344 Citations
Melanocortin receptor MC -R antagonists are actively sought for treating cancer cachexia We determined the structures of complexes with PG- and SBL-MC- These peptides differ from SHU by substituting His with Pro and inserting Gly or Arg The structures revealed two subpockets at the TM -TM -TM domains separated by N Two peptide series based on the complexed peptides led to an antagonist activity and selectivity SAR study Most ligands retained the SHU potency but several SBL-MC- -derived peptides significantly enhanced MC -R selectivity over MC -R by - to -fold We also investigated MC -R coupling to the K ... More
Melanocortin 4 receptor (MC4-R) antagonists are actively sought for treating cancer cachexia. We determined the structures of complexes with PG-934 and SBL-MC-31. These peptides differ from SHU9119 by substituting His6 with Pro6 and inserting Gly10 or Arg10. The structures revealed two subpockets at the TM7-TM1-TM2 domains, separated by N2857.36. Two peptide series based on the complexed peptides led to an antagonist activity and selectivity SAR study. Most ligands retained the SHU9119 potency, but several SBL-MC-31-derived peptides significantly enhanced MC4-R selectivity over MC1-R by 60- to 132-fold. We also investigated MC4-R coupling to the K+ channel, Kir7.1. Some peptides activated the channel, whereas others induced channel closure independently of G protein coupling. In cell culture studies, channel activation correlated with increased feeding, while a peptide with Kir7.1 inhibitory activity reduced eating. These results highlight the potential for targeting the MC4-R:Kir7.1 complex for treating positive and restrictive eating disorders. Less
Osteoclasts are over-activated as we age which results in bone loss Src deficiency in mice leads to severe osteopetrosis due to a functional defect in osteoclasts indicating that Src function is essential in osteoclasts G-protein-coupled receptors GPCRs are the targets for of approved drugs but it is still unclear how GPCRs regulate Src kinase activity Here we reveal that GPR activation by its natural ligand Kisspeptin- Kp- causes Dusp to dephosphorylate Src at Tyr Mechanistically Gpr recruits both active Src and the Dusp phosphatase at its proline arginine-rich motif in its C terminus We show that Kp- binding to Gpr ... More
Osteoclasts are over-activated as we age, which results in bone loss. Src deficiency in mice leads to severe osteopetrosis due to a functional defect in osteoclasts, indicating that Src function is essential in osteoclasts. G-protein-coupled receptors (GPCRs) are the targets for ∼35% of approved drugs but it is still unclear how GPCRs regulate Src kinase activity. Here, we reveal that GPR54 activation by its natural ligand Kisspeptin-10 (Kp-10) causes Dusp18 to dephosphorylate Src at Tyr 416. Mechanistically, Gpr54 recruits both active Src and the Dusp18 phosphatase at its proline/arginine-rich motif in its C terminus. We show that Kp-10 binding to Gpr54 leads to the up-regulation of Dusp18. Kiss1, Gpr54 and Dusp18 knockout mice all exhibit osteoclast hyperactivation and bone loss, and Kp-10 abrogated bone loss by suppressing osteoclast activity in vivo. Therefore, Kp-10/Gpr54 is a promising therapeutic target to abrogate bone resorption by Dusp18-mediated Src dephosphorylation. Less
The adenosine subfamily G protein-coupled receptors A AR and A BR have been identified as promising cancer immunotherapy candidates One of the A AR A BR dual antagonists AB has progressed to a phase II clinical trial to treat rectal cancer However the precise mechanism underlying its dual-antagonistic properties remains elusive Herein we report crystal structures of the A AR complexed with AB and a selective A AR antagonist The structures revealed a common binding mode on A AR wherein the ligands established extensive interactions with residues from the orthosteric and secondary pockets In contrast the cAMP assay and A ... More
The adenosine subfamily G protein-coupled receptors A2AR and A2BR have been identified as promising cancer immunotherapy candidates. One of the A2AR/A2BR dual antagonists, AB928, has progressed to a phase II clinical trial to treat rectal cancer. However, the precise mechanism underlying its dual-antagonistic properties remains elusive. Herein, we report crystal structures of the A2AR complexed with AB928 and a selective A2AR antagonist 2–118. The structures revealed a common binding mode on A2AR, wherein the ligands established extensive interactions with residues from the orthosteric and secondary pockets. In contrast, the cAMP assay and A2AR and A2BR molecular dynamics simulations indicated that the ligands adopted distinct binding modes on A2BR. Detailed analysis of their chemical structures suggested that AB928 readily adapted to the A2BR pocket, while 2–118 did not due to intrinsic differences. This disparity potentially accounted for the difference in inhibitory efficacy between A2BR and A2AR. This study serves as a valuable structural template for the future development of selective or dual inhibitors targeting A2AR/A2BR for cancer therapy. Less
Plasmodium falciparum pathology is driven by the accumulation of parasite-infected erythrocytes in microvessels This process is mediated by the parasite s polymorphic erythrocyte membrane protein PfEMP adhesion proteins A subset of PfEMP variants that bind human endothelial protein C receptor EPCR through their CIDR domains is responsible for severe malaria pathogenesis A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP variants Here we describe two broadly reactive and binding-inhibitory human monoclonal antibodies against CIDR The antibodies isolated from two different individuals exhibited a similar and consistent EPCR-binding inhibition of CIDR domains representing five of ... More
Plasmodium falciparum pathology is driven by the accumulation of parasite-infected erythrocytes in microvessels. This process is mediated by the parasite’s polymorphic erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. A subset of PfEMP1 variants that bind human endothelial protein C receptor (EPCR) through their CIDRα1 domains is responsible for severe malaria pathogenesis. A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP1 variants. Here, we describe two broadly reactive and binding-inhibitory human monoclonal antibodies against CIDRα1. The antibodies isolated from two different individuals exhibited a similar and consistent EPCR-binding inhibition of 34 CIDRα1 domains, representing five of the six subclasses of CIDRα1. Both antibodies inhibited EPCR binding of both recombinant full-length and native PfEMP1 proteins as well as parasite sequestration in bioengineered 3D brain microvessels under physiologically relevant flow conditions. Structural analyses of the two antibodies in complex with two different CIDRα1 antigen variants reveal similar binding mechanisms that depend on interactions with three highly conserved amino acid residues of the EPCR-binding site in CIDRα1. These broadly reactive antibodies likely represent a common mechanism of acquired immunity to severe malaria and offer novel insights for the design of a vaccine or treatment targeting severe malaria. Less
Monoolein-based liquid crystal phases are established media that are researched for various biological applications including drug delivery While water is the most common solvent for self-assembly some ionic liquids ILs can support lipidic self-assembly However currently there is limited knowledge of IL-lipid phase behavior in ILs In this study the lyotropic liquid crystal phase behavior of monoolein was investigated in six protic ILs known to support amphiphile self-assembly namely ethylammonium nitrate ethanolammonium nitrate ethylammonium formate ethanolammonium formate ethylammonium acetate and ethanolammonium acetate These ILs were selected to identify specific ion effects on monoolein self-assembly specifically increasing the alkyl chain length ... More
Monoolein-based liquid crystal phases are established media that are researched for various biological applications, including drug delivery. While water is the most common solvent for self-assembly, some ionic liquids (ILs) can support lipidic self-assembly. However, currently, there is limited knowledge of IL-lipid phase behavior in ILs. In this study, the lyotropic liquid crystal phase behavior of monoolein was investigated in six protic ILs known to support amphiphile self-assembly, namely ethylammonium nitrate, ethanolammonium nitrate, ethylammonium formate, ethanolammonium formate, ethylammonium acetate, and ethanolammonium acetate. These ILs were selected to identify specific ion effects on monoolein self-assembly, specifically increasing the alkyl chain length of the cation or anion, the presence of a hydroxyl group in the cation, and varying the anion. The lyotropic liquid crystal phases with 20–80 wt. % of monoolein were characterized over a temperature range from 25 to 65 °C using synchrotron small angle x-ray scattering and cross-polarized optical microscopy. These results were used to construct partial phase diagrams of monoolein in each of the six protic ILs, with inverse hexagonal, bicontinuous cubic, and lamellar phases observed. Protic ILs containing the ethylammonium cation led to monoolein forming lamellar and bicontinuous cubic phases, while those containing the ethanolammonium cation formed inverse hexagonal and bicontinuous cubic phases. Protic ILs containing formate and acetate anions favored bicontinuous cubic phases across a broader range of protic IL concentrations than those containing the nitrate anion. Less
TP is the most frequently mutated gene in human cancer While it is well understood that the ability of p to act as a transcription factor is required for tumor suppression the key target genes downstream of p required for tumor suppression are still incompletely understood We first set out to characterize a rare African-specific germline variant of TP in the DNA binding domain Tyr His Y H Although we find that Y H can suppress tumor colony formation and is impaired for the transactivation of only a small subset of p target genes Y H mice develop spontaneous cancers ... More
TP53 is the most frequently mutated gene in human cancer. While it is well understood that the ability of p53 to act as a transcription factor is required for tumor suppression, the key target genes downstream of p53 required for tumor suppression are still incompletely understood. We first set out to characterize a rare, African-specific, germline variant of TP53 in the DNA binding domain, Tyr107His (Y107H). Although we find that Y107H can suppress tumor colony formation and is impaired for the transactivation of only a small subset of p53 target genes, Y107H mice develop spontaneous cancers and metastases. We identified the p53 target gene, PADI4, to be exquisitely sensitive to p53 mutation, and loss of PADI4 is seen in Y107H and other transcriptionally competent p53 hypomorphs. PADI4 is a regulator of histone modification and gene transcription via citrullination, which is the process of deiminating arginine to the non-natural amino acid citrulline. Our TCGA analysis reveals PADI4 is downregulated or mutated in multiple human cancers. Surprisingly, we show that PADI4 is sufficient to suppress tumor growth and sensitize wild-type p53 cells to chemotherapeutics. We further show that PADI4 is potently tumor suppressive in vivo, and complete tumor suppression by PADI4 requires an intact immune system. We find PADI4 enhances the transactivation of p53 targets and genes involved in immune activation. In addition, we identify a p53–PADI4 gene signature that is predictive of survival and the efficacy of immune-checkpoint inhibitors. We have further found that PADI4 interacts and modifies p53 via citrullination at key residues within the oligomerization and C-terminal domain of p53. PADI4 colocalizes with p53 on chromatin at non-canonical p53 target genes and genes devoid of a p53 response element. Citrullination of p53 may alter p53 function through enhanced oligomerization or binding of p53 to DNA. The findings from this study reveal PADI4 as not only a key target gene of p53, but a core regulator of p53 activity and target specificity through a novel protein modification. This work highlights the need to reassess the role of PADI4 in cancer, and provides insight into critical downstream target genes important for tumor suppression by p53. Less
Membrane proteins are indispensable for every living organism yet their structural organization remains underexplored Despite the recent advancements in single-particle cryogenic electron microscopy and cryogenic electron tomography which have significantly increased the structural coverage of membrane proteins across various kingdoms certain scientific methods such as time-resolved crystallography still mostly rely on crystallization techniques such as lipidic cubic phase LCP or in meso crystallization In this study we present an open-access blueprint for a humidity control chamber designed for LCP in meso crystallization experiments using a Gryphon crystallization robot Using this chamber we have obtained crystals of a transmembrane aspartate transporter ... More
Membrane proteins are indispensable for every living organism, yet their structural organization remains underexplored. Despite the recent advancements in single-particle cryogenic electron microscopy and cryogenic electron tomography, which have significantly increased the structural coverage of membrane proteins across various kingdoms, certain scientific methods, such as time-resolved crystallography, still mostly rely on crystallization techniques, such as lipidic cubic phase (LCP) or in meso crystallization. In this study, we present an open-access blueprint for a humidity control chamber designed for LCP/in meso crystallization experiments using a Gryphon crystallization robot. Using this chamber, we have obtained crystals of a transmembrane aspartate transporter GltTk from Thermococcus kodakarensis in a lipidic environment using in meso crystallization. The data collected from these crystals allowed us to perform an analysis of lipids bound to the transporter. With this publication of our open-access design of a humidity chamber, we aim to improve the accessibility of in meso protein crystallization for the scientific community. Less
StayGold is an exceptionally bright and stable fluorescent protein that is highly resistant to photobleaching Despite favorable fluorescence properties use of StayGold as a fluorescent tag is limited because it forms a natural dimer Here we report the structure of StayGold and generate a derivative mStayGold that retains the brightness and photostability of the original protein while being fully monomeric
NADH cytochrome b oxidoreductase Ncb or is a cytosolic ferric reductase implicated in diabetes and neurological conditions Ncb or comprises cytochrome b b and cytochrome b reductase b R domains separated by a CHORD-Sgt CS linker domain Ncb or redox activity depends on proper inter-domain interactions to mediate electron transfer from NADH or NADPH via FAD to heme While full-length human Ncb or has proven resistant to crystallization we have succeeded in obtaining high-resolution atomic structures of the b domain and a construct containing the CS and b R domains CS b R Ncb or also contains an N-terminal intrinsically ... More
NADH cytochrome b5 oxidoreductase (Ncb5or) is a cytosolic ferric reductase implicated in diabetes and neurological conditions. Ncb5or comprises cytochrome b5 (b5) and cytochrome b5 reductase (b5R) domains separated by a CHORD-Sgt1 (CS) linker domain. Ncb5or redox activity depends on proper inter-domain interactions to mediate electron transfer from NADH or NADPH via FAD to heme. While full-length human Ncb5or has proven resistant to crystallization, we have succeeded in obtaining high-resolution atomic structures of the b5 domain and a construct containing the CS and b5R domains (CS/b5R). Ncb5or also contains an N-terminal intrinsically disordered region of 50 residues that has no homologs in other protein families in animals but features a distinctive, conserved L34MDWIRL40 motif also present in reduced lateral root formation (RLF) protein in rice and increased recombination center 21 in baker's yeast, all attaching to a b5 domain. After unsuccessful attempts at crystallizing a human Ncb5or construct comprising the N-terminal region naturally fused to the b5 domain, we were able to obtain a high-resolution atomic structure of a recombinant rice RLF construct corresponding to residues 25–129 of human Ncb5or (52% sequence identity; 74% similarity). The structure reveals Trp120 (corresponding to invariant Trp37 in Ncb5or) to be part of an 11-residue α-helix (S116QMDWLKLTRT126) packing against two of the four helices in the b5 domain that surround heme (α2 and α5). The Trp120 side chain forms a network of interactions with the side chains of four highly conserved residues corresponding to Tyr85 and Tyr88 (α2), Cys124 (α5), and Leu47 in Ncb5or. Circular dichroism measurements of human Ncb5or fragments further support a key role of Trp37 in nucleating the formation of the N-terminal helix, whose location in the N/b5 module suggests a role in regulating the function of this multi-domain redox enzyme. This study revealed for the first time an ancient origin of a helical motif in the N/b5 module as reflected by its existence in a class of cytochrome b5 proteins from three kingdoms among eukaryotes. Less
Flavins such as flavin mononucleotide or flavin adenine dinucleotide are bound by diverse proteins yet have very similar spectra when in the oxidized state Recently we developed new variants of flavin-binding protein CagFbFP exhibiting notable blue Q V or red I V A Q shifts of fluorescence emission maxima Here we use time-resolved and low-temperature spectroscopy to show that whereas the chromophore environment is static in Q V an additional protein-flavin hydrogen bond is formed upon photoexcitation in the I V A Q variant Consequently in Q V excitation emission and phosphorescence spectra are shifted whereas in I V A ... More
Flavins such as flavin mononucleotide or flavin adenine dinucleotide are bound by diverse proteins, yet have very similar spectra when in the oxidized state. Recently, we developed new variants of flavin-binding protein CagFbFP exhibiting notable blue (Q148V) or red (I52V A85Q) shifts of fluorescence emission maxima. Here, we use time-resolved and low-temperature spectroscopy to show that whereas the chromophore environment is static in Q148V, an additional protein-flavin hydrogen bond is formed upon photoexcitation in the I52V A85Q variant. Consequently, in Q148V, excitation, emission, and phosphorescence spectra are shifted, whereas in I52V A85Q, excitation and low-temperature phosphorescence spectra are relatively unchanged, while emission spectrum is altered. We also determine the x-ray structures of the two variants to reveal the flavin environment and complement the spectroscopy data. Our findings illustrate two distinct color-tuning mechanisms of flavin-binding proteins and could be helpful for the engineering of new variants with improved optical properties. Less
Cryptophyte algae have a unique phycobiliprotein light-harvesting antenna that fills a spectral gap in chlorophyll absorption from photosystems However it is unclear how the antenna transfers energy efficiently to these photosystems We show that the cryptophyte Hemiselmis andersenii expresses an energetically complex antenna comprising three distinct spectrotypes of phycobiliprotein each composed of two protomers but with different quaternary structures arising from a diverse subunit family We report crystal structures of the major phycobiliprotein from each spectrotype Two-thirds of the antenna consists of open quaternary form phycobiliproteins acting as primary photon acceptors These are supplemented by a newly discovered open-braced form ... More
Cryptophyte algae have a unique phycobiliprotein light-harvesting antenna that fills a spectral gap in chlorophyll absorption from photosystems. However, it is unclear how the antenna transfers energy efficiently to these photosystems. We show that the cryptophyte Hemiselmis andersenii expresses an energetically complex antenna comprising three distinct spectrotypes of phycobiliprotein, each composed of two αβ protomers but with different quaternary structures arising from a diverse α subunit family. We report crystal structures of the major phycobiliprotein from each spectrotype. Two-thirds of the antenna consists of open quaternary form phycobiliproteins acting as primary photon acceptors. These are supplemented by a newly discovered open-braced form (~15%), where an insertion in the α subunit produces ~10 nm absorbance red-shift. The final components (~15%) are closed forms with a long wavelength spectral feature due to substitution of a single chromophore. This chromophore is present on only one β subunit where asymmetry is dictated by the corresponding α subunit. This chromophore creates spectral overlap with chlorophyll, thus bridging the energetic gap between the phycobiliprotein antenna and the photosystems. We propose that the macromolecular organization of the cryptophyte antenna consists of bulk open and open-braced forms that transfer excitations to photosystems via this bridging closed form phycobiliprotein. Less
Kinases are important therapeutic targets and their inhibitors are classified according to their mechanism of action which range from blocking ATP binding to covalent inhibition Here a mechanism of inhibition is highlighted by capturing p -activated kinase PAK in an intermediate state of activation using an Affimer reagent that binds in the P pocket PAK was identified from a non-hypothesis-driven high-content imaging RNAi screen in urothelial cancer cells Silencing of PAK resulted in reduced cell number G S arrest and enlargement of cells suggesting it to be important in urothelial cancer cell line survival and proliferation Affimer reagents were isolated ... More
Kinases are important therapeutic targets, and their inhibitors are classified according to their mechanism of action, which range from blocking ATP binding to covalent inhibition. Here, a mechanism of inhibition is highlighted by capturing p21-activated kinase 5 (PAK5) in an intermediate state of activation using an Affimer reagent that binds in the P+1 pocket. PAK5 was identified from a non-hypothesis-driven high-content imaging RNAi screen in urothelial cancer cells. Silencing of PAK5 resulted in reduced cell number, G1/S arrest, and enlargement of cells, suggesting it to be important in urothelial cancer cell line survival and proliferation. Affimer reagents were isolated to identify mechanisms of inhibition. The Affimer PAK5-Af17 recapitulated the phenotype seen with siRNA. Co-crystallization revealed that PAK5-Af17 bound in the P+1 pocket of PAK5, locking the kinase into a partial activation state. This mechanism of inhibition indicates that another class of kinase inhibitors is possible. Less
Poly ADP-ribose polymerase PARP inhibitors are used in the clinic to treat BRCA-deficient breast ovarian and prostate cancers As their efficacy is potentiated by loss of the nucleotide salvage factor DNPH there is considerable interest in the development of highly specific small molecule DNPH inhibitors Here we present X-ray crystal structures of dimeric DNPH bound to its substrate hydroxymethyl deoxyuridine monophosphate hmdUMP Direct interaction with the hydroxymethyl group is important for substrate positioning while conserved residues surrounding the base facilitate target discrimination Glycosidic bond cleavage is driven by a conserved catalytic triad and proceeds via a two-step mechanism involving formation ... More
Poly(ADP-ribose) polymerase (PARP) inhibitors are used in the clinic to treat BRCA-deficient breast, ovarian and prostate cancers. As their efficacy is potentiated by loss of the nucleotide salvage factor DNPH1 there is considerable interest in the development of highly specific small molecule DNPH1 inhibitors. Here, we present X-ray crystal structures of dimeric DNPH1 bound to its substrate hydroxymethyl deoxyuridine monophosphate (hmdUMP). Direct interaction with the hydroxymethyl group is important for substrate positioning, while conserved residues surrounding the base facilitate target discrimination. Glycosidic bond cleavage is driven by a conserved catalytic triad and proceeds via a two-step mechanism involving formation and subsequent disruption of a covalent glycosyl-enzyme intermediate. Mutation of a previously uncharacterised yet conserved glutamate traps the intermediate in the active site, demonstrating its role in the hydrolytic step. These observations define the enzyme’s catalytic site and mechanism of hydrolysis, and provide important insights for inhibitor discovery. Less
Biochemical Properties of a Promising Milk-Clotting Enzyme, Moose (Alces alces) Recombinant Chymosin
Moose Alces alces recombinant chymosin with a milk-clotting activity of AU mL was synthesized in the Kluyveromyces lactis expression system After precipitation with ammonium sulfate and chromatographic purification a sample of genetically engineered moose chymosin with a specific milk-clotting activity of AU mg was obtained which was used for extensive biochemical characterization of the enzyme The threshold of the thermal stability of moose chymosin was C its complete inactivation occurred after heating at C The total proteolytic activity of moose chymosin was A units The ratio of milk-clotting and total proteolytic activities of the enzyme was The Km kcat and ... More
Moose (Alces alces) recombinant chymosin with a milk-clotting activity of 86 AU/mL was synthesized in the Kluyveromyces lactis expression system. After precipitation with ammonium sulfate and chromatographic purification, a sample of genetically engineered moose chymosin with a specific milk-clotting activity of 15,768 AU/mg was obtained, which was used for extensive biochemical characterization of the enzyme. The threshold of the thermal stability of moose chymosin was 55 °C; its complete inactivation occurred after heating at 60 °C. The total proteolytic activity of moose chymosin was 0.332 A280 units. The ratio of milk-clotting and total proteolytic activities of the enzyme was 0.8. The Km, kcat and kcat/Km values of moose chymosin were 4.7 μM, 98.7 s−1, and 21.1 μM−1 s−1, respectively. The pattern of change in the coagulation activity as a function of pH and Ca2+ concentration was consistent with the requirements for milk coagulants for cheese making. The optimum temperature of the enzyme was 50–55 °C. The introduction of Mg2+, Zn2+, Co2+, Ba2+, Fe2+, Mn2+, Ca2+, and Cu2+ into milk activated the coagulation ability of moose chymosin, while Ni ions on the contrary inhibited its activity. Using previously published data, we compared the biochemical properties of recombinant moose chymosin produced in bacterial (Escherichia coli) and yeast (K. lactis) producers. Less
The thermophilic bacterium Clostridium thermocellum efficiently degrades polysaccharides into oligosaccharides The metabolism of - -linked cello-oligosaccharides is initiated by three enzymes i e the cellodextrin phosphorylase Cdp the cellobiose phosphorylase Cbp and the -glucosidase A BglA in C thermocellum In comparison how the oligosaccharides containing other kinds of linkage are utilized is rarely understood In this study we found that BglA could hydrolyze the - -disaccharide laminaribiose with much higher activity than that against the - -disaccharide cellobiose The structural basis of the substrate specificity was analyzed by crystal structure determination and molecular docking Genetic deletions of BglA and Cbp ... More
The thermophilic bacterium Clostridium thermocellum efficiently degrades polysaccharides into oligosaccharides. The metabolism of β-1,4-linked cello-oligosaccharides is initiated by three enzymes, i.e., the cellodextrin phosphorylase (Cdp), the cellobiose phosphorylase (Cbp), and the β-glucosidase A (BglA), in C. thermocellum. In comparison, how the oligosaccharides containing other kinds of linkage are utilized is rarely understood. In this study, we found that BglA could hydrolyze the β-1,3-disaccharide laminaribiose with much higher activity than that against the β-1,4-disaccharide cellobiose. The structural basis of the substrate specificity was analyzed by crystal structure determination and molecular docking. Genetic deletions of BglA and Cbp, respectively, and enzymatic analysis of cell extracts demonstrated that BglA is the key enzyme responsible for laminaribiose metabolism. Furthermore, the deletion of BglA can suppress the expression of Cbp and the deletion of Cbp can up-regulate the expression of BglA, indicating that BglA and Cbp have cross-regulation and BglA is also critical for cellobiose metabolism. These insights pave the way for both a fundamental understanding of metabolism and regulation in C. thermocellum and emphasize the importance of the degradation and utilization of polysaccharides containing β-1,3-linked glycosidic bonds in lignocellulose biorefinery. Less
Laboratory production of recombinant mammalian proteins particularly antibodies requires an expression pipeline assuring sufficient yield and correct folding with appropriate posttranslational modifications Transient gene expression TGE in the suspension-adapted Chinese Hamster Ovary CHO cell lines has become the method of choice for this task The antibodies can be secreted into the media which facilitates subsequent purification and can be glycosylated However in general protein production in CHO cells is expensive and may provide variable outcomes namely in laboratories without previous experience While achievable yields may be influenced by the nucleotide sequence there are other aspects of the process which offer ... More
Laboratory production of recombinant mammalian proteins, particularly antibodies, requires an expression pipeline assuring sufficient yield and correct folding with appropriate posttranslational modifications. Transient gene expression (TGE) in the suspension-adapted Chinese Hamster Ovary (CHO) cell lines has become the method of choice for this task. The antibodies can be secreted into the media, which facilitates subsequent purification, and can be glycosylated. However, in general, protein production in CHO cells is expensive and may provide variable outcomes, namely in laboratories without previous experience. While achievable yields may be influenced by the nucleotide sequence, there are other aspects of the process which offer space for optimization, like gene delivery method, cultivation process or expression plasmid design. Polyethylenimine (PEI)-mediated gene delivery is frequently employed as a low-cost alternative to liposome-based methods. In this work, we are proposing a TGE platform for universal medium-scale production of antibodies and other proteins in CHO cells, with a novel expression vector allowing fast and flexible cloning of new genes and secretion of translated proteins. The production cost has been further reduced using recyclable labware. Nine days after transfection, we routinely obtain milligrams of antibody Fabs or human lactoferrin in a 25 mL culture volume. Potential of the platform is established based on the production and crystallization of antibody Fabs and their complexes. Less
The use of antibiotics has undoubtedly been a boon for humanity in combating infections and microbial threats However their widespread utilization has contributed to the emergence and spread of antibiotic resistance which now poses a significant public health challenge Streptomyces bacterium produce diverse secondary metabolites with antibacterial antifungal antiviral antitumoral and immunosuppressant activities Among these compounds is echinomycin a nonribosomal peptide antibiotic synthesized by Streptomyces lasalocidi which inhibits DNA replication and transcription by intercalating the DNA duplex at CpG steps A gene called ecm was identified in the echinomycin biosynthetic gene cluster which provides echinomycin self-resistance Ecm recognizes DNA duplexes ... More
The use of antibiotics has undoubtedly been a boon for humanity in combating infections and microbial threats. However, their widespread utilization has contributed to the emergence and spread of antibiotic resistance, which now poses a significant public health challenge. Streptomyces bacterium, produce diverse secondary metabolites with antibacterial, antifungal, antiviral, antitumoral, and immunosuppressant activities. Among these compounds is echinomycin, a nonribosomal peptide antibiotic synthesized by Streptomyces lasalocidi, which inhibits DNA replication and transcription by intercalating the DNA duplex at CpG steps. A gene called ecm16 was identified in the echinomycin biosynthetic gene cluster, which provides echinomycin self-resistance. Ecm16 recognizes DNA duplexes that contain echinomycin, and neutralizes its toxicity through a yet undetermined mechanism. To shed light on this process, we are determining the crystal structure of Ecm16 containing ATP. We introduced strategic mutations in the nucleotide binding site of Ecm16 to prevent ATP hydrolysis. We have successfully cloned, expressed, and purified the recombinant Ecm16 E399Q,E708Q double mutant protein. We have solved the structure of this protein using X-ray Crystallography at a resolution of 2.07 Å. However, our crystal structure contained ADP instead of the expected ATP. We propose and alternative experimental strategies for structure determination of ATP-containing Ecm16. Less
The Dictyostelium discoideum dye-decolorizing peroxidase DdDyP is a newly discovered peroxidase which belongs to a unique class of heme peroxidase family that lacks homology to the known members of plant peroxidase superfamily DdDyP catalyzes the H O -dependent oxidation of a wide-spectrum of substrates ranging from polycyclic dyes to lignin biomass holding promise for potential industrial and biotechnological applications To study the molecular mechanism of DdDyP highly pure and functional protein with a natively incorporated heme is required however obtaining a functional DyP-type peroxidase with a natively bound heme is challenging and often requires addition of expensive biosynthesis precursors Alternatively ... More
The Dictyostelium discoideum dye-decolorizing peroxidase (DdDyP) is a newly discovered peroxidase, which belongs to a unique class of heme peroxidase family that lacks homology to the known members of plant peroxidase superfamily. DdDyP catalyzes the H2O2-dependent oxidation of a wide-spectrum of substrates ranging from polycyclic dyes to lignin biomass, holding promise for potential industrial and biotechnological applications. To study the molecular mechanism of DdDyP, highly pure and functional protein with a natively incorporated heme is required, however, obtaining a functional DyP-type peroxidase with a natively bound heme is challenging and often requires addition of expensive biosynthesis precursors. Alternatively, a heme in vitro reconstitution approach followed by a chromatographic purification step to remove the excess heme is often used. Here, we show that expressing the DdDyP peroxidase in ×2 YT enriched medium at low temperature (20°C), without adding heme supplement or biosynthetic precursors, allows for a correct native incorporation of heme into the apo-protein, giving rise to a stable protein with a strong Soret peak at 402 nm. Further, we crystallized and determined the native structure of DdDyP at a resolution of 1.95 Å, which verifies the correct heme binding and its geometry. The structural analysis also reveals a binding of two water molecules at the distal site of heme plane bridging the catalytic residues (Arg239 and Asp149) of the GXXDG motif to the heme-Fe(III) via hydrogen bonds. Our results provide new insights into the geometry of native DdDyP active site and its implication on DyP catalysis. Less
TP is the most frequently mutated gene in cancer yet key target genes for p -mediated tumor suppression remain unidentified Here we characterize a rare African-specific germline variant of TP in the DNA-binding domain Tyr His Y H Nuclear magnetic resonance and crystal structures reveal that Y H is structurally similar to wild-type p Consistent with this we find that Y H can suppress tumor colony formation and is impaired for the transactivation of only a small subset of p target genes this includes the epigenetic modifier PADI which deiminates arginine to the nonnatural amino acid citrulline Surprisingly we show ... More
TP53 is the most frequently mutated gene in cancer, yet key target genes for p53-mediated tumor suppression remain unidentified. Here, we characterize a rare, African-specific germline variant of TP53 in the DNA-binding domain Tyr107His (Y107H). Nuclear magnetic resonance and crystal structures reveal that Y107H is structurally similar to wild-type p53. Consistent with this, we find that Y107H can suppress tumor colony formation and is impaired for the transactivation of only a small subset of p53 target genes; this includes the epigenetic modifier PADI4, which deiminates arginine to the nonnatural amino acid citrulline. Surprisingly, we show that Y107H mice develop spontaneous cancers and metastases and that Y107H shows impaired tumor suppression in two other models. We show that PADI4 is itself tumor suppressive and that it requires an intact immune system for tumor suppression. We identify a p53–PADI4 gene signature that is predictive of survival and the efficacy of immune-checkpoint inhibitors. Less
The small size and flexibility of G protein-coupled receptors GPCRs have long posed a significant challenge to determining their structures for research and therapeutic applications Single particle cryogenic electron microscopy cryoEM is often out of reach due to the small size of the receptor without a signaling partner Crystallization of GPCRs in lipidic cubic phase LCP often results in crystals that may be too small and difficult to analyze using X-ray microcrystallography at synchrotron sources or even serial femtosecond crystallography at X-ray free electron lasers Here we determine the previously unknown structure of the human vasopressin B receptor V BR ... More
The small size and flexibility of G protein-coupled receptors (GPCRs) have long posed a significant challenge to determining their structures for research and therapeutic applications. Single particle cryogenic electron microscopy (cryoEM) is often out of reach due to the small size of the receptor without a signaling partner. Crystallization of GPCRs in lipidic cubic phase (LCP) often results in crystals that may be too small and difficult to analyze using X-ray microcrystallography at synchrotron sources or even serial femtosecond crystallography at X-ray free electron lasers. Here, we determine the previously unknown structure of the human vasopressin 1B receptor (V1BR) using microcrystal electron diffraction (MicroED). To achieve this, we grew V1BR microcrystals in LCP and transferred the material directly onto electron microscopy grids. The protein was labeled with a fluorescent dye prior to crystallization to locate the microcrystals using cryogenic fluorescence microscopy, and then the surrounding material was removed using a plasma-focused ion beam to thin the sample to a thickness amenable to MicroED. MicroED data from 14 crystalline lamellae were used to determine the 3.2 Å structure of the receptor in the crystallographic space group P 1. These results demonstrate the use of MicroED to determine previously unknown GPCR structures that, despite significant effort, were not tractable by other methods. Less
Histamine dehydrogenase from the gram-negative bacterium Rhizobium sp - HaDHR is a member of a small family of dehydrogenases containing a covalently attached FMN and the only member so far identified to date that does not exhibit substrate inhibition In this study we present the resolution crystal structure of HaDHR This new structure allowed for the identification of the internal electron transfer pathway to abiological ferrocene-based mediators Alanine was identified as the exit point of electrons from the Fe S cluster The enzyme was modified with a Ser Cys mutation to facilitate covalent attachment of a ferrocene moiety When modified ... More
Histamine dehydrogenase from the gram-negative bacterium Rhizobium sp. 4-9 (HaDHR) is a member of a small family of dehydrogenases containing a covalently attached FMN, and the only member so far identified to date that does not exhibit substrate inhibition. In this study, we present the 2.1 Å resolution crystal structure of HaDHR. This new structure allowed for the identification of the internal electron transfer pathway to abiological ferrocene-based mediators. Alanine 437 was identified as the exit point of electrons from the Fe4S4 cluster. The enzyme was modified with a Ser436Cys mutation to facilitate covalent attachment of a ferrocene moiety. When modified with Fc-maleimide, this new construct demonstrated direct electron transfer from the enzyme to a gold electrode in a histamine concentration-dependent manner without the need for any additional electron mediators. Less
Cold-adapted enzymes are characterized both by a higher catalytic activity at low temperatures and by having their temperature optimum down-shifted compared to mesophilic orthologs In several cases the optimum does not coincide with the onset of protein melting but reflects some other type of inactivation In the psychrophilic -amylase from an Antarctic bacterium the inactivation is thought to originate from a specific enzyme-substrate interaction that breaks around room temperature Here we report a computational redesign of this enzyme aimed at shifting its temperature optimum upward A set of mutations designed to stabilize the enzyme-substrate interaction were predicted by computer simulations ... More
Cold-adapted enzymes are characterized both by a higher catalytic activity at low temperatures and by having their temperature optimum down-shifted, compared to mesophilic orthologs. In several cases, the optimum does not coincide with the onset of protein melting but reflects some other type of inactivation. In the psychrophilic α-amylase from an Antarctic bacterium, the inactivation is thought to originate from a specific enzyme-substrate interaction that breaks around room temperature. Here, we report a computational redesign of this enzyme aimed at shifting its temperature optimum upward. A set of mutations designed to stabilize the enzyme-substrate interaction were predicted by computer simulations of the catalytic reaction at different temperatures. The predictions were verified by kinetic experiments and crystal structures of the redesigned α-amylase, showing that the temperature optimum is indeed markedly shifted upward and that the critical surface loop controlling the temperature dependence approaches the target conformation observed in a mesophilic ortholog. Less
Protonation of key histidine residues has been long implicated in the acid-mediated cellular action of the diphtheria toxin translocation T- domain responsible for the delivery of the catalytic domain into the cell Here we use a combination of computational constant-pH Molecular Dynamics simulations and experimental NMR circular dichroism and fluorescence spectroscopy along with the X-ray crystallography approaches to characterize the initial stages of conformational change happening in solution in the wild-type T-domain and in the H Q H Q double mutant This replacement suppresses the acid-induced transition resulting in the retention of a more stable protein structure in solutions at ... More
Protonation of key histidine residues has been long implicated in the acid-mediated cellular action of the diphtheria toxin translocation (T-) domain, responsible for the delivery of the catalytic domain into the cell. Here, we use a combination of computational (constant-pH Molecular Dynamics simulations) and experimental (NMR, circular dichroism, and fluorescence spectroscopy along with the X-ray crystallography) approaches to characterize the initial stages of conformational change happening in solution in the wild-type T-domain and in the H223Q/H257Q double mutant. This replacement suppresses the acid-induced transition, resulting in the retention of a more stable protein structure in solutions at pH 5.5 and, consequently, in reduced membrane-disrupting activity. Here, for the first time, we report the pKa values of the histidine residues of the T-domain, measured by NMR-monitored pH titrations. Most peaks in the histidine side chain spectral region are titrated with pKas ranging from 6.2 to 6.8. However, the two most up-field peaks display little change down to pH 6, which is a limiting pH for this protein in solution at concentrations required for NMR. These peaks are absent in the double mutant, suggesting they belong to H223 and H257. The constant-pH simulations indicate that for the T-domain in solution, the pKa values for histidine residues range from 3.0 to 6.5, with those most difficult to protonate being H251 and H257. Taken together, our experimental and computational data demonstrate that previously suggested cooperative protonation of all six histidines in the T-domain does not occur. Less
We recently converted the GAF domain of NpR cyanobacteriochrome into near-infrared NIR fluorescent proteins FPs Unlike cyanobacterichrome which incorporates phycocyanobilin tetrapyrrole engineered NIR FPs bind biliverdin abundant in mammalian cells thus being the smallest scaffold for it Here we determined the crystal structure of the brightest blue-shifted protein of the series miRFP nano at resolution characterized its chromophore environment and explained the molecular basis of its spectral properties Using the determined structure we have rationally designed a red-shifted NIR FP termed miRFP nano with excitation at nm and emission at nm miRFP nano exhibits a small size of kDa enhanced ... More
We recently converted the GAF domain of NpR3784 cyanobacteriochrome into near-infrared (NIR) fluorescent proteins (FPs). Unlike cyanobacterichrome, which incorporates phycocyanobilin tetrapyrrole, engineered NIR FPs bind biliverdin abundant in mammalian cells, thus being the smallest scaffold for it. Here, we determined the crystal structure of the brightest blue-shifted protein of the series, miRFP670nano3, at 1.8 Å resolution, characterized its chromophore environment and explained the molecular basis of its spectral properties. Using the determined structure, we have rationally designed a red-shifted NIR FP, termed miRFP704nano, with excitation at 680 nm and emission at 704 nm. miRFP704nano exhibits a small size of 17 kDa, enhanced molecular brightness, photostability and pH-stability. miRFP704nano performs well in various protein fusions in live mammalian cells and should become a versatile genetically-encoded NIR probe for multiplexed imaging across spatial scales in different modalities. Less
The high morbidity and mortality associated with SARS-CoV- infection the etiological agent of COVID- has had a major impact on global public health Significant progress has been made in the development of an array of vaccines and biologics however the emergence of SARS-CoV- variants and breakthrough infections are an ongoing major concern Furthermore there is an existing paucity of small-molecule host and virus-directed therapeutics and prophylactics that can be used to counter the spread of SARS-CoV- and any emerging and re-emerging coronaviruses We describe herein our efforts to address this urgent need by focusing on the structure-guided design of potent ... More
The high morbidity and mortality associated with SARS-CoV-2 infection, the etiological agent of COVID-19, has had a major impact on global public health. Significant progress has been made in the development of an array of vaccines and biologics, however, the emergence of SARS-CoV-2 variants and breakthrough infections are an ongoing major concern. Furthermore, there is an existing paucity of small-molecule host and virus-directed therapeutics and prophylactics that can be used to counter the spread of SARS-CoV-2, and any emerging and re-emerging coronaviruses. We describe herein our efforts to address this urgent need by focusing on the structure-guided design of potent broad-spectrum inhibitors of SARS-CoV-2 3C-like protease (3CLpro or Main protease), an enzyme essential for viral replication. The inhibitors exploit the directional effects associated with the presence of a gem-dimethyl group that allow the inhibitors to optimally interact with the S4 subsite of the enzyme. Several compounds were found to potently inhibit SARS-CoV-2 and MERS-CoV 3CL proteases in biochemical and cell-based assays. Specifically, the EC50 values of aldehyde 1c and its corresponding bisulfite adduct 1d against SARS-CoV-2 were found to be 12 and 10 nM, respectively, and their CC50 values were >50 μM. Furthermore, deuteration of these compounds yielded compounds 2c/2d with EC50 values 11 and 12 nM, respectively. Replacement of the aldehyde warhead with a nitrile (CN) or an α-ketoamide warhead or its corresponding bisulfite adduct yielded compounds 1g, 1e and 1f with EC50 values 60, 50 and 70 nM, respectively. High-resolution cocrystal structures have identified the structural determinants associated with the binding of the inhibitors to the active site of the enzyme and, furthermore, have illuminated the mechanism of action of the inhibitors. Overall, the high Safety Index (SI) (SI=CC50/EC50) displayed by these compounds suggests that they are well-suited to conducting further preclinical studies. Less
The Gs protein-coupled adenosine A A receptor A AAR represents an emerging drug target for cancer immunotherapy The clinical candidate Etrumadenant was developed as an A AAR antagonist with ancillary blockade of the A BAR subtype It constitutes a unique chemotype featuring a poly-substituted -amino- -phenyl- -triazolylpyrimidine core structure Herein we report two crystal structures of the A AAR in complex with Etrumadenant obtained with differently thermostabilized A AAR constructs This led to the discovery of an unprecedented interaction a hydrogen bond of T with the cyano group of Etrumadenant T is mutated in most A AAR constructs used for ... More
The Gs protein-coupled adenosine A2A receptor (A2AAR) represents an emerging drug target for cancer immunotherapy. The clinical candidate Etrumadenant was developed as an A2AAR antagonist with ancillary blockade of the A2BAR subtype. It constitutes a unique chemotype featuring a poly-substituted 2-amino-4-phenyl-6-triazolylpyrimidine core structure. Herein, we report two crystal structures of the A2AAR in complex with Etrumadenant, obtained with differently thermostabilized A2AAR constructs. This led to the discovery of an unprecedented interaction, a hydrogen bond of T883.36 with the cyano group of Etrumadenant. T883.36 is mutated in most A2AAR constructs used for crystallization, which has prevented the discovery of its interactions. In-vitro characterization of Etrumadenant indicated low selectivity versus the A1AR subtype, which can be rationalized by the structural data. These results will facilitate the future design of AR antagonists with desired selectivity. Moreover, they highlight the advantages of the employed A2AAR crystallization construct that is devoid of ligand binding site mutations. Less
Cytotoxic-T-lymphocyte CTL mediated control of HIV- is enhanced by targeting highly networked epitopes in complex with human-leukocyte-antigen-class-I HLA-I However the extent to which the presenting HLA allele contributes to this process is unknown Here we examine the CTL response to QW a highly networked epitope presented by the disease-protective HLA-B and disease-neutral HLA-B Despite robust targeting of QW in persons expressing either allele T cell receptor TCR cross-recognition of the naturally occurring variant QW S T is consistently reduced when presented by HLA-B but not by HLA-B Crystal structures show substantial conformational changes from QW -HLA to QW S T-HLA ... More
Cytotoxic-T-lymphocyte (CTL) mediated control of HIV-1 is enhanced by targeting highly networked epitopes in complex with human-leukocyte-antigen-class-I (HLA-I). However, the extent to which the presenting HLA allele contributes to this process is unknown. Here we examine the CTL response to QW9, a highly networked epitope presented by the disease-protective HLA-B57 and disease-neutral HLA-B53. Despite robust targeting of QW9 in persons expressing either allele, T cell receptor (TCR) cross-recognition of the naturally occurring variant QW9_S3T is consistently reduced when presented by HLA-B53 but not by HLA-B57. Crystal structures show substantial conformational changes from QW9-HLA to QW9_S3T-HLA by both alleles. The TCR-QW9-B53 ternary complex structure manifests how the QW9-B53 can elicit effective CTLs and suggests sterically hindered cross-recognition by QW9_S3T-B53. We observe populations of cross-reactive TCRs for B57, but not B53 and also find greater peptide-HLA stability for B57 in comparison to B53. These data demonstrate differential impacts of HLAs on TCR cross-recognition and antigen presentation of a naturally arising variant, with important implications for vaccine design. Less
In the DarTG toxin-antitoxin system the DarT toxin ADP-ribosylates single-stranded DNA ssDNA which stalls DNA replication and plays a crucial role in controlling bacterial growth and bacteriophage infection This toxic activity is reversed by the N-terminal macrodomain of the cognate antitoxin DarG DarG also binds DarT but the role of these interactions in DarT neutralization is unknown Here we report that the C-terminal domain of DarG DarG toxin-binding domain DarGTBD interacts with DarT to form a stoichiometric heterodimeric complex We determined the resolution crystal structure of the Mycobacterium tuberculosis DarT-DarGTBD complex The comparative structural analysis reveals that DarGTBD interacts with ... More
In the DarTG toxin-antitoxin system, the DarT toxin ADP-ribosylates single-stranded DNA (ssDNA), which stalls DNA replication and plays a crucial role in controlling bacterial growth and bacteriophage infection. This toxic activity is reversed by the N-terminal macrodomain of the cognate antitoxin DarG. DarG also binds DarT, but the role of these interactions in DarT neutralization is unknown. Here, we report that the C-terminal domain of DarG (DarG toxin-binding domain [DarGTBD]) interacts with DarT to form a 1:1 stoichiometric heterodimeric complex. We determined the 2.2 Å resolution crystal structure of the Mycobacterium tuberculosis DarT-DarGTBD complex. The comparative structural analysis reveals that DarGTBD interacts with DarT at the DarT/ssDNA interaction interface, thus sterically occluding substrate ssDNA binding and consequently inactivating toxin by direct protein-protein interactions. Our data support a unique two-layered DarT toxin neutralization mechanism of DarG, which is important in keeping the toxin molecules in check under normal growth conditions. Less
Proteorhodopsins PRs bacterial light-driven outward proton pumps comprise the first discovered and largest family of rhodopsins they play a significant role in life on the Earth A big remaining mystery was that up-to-date there was no described bacterial rhodopsins pumping protons at acidic pH despite the fact that bacteria live in different pH environment Here we describe conceptually new bacterial rhodopsins which are operating as outward proton pumps at acidic pH A comprehensive function-structure study of a representative of a new clade of proton pumping rhodopsins which we name mirror proteorhodopsins from Sphingomonas paucimobilis SpaR shows cavity gate architecture of ... More
Proteorhodopsins (PRs), bacterial light-driven outward proton pumps comprise the first discovered and largest family of rhodopsins, they play a significant role in life on the Earth. A big remaining mystery was that up-to-date there was no described bacterial rhodopsins pumping protons at acidic pH despite the fact that bacteria live in different pH environment. Here we describe conceptually new bacterial rhodopsins which are operating as outward proton pumps at acidic pH. A comprehensive function-structure study of a representative of a new clade of proton pumping rhodopsins which we name “mirror proteorhodopsins”, from Sphingomonas paucimobilis (SpaR) shows cavity/gate architecture of the proton translocation pathway rather resembling channelrhodopsins than the known rhodopsin proton pumps. Another unique property of mirror proteorhodopsins is that proton pumping is inhibited by a millimolar concentration of zinc. We also show that mirror proteorhodopsins are extensively represented in opportunistic multidrug resistant human pathogens, plant growth-promoting and zinc solubilizing bacteria. They may be of optogenetic interest. Less
SARS-CoV- and MERS-CoV president the human coronaviruses and zoonotic coronaviruses due to tremendous repercussions to wellbeing of the world population Investigational efforts focusing on the disease pathology and viral lifecycle have led to identifying multiple druggable targets of both viruses including CL protease PL protease and RNA dependent RNA polymerase This dissertation summarizes the research findings related to CL protease inhibitors of SARS-CoV- and MERS-CoV A structure-guided design approach was used with the aid of X-ray crystallography of inhibitor- CL protease complexes The iterative optimization of potency in enzyme assays and cell-based assays yielded inhibitors with nanomolar potency Multiple inhibitors ... More
SARS-CoV-2 and MERS-CoV president the human coronaviruses and zoonotic coronaviruses due to tremendous repercussions to wellbeing of the world population. Investigational efforts focusing on the disease pathology and viral lifecycle have led to identifying multiple druggable targets of both viruses including 3CL protease, PL protease and RNA dependent RNA polymerase.
This dissertation summarizes the research findings related to 3CL protease inhibitors of SARS-CoV-2 and MERS-CoV. A structure-guided design approach was used with the aid of X-ray crystallography of inhibitor-3CL protease complexes. The iterative optimization of potency in enzyme assays and cell-based assays yielded inhibitors with nanomolar potency. Multiple inhibitors were found to be highly potent (IC50 < 100 nM) against 3CL proteases of SARS-CoV-2 and MERS-CoV. Inhibitors with dramatically improved antiviral activity in cell-based assays (EC50 = 11-13 nM) against SARS-CoV-2 (chapter 5, compounds 5c/d and 11c/d) were developed. Importantly, these inhibitors specifically target viral lifecycle without causing harmful effects on healthy cells as evidenced by having high safety indices (CC50/EC50 > 1000).
Bioisosteric replacement of metabolically vulnerable protons of GC376 with deuterium has led to identification of compound 2 (chapter 1) which showed significantly enhanced survival of SARS-CoV-2 infected K18-hACE2 mice 83 – 100 % compared to 0 % when untreated. Several other drug candidates suitable for conducting further preclinical studies have been identified Less
This dissertation summarizes the research findings related to 3CL protease inhibitors of SARS-CoV-2 and MERS-CoV. A structure-guided design approach was used with the aid of X-ray crystallography of inhibitor-3CL protease complexes. The iterative optimization of potency in enzyme assays and cell-based assays yielded inhibitors with nanomolar potency. Multiple inhibitors were found to be highly potent (IC50 < 100 nM) against 3CL proteases of SARS-CoV-2 and MERS-CoV. Inhibitors with dramatically improved antiviral activity in cell-based assays (EC50 = 11-13 nM) against SARS-CoV-2 (chapter 5, compounds 5c/d and 11c/d) were developed. Importantly, these inhibitors specifically target viral lifecycle without causing harmful effects on healthy cells as evidenced by having high safety indices (CC50/EC50 > 1000).
Bioisosteric replacement of metabolically vulnerable protons of GC376 with deuterium has led to identification of compound 2 (chapter 1) which showed significantly enhanced survival of SARS-CoV-2 infected K18-hACE2 mice 83 – 100 % compared to 0 % when untreated. Several other drug candidates suitable for conducting further preclinical studies have been identified Less
Influenza virus IV causes several outbreaks of the flu each year resulting in an economic burden to the healthcare system in the billions of dollars Several influenza pandemics have occurred during the last century and estimated to have caused million deaths There are four genera of IV A IVA B IVB C IVC and D IVD with IVA being the most virulent to the human population Hemagglutinin HA is an IVA surface protein that allows the virus to attach to host cell receptors and enter the cell Here we have characterised the high-resolution structures of seven IVA HAs with one ... More
Influenza virus (IV) causes several outbreaks of the flu each year resulting in an economic burden to the healthcare system in the billions of dollars. Several influenza pandemics have occurred during the last century and estimated to have caused 100 million deaths. There are four genera of IV, A (IVA), B (IVB), C (IVC), and D (IVD), with IVA being the most virulent to the human population. Hemagglutinin (HA) is an IVA surface protein that allows the virus to attach to host cell receptors and enter the cell. Here we have characterised the high-resolution structures of seven IVA HAs, with one in complex with the anti-influenza head-binding antibody C05. Our analysis revealed conserved receptor binding residues in all structures, as seen in previously characterised IV HAs. Amino acid conservation is more prevalent on the stalk than the receptor binding domain (RBD; also called the head domain), allowing the virus to escape from antibodies targeting the RBD. The equivalent site of C05 antibody binding to A/Denver/57 HA appears hypervariable in the other H1N1 IV HAs. Modifications within this region appear to disrupt binding of the C05 antibody, as these HAs no longer bind the C05 antibody by analytical SEC. Our study brings new insights into the structural and functional recognition of IV HA proteins and can contribute to further development of anti-influenza vaccines. Less
Destabilase from the medical leech Hirudo medicinalis belongs to the family of i-type lysozymes It has two different enzymatic activities microbial cell walls destruction muramidase activity and dissolution of the stabilized fibrin isopeptidase activity Both activities are known to be inhibited by sodium chloride at near physiological concentrations but the structural basis remains unknown Here we present two crystal structures of destabilase including a -resolution structure in complex with sodium ion Our structures reveal the location of sodium ion between Glu Asp residues which were previously recognized as a glycosidase active site While sodium coordination with these amino acids may ... More
Destabilase from the medical leech Hirudo medicinalis belongs to the family of i-type lysozymes. It has two different enzymatic activities: microbial cell walls destruction (muramidase activity), and dissolution of the stabilized fibrin (isopeptidase activity). Both activities are known to be inhibited by sodium chloride at near physiological concentrations, but the structural basis remains unknown. Here we present two crystal structures of destabilase, including a 1.1 Å-resolution structure in complex with sodium ion. Our structures reveal the location of sodium ion between Glu34/Asp46 residues, which were previously recognized as a glycosidase active site. While sodium coordination with these amino acids may explain inhibition of the muramidase activity, its influence on previously suggested Ser49/Lys58 isopeptidase activity dyad is unclear. We revise the Ser49/Lys58 hypothesis and compare sequences of i-type lysozymes with confirmed destabilase activity. We suggest that the general base for the isopeptidase activity is His112 rather than Lys58. pKa calculations of these amino acids, assessed through the 1 μs molecular dynamics simulation, confirm the hypothesis. Our findings highlight the ambiguity of destabilase catalytic residues identification and build foundations for further research of structure–activity relationship of isopeptidase activity as well as structure-based protein design for potential anticoagulant drug development. Less
Transporters of the Nramp Natural resistance-associated macrophage protein family import divalent transition metal ions into cells of most organisms By supporting metal homeostasis Nramps prevent diseases and disorders related to metal insufficiency or overload Previous studies revealed that Nramps take on a LeuT fold and identified the metal-binding site We present high-resolution structures of Deinococcus radiodurans Dra Nramp in three stable conformations of the transport cycle revealing that global conformational changes are supported by distinct coordination geometries of its physiological substrate Mn across conformations and by conserved networks of polar residues lining the inner and outer gates In addition a ... More
Transporters of the Nramp (Natural resistance-associated macrophage protein) family import divalent transition metal ions into cells of most organisms. By supporting metal homeostasis, Nramps prevent diseases and disorders related to metal insufficiency or overload. Previous studies revealed that Nramps take on a LeuT fold and identified the metal-binding site. We present high-resolution structures of Deinococcus radiodurans (Dra)Nramp in three stable conformations of the transport cycle revealing that global conformational changes are supported by distinct coordination geometries of its physiological substrate, Mn2+, across conformations, and by conserved networks of polar residues lining the inner and outer gates. In addition, a high-resolution Cd2+-bound structure highlights differences in how Cd2+ and Mn2+ are coordinated by DraNramp. Complementary metal binding studies using isothermal titration calorimetry with a series of mutated DraNramp proteins indicate that the thermodynamic landscape for binding and transporting physiological metals like Mn2+ is different and more robust to perturbation than for transporting the toxic Cd2+ metal. Overall, the affinity measurements and high-resolution structural information on metal substrate binding provide a foundation for understanding the substrate selectivity of essential metal ion transporters like Nramps. Less
Hydromedusan photoproteins responsible for the bioluminescence of a variety of marine jellyfish and hydroids are a unique biochemical system recognized as a stable enzyme-substrate complex consisting of apoprotein and preoxygenated coelenterazine which is tightly bound in the protein inner cavity The binding of calcium ions to the photoprotein molecule is only required to initiate the light emission reaction Although numerous experimental and theoretical studies on the bioluminescence of these photoproteins were performed many features of their functioning are yet unclear In particular which ionic state of dioxetanone intermediate decomposes to yield a coelenteramide in an excited state and the role ... More
Hydromedusan photoproteins responsible for the bioluminescence of a variety of marine jellyfish and hydroids are a unique biochemical system recognized as a stable enzyme-substrate complex consisting of apoprotein and preoxygenated coelenterazine, which is tightly bound in the protein inner cavity. The binding of calcium ions to the photoprotein molecule is only required to initiate the light emission reaction. Although numerous experimental and theoretical studies on the bioluminescence of these photoproteins were performed, many features of their functioning are yet unclear. In particular, which ionic state of dioxetanone intermediate decomposes to yield a coelenteramide in an excited state and the role of the water molecule residing in a proximity to the N1 atom of 2-hydroperoxycoelenterazine in the bioluminescence reaction are still under discussion. With the aim to elucidate the function of this water molecule as well as to pinpoint the amino acid residues presumably involved in the protonation of the primarily formed dioxetanone anion, we constructed a set of single and double obelin and aequorin mutants with substitutions of His, Trp, Tyr, and Ser to residues with different properties of side chains and investigated their bioluminescence properties (specific activity, bioluminescence spectra, stopped-flow kinetics, and fluorescence spectra of Ca2+-discharged photoproteins). Moreover, we determined the spatial structure of the obelin mutant with a substitution of His64, the key residue of the presumable proton transfer, to Phe. On the ground of the bioluminescence properties of the obelin and aequorin mutants as well as the spatial structures of the obelin mutants with the replacements of His64 and Tyr138, the conclusion was made that, in fact, His residue of the Tyr-His-Trp triad and the water molecule perform the “catalytic function” by transferring the proton from solvent to the dioxetanone anion to generate its neutral ionic state in complex with water, as only the decomposition of this form of dioxetanone can provide the highest light output in the light-emitting reaction of the hydromedusan photoproteins. Less
An oxalate-degrading bacterium in the gut microbiota absorbs food-derived oxalate to use this as a carbon and energy source thereby reducing the risk of kidney stone formation in host animals The bacterial oxalate transporter OxlT selectively uptakes oxalate from the gut to bacterial cells with a strict discrimination from other nutrient carboxylates Here we present crystal structures of oxalate-bound and ligand-free OxlT in two distinct conformations occluded and outward-facing states The ligand-binding pocket contains basic residues that form salt bridges with oxalate while preventing the conformational switch to the occluded state without an acidic substrate The occluded pocket can accommodate ... More
An oxalate-degrading bacterium in the gut microbiota absorbs food-derived oxalate to use this as a carbon and energy source, thereby reducing the risk of kidney stone formation in host animals. The bacterial oxalate transporter OxlT selectively uptakes oxalate from the gut to bacterial cells with a strict discrimination from other nutrient carboxylates. Here, we present crystal structures of oxalate-bound and ligand-free OxlT in two distinct conformations, occluded and outward-facing states. The ligand-binding pocket contains basic residues that form salt bridges with oxalate while preventing the conformational switch to the occluded state without an acidic substrate. The occluded pocket can accommodate oxalate but not larger dicarboxylates, such as metabolic intermediates. The permeation pathways from the pocket are completely blocked by extensive interdomain interactions, which can be opened solely by a flip of a single side chain neighbouring the substrate. This study shows the structural basis underlying metabolic interactions enabling favourable symbiosis. Less
Clostridioides difficile is a Gram-positive opportunistic human pathogen that causes deaths annually in the United States prompting a need for vaccine development In addition to the important toxins TcdA and TcdB binary toxin CDT plays a significant role in the pathogenesis of certain C difficile ribotypes by catalyzing the ADP-ribosylation of actin in host cells However the mechanisms of CDT neutralization by antibodies have not been studied limiting our understanding of key epitopes for CDT antigen design Therefore we isolated neutralizing monoclonal antibodies against CDT and characterized their mechanisms of neutralization structurally and biochemically Here - and - resolution X-ray ... More
Clostridioides difficile is a Gram-positive opportunistic human pathogen that causes 15,000 deaths annually in the United States, prompting a need for vaccine development. In addition to the important toxins TcdA and TcdB, binary toxin (CDT) plays a significant role in the pathogenesis of certain C. difficile ribotypes by catalyzing the ADP-ribosylation of actin in host cells. However, the mechanisms of CDT neutralization by antibodies have not been studied, limiting our understanding of key epitopes for CDT antigen design. Therefore, we isolated neutralizing monoclonal antibodies against CDT and characterized their mechanisms of neutralization structurally and biochemically. Here, 2.5-Å and 2.6-Å resolution X-ray crystal structures of the antibodies BINTOXB/22 and BINTOXB/9, respectively, in complex with CDTb—the CDT subunit that forms a heptameric pore for the delivery of toxic CDTa enzyme into the host cytosol—showed that both antibodies sterically clash with adjacent protomers in the assembled heptamer. Assessment of trypsin-induced oligomerization of the purified CDTb protoxin in vitro showed that BINTOXB/22 and BINTOXB/9 prevented the assembly of di-heptamers upon prodomain cleavage. This work suggests that the CDT oligomerization process can be effectively targeted by antibodies, which will aid in the development of C. difficile vaccines and therapeutics. Less
We report the structural biochemical and functional characterization of the product of gene PA from Pseudomonas aeruginosa PAO The protein termed Pa Dps adopts the Dps subunit fold and oligomerizes into a nearly spherical -mer quaternary structure at pH or in the presence of divalent cations at neutral pH and above The -Mer Pa Dps contains two di-iron centers at the interface of each subunit dimer coordinated by conserved His Glu and Asp residues In vitro the di-iron centers catalyze the oxidation of Fe utilizing H O not O as an oxidant suggesting Pa Dps functions to aid P aeruginosa ... More
We report the structural, biochemical, and functional characterization of the product of gene PA0962 from Pseudomonas aeruginosa PAO1. The protein, termed Pa Dps, adopts the Dps subunit fold and oligomerizes into a nearly spherical 12-mer quaternary structure at pH 6.0 or in the presence of divalent cations at neutral pH and above. The 12-Mer Pa Dps contains two di-iron centers at the interface of each subunit dimer, coordinated by conserved His, Glu, and Asp residues. In vitro, the di-iron centers catalyze the oxidation of Fe2+ utilizing H2O2 (not O2) as an oxidant, suggesting Pa Dps functions to aid P. aeruginosa to survive H2O2-mediated oxidative stress. In agreement, a P. aeruginosa Δdps mutant is significantly more susceptible to H2O2 than the parent strain. The Pa Dps structure harbors a novel network of Tyr residues at the interface of each subunit dimer between the two di-iron centers, which captures radicals generated during Fe2+ oxidation at the ferroxidase centers and forms di-tyrosine linkages, thus effectively trapping the radicals within the Dps shell. Surprisingly, incubating Pa Dps and DNA revealed unprecedented DNA cleaving activity that is independent of H2O2 or O2 but requires divalent cations and 12-mer Pa Dps. Less
Tyrosine kinases TKs play essential roles in signaling processes that regulate cell survival migration and proliferation Dysregulation of tyrosine kinases underlies many disorders including cancer cardiovascular and developmental diseases as well as pathologies of the immune system Ack and Brk are nonreceptor tyrosine kinases NRTKs best known for their roles in cancer Here we have biochemically characterized novel Ack and Brk mutations identified in patients with systemic lupus erythematosus SLE These mutations are the first SLE-linked polymorphisms found among NRTKs We show that two of the mutants are catalytically inactive while the other three have reduced activity To understand the ... More
Tyrosine kinases (TKs) play essential roles in signaling processes that regulate cell survival, migration, and proliferation. Dysregulation of tyrosine kinases underlies many disorders, including cancer, cardiovascular and developmental diseases, as well as pathologies of the immune system. Ack1 and Brk are nonreceptor tyrosine kinases (NRTKs) best known for their roles in cancer. Here, we have biochemically characterized novel Ack1 and Brk mutations identified in patients with systemic lupus erythematosus (SLE). These mutations are the first SLE-linked polymorphisms found among NRTKs. We show that two of the mutants are catalytically inactive, while the other three have reduced activity. To understand the structural changes associated with the loss-of-function phenotype, we solved the crystal structure of one of the Ack1 kinase mutants, K161Q. Furthermore, two of the mutated residues (Ack1 A156 and K161) critical for catalytic activity are highly conserved among other TKs, and their substitution in other members of the kinase family could have implications in cancer. In contrast to canonical gain-of-function mutations in TKs observed in many cancers, we report loss-of-function mutations in Ack1 and Brk, highlighting the complexity of TK involvement in human diseases. Less
PT Formulatrix is one of the industries that produce automation tools or robots therefore companies are required to produce automation products that meet the needs of the world market PT Formulatrix has production divisions LH Liquid Handling and RI Rock Imager Examples of the production of automation equipment include NT Formulator for liquid handling and RI RI RI for rock imagers The difference lies in the shape and function of each product The problem focuses on the rock imager production process where production is made with a make to order system and the assembly process is parallel making the assembly ... More
PT. Formulatrix is one of the industries that produce automation tools or robots, therefore companies are required to produce automation products that meet the needs of the world market. PT. Formulatrix has 2 production divisions LH (Liquid Handling) and RI (Rock Imager). Examples of the production of automation equipment include, NT8, Formulator for liquid handling and RI 1000, RI 182, RI 54 for rock imagers. The difference lies in the shape and function of each product. The problem focuses on the rock imager production process, where production is made with a make to order system and the assembly process is parallel, making the assembly not smooth because you have to choose components, waiting for replacement components because they are damaged or missing to assemble. To improve the effective and efficient production process, replace the better production process. While production in liquid handling is said to be smooth because the production is small and the product is small compared to the rock imager. Assembling in parallel makes assemblers a hassle when assembling robots, because they have to sort out robot components in one container. Making assembly inefficient and ineffective. After collecting 5S data to support the kitting process in the rock imager assembling division, the process of assembling a one-table robot with components. As for the application of the 5S method applied at PT. Formulatrix Indonesia. The seiri method is applied to every division without exception, because in each division many mechanical equipment is found scattered on tables or on the floor. Seiton is a continuation of seiri, where the sorting results that have been carried out will be followed by the process of arranging the sorted equipment. Seiso at this stage, what is being done is the cleaning process. The cleaning that is done is cleaning the work area, such as the floor of the equipment used for the production process. At this stage it is more directed at the process of monitoring the 5S method that has been implemented. This stage is the last part of the 5S method. This section focuses more on how to get used to the application of this method. With the kitting process and the 5S method, it is hoped that assembling robots will be more efficient and effective, because waiting, sorting out parts, and looking for tools to assemble will be more optimal than the old process. With the kitting process, the efficiency value is better than the old process, from the previous 83% to 92%, which the researchers got from a trial at PT. Formulatrix Indonesia. Keywords: Rock Imager production process, 5S, Kitting. Less
Enzymatic pockets such as those of histone deacetylases HDACs are among the most favored targets for drug development However enzymatic inhibitors often exhibit low selectivity and high toxicity due to targeting multiple enzyme paralogs which are often involved in distinct multisubunit complexes Here we report the discovery and characterization of a non-enzymatic small molecule inhibitor of HDAC transcriptional repression functions with comparable anti-tumor activity to the enzymatic HDAC inhibitor Vorinostat and anti-psychedelic activity of an HDAC knockout in vivo We highlight that these phenotypes are achieved while modulating the expression of - and -fold fewer genes than enzymatic and genetic ... More
Enzymatic pockets such as those of histone deacetylases (HDACs) are among the most favored targets for drug development. However, enzymatic inhibitors often exhibit low selectivity and high toxicity due to targeting multiple enzyme paralogs, which are often involved in distinct multisubunit complexes. Here, we report the discovery and characterization of a non-enzymatic small molecule inhibitor of HDAC transcriptional repression functions with comparable anti-tumor activity to the enzymatic HDAC inhibitor Vorinostat, and anti-psychedelic activity of an HDAC2 knockout in vivo. We highlight that these phenotypes are achieved while modulating the expression of 20- and 80-fold fewer genes than enzymatic and genetic inhibition in the respective models. Thus, by achieving the same biological outcomes as established therapeutics while impacting a dramatically smaller number of genes, inhibitors of protein-protein interactions can offer important advantages in improving the selectivity of epigenetic modulators. Less
Opioid-related fatal overdoses have reached epidemic proportions Because existing treatments for opioid use disorders offer limited long-term protection accelerating the development of newer approaches is critical Monoclonal antibodies mAbs are an emerging treatment strategy that targets and sequesters selected opioids in the bloodstream reducing drug distribution across the blood-brain barrier thus preventing or reversing opioid toxicity We previously identified a series of murine mAbs with high affinity and selectivity for oxycodone morphine fentanyl and nicotine To determine their binding mechanism we used X-ray crystallography to solve the structures of mAbs bound to their respective targets to resolution or higher Structural ... More
Opioid-related fatal overdoses have reached epidemic proportions. Because existing treatments for opioid use disorders offer limited long-term protection, accelerating the development of newer approaches is critical. Monoclonal antibodies (mAbs) are an emerging treatment strategy that targets and sequesters selected opioids in the bloodstream, reducing drug distribution across the blood-brain barrier, thus preventing or reversing opioid toxicity. We previously identified a series of murine mAbs with high affinity and selectivity for oxycodone, morphine, fentanyl, and nicotine. To determine their binding mechanism, we used X-ray crystallography to solve the structures of mAbs bound to their respective targets, to 2.2 Å resolution or higher. Structural analysis showed a critical convergent hydrogen bonding mode that is dependent on a glutamic acid residue in the mAbs’ heavy chain and a tertiary amine of the ligand. Characterizing drug-mAb complexes represents a significant step toward rational antibody engineering and future manufacturing activities to support clinical evaluation. Less
Chemical modifications of RNA have key roles in many biological processes N -methylguanosine m G is required for integrity and stability of a large subset of tRNAs The methyltransferase WD repeat-containing protein METTL WDR complex is the methyltransferase that modifies G in the variable loop of certain tRNAs and its dysregulation drives tumorigenesis in numerous cancer types Mutations in WDR cause human developmental phenotypes including microcephaly How METTL WDR modifies tRNA substrates and is regulated remains elusive Here we show through structural biochemical and cellular studies of human METTL WDR that WDR serves as a scaffold for METTL and the ... More
Chemical modifications of RNA have key roles in many biological processes1,2,3. N7-methylguanosine (m7G) is required for integrity and stability of a large subset of tRNAs4,5,6,7. The methyltransferase 1–WD repeat-containing protein 4 (METTL1–WDR4) complex is the methyltransferase that modifies G46 in the variable loop of certain tRNAs, and its dysregulation drives tumorigenesis in numerous cancer types8,9,10,11,12,13,14. Mutations in WDR4 cause human developmental phenotypes including microcephaly15,16,17. How METTL1–WDR4 modifies tRNA substrates and is regulated remains elusive18. Here we show, through structural, biochemical and cellular studies of human METTL1–WDR4, that WDR4 serves as a scaffold for METTL1 and the tRNA T-arm. Upon tRNA binding, the αC region of METTL1 transforms into a helix, which together with the α6 helix secures both ends of the tRNA variable loop. Unexpectedly, we find that the predicted disordered N-terminal region of METTL1 is part of the catalytic pocket and essential for methyltransferase activity. Furthermore, we reveal that S27 phosphorylation in the METTL1 N-terminal region inhibits methyltransferase activity by locally disrupting the catalytic centre. Our results provide a molecular understanding of tRNA substrate recognition and phosphorylation-mediated regulation of METTL1–WDR4, and reveal the presumed disordered N-terminal region of METTL1 as a nexus of methyltransferase activity. Less
Linear IgE epitopes play essential roles in persistent allergies including peanut and tree nut allergies Using chemically synthesized peptides attached to membranes and microarray experiments is one approach for determining predominant epitopes that has seen success However the overall expense of this approach and the inherent challenges in scaling up the production and purification of synthetic peptides precludes the general application of this approach To overcome this problem we have constructed a plasmid vector for expressing peptides sandwiched between an N-terminal His-tag and a trimeric protein The vector was used to make overlapping peptides derived from peanut allergens Ara h ... More
Linear IgE epitopes play essential roles in persistent allergies, including peanut and tree nut allergies. Using chemically synthesized peptides attached to membranes and microarray experiments is one approach for determining predominant epitopes that has seen success. However, the overall expense of this approach and the inherent challenges in scaling up the production and purification of synthetic peptides precludes the general application of this approach. To overcome this problem, we have constructed a plasmid vector for expressing peptides sandwiched between an N-terminal His-tag and a trimeric protein. The vector was used to make overlapping peptides derived from peanut allergens Ara h 2. All the peptides were successfully expressed and purified. The resulting peptides were applied to identify IgE binding epitopes of Ara h 2 using four sera samples from individuals with known peanut allergies. New and previously defined dominant IgE binding epitopes of Ara h 2 were identified. This system may be readily applied to produce agents for component- and epitope-resolved food allergy diagnosis. Less
The advent of SARS-CoV- the causative agent of COVID- and its worldwide impact on global health have provided the impetus for the development of effective countermeasures that can be deployed against the virus including vaccines monoclonal antibodies and direct-acting antivirals DAAs Despite these efforts the current paucity of DAAs has created an urgent need for the creation of an enhanced and diversified portfolio of broadly acting agents with different mechanisms of action that can effectively abrogate viral infection SARS-CoV- C-like protease CLpro an enzyme essential for viral replication is a validated target for the discovery of SARS-CoV- therapeutics In this ... More
The advent of SARS-CoV-2, the causative agent of COVID-19, and its worldwide impact on global health, have provided the impetus for the development of effective countermeasures that can be deployed against the virus, including vaccines, monoclonal antibodies, and direct-acting antivirals (DAAs). Despite these efforts, the current paucity of DAAs has created an urgent need for the creation of an enhanced and diversified portfolio of broadly acting agents with different mechanisms of action that can effectively abrogate viral infection. SARS-CoV-2 3C-like protease (3CLpro), an enzyme essential for viral replication, is a validated target for the discovery of SARS-CoV-2 therapeutics. In this report, we describe the structure-guided utilization of the cyclopropane moiety in the design of highly potent inhibitors of SARS-CoV-2 3CLpro, SARS-CoV-1 3CLpro, and MERS-CoV 3CLpro. High-resolution cocrystal structures were used to identify the structural determinants associated with the binding of the inhibitors to the active site of the enzyme and unravel the mechanism of action. Aldehydes 5c and 11c inhibited SARS-CoV-2 replication with EC50 values of 12 and 11 nM, respectively. Furthermore, the corresponding aldehyde bisulfite adducts 5d and 11d were equipotent with EC50 values of 13 and 12 nM, respectively. The safety index (SI) values for compounds 5c/11c and 5d/11d ranged between 7692 and 9090. Importantly, aldehydes 5c/11c and bisulfite adducts 5d/11d potently inhibited MERS-CoV 3CLpro with IC50 values of 80 and 120 nM, and 70 and 70 nM, respectively. Likewise, compounds 5c/11c and 5d/11d inhibited SARS-CoV-1 with IC50 values of 960 and 350 nM and 790 and 240 nM, respectively. Taken together, these studies suggest that the inhibitors described herein have low cytotoxicity and high potency and are promising candidates for further development as broad-spectrum direct-acting antivirals against highly pathogenic coronaviruses. Less
Crystallization of proteins is a critical step in structural biology biopharmaceutical industry and materials science Microfluidic technology has emerged as a promising tool for screening the crystallization conditions with advantages of increased throughput reduced consumption of reagents and lower cost However current mirofluidic approches generally lack the module for high-speed data analysis ignore the time-resolved changes of protein crystalline states or morphologies in the crystallization process and suffer from inconsistency after scaling up due to the subnano- nano-liter-scaled volume To address the issues we propose a deep learning-aided programmable microliter-droplet system which allows the high-throughput screening of time-resolved protein crystallization ... More
Crystallization of proteins is a critical step in structural biology, biopharmaceutical industry and materials science. Microfluidic technology has emerged as a promising tool for screening the crystallization conditions with advantages of increased throughput, reduced consumption of reagents and lower cost. However, current mirofluidic approches generally lack the module for high-speed data analysis, ignore the time-resolved changes of protein crystalline states or morphologies in the crystallization process and suffer from inconsistency after scaling up due to the subnano-/nano-liter-scaled volume. To address the issues, we propose a deep learning-aided programmable microliter-droplet system, which allows the high-throughput screening of time-resolved protein crystallization in microliter scale. Based on the system, a series of temporal phase diagrams are acquired, which reveal the time-resolved crystallization of target proteins under different crystallization conditions. They provide precise guidance on the scale-up experiment (∼93 % in consistency), and help gain insight into the kinetic characteristics of protein crystallization. Less
Mitogen-activated protein kinase MAPK phosphatase MKP is responsible for regulating the activity of the stress-responsive MAPKs and has been put forth as a potential therapeutic target for a number of diseases including dystrophic muscle disease a fatal rare disease which has neither a treatment nor cure In previous work we identified Compound -dimethyl- - - methylthio - -dihydrothieno -h quinazolin- -yl thio butan- -one as the lead compound of a novel class of MKP inhibitors In this work we explore the structure-activity relationship for inhibition of MKP through modifications to the scaffold and functional groups present in A series of ... More
Mitogen-activated protein kinase (MAPK) phosphatase 5 (MKP5) is responsible for regulating the activity of the stress-responsive MAPKs and has been put forth as a potential therapeutic target for a number of diseases, including dystrophic muscle disease a fatal rare disease which has neither a treatment nor cure. In previous work, we identified Compound 1 (3,3-dimethyl-1-((9-(methylthio)-5,6-dihydrothieno[3,4-h]quinazolin-2-yl)thio)butan-2-one) as the lead compound of a novel class of MKP5 inhibitors. In this work, we explore the structure-activity relationship for inhibition of MKP5 through modifications to the scaffold and functional groups present in 1. A series of derivative compounds was designed, synthesized, and evaluated for inhibition of MKP5. In addition, the X-ray crystal structures of six enzyme-inhibitor complexes were solved, further elucidating the necessary requirements for MKP5 inhibition. We found that the parallel-displaced π-π interaction between the inhibitor three-ring core and Tyr435 is critical for modulating potency, and that modifications to the core and functionalization at the C-9 position are essential for ensuring proper positioning of the core for this interaction. These results lay the foundation from which more potent MKP5 allosteric inhibitors can be developed for potential therapeutics towards the treatment of dystrophic muscle disease. Less
Applying rational design we developed kDa cyanobacteriochrome-based near-infrared NIR-I fluorescent protein miRFP nano miRFP nano efficiently binds endogenous biliverdin chromophore and brightly fluoresces in mammalian cells and tissues miRFP nano has maximal emission at nm and an emission tail in the short-wave infrared SWIR region allowing deep-penetrating off-peak fluorescence imaging in vivo The miRFP nano structure reveals the molecular basis of its red shift We demonstrate superiority of miRFP nano-enabled SWIR imaging over NIR-I imaging of microbes in the mouse digestive tract mammalian cells injected into the mouse mammary gland and NF-kB activity in a mouse model of liver inflammation
Coelenterazine-v CTZ-v a synthetic vinylene-bridged -extended derivative is able to significantly alter bioluminescence spectra of different CTZ-dependent luciferases and photoproteins by shifting them towards longer wavelengths However Ca -regulated photoproteins activated with CTZ-v display very low bioluminescence activities that hampers its usage as a substrate of photoprotein bioluminescence Here we report the crystal structure of semi-synthetic Ca -discharged obelin-v bound with the reaction product determined at resolution Comparison of the crystal structure of Ca -discharged obelin-v with those of other obelins before and after bioluminescence reaction reveals no considerable changes in the overall structure However the drastic changes in CTZ-binding ... More
Coelenterazine-v (CTZ-v), a synthetic vinylene-bridged π-extended derivative, is able to significantly alter bioluminescence spectra of different CTZ-dependent luciferases and photoproteins by shifting them towards longer wavelengths. However, Ca2+-regulated photoproteins activated with CTZ-v display very low bioluminescence activities that hampers its usage as a substrate of photoprotein bioluminescence. Here, we report the crystal structure of semi-synthetic Ca2+-discharged obelin-v bound with the reaction product determined at 2.1 Å resolution. Comparison of the crystal structure of Ca2+-discharged obelin-v with those of other obelins before and after bioluminescence reaction reveals no considerable changes in the overall structure. However, the drastic changes in CTZ-binding cavity are observed owing to the completely different reaction product, coelenteramine-v (CTM-v). Since CTM-v is certainly the main product of obelin-v bioluminescence and is considered to be a product of the “dark” pathway of dioxetanone intermediate decomposition, it explains the low bioluminescence activity of obelin and apparently of other photoproteins with CTZ-v. Less
The past fifty years have been marked by the surge of neurodegenerative diseases Unfortunately current treatments are only symptomatic Hence the search for new and innovative therapeutic targets for curative treatments becomes a major challenge Among these targets the adenosine A A receptor A AAR has been the subject of much research in recent years In this paper we report the design synthesis and pharmacological analysis of quinazoline derivatives as A AAR antagonists with high ligand efficiency This class of molecules has been discovered by a virtual screening and bears no structural semblance with reference antagonist ZM- More precisely we ... More
The past fifty years have been marked by the surge of neurodegenerative diseases. Unfortunately, current treatments are only symptomatic. Hence, the search for new and innovative therapeutic targets for curative treatments becomes a major challenge. Among these targets, the adenosine A2A receptor (A2AAR) has been the subject of much research in recent years. In this paper, we report the design, synthesis and pharmacological analysis of quinazoline derivatives as A2AAR antagonists with high ligand efficiency. This class of molecules has been discovered by a virtual screening and bears no structural semblance with reference antagonist ZM-241385. More precisely, we identified a series of 2-aminoquinazoline as promising A2AAR antagonists. Among them, one compound showed a high affinity towards A2AAR (21a, Ki = 20 nM). We crystallized this ligand in complex with A2AAR, confirming one of our predicted docking poses and opening up possibilities for further optimization to derive selective ligands for specific adenosine receptor subtypes. Less
Transmembrane ion transport is a key process in living cells Active transport of ions is carried out by various ion transporters including microbial rhodopsins MRs MRs perform diverse functions such as active and passive ion transport photo-sensing and others In particular MRs can pump various monovalent ions like Na K Cl I NO The only characterized MR proposed to pump sulfate in addition to halides belongs to the cyanobacterium Synechocystis sp PCC and is named Synechocystis halorhodopsin SyHR The structural study of SyHR may help to understand what makes an MR pump divalent ions Here we present the crystal structure ... More
Transmembrane ion transport is a key process in living cells. Active transport of ions is carried out by various ion transporters including microbial rhodopsins (MRs). MRs perform diverse functions such as active and passive ion transport, photo-sensing, and others. In particular, MRs can pump various monovalent ions like Na+, K+, Cl−, I−, NO3−. The only characterized MR proposed to pump sulfate in addition to halides belongs to the cyanobacterium Synechocystis sp. PCC 7509 and is named Synechocystis halorhodopsin (SyHR). The structural study of SyHR may help to understand what makes an MR pump divalent ions. Here we present the crystal structure of SyHR in the ground state, the structure of its sulfate-bound form as well as two photoreaction intermediates, the K and O states. These data reveal the molecular origin of the unique properties of the protein (exceptionally strong chloride binding and proposed pumping of divalent anions) and sheds light on the mechanism of anion release and uptake in cyanobacterial halorhodopsins. The unique properties of SyHR highlight its potential as an optogenetics tool and may help engineer different types of anion pumps with applications in optogenetics. Less