Crystal structures reveal an induced-fit binding of a substrate-like Aza-peptide epoxide to SARS coronavirus main peptidase.
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ABSTRACT: The SARS coronavirus main peptidase (SARS-CoV M(pro)) plays an essential role in the life-cycle of the virus and is a primary target for the development of anti-SARS agents. Here, we report the crystal structure of M(pro) at a resolution of 1.82 Angstroms, in space group P2(1) at pH 6.0. In contrast to the previously reported structure of M(pro) in the same space group at the same pH, the active sites and the S1 specificity pockets of both protomers in the structure of M(pro) reported here are in the catalytically competent conformation, suggesting their conformational flexibility. We report two crystal structures of M(pro) having an additional Ala at the N terminus of each protomer (M(+A(-1))(pro)), both at a resolution of 2.00 Angstroms, in space group P4(3)2(1)2: one unbound and one bound by a substrate-like aza-peptide epoxide (APE). In the unbound form, the active sites and the S1 specificity pockets of both protomers of M(+A(-1))(pro) are observed in a collapsed (catalytically incompetent) conformation; whereas they are in an open (catalytically competent) conformation in the APE-bound form. The observed conformational flexibility of the active sites and the S1 specificity pockets suggests that these parts of M(pro) exist in dynamic equilibrium. The structural data further suggest that the binding of APE to M(pro) follows an induced-fit model. The substrate likely also binds in an induced-fit manner in a process that may help drive the catalytic cycle.
Project description:The main peptidase (M(pro)) from the coronavirus (CoV) causing severe acute respiratory syndrome (SARS) is one of the most attractive molecular targets for the development of anti-SARS agents. We report the irreversible inhibition of SARS-CoV M(pro) by an aza-peptide epoxide (APE; k(inact)/K(i) = 1900(+/-400) M(-1) s(-1)). The crystal structures of the M(pro):APE complex in the space groups C2 and P2(1)2(1)2(1) revealed the formation of a covalent bond between the catalytic Cys145 S(gamma) atom of the peptidase and the epoxide C3 atom of the inhibitor, substantiating the mode of action of this class of cysteine-peptidase inhibitors. The aza-peptide component of APE binds in the substrate-binding regions of M(pro) in a substrate-like manner, with excellent structural and chemical complementarity. In addition, the crystal structure of unbound M(pro) in the space group C2 revealed that the "N-fingers" (N-terminal residues 1 to 7) of both protomers of M(pro) are well defined and the substrate-binding regions of both protomers are in the catalytically competent conformation at the crystallization pH of 6.5, contrary to the previously determined crystal structures of unbound M(pro) in the space group P2(1).
Project description:Dipeptidyl peptidase IV (DPP IV, DPP4, or DAP IV) preferentially cleaves substrate peptides with Pro or Ala at the P1 position. The substrate recognition mechanism has been fully elucidated for mammalian DPP IV by crystal structure analyses but not for bacterial orthologues. Here, we report the crystal structures of a bacterial DPP IV (PmDAP IV) in its free form and in complexes with two kinds of dipeptides as well as with a non-peptidyl inhibitor at 1.90 to 2.47 Å resolution. Acyl-enzyme intermediates were observed for the dipeptide complexes of PmDAP IV, whereas tetrahedral intermediates were reported for the oligopeptide complexes of mammalian DPP IVs. This variation reflects the different structural environments of the active site Arg residues, which are involved in the recognition of a substrate carbonyl group, of mammalian and bacterial enzymes. A phylogenetic analysis revealed that PmDAP IV is a closer relative of dipeptidyl peptidases 8 and 9 (DPP8 and DPP9, DPP IV-family enzymes) than DPP IV. These results provide new insights into the substrate recognition mechanism of bacterial DAP IVs and may assist in the development of selective inhibitors for DAP IVs from pathogenic asaccharolytic bacteria, which utilise proteins or peptides as an energy source.
Project description:BackgroundArchaemetzincins are metalloproteases occurring in archaea and some mammalia. They are distinct from all the other metzincins by their extended active site consensus sequence HEXXHXXGXXHCX(4)CXMX(17)CXXC featuring four conserved cysteine residues. Very little is known about their biological importance and structure-function relationships.Principal findingsHere we present three crystal structures of the archaemetzincin AfAmzA (Uniprot O29917) from Archaeoglobus fulgidus, revealing a metzincin architecture featuring a zinc finger-like structural element involving the conserved cysteines of the consensus motif. The active sites in all three structures are occluded to different extents rendering the enzymes proteolytically inactive against a large variety of tested substrates. Owing to the different ligand binding there are significant differences in active site architecture, revealing a large flexibility of the loops covering the active site cleft.ConclusionsThe crystal structures of AfAmzA provide the structural basis for the lack of activity in standard proteolytic assays and imply a triggered activity onset upon opening of the active site cleft.
Project description:Coronaviruses (CoVs) can infect humans and multiple species of animals, causing a wide spectrum of diseases. The coronavirus main protease (M(pro)), which plays a pivotal role in viral gene expression and replication through the proteolytic processing of replicase polyproteins, is an attractive target for anti-CoV drug design. In this study, the crystal structures of infectious bronchitis virus (IBV) M(pro) and a severe acute respiratory syndrome CoV (SARS-CoV) M(pro) mutant (H41A), in complex with an N-terminal autocleavage substrate, were individually determined to elucidate the structural flexibility and substrate binding of M(pro). A monomeric form of IBV M(pro) was identified for the first time in CoV M(pro) structures. A comparison of these two structures to other available M(pro) structures provides new insights for the design of substrate-based inhibitors targeting CoV M(pro)s. Furthermore, a Michael acceptor inhibitor (named N3) was cocrystallized with IBV M(pro) and was found to demonstrate in vitro inactivation of IBV M(pro) and potent antiviral activity against IBV in chicken embryos. This provides a feasible animal model for designing wide-spectrum inhibitors against CoV-associated diseases. The structure-based optimization of N3 has yielded two more efficacious lead compounds, N27 and H16, with potent inhibition against SARS-CoV M(pro).
Project description:The ?-1,4-galactosyltransferase 7 (?4GalT7) enzyme is involved in proteoglycan synthesis. In the presence of a manganese ion, it transfers galactose from UDP-galactose to xylose on a proteoglycan acceptor substrate. We present here the crystal structures of human ?4GalT7 in open and closed conformations. A comparison of these crystal structures shows that, upon manganese and UDP or UDP-Gal binding, the enzyme undergoes conformational changes involving a small and a long loop. We also present the crystal structures of Drosophila wild-type ?4GalT7 and D211N ?4GalT7 mutant enzymes in the closed conformation in the presence of the acceptor substrate xylobiose and the donor substrate UDP-Gal, respectively. To understand the catalytic mechanism, we have crystallized the ternary complex of D211N ?4GalT7 mutant enzyme in the presence of manganese with the donor and the acceptor substrates together in the same crystal structure. The galactose moiety of the bound UDP-Gal molecule forms seven hydrogen bonds with the protein molecule. The nonreducing end of the xylose moiety of xylobiose binds to the hydrophobic acceptor sugar binding pocket created by the conformational changes, whereas its extended xylose moiety forms hydrophobic interactions with a Tyr residue. In the ternary complex crystal structure, the nucleophile O4 oxygen atom of the xylose molecule is found in close proximity to the C1 and O5 atoms of the galactose moiety. This is the first time that a Michaelis complex of a glycosyltransferase has been described, and it clearly suggests an SN2 type catalytic mechanism for the ?4GalT7 enzyme.
Project description:N(6)-Methylation of adenosine is the most ubiquitous and abundant modification of nucleoside in eukaryotic mRNA and long non-coding RNA. This modification plays an essential role in the regulation of mRNA translation and RNA metabolism. Recently, human AlkB homolog 5 (Alkbh5) and fat mass- and obesity-associated protein (FTO) were shown to erase this methyl modification on mRNA. Here, we report five high resolution crystal structures of the catalytic core of Alkbh5 in complex with different ligands. Compared with other AlkB proteins, Alkbh5 displays several unique structural features on top of the conserved double-stranded β-helix fold typical of this protein family. Among the unique features, a distinct "lid" region of Alkbh5 plays a vital role in substrate recognition and catalysis. An unexpected disulfide bond between Cys-230 and Cys-267 is crucial for the selective binding of Alkbh5 to single-stranded RNA/DNA by bringing a "flipping" motif toward the central β-helix fold. We generated a substrate binding model of Alkbh5 based on a demethylation activity assay of several structure-guided site-directed mutants. Crystallographic and biochemical studies using various analogs of α-ketoglutarate revealed that the active site cavity of Alkbh5 is much smaller than that of FTO and preferentially binds small molecule inhibitors. Taken together, our findings provide a structural basis for understanding the substrate recognition specificity of Alkbh5 and offer a foundation for selective drug design against AlkB members.
Project description:The bifunctional human enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthetase (PAICS) catalyzes two essential steps in the de novo purine biosynthesis pathway. PAICS is overexpressed in many cancers and could be a promising target for the development of cancer therapeutics. Here, using gene knockdowns and clonogenic survival and cell viability assays, we demonstrate that PAICS is required for growth and survival of prostate cancer cells. PAICS catalyzes the carboxylation of aminoimidazole ribonucleotide (AIR) and the subsequent conversion of carboxyaminoimidazole ribonucleotide (CAIR) and l-aspartate to N-succinylcarboxamide-5-aminoimidazole ribonucleotide (SAICAR). Of note, we present the first structures of human octameric PAICS in complexes with native ligands. In particular, we report the structure of PAICS with CAIR bound in the active sites of both domains and SAICAR bound in one of the SAICAR synthetase domains. Moreover, we report the PAICS structure with SAICAR and an ATP analog occupying the SAICAR synthetase active site. These structures provide insight into substrate and product binding and the architecture of the active sites, disclosing important structural information for rational design of PAICS inhibitors as potential anticancer drugs.
Project description:SulE, an esterase, which detoxifies a variety of sulfonylurea herbicides through de-esterification, provides an attractive approach to remove environmental sulfonylurea herbicides and develop herbicide-tolerant crops. Here, we determined the crystal structures of SulE and an activity improved mutant P44R. Structural analysis revealed that SulE is a dimer with spacious binding pocket accommodating the large sulfonylureas substrate. Particularly, SulE contains a protruding β hairpin with a lid loop covering the active site of the other subunit of the dimer. The lid loop participates in substrate recognition and binding. P44R mutation altered the lid loop flexibility, resulting in the sulfonylurea heterocyclic ring repositioning to a relative stable conformation thus leading to dramatically increased activity. Our work provides important insights into the molecular mechanism of SulE, and establish a solid foundation for further improving the enzyme activity to various sulfonylurea herbicides through rational design.
Project description:Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic virus that causes severe respiratory illness accompanied by multi-organ dysfunction, resulting in a case fatality rate of approximately 40%. As found in other coronaviruses, the majority of the positive-stranded RNA MERS-CoV genome is translated into two polyproteins, one created by a ribosomal frameshift, that are cleaved at three sites by a papain-like protease and at 11 sites by a 3C-like protease (3 CL(pro)). Since 3 CL(pro) is essential for viral replication, it is a leading candidate for therapeutic intervention. To accelerate the development of 3 CL(pro) inhibitors, three crystal structures of a catalytically inactive variant (C148A) of the MERS-CoV 3 CL(pro) enzyme were determined. The aim was to co-crystallize the inactive enzyme with a peptide substrate. Fortuitously, however, in two of the structures the C-terminus of one protomer is bound in the active site of a neighboring molecule, providing a snapshot of an enzyme-product complex. In the third structure, two of the three protomers in the asymmetric unit form a homodimer similar to that of SARS-CoV 3 CL(pro); however, the third protomer adopts a radically different conformation that is likely to correspond to a crystallographic monomer, indicative of substantial structural plasticity in the enzyme. The results presented here provide a foundation for the structure-based design of small-molecule inhibitors of the MERS-CoV 3 CL(pro) enzyme.
Project description:AspH is an endoplasmic reticulum (ER) membrane-anchored 2-oxoglutarate oxygenase whose C-terminal oxygenase and tetratricopeptide repeat (TPR) domains present in the ER lumen. AspH catalyses hydroxylation of asparaginyl- and aspartyl-residues in epidermal growth factor-like domains (EGFDs). Here we report crystal structures of human AspH, with and without substrate, that reveal substantial conformational changes of the oxygenase and TPR domains during substrate binding. Fe(II)-binding by AspH is unusual, employing only two Fe(II)-binding ligands (His679/His725). Most EGFD structures adopt an established fold with a conserved Cys1-3, 2-4, 5-6 disulfide bonding pattern; an unexpected Cys3-4 disulfide bonding pattern is observed in AspH-EGFD substrate complexes, the catalytic relevance of which is supported by studies involving stable cyclic peptide substrate analogues and by effects of Ca(II) ions on activity. The results have implications for EGFD disulfide pattern processing in the ER and will enable medicinal chemistry efforts targeting human 2OG oxygenases.