Jiang Yin

Dual modes of modification of hepatitis A virus 3C protease by a serine-derived beta-lactone: selective crystallization and formation of a functional catalytic triad in the active site.

Hepatitis A virus (HAV) 3C proteinase is a member of the picornain cysteine proteases responsible for the processing of the viral polyprotein, a function essential for viral maturation and infectivity. This and its structural similarity to other 3C and 3C-like proteases make it an attractive target for the development of antiviral drugs. Previous solution NMR studies have shown that a Cys24Ser (C24S) variant of HAV 3C protein, which displays catalytic properties indistinguishable from the native enzyme, is irreversibly inactivated by N-benzyloxycarbonyl-l-serine-β-lactone (1a) through alkylation of the sulfur atom at the active site Cys172. However, crystallization of an enzyme–inhibitor adduct from the reaction mixture followed by X-ray structural analysis shows only covalent modification of the ε2-nitrogen of the surface His102 by the β-lactone with no reaction at Cys172. Re-examination of the heteronuclear multiple quantum coherence (HMQC) NMR spectra of the enzyme–inhibitor mixture indicates that dual modes of single covalent modification occur with a ≥3:1 ratio of S-alkylation of Cys172 to N-alkylation of His102. The latter product crystallizes readily, probably due to the interaction between the phenyl ring of the N-benzyloxycarbonyl (N-Cbz) moiety and a hydrophobic pocket of a neighboring protein molecule in the crystal. Furthermore, significant structural changes are observed in the active site of the 3C protease, which lead to the formation of a functional catalytic triad with Asp84 accepting one hydrogen bond from His44. Although the 3C protease modified at Cys172 is catalytically inactive, the singly modified His102 Nε2-alkylated protein displays a significant level of enzymatic activity, which can be further modified/inhibited by N-iodoacetyl-valine-phenylalanine-amide (IVF) (in solution and in crystal) or excessive amount of the same β-lactone inhibitor (in solution). The success of soaking IVF into HAV 3C–1a crystals demonstrates the usefulness of this new crystal form in the study of enzyme–inhibitor interactions in the proteolytic active site.

Insights into mucopolysaccharidosis I from the structure and action of α-L-iduronidase

Mucopolysaccharidosis type I (MPS I), caused by mutations in the gene encoding α-L-iduronidase (IDUA), is one of approximately 70 genetic disorders collectively known as the lysosomal storage diseases. To gain insight into the basis for MPS I, we have crystallized human IDUA produced in an Arabidopsis thaliana cgl mutant. IDUA consists of a TIM barrel domain containing the catalytic site, a β-sandwich domain and a fibronectin-like domain. Structures of IDUA bound to induronate analogues illustrate the Michaelis complex and reveal a 2,5B conformation in the glycosyl-enzyme intermediate, that suggest a retaining double displacement reaction employing the nucleophilic Glu299 and the general acid/base Glu182. Surprisingly, the N-glycan attached to Asn372 interacts with iduronate analogues in the active site and is required for enzymatic activity. Finally, these IDUA structures and biochemical analysis of the disease-relevant Pro533Arg mutation have enabled us to correlate the effects of mutations in IDUA to clinical phenotypes.

A mechanistic view of enzyme inhibition and peptide hydrolysis in the active site of the SARS-CoV 3C-like peptidase

Abstract The 3C-like main peptidase 3CLpro is a viral polyprotein processing enzyme essential for the viability of the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV). While it is generalized that 3CLpro and the structurally related 3Cpro viral peptidases cleave their substrates via a mechanism similar to that underlying the peptide hydrolysis by chymotrypsin-like serine proteinases (CLSPs), some of the hypothesized key intermediates have not been structurally characterized. Here, we present three crystal structures of SARS 3CLpro in complex with each of two members of a new class of peptide-based phthalhydrazide inhibitors. Both inhibitors form an unusual thiiranium ring with the nucleophilic sulfur atom of Cys145, trapping the enzymes catalytic residues in configurations similar to the intermediate states proposed to exist during the hydrolysis of native substrates. Most significantly, our crystallographic data are consistent with a scenario in which a water molecule, possibly via indirect coordination from the carbonyl oxygen of Thr26, has initiated nucleophilic attack on the enzyme-bound inhibitor. Our data suggest that this structure resembles that of the proposed tetrahedral intermediate during the deacylation step of normal peptidyl cleavage.

Crystal Structure of the Vitamin B12 Biosynthetic Cobaltochelatase, CbiXS, from Archaeoglobus Fulgidus

The Archaeoglobus fulgidus gene af0721 encodes CbiXS, a small cobaltochelatase associated with the anaerobic biosynthesis of vitamin B12 (cobalamin). The protein was shown to have activity both in vivo and in vitro, catalyzing the insertion of Co2+ into sirohydrochlorin. The structure of CbiXS was determined in two different crystal forms and was shown to consist of a central mixed β-sheet flanked by four α-helices, one of which originates in the C-terminus of a neighboring molecule. CbiXS is about half the size of other Class II tetrapyrrole chelatases. The overall topography of CbiXS exhibits substantial resemblance to both the N- and C-terminal regions of several members of the Class II metal chelatases involved in tetrapyrrole biosynthesis. Two histidines (His10 and His74), are in similar positions as the catalytic histidine residues in the anaerobic cobaltochelatase CbiK (His145 and His207). In light of the hypothesis that suggests the larger chelatases evolved via gene duplication and fusion from a CbiXS-like enzyme, the structure of AF0721 may represent that of an “ancestral” precursor of class II metal chelatases.

The crystal structure of shiga toxin type 2 with bound disaccharide guides the design of a heterobifunctional toxin inhibitor.

Background: E. coli Shiga like toxin type 2 (Stx2a) is responsible for serious clinical outcomes. Results: The crystal structure of Stx2a with bound disaccharide was solved and used to design a potent toxin inhibitor. Conclusion: The primary binding site of Stx2a is able to accommodate extended structural elements. Significance: Knowledge of the toxin binding site can guide discovery of therapeutics to treat E. coli food poisoning. Shiga toxin type 2 (Stx2a) is clinically most closely associated with enterohemorrhagic E. coli O157:H7-mediated hemorrhagic colitis that sometimes progresses to hemolytic-uremic syndrome. The ability to express the toxin has been acquired by other Escherichia coli strains, and outbreaks of food poisoning have caused significant mortality rates as, for example, in the 2011 outbreak in northern Germany. Stx2a, an AB5 toxin, gains entry into human cells via the glycosphingolipid receptor Gb3. We have determined the first crystal structure of a disaccharide analog of Gb3 bound to the B5 pentamer of Stx2a holotoxin. In this Gb3 analog, α-GalNAc replaces the terminal α-Gal residue. This co-crystal structure confirms previous inferences that two of the primary binding sites identified in the B5 pentamer of Stx1 are also functional in Stx2a. This knowledge provides a rationale for the synthesis and evaluation of heterobifunctional antagonists for E. coli toxins that target Stx2a. Incorporation of GalNAc Gb3 trisaccharide in a heterobifunctional ligand with an attached pyruvate acetal, a ligand for human amyloid P component, and conjugation to poly[acrylamide-co-(3-azidopropylmethacrylamide)] produced a polymer that neutralized Stx2a in a mouse model of Shigatoxemia.

Aryl methylene ketones and fluorinated methylene ketones as reversible inhibitors for severe acute respiratory syndrome (SARS) 3C-like proteinase.

Abstract The severe acute respiratory syndrome (SARS) virus depends on a chymotrypsin-like cysteine proteinase (3CLpro) to process the translated polyproteins to functional viral proteins. This enzyme is a target for the design of potential anti-SARS drugs. A series of ketones and corresponding mono- and di-fluoro ketones having two or three aromatic rings were synthesized as possible reversible inhibitors of SARS 3CLpro. The design was based on previously established potent inhibition of the enzyme by oxa analogues (esters), which also act as substrates. Structure–activity relationships and modeling studies indicate that three aromatic rings, including a 5-bromopyridin-3-yl moiety, are key features for good inhibition of SARS 3CLpro. Compound 11d, 2-(5-bromopyridin-3-yl)-1-(5-(4-chlorophenyl)furan-2-yl)ethanone and its α-monofluorinated analogue 12d, gave the best reversible inhibition with IC50 values of 13μM and 28μM, respectively. In contrast to inhibitors having two aromatic rings, α-fluorination of compounds with three rings unexpectedly decreased the inhibitory activity.

Heteroaromatic ester inhibitors of hepatitis A virus 3C proteinase : Evaluation of mode of action

Abstract The related 3C and 3C-like proteinase (3Cpro and 3CLpro) of picornaviruses and coronaviruses, respectively, are good drug targets. As part of an effort to generate broad-spectrum inhibitors of these enzymes, we screened a library of inhibitors based on a halopyridinyl ester from a previous study of the severe acute respiratory syndrome (SARS) 3CL proteinase against Hepatitis A virus (HAV) 3Cpro. Three of the compounds, which also had furan rings, inhibited the cleavage activity of HAV 3Cpro with K ics of 120–240nM. HPLC-based assays revealed that the inhibitors were slowly hydrolyzed by both HAV 3Cpro and SARS 3CLpro, confirming the identity of the expected products. Mass spectrometric analyses indicated that this hydrolysis proceeded via an acyl-enzyme intermediate. Modeling studies indicated that the halopyridinyl moiety of the inhibitor fits tightly into the S1-binding pocket, consistent with the lack of tolerance of the inhibitors to modification in this portion of the molecule. These compounds are among the most potent non-peptidic inhibitors reported to date against a 3Cpro.

Molecular docking identifies the binding of 3-chloropyridine moieties specifically to the S1 pocket of SARS-CoV Mpro.

Abstract The 3C-like main proteinase of the severe acute respiratory syndrome (SARS) coronavirus, SARS-CoV Mpro, is widely considered to be a major drug target for the development of anti-SARS treatment. Based on the chemical structure of a lead compound from a previous screening, we have designed and synthesized a number of non-peptidyl inhibitors, some of which have shown significantly improved inhibitory activity against SARS-CoV Mpro with IC50 values of ∼60nM. In the absence of SARS-CoV Mpro crystal structures in complex with these synthetic inhibitors, molecular docking tools have been employed to study possible interactions between these inhibitors and SARS-CoV Mpro. The docking results suggest two major modes for the initial binding of these inhibitors to the active site of SARS-CoV Mpro. They also establish a structural basis for the ‘core design’ of these inhibitors by showing that the 3-chloropyridine functions common to all of the present inhibitors tend to cluster in the S1 specificity pocket. In addition, intrinsic flexibility in the S4 pocket allows for the accommodation of bulky groups such as benzene rings, suggesting that this structural plasticity can be further exploited for optimizing inhibitor–enzyme interactions that should promote a tighter binding mode. Most importantly, our results provide the structural basis for rational design of wide-spectrum antiviral drugs targeting the chymotrypsin-like cysteine proteinases from coronaviruses and picornaviruses.

Expression, purification and preliminary crystallographic analysis of Rv2247, the β subunit of acyl-CoA carboxylase (ACCD6) from Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) acyl-CoA carboxylase is involved in the biosynthesis of mycolic acids, which are a key component of the bacillus cell wall. The Mtb genome encodes six acyl-CoA carboxylase β subunits (ACCD1-6), three of which (ACCD4-6) are essential for survival of the pathogen on minimal medium. Mtb ACCD6 has been expressed, purified and crystallized. The two forms of Mtb ACCD6 crystals belonged to space groups P4(1)2(1)2 and P2(1)2(1)2(1) and diffracted to 2.9 and 2.5 Å resolution, respectively, at a synchrotron-radiation source.

The Ubiquitination of PINK1 Is Restricted to Its Mature 52-kDa Form

SUMMARY Along with Parkin, PINK1 plays a critical role in maintaining mitochondrial quality control. Although PINK1 is expressed constitutively, its level is kept low in healthy mitochondria by polyubiquitination and ensuing proteasomal degradation of its mature, 52 kDa, form. We show here that the target of PINK1 polyubiquitination is the mature form and is mediated by ubiquitination of a conserved lysine at position 137. Notably, the full-length protein also contains Lys-137 but is not ubiquitinated. On the basis of our data, we propose that cleavage of full-length PINK1 at Phe-104 disrupts the major hydrophobic membrane-spanning domain in the protein, inducing a conformation change in the resultant mature form that exposes Lys-137 to the cytosol for subsequent modification by the ubiquitination machinery. Thus, the balance between the full-length and mature PINK1 allows its levels to be regulated via ubiquitination of the mature form and ensures that PINK1 functions as a mitochondrial quality control factor.