm at 2.8 Å resolution. The structure adopts an active conformation and exhibits a deep ligand-binding pocket. Combined with mutagenesis, ligand binding and signaling studies, key interactions between the agonist and FPR2 that govern ligand recognition and receptor activation are identified. Furthermore, molecular docking and functional assays reveal key factors that may define binding affinity and agonist potency of formyl peptides. These findings deepen our understanding about ligand recognition and selectivity mechanisms of the formyl peptide receptor family. Less
One central question surrounding the biosynthesis of fatty acids and polyketide-derived natural products is how the -phosphopantetheinyl transferase PPTase interrogates the essential acyl carrier protein ACP domain to fulfill the initial activation step The triggering factor of this study was the lack of structural information on PPTases at physiological pH which could bias our comprehension of the mechanism of action of these important enzymes Structural and functional studies on the family II PPTase PptAb of Mycobacterium abscessus show that pH has a profound effect on the coordination of metal ions and on the conformation of endogenously bound coenzyme A CoA ... More
One central question surrounding the biosynthesis of fatty acids and polyketide-derived natural products is how the 4′-phosphopantetheinyl transferase (PPTase) interrogates the essential acyl carrier protein (ACP) domain to fulfill the initial activation step. The triggering factor of this study was the lack of structural information on PPTases at physiological pH, which could bias our comprehension of the mechanism of action of these important enzymes. Structural and functional studies on the family II PPTase PptAb of Mycobacterium abscessus show that pH has a profound effect on the coordination of metal ions and on the conformation of endogenously bound coenzyme A (CoA). The observed conformational flexibility of CoA at physiological pH is accompanied by a disordered 4′-phosphopantetheine (Ppant) moiety. Finally, structural and dynamical information on an isolated mycobacterial ACP domain, in its apo form and in complex with the activator PptAb, suggests an alternate mechanism for the post-translational modification of modular megasynthases. Less
The lipid cubic phases LCP have enabled the determination of many important high-resolution structures of membrane proteins such as G-protein-coupled receptors photosensitive proteins enzymes channels and transporters However harvesting the crystals from the glass or plastic plates in which crystals grow is challenging The in meso in situ serial X-ray crystallography IMISX method uses thin plastic windowed plates that minimize LCP crystal manipulation The method which is compatible with high-throughput in situ measurements allows systematic diffraction screening and rapid data collection from hundreds of microcrystals in in meso crystallization wells without direct crystal harvesting In this chapter we describe an ... More
The lipid cubic phases (LCP) have enabled the determination of many important high-resolution structures of membrane proteins such as G-protein-coupled receptors, photosensitive proteins, enzymes, channels, and transporters. However, harvesting the crystals from the glass or plastic plates in which crystals grow is challenging. The in meso in situ serial X-ray crystallography (IMISX) method uses thin plastic windowed plates that minimize LCP crystal manipulation. The method, which is compatible with high-throughput in situ measurements, allows systematic diffraction screening and rapid data collection from hundreds of microcrystals in in meso crystallization wells without direct crystal harvesting. In this chapter, we describe an IMISX protocol for in situ serial X-ray data collection of LCP-grown crystals at both cryogenic and room temperatures which includes the crystallization setup, sample delivery, automated serial diffraction data collection, and experimental phasing. We also detail how the IMISX method was applied successfully for the structure determination of two novel targets—the undecaprenyl-pyrophosphate phosphatase BacA and the chemokine G-protein-coupled receptor CCR2A. Less
Membrane proteins are highly interesting targets due to their pivotal role in cell function and disease They are inserted in cell membranes are often intrinsically flexible and can adopt several conformational states to carry out their function Although most overall folds of membrane proteins are known many questions remain about specific functionally relevant intramolecular rearrangements that require experimental structure determination Here using the example of rhodopsin we describe how to prepare and analyze membrane protein crystals for serial crystallography at room temperature a new technique allowing to merge diffraction data from thousands of injector-delivered crystals that are too tiny for ... More
Membrane proteins are highly interesting targets due to their pivotal role in cell function and disease. They are inserted in cell membranes, are often intrinsically flexible, and can adopt several conformational states to carry out their function. Although most overall folds of membrane proteins are known, many questions remain about specific functionally relevant intramolecular rearrangements that require experimental structure determination. Here, using the example of rhodopsin, we describe how to prepare and analyze membrane protein crystals for serial crystallography at room temperature, a new technique allowing to merge diffraction data from thousands of injector-delivered crystals that are too tiny for classical single-crystal analysis even in cryogenic conditions. The application of serial crystallography for studying protein dynamics is mentioned. Less
The determination of distances between specific points in nucleic acids is essential to understanding their behaviour at the molecular level The ability to measure distances of nm is particularly important deformations arising from protein binding commonly fall within this range but the reliable measurement of such distances for a conformational ensemble remains a significant challenge Using several techniques we show that electron paramagnetic resonance EPR spectroscopy of oligonucleotides spin-labelled with triazole-appended nitroxides at the position offers a robust and minimally perturbing tool for obtaining such measurements For two nitroxides we present results from EPR spectroscopy X-ray crystal structures of B-form ... More
The determination of distances between specific points in nucleic acids is essential to understanding their behaviour at the molecular level. The ability to measure distances of 2–10 nm is particularly important: deformations arising from protein binding commonly fall within this range, but the reliable measurement of such distances for a conformational ensemble remains a significant challenge. Using several techniques, we show that electron paramagnetic resonance (EPR) spectroscopy of oligonucleotides spin-labelled with triazole-appended nitroxides at the 2′ position offers a robust and minimally perturbing tool for obtaining such measurements. For two nitroxides, we present results from EPR spectroscopy, X-ray crystal structures of B-form spin-labelled DNA duplexes, molecular dynamics simulations and nuclear magnetic resonance spectroscopy. These four methods are mutually supportive, and pinpoint the locations of the spin labels on the duplexes. In doing so, this work establishes 2′-alkynyl nitroxide spin-labelling as a minimally perturbing method for probing DNA conformation. Less
Trematode infections such as schistosomiasis and fascioliasis cause significant morbidity in an estimated million people worldwide and the associated agricultural losses are estimated at more than US billion per year Current chemotherapy is limited Triosephosphate isomerase TIM an enzyme of the glycolytic pathway has emerged as a useful drug target in many parasites including Fasciola hepatica TIM FhTIM We identified novel compounds that selectively inhibit this enzyme Using microscale thermophoresis we explored the interaction between target and compounds and identified a potent interaction between the sulfonyl- -thiadiazole compound and FhTIM which showed an IC of M and a Kd of ... More
Trematode infections such as schistosomiasis and fascioliasis cause significant morbidity in an estimated 250 million people worldwide and the associated agricultural losses are estimated at more than US$ 6 billion per year. Current chemotherapy is limited. Triosephosphate isomerase (TIM), an enzyme of the glycolytic pathway, has emerged as a useful drug target in many parasites, including Fasciola hepatica TIM (FhTIM). We identified 21 novel compounds that selectively inhibit this enzyme. Using microscale thermophoresis we explored the interaction between target and compounds and identified a potent interaction between the sulfonyl-1,2,4-thiadiazole (compound 187) and FhTIM, which showed an IC50 of 5 µM and a Kd of 66 nM. In only 4 hours, this compound killed the juvenile form of F. hepatica with an IC50 of 3 µM, better than the reference drug triclabendazole (TCZ). Interestingly, we discovered in vitro inhibition of FhTIM by TCZ, with an IC50 of 7 µM suggesting a previously uncharacterized role of FhTIM in the mechanism of action of this drug. Compound 187 was also active against various developmental stages of Schistosoma mansoni. The low toxicity in vitro in different cell types and lack of acute toxicity in mice was demonstrated for this compound, as was demonstrated the efficacy of 187 in vivo in F. hepatica infected mice. Finally, we obtained the first crystal structure of FhTIM at 1.9 Å resolution which allows us using docking to suggest a mechanism of interaction between compound 187 and TIM. In conclusion, we describe a promising drug candidate to control neglected trematode infections in human and animal health. Less
The process of macromolecular crystallisation almost always begins by setting up crystallisation trials using commercial or other premade screens followed by cycles of optimisation where the crystallisation cocktails are focused towards a particular small region of chemical space The screening process is relatively straightforward but still requires an understanding of the plethora of commercially available screens Optimisation is complicated by requiring both the design and preparation of the appropriate secondary screens Software has been developed in the C lab to aid the process of choosing initial screens to analyse the results of the initial trials and to design and describe ... More
The process of macromolecular crystallisation almost always begins by setting up crystallisation trials using commercial or other premade screens, followed by cycles of optimisation where the crystallisation cocktails are focused towards a particular small region of chemical space. The screening process is relatively straightforward, but still requires an understanding of the plethora of commercially available screens. Optimisation is complicated by requiring both the design and preparation of the appropriate secondary screens. Software has been developed in the C3 lab to aid the process of choosing initial screens, to analyse the results of the initial trials, and to design and describe how to prepare optimisation screens. Less
Developing antibody agonists targeting the human apelin receptor APJ is a promising therapeutic approach for the treatment of chronic heart failure Here we report the structure-guided discovery of a single-domain antibody sdAb agonist JN - based on the cocrystal structure of APJ with an sdAb antagonist JN the first cocrystal structure of a class A G protein coupled receptor GPCR with a functional antibody As revealed by the structure JN binds to the extracellular side of APJ makes critical contacts with the second extracellular loop and inserts the CDR into the ligand-binding pocket We converted JN into a full agonist ... More
Developing antibody agonists targeting the human apelin receptor (APJ) is a promising therapeutic approach for the treatment of chronic heart failure. Here, we report the structure-guided discovery of a single-domain antibody (sdAb) agonist JN241-9, based on the cocrystal structure of APJ with an sdAb antagonist JN241, the first cocrystal structure of a class A G protein–coupled receptor (GPCR) with a functional antibody. As revealed by the structure, JN241 binds to the extracellular side of APJ, makes critical contacts with the second extracellular loop, and inserts the CDR3 into the ligand-binding pocket. We converted JN241 into a full agonist JN241-9 by inserting a tyrosine into the CDR3. Modeling and molecular dynamics simulation shed light on JN241-9–stimulated receptor activation, providing structural insights for finding agonistic antibodies against class A GPCRs. Less
Antimicrobial resistance is a major global threat that calls for new antibiotics Globomycin and myxovirescin are two natural antibiotics that target the lipoprotein-processing enzyme LspA thereby compromising the integrity of the bacterial cell envelope As part of a project aimed at understanding their mechanism of action and for drug development we provide high-resolution crystal structures of the enzyme from the human pathogen methicillin-resistant Staphylococcus aureus MRSA complexed with globomycin and with myxovirescin Our results reveal an instance of convergent evolution The two antibiotics possess different molecular structures Yet they appear to inhibit identically as non-cleavable tetrahedral intermediate analogs Remarkably the ... More
Antimicrobial resistance is a major global threat that calls for new antibiotics. Globomycin and myxovirescin are two natural antibiotics that target the lipoprotein-processing enzyme, LspA, thereby compromising the integrity of the bacterial cell envelope. As part of a project aimed at understanding their mechanism of action and for drug development, we provide high-resolution crystal structures of the enzyme from the human pathogen methicillin-resistant Staphylococcus aureus (MRSA) complexed with globomycin and with myxovirescin. Our results reveal an instance of convergent evolution. The two antibiotics possess different molecular structures. Yet, they appear to inhibit identically as non-cleavable tetrahedral intermediate analogs. Remarkably, the two antibiotics superpose along nineteen contiguous atoms that interact similarly with LspA. This 19-atom motif recapitulates a part of the substrate lipoprotein in its proposed binding mode. Incorporating this motif into a scaffold with suitable pharmacokinetic properties should enable the development of effective antibiotics with built-in resistance hardiness. Less
The most abundant member of the collagen protein family collagen I also known as type I collagen COL is composed of one unique chain B and two similar chain A polypeptides that self-assemble with one amino acid offset into a heterotrimeric triple helix Given the offset chain B can occupy either the leading BAA middle ABA or trailing AAB position of the triple helix yielding three isomeric biomacromolecules with different protein recognition properties Despite five decades of intensive research there is no consensus on the position of chain B in COL Here three triple-helical heterotrimers that each contain a putative ... More
The most abundant member of the collagen protein family, collagen I (also known as type I collagen; COL1), is composed of one unique (chain B) and two similar (chain A) polypeptides that self-assemble with one amino acid offset into a heterotrimeric triple helix. Given the offset, chain B can occupy either the leading (BAA), middle (ABA) or trailing (AAB) position of the triple helix, yielding three isomeric biomacromolecules with different protein recognition properties. Despite five decades of intensive research, there is no consensus on the position of chain B in COL1. Here, three triple-helical heterotrimers that each contain a putative von Willebrand factor (VWF) and discoidin domain receptor (DDR) recognition sequence from COL1 were designed with chain B permutated in all three positions. AAB demonstrated a strong preference for both VWF and DDR, and also induced higher levels of cellular DDR phosphorylation. Thus, we resolve this long-standing mystery and show that COL1 adopts an AAB register. Less
Direct soaking of protein crystals with small-molecule fragments grouped into complementary clusters is a useful technique when assessing the potential of a new crystal system to support structure-guided drug discovery It provides a robustness check prior to any extensive crystal screening a double check for assay binding cutoffs and structural data for binding pockets that may or may not be picked out in assay measurements The structural output from this technique for three novel fragment molecules identified to bind to the antibacterial target Acinetobacter baumannii undecaprenyl pyrophosphate synthase are reported and the different physicochemical requirements of a successful antibiotic are ... More
Direct soaking of protein crystals with small-molecule fragments grouped into complementary clusters is a useful technique when assessing the potential of a new crystal system to support structure-guided drug discovery. It provides a robustness check prior to any extensive crystal screening, a double check for assay binding cutoffs and structural data for binding pockets that may or may not be picked out in assay measurements. The structural output from this technique for three novel fragment molecules identified to bind to the antibacterial target Acinetobacter baumannii undecaprenyl pyrophosphate synthase are reported, and the different physicochemical requirements of a successful antibiotic are compared with traditional medicines. Less
Serial crystallography at both synchrotron and X-ray free-electron laser light sources is becoming increasingly popular However the tools in the majority of crystallization laboratories are focused on producing large single crystals by vapour diffusion that fit the cryo-cooled paradigm of modern synchrotron crystallography This paper presents several case studies and some ideas and strategies on how to perform the conversion from a single crystal grown by vapour diffusion to the many thousands of micro-crystals required for modern serial crystallography grown by batch crystallization These case studies aim to show i how vapour diffusion conditions can be converted into batch by ... More
Serial crystallography, at both synchrotron and X-ray free-electron laser light sources, is becoming increasingly popular. However, the tools in the majority of crystallization laboratories are focused on producing large single crystals by vapour diffusion that fit the cryo-cooled paradigm of modern synchrotron crystallography. This paper presents several case studies and some ideas and strategies on how to perform the conversion from a single crystal grown by vapour diffusion to the many thousands of micro-crystals required for modern serial crystallography grown by batch crystallization. These case studies aim to show (i) how vapour diffusion conditions can be converted into batch by optimizing the length of time crystals take to appear; (ii) how an understanding of the crystallization phase diagram can act as a guide when designing batch crystallization protocols; and (iii) an accessible methodology when attempting to scale batch conditions to larger volumes. These methods are needed to minimize the sample preparation gap between standard rotation crystallography and dedicated serial laboratories, ultimately making serial crystallography more accessible to all crystallographers. Less
NEMO is a scaffolding protein which plays an essential role in the NF- B pathway by assembling the IKK-complex with the kinases IKK and IKK Upon activation the IKK complex phosphorylates the I B molecules leading to NF- B nuclear translocation and activation of target genes Inhibition of the NEMO IKK interaction is an attractive therapeutic paradigm for the modulation of NF- B pathway activity making NEMO a target for inhibitors design and discovery To facilitate the process of discovery and optimization of NEMO inhibitors we engineered an improved construct of the IKK-binding domain of NEMO that would allow for ... More
NEMO is a scaffolding protein which plays an essential role in the NF-κB pathway by assembling the IKK-complex with the kinases IKKα and IKKβ. Upon activation, the IKK complex phosphorylates the IκB molecules leading to NF-κB nuclear translocation and activation of target genes. Inhibition of the NEMO/IKK interaction is an attractive therapeutic paradigm for the modulation of NF-κB pathway activity, making NEMO a target for inhibitors design and discovery. To facilitate the process of discovery and optimization of NEMO inhibitors, we engineered an improved construct of the IKK-binding domain of NEMO that would allow for structure determination of the protein in the apo form and while bound to small molecular weight inhibitors. Here, we present the strategy utilized for the design, expression and structural characterization of the IKK-binding domain of NEMO. The protein is expressed in E. coli cells, solubilized under denaturing conditions and purified through three chromatographic steps. We discuss the protocols for obtaining crystals for structure determination and describe data acquisition and analysis strategies. The protocols will find wide applicability to the structure determination of complexes of NEMO and small molecule inhibitors. Less
The hormone melatonin secreted from the pineal gland mediates multiple physiological effects including modulation of Wnt -catenin signalling The Wnt palmitoleate lipid modification is essential for its signalling activity while the carboxylesterase Notum can remove the lipid from Wnt and inactivate it Notum enzyme inhibition can therefore upregulate Wnt signalling While searching for Notum inhibitors by crystallographic fragment screening a hit compound N- - -fluoro- H-indol- -yl ethyl acetamide that is structurally similar to melatonin came to our attention We then soaked melatonin and its precursor N-acetylserotonin into Notum crystals and obtained high-resolution structures of their complexes In each of ... More
The hormone melatonin, secreted from the pineal gland, mediates multiple physiological effects including modulation of Wnt/β-catenin signalling. The Wnt palmitoleate lipid modification is essential for its signalling activity, while the carboxylesterase Notum can remove the lipid from Wnt and inactivate it. Notum enzyme inhibition can therefore upregulate Wnt signalling. While searching for Notum inhibitors by crystallographic fragment screening, a hit compound N-[2-(5-fluoro-1H-indol-3-yl)ethyl]acetamide that is structurally similar to melatonin came to our attention. We then soaked melatonin and its precursor N-acetylserotonin into Notum crystals and obtained high-resolution structures (≤1.5 Å) of their complexes. In each of the structures, two compound molecules bind with Notum: one at the enzyme's catalytic pocket, overlapping the space occupied by the acyl tail of the Wnt palmitoleate lipid, and the other at the edge of the pocket opposite the substrate entrance. Although the inhibitory activity of melatonin shown by in vitro enzyme assays is low (IC50 75 µmol/L), the structural information reported here provides a basis for the design of potent and brain accessible drugs for neurodegenerative diseases such as Alzheimer's disease, in which upregulation of Wnt signalling may be beneficial. Less
The selective downregulation of activated intracellular proteins is a key challenge in cell biology RHO small GTPases switch between a guanosine diphosphate GDP -bound and a guanosine triphosphate GTP -bound state that drives downstream signaling At present no tool is available to study endogenous RHO-GTPinduced conformational changes in live cells Here we established a cell-based screen to selectively degrade RHOB-GTP using F-box-intracellular single-domain antibody fusion We identified one intracellular antibody intrabody that shows selective targeting of endogenous RHOB-GTP mediated by interactions between the CDR loop of the domain antibody and the GTP-binding pocket of RHOB Our results suggest that while ... More
The selective downregulation of activated intracellular proteins is a key challenge in cell biology. RHO small GTPases switch between a guanosine diphosphate (GDP)-bound and a guanosine triphosphate (GTP)-bound state that drives downstream signaling. At present, no tool is available to study endogenous RHO-GTPinduced conformational changes in live cells. Here, we established a cell-based screen to selectively degrade RHOB-GTP using F-box-intracellular single-domain antibody fusion. We identified one intracellular antibody (intrabody) that shows selective targeting of endogenous RHOB-GTP mediated by interactions between the CDR3 loop of the domain antibody and the GTP-binding pocket of RHOB. Our results suggest that, while RHOB is highly regulated at the expression level, only the GTP-bound pool, but not its global expression, mediates RHOB functions in genomic instability and in cell invasion. The F-box/intrabody-targeted protein degradation represents a unique approach to knock down the active form of small GTPases or other proteins with multiple cellular activities. Less
Short-term parameters correlating to long-term protein stability such as the protein cloud point temperature Tcloud are of interest to improve efficiency during protein product development Such efficiency is reached if short-term parameters are obtained in a low volume and high-throughput HT manner This study presents a low volume HT detection method for sub-zero Tcloud determination of lysozyme as such an experimental method is not available yet The setup consists of a cryogenic device with an automated imaging system Measurement reproducibility median absolute deviation of C and literature-based parameter validation Pearson correlation coefficient of were shown by a robustness and validation ... More
Short-term parameters correlating to long-term protein stability, such as the protein cloud point temperature (Tcloud), are of interest to improve efficiency during protein product development. Such efficiency is reached if short-term parameters are obtained in a low volume and high-throughput (HT) manner. This study presents a low volume HT detection method for (sub-zero) Tcloud determination of lysozyme, as such an experimental method is not available yet. The setup consists of a cryogenic device with an automated imaging system. Measurement reproducibility (median absolute deviation of 0.2 °C) and literature-based parameter validation (Pearson correlation coefficient of 0.996) were shown by a robustness and validation study. The subsequent case study demonstrated a partial correlation between the obtained apparent Tcloud parameter and long-term protein stability as a function of lysozyme concentration, ion type, ionic strength, and freeze/thaw stress. The presented experimental setup demonstrates its ability to advance short-term strategies for efficient protein formulation development. Less
A multiprotein complex polarisome nucleates actin cables for polarized cell growth in budding yeast and filamentous fungi However the dynamic regulations of polarisome proteins in polymerizing actin under physiological and stress conditions remains unknown We identify a previously functionally unknown polarisome member actin-interacting-protein Aip which promotes actin assembly synergistically with formin Bni Aip -C terminus is responsible for its activities by interacting with G-actin and Bni Through N-terminal intrinsically disordered region Aip forms high-order oligomers and generate cytoplasmic condensates under the stresses conditions The molecular dynamics and reversibility of Aip condensates are regulated by scaffolding protein Spa via liquid-liquid phase ... More
A multiprotein complex polarisome nucleates actin cables for polarized cell growth in budding yeast and filamentous fungi. However, the dynamic regulations of polarisome proteins in polymerizing actin under physiological and stress conditions remains unknown. We identify a previously functionally unknown polarisome member, actin-interacting-protein 5 (Aip5), which promotes actin assembly synergistically with formin Bni1. Aip5-C terminus is responsible for its activities by interacting with G-actin and Bni1. Through N-terminal intrinsically disordered region, Aip5 forms high-order oligomers and generate cytoplasmic condensates under the stresses conditions. The molecular dynamics and reversibility of Aip5 condensates are regulated by scaffolding protein Spa2 via liquid-liquid phase separation both in vitro and in vivo. In the absence of Spa2, Aip5 condensates hamper cell growth and actin cable structures under stress treatment. The present study reveals the mechanisms of actin assembly for polarity establishment and the adaptation in stress conditions to protect actin assembly by protein phase separation. Less
Redesigning existing food protein formulations is necessary in situations where food authorities propose dose adjustments or removal of currently employed additives Redesigning formulations involves evaluating substitute additives to obtain similar long-term physical stability as the original formulation Such formulation screening experiments benefit from comprehensive data visualization understanding the effects of substitute additives on long-term physical stability and identification of short-term optimization targets This work employs empirical phase diagrams to reach these benefits by combining multidimensional long-term protein physical stability data with short-term empirical protein properties A case study was performed where multidimensional protein phase diagrams formulations allowed for identification of ... More
Redesigning existing food protein formulations is necessary in situations where food authorities propose dose adjustments or removal of currently employed additives. Redesigning formulations involves evaluating substitute additives to obtain similar long-term physical stability as the original formulation. Such formulation screening experiments benefit from comprehensive data visualization, understanding the effects of substitute additives on long-term physical stability, and identification of short-term optimization targets. This work employs empirical phase diagrams to reach these benefits by combining multidimensional long-term protein physical stability data with short-term empirical protein properties. A case study was performed where multidimensional protein phase diagrams (1152 formulations) allowed for identification of stabilizing effects as a result of pH, methionine, sugars, salt, and minimized glycerol content. Corresponding empirical protein property diagrams (144 formulations) resulted in the identification of normalized surface tension as a short-term empirical protein property to reach long-term physical stability presumably similar to the original product, namely via preferential hydration. Additionally, changes in pH and salt were identified as environmental optimization targets to reach stability via repulsive electrostatic forces. This case study shows the applicability of the empirical phase diagram method to rationally perform formulation redesign screenings, while simultaneously expanding knowledge on protein long-term physical stability. Less
Co-inhibitory immune receptors can contribute to T cell dysfunction in patients with cancer Blocking antibodies against cytotoxic T-lymphocyte-associated protein CTLA- and programmed cell death PD- partially reverse this effect and are becoming standard of care in an increasing number of malignancies However many of the other axes by which tumours become inhospitable to T cells are not fully understood Here we report that V-domain immunoglobulin suppressor of T cell activation VISTA engages and suppresses T cells selectively at acidic pH such as that found in tumour microenvironments Multiple histidine residues along the rim of the VISTA extracellular domain mediate binding ... More
Co-inhibitory immune receptors can contribute to T cell dysfunction in patients with cancer1,2. Blocking antibodies against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) partially reverse this effect and are becoming standard of care in an increasing number of malignancies3. However, many of the other axes by which tumours become inhospitable to T cells are not fully understood. Here we report that V-domain immunoglobulin suppressor of T cell activation (VISTA) engages and suppresses T cells selectively at acidic pH such as that found in tumour microenvironments. Multiple histidine residues along the rim of the VISTA extracellular domain mediate binding to the adhesion and co-inhibitory receptor P-selectin glycoprotein ligand-1 (PSGL-1). Antibodies engineered to selectively bind and block this interaction in acidic environments were sufficient to reverse VISTA-mediated immune suppression in vivo. These findings identify a mechanism by which VISTA may engender resistance to anti-tumour immune responses, as well as an unexpectedly determinative role for pH in immune co-receptor engagement. Less
The tricarboxylic acid cycle intermediate succinate is involved in metabolic processes and plays a crucial role in the homeostasis of mitochondrial reactive oxygen species The receptor responsible for succinate signalling SUCNR also known as GPR is a member of the G-protein-coupled-receptor family and links succinate signalling to renin-induced hypertension retinal angiogenesis and inflammation Because SUCNR senses succinate as an immunological danger signal which has relevance for diseases including ulcerative colitis liver fibrosis diabetes and rheumatoid arthritis it is of interest as a therapeutic target Here we report the high-resolution crystal structure of rat SUCNR in complex with an intracellular binding ... More
The tricarboxylic acid cycle intermediate succinate is involved in metabolic processes and plays a crucial role in the homeostasis of mitochondrial reactive oxygen species1. The receptor responsible for succinate signalling, SUCNR1 (also known as GPR91), is a member of the G-protein-coupled-receptor family2 and links succinate signalling to renin-induced hypertension, retinal angiogenesis and inflammation3,4,5. Because SUCNR1 senses succinate as an immunological danger signal6—which has relevance for diseases including ulcerative colitis, liver fibrosis7, diabetes and rheumatoid arthritis3,8—it is of interest as a therapeutic target. Here we report the high-resolution crystal structure of rat SUCNR1 in complex with an intracellular binding nanobody in the inactive conformation. Structure-based mutagenesis and radioligand-binding studies, in conjunction with molecular modelling, identified key residues for species-selective antagonist binding and enabled the determination of the high-resolution crystal structure of a humanized rat SUCNR1 in complex with a high-affinity, human-selective antagonist denoted NF-56-EJ40. We anticipate that these structural insights into the architecture of the succinate receptor and its antagonist selectivity will enable structure-based drug discovery and will further help to elucidate the function of SUCNR1 in vitro and in vivo. Less
Influenza A virus IAV nonstructural protein NS a potent antagonist of the host immune response is capable of interacting with RNA and a wide range of cellular proteins NS consists of an RNA-binding domain RBD and an effector domain ED separated by a flexible linker region LR H N -NS has a characteristic -residue deletion in the LR with either G minor group or E major group at the st position and non-H N -NS contains E with an intact linker Based on the orientation of the ED with respect to the RBD previous crystallographic studies have shown that minor ... More
Influenza A virus (IAV) nonstructural protein 1 (NS1), a potent antagonist of the host immune response, is capable of interacting with RNA and a wide range of cellular proteins. NS1 consists of an RNA-binding domain (RBD) and an effector domain (ED) separated by a flexible linker region (LR). H5N1-NS1 has a characteristic 5-residue deletion in the LR, with either G (minor group) or E (major group) at the 71st position, and non-H5N1-NS1 contains E71 with an intact linker. Based on the orientation of the ED with respect to the RBD, previous crystallographic studies have shown that minor group H5N1-NS1(G71), a non-H5N1-NS1 [H6N6-NS1(E71)], and the LR deletion mutant H6N6-NS1(Δ80-84/E71) mimicking the major group H5N1-NS1 exhibit “open,” “semiopen,” and “closed” conformations, respectively, suggesting that NS1 exhibits a strain-dependent conformational preference. Here we report the first crystal structure of a naturally occurring H5N1-NS1(E71) and show that it adopts an open conformation similar to that of the minor group of H5N1-NS1 [H5N1-NS1(G71)]. We also show that H6N6-NS1(Δ80-84/E71) under a different crystallization condition and H6N6-NS1(Δ80-84/G71) also exhibit open conformations, suggesting that NS1 can adopt an open conformation irrespective of E or G at the 71st position. Our single-molecule fluorescence resonance energy transfer (FRET) analysis to investigate the conformational preference of NS1 in solution showed that all NS1 constructs predominantly exist in an open conformation. Further, our coimmunoprecipitation and binding studies showed that they all bind to cellular factors with similar affinities. Taken together, our studies suggest that NS1 exhibits strain-independent structural plasticity that allows it to interact with a wide variety of cellular ligands during viral infection. Less
Protein-crystallization imaging and classification is a labor-intensive process typically performed either by humans or by instruments that currently cost well over This cost puts the use of crystallization-trial imaging outside the reach of most academic laboratories and also start-up biotechnology firms where resources are scarce An imaging system has been designed and prototyped which automatically captures images from multi-well protein-crystallization experiments using both standard and fluorescent imaging techniques at a cost times lower than current market rates The machine uses a Panowin F D printer as a base and controls it using G-code commands sent from a Python script running ... More
Protein-crystallization imaging and classification is a labor-intensive process typically performed either by humans or by instruments that currently cost well over $100 000. This cost puts the use of crystallization-trial imaging outside the reach of most academic laboratories, and also start-up biotechnology firms, where resources are scarce. An imaging system has been designed and prototyped which automatically captures images from multi-well protein-crystallization experiments using both standard and fluorescent imaging techniques at a cost 28 times lower than current market rates. The machine uses a Panowin F1 3D printer as a base and controls it using G-code commands sent from a Python script running on a desktop computer. A graphical user interface (GUI) was developed to enable users to control the machine and facilitate image capture, classification and editing. A 488 nm laser diode and a 525 nm filter were incorporated to allow in situ fluorescent imaging of proteins trace-labeled with a fluorophore, Alexa Fluor 488. The instrument was primarily designed using a 3D printer and augmented using commercially available parts, and this publication aims to serve as a guide for comparable in-laboratory robotics projects. Less
Protein trafficking requires coat complexes that couple recognition of sorting motifs in transmembrane cargoes with biogenesis of transport carriers The mechanisms of cargo transport through the endosomal network are poorly understood Here we identify a sorting motif for endosomal recycling of cargoes including the cation-independent mannose- -phosphate receptor and semaphorin C by the membrane tubulating BAR domain-containing sorting nexins SNX and SNX Crystal structures establish that this motif folds into a -hairpin which binds a site in the SNX SNX phox homology domains Over sixty cargoes share this motif and require SNX SNX for their recycling These include cargoes involved ... More
Protein trafficking requires coat complexes that couple recognition of sorting motifs in transmembrane cargoes with biogenesis of transport carriers. The mechanisms of cargo transport through the endosomal network are poorly understood. Here, we identify a sorting motif for endosomal recycling of cargoes, including the cation-independent mannose-6-phosphate receptor and semaphorin 4C, by the membrane tubulating BAR domain-containing sorting nexins SNX5 and SNX6. Crystal structures establish that this motif folds into a β-hairpin, which binds a site in the SNX5/SNX6 phox homology domains. Over sixty cargoes share this motif and require SNX5/SNX6 for their recycling. These include cargoes involved in neuronal migration and a Drosophila snx6 mutant displays defects in axonal guidance. These studies identify a sorting motif and provide molecular insight into an evolutionary conserved coat complex, the ‘Endosomal SNX–BAR sorting complex for promoting exit 1’ (ESCPE-1), which couples sorting motif recognition to the BAR-domain-mediated biogenesis of cargo-enriched tubulo-vesicular transport carriers. Less
The influence of process parameters during freeze thaw FT operations is essential for the preservation of the protein stability activity during production and storage processes in the biopharmaceutical industry Process parameters such as FT ramps the final storage time and temperature affect the occurring FT stress onto the target protein in different ways FT stress includes cold denaturation freeze concentration and ice crystal formation which can result in protein aggregation To visualize the impact of variations in FT ramps descriptors such as solubility phase behavior and crystal morphology were evaluated The phase diagram-based toolbox in combination with an HTS-compatible cryo-device ... More
The influence of process parameters during freeze/thaw (FT) operations is essential for the preservation of the protein stability/activity during production and storage processes in the biopharmaceutical industry. Process parameters, such as FT ramps, the final storage time and temperature, affect the occurring FT stress onto the target protein in different ways. FT stress includes cold denaturation, freeze concentration, and ice crystal formation which can result in protein aggregation. To visualize the impact of variations in FT ramps, descriptors such as solubility, phase behavior and crystal morphology were evaluated. The phase diagram-based toolbox in combination with an HTS-compatible cryo-device allowed the identification of suitable ramping schemes during FT operations. It could be clearly shown that rapid operations are needed above the glass transition temperature of the target protein to circumvent precipitation during FT cycles. Finally, a stability index is introduced which allows ranking of the systems investigated. Less
Yersinia pestis the causative agent of bubonic plague is one of the most lethal pathogens in recorded human history Today the concern is the possible misuse of Y pestis as an agent in bioweapons and bioterrorism Current therapies for the treatment of plague include the use of a small number of antibiotics but clinical cases of antibiotic resistance have been reported in some areas of the world Therefore the discovery of new drugs is required to combat potential Y pestis infection Here the crystal structure of the Y pestis UDP-glucose pyrophosphorylase UGP a metabolic enzyme implicated in the survival of ... More
Yersinia pestis, the causative agent of bubonic plague, is one of the most lethal pathogens in recorded human history. Today, the concern is the possible misuse of Y. pestis as an agent in bioweapons and bioterrorism. Current therapies for the treatment of plague include the use of a small number of antibiotics, but clinical cases of antibiotic resistance have been reported in some areas of the world. Therefore, the discovery of new drugs is required to combat potential Y. pestis infection. Here, the crystal structure of the Y. pestis UDP-glucose pyrophosphorylase (UGP), a metabolic enzyme implicated in the survival of Y. pestis in mouse macrophages, is described at 2.17 Å resolution. The structure provides a foundation that may enable the rational design of inhibitors and open new avenues for the development of antiplague therapeutics. Less
Curli amyloid fibrils secreted by Enterobacteriaceae mediate host cell adhesion and contribute to biofilm formation thereby promoting bacterial resistance to environmental stressors Here we present crystal structures of amyloid-forming segments from the major curli subunit CsgA revealing steric zipper fibrils of tightly mated -sheets demonstrating a structural link between curli and human pathological amyloids D-enantiomeric peptides originally developed to interfere with Alzheimer s disease-associated amyloid- inhibited CsgA fibrillation and reduced biofilm formation in Salmonella typhimurium Moreover as previously shown CsgA fibrils cross-seeded fibrillation of amyloid- providing support for the proposed structural resemblance and potential for cross-species amyloid interactions The presented ... More
Curli amyloid fibrils secreted by Enterobacteriaceae mediate host cell adhesion and contribute to biofilm formation, thereby promoting bacterial resistance to environmental stressors. Here, we present crystal structures of amyloid-forming segments from the major curli subunit, CsgA, revealing steric zipper fibrils of tightly mated β-sheets, demonstrating a structural link between curli and human pathological amyloids. D-enantiomeric peptides, originally developed to interfere with Alzheimer’s disease-associated amyloid-β, inhibited CsgA fibrillation and reduced biofilm formation in Salmonella typhimurium. Moreover, as previously shown, CsgA fibrils cross-seeded fibrillation of amyloid-β, providing support for the proposed structural resemblance and potential for cross-species amyloid interactions. The presented findings provide structural insights into amyloidogenic regions important for curli formation, suggest a novel strategy for disrupting amyloid-structured biofilms, and hypothesize on the formation of self-propagating prion-like species originating from a microbial source that could influence neurodegenerative diseases. Less
Phosphate acquisition by plants is an essential process that is directly implicated in the optimization of crop yields Purple acid phosphatases PAPs are ubiquitous metalloenzymes which catalyze the hydrolysis of a wide range of phosphate esters and anhydrides While some plant PAPs display a preference for ATP as the substrate others are efficient in hydrolyzing phytate or -phosphoenolpyruvate PEP PAP from red kidney bean rkbPAP is an efficient ATP- and ADPase but has no activity towards phytate The crystal structure of this enzyme in complex with an ATP analogue to resolution provides insight into the amino acid residues that play ... More
Phosphate acquisition by plants is an essential process that is directly implicated in the optimization of crop yields. Purple acid phosphatases (PAPs) are ubiquitous metalloenzymes, which catalyze the hydrolysis of a wide range of phosphate esters and anhydrides. While some plant PAPs display a preference for ATP as the substrate, others are efficient in hydrolyzing phytate or 2-phosphoenolpyruvate (PEP). PAP from red kidney bean (rkbPAP) is an efficient ATP- and ADPase, but has no activity towards phytate. The crystal structure of this enzyme in complex with an ATP analogue (to 2.20 Å resolution) provides insight into the amino acid residues that play an essential role in binding this substrate. Homology modelling was used to generate three-dimensional structures for the active sites of PAPs from tobacco (NtPAP) and Arabidopsis thaliana (AtPAP12 and AtPAP26) that are efficient in hydrolyzing phytate and PEP as substrates, respectively. In combination with substrate docking simulations and a phylogenetic analysis of 49 plant PAP sequences (including the first PAP sequences reported from Eucalyptus), several active site residues were identified that are important in defining the substrate specificities of plant PAPs. These results may inform bioengineering studies aimed at identifying and incorporating suitable plant PAP genes into crops to improve phosphorus use efficiency. Organic phosphorus sources increasingly supplement or replace inorganic fertilizer, and efficient phosphorus use of crops will lower the environmental footprint of agriculture while enhancing food production. Less
The CC chemokine receptor CCR balances immunity and tolerance by homeostatic trafficking of immune cells In cancer CCR -mediated trafficking leads to lymph node metastasis suggesting the receptor as a promising therapeutic target Here we present the crystal structure of human CCR fused to the protein Sialidase NanA by using data up to resolution The structure shows the ligand Cmp bound to an intracellular allosteric binding pocket A sulfonamide group characteristic for various chemokine receptor ligands binds to a patch of conserved residues in the Gi protein binding region between transmembrane helix and helix We demonstrate how structural data can ... More
The CC chemokine receptor 7 (CCR7) balances immunity and tolerance by homeostatic trafficking of immune cells. In cancer, CCR7-mediated trafficking leads to lymph node metastasis, suggesting the receptor as a promising therapeutic target. Here, we present the crystal structure of human CCR7 fused to the protein Sialidase NanA by using data up to 2.1 Å resolution. The structure shows the ligand Cmp2105 bound to an intracellular allosteric binding pocket. A sulfonamide group, characteristic for various chemokine receptor ligands, binds to a patch of conserved residues in the Gi protein binding region between transmembrane helix 7 and helix 8. We demonstrate how structural data can be used in combination with a compound repository and automated thermal stability screening to identify and modulate allosteric chemokine receptor antagonists. We detect both novel (CS-1 and CS-2) and clinically relevant (CXCR1-CXCR2 phase-II antagonist Navarixin) CCR7 modulators with implications for multi-target strategies against cancer. Less
We previously generated rotavirus-specific RV-specific recombinant monoclonal antibodies mAbs derived from B cells isolated from human intestinal resections Twenty-four of these mAbs were specific for the VP fragment of RV VP and most of were non-neutralizing when tested in the conventional MA cell based assay We reexamined the ability of these mAbs to neutralize RVs in human intestinal epithelial cells including ileal enteroids and HT- cells Most of of the non-neutralizing VP mAbs efficiently neutralized human RV in HT- cells or enteroids Serum RV neutralization titers in adults and infants were significantly higher in HT- than MA cells and adsorption ... More
We previously generated 32 rotavirus-specific (RV-specific) recombinant monoclonal antibodies (mAbs) derived from B cells isolated from human intestinal resections. Twenty-four of these mAbs were specific for the VP8* fragment of RV VP4, and most (20 of 24) were non-neutralizing when tested in the conventional MA104 cell–based assay. We reexamined the ability of these mAbs to neutralize RVs in human intestinal epithelial cells, including ileal enteroids and HT-29 cells. Most (18 of 20) of the “non-neutralizing” VP8* mAbs efficiently neutralized human RV in HT-29 cells or enteroids. Serum RV neutralization titers in adults and infants were significantly higher in HT-29 than MA104 cells and adsorption of these sera with recombinant VP8* lowered the neutralization titers in HT-29 but not MA104 cells. VP8* mAbs also protected suckling mice from diarrhea in an in vivo challenge model. X-ray crystallographic analysis of one VP8* mAb (mAb9) in complex with human RV VP8* revealed that the mAb interaction site was distinct from the human histo-blood group antigen binding site. Since MA104 cells are the most commonly used cell line to detect anti-RV neutralization activity, these findings suggest that prior vaccine and other studies of human RV neutralization responses may have underestimated the contribution of VP8* antibodies to the overall neutralization titer. Less
Image-based protein phase diagram analysis is key for understanding and exploiting protein phase behavior in the biopharmaceutical field However required data analysis has become a notorious time-consuming task since high-throughput screening approaches were implemented A variety of computational tools have been developed to support analysis but these tools primarily use end point visible light images This study investigates the combined effect of end point and time-dependent image features obtained from cross-polarized and ultraviolet light features supplementary to visible light on protein phase diagram image classification In addition external validation was performed to evaluate the classification algorithm s applicability to support ... More
Image-based protein phase diagram analysis is key for understanding and exploiting protein phase behavior in the biopharmaceutical field. However, required data analysis has become a notorious time-consuming task since high-throughput screening approaches were implemented. A variety of computational tools have been developed to support analysis, but these tools primarily use end point visible light images. This study investigates the combined effect of end point and time-dependent image features obtained from cross-polarized and ultraviolet light features, supplementary to visible light, on protein phase diagram image classification. In addition, external validation was performed to evaluate the classification algorithm’s applicability to support protein phase diagram scoring. The predicted protein phase behavior classes were subsequently used to automatically construct multidimensional protein phase diagrams to prevent image information loss without complicating the used image classification algorithm. Combining end point and time-dependent features from 3 light sources resulted in a balanced accuracy of 86.4 ± 4.3%, which is comparable to or better than more complex classifiers reported in literature. External validation resulted in a correct formulation classification rate of 91.7%. Subsequent automated construction of the multidimensional protein phase diagrams, using predicted classes, allowed visualization of details such as crystallization rate and protein phase behavior type coexistence. Less