A theoretical study of the active sites of papain and S195C rat trypsin: implications for the low reactivity of mutant serine proteinases.

The serine and cysteine proteinases represent two important classes of enzymes that use a catalytic triad to hydrolyze peptides and esters. The active site of the serine proteinases consists of three key residues, Asp...His...Ser. The hydroxyl group of serine functions as a nucleophile and the imidazole ring of histidine functions as a general acid/general base during catalysis. Similarly, the active site of the cysteine proteinases also involves three key residues: Asn, His, and Cys. The active site of the cysteine proteinases is generally believed to exist as a zwitterion (Asn...His+...Cys-) with the thiolate anion of the cysteine functioning as a nucleophile during the initial stages of catalysis. Curiously, the mutant serine proteinases, thiol subtilisin and thiol trypsin, which have the hybrid Asp...His...Cys triad, are almost catalytically inert. In this study, ab initio Hartree-Fock calculations have been performed on the active sites of papain and the mutant serine proteinase S195C rat trypsin. These calculations predict that the active site of papain exists predominately as a zwitterion (Cys-...His+...Asn). However, similar calculations on S195C rat trypsin demonstrate that the thiol mutant is unable to form a reactive thiolate anion prior to catalysis. Furthermore, structural comparisons between native papain and S195C rat trypsin have demonstrated that the spatial juxtapositions of the triad residues have been inverted in the serine and cysteine proteinases and, on this basis, I argue that it is impossible to convert a serine proteinase to a cysteine proteinase by site-directed mutagenesis.     

Molecular cloning and structural and functional characterization of human cathepsin F, a new cysteine proteinase of the papain family with a long propeptide domain.

A cDNA encoding a new cysteine proteinase belonging to the papain family and called cathepsin F has been cloned from a human prostate cDNA library. This cDNA encodes a polypeptide of 484 amino acids, with the same domain organization as other cysteine proteinases, including a hydrophobic signal sequence, a prodomain, and a catalytic region. However, this propeptide domain is unusually long and distinguishes cathepsin F from other proteinases of the papain family. Cathepsin F also shows all structural motifs characteristic of these proteinases, including the essential cysteine residue of the active site. Consistent with these structural features, cathepsin F produced in Escherichia coli as a fusion protein with glutathione S-transferase degrades the synthetic peptide benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin, a substrate commonly used for functional characterization of cysteine proteinases. Furthermore, this proteolytic activity is blocked by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, an inhibitor of cysteine proteinases. The gene encoding cathepsin F maps to chromosome 11q13, close to that encoding cathepsin W. Cathepsin F is widely expressed in human tissues, suggesting a role in normal protein catabolism. Northern blot analysis also revealed a significant level of expression in some cancer cell lines opening the possibility that this enzyme could be involved in degradative processes occurring during tumor progression.     

Sequence conservation in the chagasin family suggests a common trend in cysteine proteinase binding by unrelated protein inhibitors

The recently described inhibitor of cysteine proteinases from Trypanosoma cruzi, chagasin, was found to have close homologs in several eukaryotes, bacteria and archaea, the first protein inhibitors of cysteine proteases in prokaryotes. These previously uncharacterized 110-130 residue-long proteins share a well-conserved sequence motif that corresponds to two adjacent beta-strands and the short loop connecting them. Chagasin-like proteins also have other conserved, mostly aromatic, residues, and share the same predicted secondary structure. These proteins adopt an all-beta fold with eight predicted beta-strands of the immunoglobulin type. The phylogenetic distribution of the chagasins generally correlates with the presence of papain-like cysteine proteases. Previous studies have uncovered similar trends in cysteine proteinase binding by two unrelated inhibitors, stefin and p41, that belong to the cystatin and thyroglobulin families, respectively. A hypothetical model of chagasin-cruzipain interaction suggests that chagasin may dock to the cruzipain active site in a similar manner with the conserved NPTTG motif of chagasin forming a loop that is similar to the wedge structures formed at the active sites of papain and cathepsin L by stefin and p41.     

Crystal structure of YnjE from Escherichia coli,a sulfurtransferase with three rhodanese domains

Rhodaneses/sulfurtransferases are ubiquitous enzymes that catalyze the transfer of sulfane sulfur from a donor molecule to a thiophilic acceptor via an active site cysteine that is modified to a persulfide during the reaction. Here, we present the first crystal structure of a triple‐domain rhodanese‐like protein, namely YnjE from Escherichia coli, in two states where its active site cysteine is either unmodified or present as a persulfide. Compared to well‐characterized tandem domain rhodaneses, which are composed of one inactive and one active domain, YnjE contains an extra N‐terminal inactive rhodanese‐like domain. Phylogenetic analysis reveals that YnjE triple‐domain homologs can be found in a variety of other γ‐proteobacteria, in addition, some single‐, tandem‐, four and even six‐domain variants exist. All YnjE rhodaneses are characterized by a highly conserved active site loop (CGTGWR) and evolved independently from other rhodaneses, thus forming their own subfamily. On the basis of structural comparisons with other rhodaneses and kinetic studies, YnjE, which is more similar to thiosulfate:cyanide sulfurtransferases than to 3‐mercaptopyruvate:cyanide sulfurtransferases, has a different substrate specificity that depends not only on the composition of the active site loop with the catalytic cysteine at the first position but also on the surrounding residues. In vitro YnjE can be efficiently persulfurated by the cysteine desulfurase IscS. The catalytic site is located within an elongated cleft, formed by the central and C‐terminal domain and is lined by bulky hydrophobic residues with the catalytic active cysteine largely shielded from the solvent.     

Structure and dynamics of coxsackievirus B4 2A proteinase, an enyzme involved in the etiology of heart disease

The 2A proteinases (2A(pro)) from the picornavirus family are multifunctional cysteine proteinases that perform essential roles during viral replication, involving viral polyprotein self-processing and shutting down host cell protein synthesis through cleavage of the eukaryotic initiation factor 4G (eIF4G) proteins. Coxsackievirus B4 (CVB4) 2A(pro) also cleaves heart muscle dystrophin, leading to cytoskeletal dysfunction and the symptoms of human acquired dilated cardiomyopathy. We have determined the solution structure of CVB4 2A(pro) (extending in an N-terminal direction to include the C-terminal eight residues of CVB4 VP1, which completes the VP1-2A(pro) substrate region). In terms of overall fold, it is similar to the crystal structure of the mature human rhinovirus serotype 2 (HRV2) 2A(pro), but the relatively low level (40%) of sequence identity leads to a substantially different surface. We show that differences in the cI-to-eI2 loop between HRV2 and CVB4 2A(pro) translate to differences in the mechanism of eIF4GI recognition. Additionally, the nuclear magnetic resonance relaxation properties of CVB4 2A(pro), particularly of residues G1 to S7, F64 to S67, and P107 to G111, reveal that the substrate region is exchanging in and out of a conformation in which it occupies the active site with association and dissociation rates in the range of 100 to 1,000 s(-1). This exchange influences the conformation of the active site and points to a mechanism for how self-processing can occur efficiently while product inhibition is avoided.     

Gene structure of rat cathepsin H

The gene structure of rat cathepsin H was determined. It comprises at least 12 exons of various lengths (32-433 bp) spanning in total more than 17.5 kbp. The gene structure does not correspond well to the functional unit of the proteinase. The region around the active site Cys residue, the most conserved region among cysteine proteinases, is split by an intron. This is a common characteristic among the gene structures of cysteine proteinases.     

Solution structures and backbone dynamics of Escherichia coli rhodanese PspE in its sulfur-free and persulfide-intermediate forms: implications for the catalytic mechanism of rhodanese

Rhodanese catalyzes the sulfur-transfer reaction that transfers sulfur from thiosulfate to cyanide by a double-displacement mechanism, in which an active cysteine residue plays a central role. Previous studies indicated that the phage-shock protein E (PspE) from Escherichia coli is a rhodanese composed of a single active domain and is the only accessible rhodanese among the three single-domain rhodaneses in E. coli. To understand the catalytic mechanism of rhodanese at the molecular level, we determined the solution structures of the sulfur-free and persulfide-intermediate forms of PspE by nuclear magnetic resonance (NMR) spectroscopy and identified the active site by NMR titration experiments. To obtain further insights into the catalytic mechanism, we studied backbone dynamics by NMR relaxation experiments. Our results demonstrated that the overall structures in both sulfur-free and persulfide-intermediate forms are highly similar, suggesting that no significant conformational changes occurred during the catalytic reaction. However, the backbone dynamics revealed that the motional properties of PspE in its sulfur-free form are different from the persulfide-intermediate state. The conformational exchanges are largely enhanced in the persulfide-intermediate form of PspE, especially around the active site. The present structural and biochemical studies in combination with backbone dynamics provide further insights in understanding the catalytic mechanism of rhodanese.     

Structural basis of cellular redox regulation by human TRP14

Thioredoxin-related protein 14 (TRP14) is involved in regulating tumor necrosis factor-alpha-induced signaling pathways in a different manner from human thioredoxin 1 (Trx1). Here, we report the crystal structure of human TRP14 determined at 1.8-A resolutions. The structure reveals a typical thioredoxin fold with characteristic structural features that account for the substrate specificity of the protein. The surface of TRP14 in the vicinity of the active site includes an extended loop and an additional alpha-helix, and the distribution of charged residues in the surface is different from Trx1. The distinctive dipeptide between the redox-active cysteines contributes to stabilizing the thiolate anion of the active site cysteine 43, increasing reactivity of the cysteine toward substrates. These structural differences in the active site suggest that TRP14 has evolved to regulate cellular redox signaling by recognizing a distinctive group of substrates that would complement the group of proteins regulated by Trx1.     

1.3 A structure of arylsulfatase from Pseudomonas aeruginosa establishes the catalytic mechanism of sulfate ester cleavage in the sulfatase family.

BACKGROUND: Sulfatases constitute a family of enzymes with a highly conserved active site region including a Calpha-formylglycine that is posttranslationally generated by the oxidation of a conserved cysteine or serine residue. The crystal structures of two human arylsulfatases, ASA and ASB, along with ASA mutants and their complexes led to different proposals for the catalytic mechanism in the hydrolysis of sulfate esters. RESULTS: The crystal structure of a bacterial sulfatase from Pseudomonas aeruginosa (PAS) has been determined at 1.3 A. Fold and active site region are strikingly similar to those of the known human sulfatases. The structure allows a precise determination of the active site region, unequivocally showing the presence of a Calpha-formylglycine hydrate as the key catalytic residue. Furthermore, the cation located in the active site is unambiguously characterized as calcium by both its B value and the geometry of its coordination sphere. The active site contains a noncovalently bonded sulfate that occupies the same position as the one in para-nitrocatecholsulfate in previously studied ASA complexes. CONCLUSIONS: The structure of PAS shows that the resting state of the key catalytic residue in sulfatases is a formylglycine hydrate. These structural data establish a mechanism for sulfate ester cleavage involving an aldehyde hydrate as the functional group that initiates the reaction through a nucleophilic attack on the sulfur atom in the substrate. The alcohol is eliminated from a reaction intermediate containing pentacoordinated sulfur. Subsequent elimination of the sulfate regenerates the aldehyde, which is again hydrated. The metal cation involved in stabilizing the charge and anchoring the substrate during catalysis is established as calcium.     

Human UDP-galactose 4-epimerase. Accommodation of UDP-N-acetylglucosamine within the active site

UDP-galactose 4-epimerase catalyzes the interconversion of UDP-galactose and UDP-glucose during normal galactose metabolism. One of the key structural features in the proposed reaction mechanism for the enzyme is the rotation of a 4'-ketopyranose intermediate within the active site pocket. Recently, the three-dimensional structure of the human enzyme with bound NADH and UDP-glucose was determined. Unlike that observed for the protein isolated from Escherichia coli, the human enzyme can also turn over UDP-GlcNAc to UDP-GalNAc and vice versa. Here we describe the three-dimensional structure of human epimerase complexed with NADH and UDP-GlcNAc. To accommodate the additional N-acetyl group at the C2 position of the sugar, the side chain of Asn-207 rotates toward the interior of the protein and interacts with Glu-199. Strikingly, in the human enzyme, the structural equivalent of Tyr-299 in the E. coli protein is replaced with a cysteine residue (Cys-307) and the active site volume for the human protein is calculated to be approximately 15% larger than that observed for the bacterial epimerase. This combination of a larger active site cavity and amino acid residue replacement most likely accounts for the inability of the E. coli enzyme to interconvert UDP-GlcNAc and UDP-GalNAc.     

Use of fluorescence resonance energy transfer to study serpin-proteinase interactions

One of the important questions in the serpin mechanism of inhibition of serine and cysteine proteinases of different specificities and structural classes is whether a common "crushing" mechanism of proteinase inactivation is used in all cases. This mechanism was seen in an X-ray structure of the complex between alpha(1)-proteinase inhibitor and trypsin and required the full insertion of the reactive center loop into beta-sheet A and translocation of the proteinase from one pole of the serpin to the other. However, it has yet to be shown to be general for serine proteinases of structural classes other than the trypsin-fold or for cysteine proteinases with the papain-fold or for the caspases. Fluorescence resonance energy transfer offers a potential means of obtaining an answer to this question for each of these classes, without the concern for the effect that increasing size has on the observed signal that applies to NMR spectroscopy. However, care must be taken to ensure that measurements made represent sufficient overdetermination that the answer obtained is unambiguous.     

Prediction of the secondary structures of stefins and cystatins, the low-molecular mass protein inhibitors of cysteine proteinases

A procedure for classifying proteins of known sequence into structurally similar groups was developed on the basis of the Argos parametric approach. It is shown that stefins and cystatins constitute two structurally well resolved, but homologous groups of proteins. Furthermore, it is very probable that segments of secondary structures within each family are conserved, although significant differences between stefins and cystatins are indicated at the level of secondary structure. Next, secondary structures of all sequenced stefins and cystatins were predicted and used in the construction of secondary structures of the "typical stefin" and the "typical cystatin". Results were interpreted in the light of evolution and inhibition mechanism: Alignment of the "typical stefin" versus the "typical cystatin" secondary structure segments suggests that the divergence of stefin and cystatin families did not occur by a gene fusion event, but only by a mechanism of substitution, insertion and/or deletion. The central region of low-molecular mass cystatins, which is assumed to interact with cysteine proteinases, is predicted to be in a beta-sheet conformation. This resembles the beta-sheet in the active site of "standard mechanism" serine proteinases inhibitors.     

Simple modifications of the serpin reactive site loop convert SCCA2 into a cysteine proteinase inhibitor: a critical role for the P3' proline in facilitating RSL cleavage

The human squamous cell carcinoma antigens (SCCA) 1 and 2 are members of the serpin family that are 92% identical in their amino acid sequence. Despite this similarity, they inhibit distinct classes of proteinases. SCCA1 neutralizes the papain-like cysteine proteinases, cathepsins (cat) S, L, and K; and SCCA2 inhibits the chymotrypsin-like serine proteinases, catG and human mast cell chymase. SCCA2 also can inhibit catS, as well as other papain-like cysteine proteinases, albeit at a rate 50-fold less than that of SCCA1. Analysis of the mechanism of inhibition by SCCA1 revealed that the reactive site loop (RSL) is important for cysteine proteinase inhibition. The inhibition of catS by a mutant SCCA2 containing the RSL of SCCA1 is comparable to that of wild-type SCCA1. This finding suggested that there were no motifs outside and only eight residues within the RSL that were directing catS-specific inhibition. The purpose of this study was to determine which of these residues might account for the marked difference in the ability of SCCA1 and SCCA2 to inhibit papain-like cysteine proteinases. SCCA2 molecules containing different RSL mutations showed that no single amino acid substitution could convert SCCA2 into a more potent cysteine proteinase inhibitor. Rather, different combinations of mutations led to incremental increases in catS inhibitory activity with residues in four positions (P1, P3', P4', and P11') accounting for 80% of the difference in activity between SCCA1 and SCCA2. Interestingly, the RSL cleavage site differed between wild-type SCCA2 and this mutant. Moreover, these data established the importance of a Pro residue in the P3' position for efficient inhibition of catS by both wild-type SCCA1 and mutated SCCA2. Molecular modeling studies suggested that this residue might facilitate positioning of the RSL within the active site of the cysteine proteinase.     

Structural basis of the endoproteinase-protein inhibitor interaction

Proteolytic enzymes are potentially hazardous to their protein environment, so that their activity must be carefully controlled. Living organisms use protein inhibitors as a major tool to regulate the proteolytic activity of proteinases. Most of the inhibitors for which 3D structures are available are directed towards serine proteinases, interacting with the active sites in a 'canonical' i.e. substrate-like manner via an exposed reactive site loop of conserved conformation. More recently, some non-canonically binding serine proteinase inhibitors directed against coagulation factors, in particular thrombin, a few cysteine proteinase inhibitors inhibitory towards papain-like proteinases, and three zinc endopeptidase inhibitors directed against metzincins and thermolysin have been characterised in the free and complexed state, displaying novel mechanisms of inhibition with their target proteinases. These different interaction modes are presented and briefly discussed with respect to the different strategies applied by nature.     

Degradation of myocardial structural proteins in myocardial infarcted dogs is reduced by Ep459, a cysteine proteinase inhibitor

The purpose of this study is to clarify whether cysteine proteinases play an important role in the degradation of myocardial proteins in the infarcted tissue. We studied the effects of a cysteine proteinase inhibitor, Ep459, on degradation of cardiac structural proteins caused by ischemia due to coronary artery ligation for 24 h. Proteolytic effects of purified cysteine proteinases on isolated cardiac tissue were also examined. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, degradation of cardiac structural proteins, particularly of myosin heavy chain, alpha-actinin and troponin-I was observed in the infarcted tissue. Treatment with Ep459 significantly reduced protein degradation and total activity of cathepsins B and L in the infarcted tissue, compared with the findings in the untreated group. The electrophoretic pattern of the infarcted myocardium was similar to that of myofibrillar proteins degraded by cathepsins B and L. These results suggest that cysteine proteinases, particularly cathepsins B and L, are involved in degradation of myofibrillar proteins in myocardial infarction.     

A test case for structure-based functional assignment: the 1.2 A crystal structure of the yjgF gene product from Escherichia coli

The YER057c/YIL051c/YjgF protein family is a set of 24 full-length homologs, each approximately 130 residues in length, and each with no known function or relationship to proteins of known structure. To determine the function of this family, the structure of one member--the YjgF protein from Escherichia coli--was solved and refined at a resolution of 1.2 A. The YjgF molecule is a homotrimer with exact threefold symmetry. Its tertiary and quaternary structures are related to that of Bacillus subtilis chorismate mutase, although their active sites are completely different. The YjgF protein has an active site curiously similar to protein tyrosine phosphatases, including a covalently modified cysteine, but it is unlikely to be functionally related. The lessons learned from this attempt to deduce function from structure may be useful to future projects in structural genomics.     

Anatomy of the E2 ligase fold: implications for enzymology and evolution of ubiquitin/Ub-like protein conjugation

The configuration of the active site of E2 ligases, central enzymes in the ubiquitin/ubiquitin-like protein (Ub/Ubl) conjugation systems, has long puzzled researchers. Taking advantage of the wealth of newly available structures and sequences of E2s from diverse organisms, we performed a large-scale comparative analysis of these proteins. As a result we identified a previously under-appreciated diversity in the active site of these enzymes, in particular, the spatial location of the catalytic cysteine and a constellation of associated conserved residues that potentially contributes to catalysis. We observed structural innovations of differing magnitudes occurring in various families across the E2 fold that might correlate in part with differences in target interaction. A key finding was the independent emergence on multiple occasions of a polar residue, often a histidine, in the vicinity of the catalytic cysteine in different E2 families. We propose that these convergently emerging polar residues have a common function, such as in the stabilization of oxyanion holes during Ub/Ubl transfer and spatial localization of the Ub/Ubl tails in the active site. Thus, the E2 ligases represent a rare example in enzyme evolution of high structural diversity of the active site and position of the catalytic residue despite all characterized members catalyzing a similar reaction. Our studies also indicated certain evolutionarily conserved features in all active members of the E2 superfamily that stabilize the unusual flap-like structure in the fold. These features are likely to form a critical mechanical element of the fold required for catalysis. The results presented here could aid in new experiments to understand E2 catalysis.     

The cystatins: protein inhibitors of cysteine proteinases.

The last decade has witnessed enormous progress of protein inhibitors of cysteine proteinases concerning their structures, functions and evolutionary relationships. Although they differ in their molecular properties and biological distribution, they are structurally related proteins. All three inhibitory families, the stefins, the cystatins and the kininogens, are members of the same superfamily. Recently determined crystal structures of chicken cystatin and human stefin B established a new mechanism of interaction between cysteine proteinases and their inhibitors which is fundamentally different from the standard mechanism for serine proteinases and their inhibitors.     

Solution NMR structure of the NlpC/P60 domain of lipoprotein Spr from Escherichia coli: structural evidence for a novel cysteine peptidase catalytic triad

Escherichia coli Spr is a membrane-anchored cell wall hydrolase. The solution NMR structure of the C-terminal NlpC/P60 domain of E. coli Spr described here reveals that the protein adopts a papain-like alpha+beta fold and identifies a substrate-binding cleft featuring several highly conserved residues. The active site features a novel Cys-His-His catalytic triad that appears to be a unique structural signature of this cysteine peptidase family. Moreover, the relative orientation of these catalytic residues is similar to that observed in the analogous Ser-His-His triad, a variant of the classic Ser-His-Asp charge relay system, suggesting the convergent evolution of a catalytic mechanism in quite distinct peptidase families.