Unexpected Specificity of a Trypsin-like Enzyme
2018; Elsevier BV; Volume: 26; Issue: 4 Linguagem: Inglês
10.1016/j.str.2018.03.007
ISSN1878-4186
AutoresZbigniew Dauter, Alexander Wlodawer,
Tópico(s)Antimicrobial Peptides and Activities
ResumoIn this issue of Structure, Stach et al., 2018Stach N. Kalinska M. Zdzalik M. Kitel R. Karim A. Serwin K. Rut W. Larsen K. Jabaiah A. Firlej M. et al.Unique substrate specificity of SpiE serine protease from Staphylococcus aureus.Structure. 2018; 26 (this issue): 572-579Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar describe the properties of SplE, a trypsin-like enzyme from Staphylococcus aureus. They report, on the basis of biochemical and structural studies, the unusual specificity requiring the presence of histidine in the P1 subsite of its substrate. In this issue of Structure, Stach et al., 2018Stach N. Kalinska M. Zdzalik M. Kitel R. Karim A. Serwin K. Rut W. Larsen K. Jabaiah A. Firlej M. et al.Unique substrate specificity of SpiE serine protease from Staphylococcus aureus.Structure. 2018; 26 (this issue): 572-579Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar describe the properties of SplE, a trypsin-like enzyme from Staphylococcus aureus. They report, on the basis of biochemical and structural studies, the unusual specificity requiring the presence of histidine in the P1 subsite of its substrate. A search of the Protein Data Bank (PDB; Berman et al., 2000Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. The protein data bank.Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (27281) Google Scholar) with the term “trypsin” identifies over 1,000 entries, with a further 500 or so found with the term “chymotrypsin.” The oldest deposited structures of these enzymes are 40 years old (PDB: 1tgb; Fehlhammer et al., 1977Fehlhammer H. Bode W. Huber R. Crystal structure of bovine trypsinogen at 1-8 A resolution. II. Crystallographic refinement, refined crystal structure and comparison with bovine trypsin.J. Mol. Biol. 1977; 111: 415-438Crossref PubMed Scopus (220) Google Scholar), so it should be a safe bet that by now all should be known about the mode of activity and about specificity of this family of enzymes. Nevertheless, a paper by Stach et al., 2018Stach N. Kalinska M. Zdzalik M. Kitel R. Karim A. Serwin K. Rut W. Larsen K. Jabaiah A. Firlej M. et al.Unique substrate specificity of SpiE serine protease from Staphylococcus aureus.Structure. 2018; 26 (this issue): 572-579Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar in this issue of Structure describes unusual properties of another trypsin-like enzyme and its specificity for the presence of histidine in the P1 subsite (Schechter and Berger, 1967Schechter I. Berger A. On the size of the active site in proteases. I. Papain.Biochem. Biophys. Res. Commun. 1967; 27: 157-162Crossref PubMed Scopus (4752) Google Scholar) of its substrate. This trypsin-like enzyme SplE from Staphylococcus aureus is a product of an operon that encodes, in total, six closely related proteases and might constitute a first example of a proteolytic system in bacteria. This operon is located on a pathogenicity island, suggesting that the produced enzymes might be involved in bacterial virulence. All six enzymes share a similar fold, whereas their specificity differs very significantly, ranging from chymotrypsin-like specificity of SplA (P1 tyrosine or phenylalanine) and glutamine, asparagine, and aspartic acid for SplB, to elastase-like specificity of SplD (small residues in P1). SplE, however, differs in its specificity not only from the other enzymes encoded by this operon but from practically all other members of the trypsin family, which rarely if ever are capable of cleaving substrates containing a histidine residue at the P1 site. Determination of the specificity of SplE was a relatively straightforward part of this project. A tetrapeptide library of the type Ac-X4-X3-X2-P1-Acc (where Acc denotes 7-amino-4-carbamoylmethylcoumarin) containing 18 sub-libraries was utilized. In each sub-library, the P1 position contained a particular natural amino acid, while the X4, X3, and X2 positions contained equimolar mixtures of tested residues. Hydrolysis of peptides in the library resulted in an increase of fluorescence, and the results were completely clear—high specificity for a histidine in P1, with much lower activity for a glutamine, and practically no activity with other amino acids. Further elaboration of the specificity of SplE was performed with a cellular library of peptide substrates, in which a large number of potential substrates (8-mer peptides) were displayed on the surface of E. coli cells. The cleavage of these peptide substrates would lead to a change in the fluorescence properties of the cells, amenable to fluorescence-activated cell sorting (FACS). The results of the latter studies have shown that the specificity of SplE was not limited to the P1 site alone, but was also influenced by other sites more distant from the cleavage point. Thus, the P2 site would preferentially contain leucine, whereas the preference at the P3 site was for a large aromatic residue (tyrosine, phenylanine, or tryptophan). The authors determined a high-resolution (1.75 Å) crystal structure of SplE to gain insight into the structural basis of the observed specificity of the enzyme. Whereas the structure is that of the apoenzyme, the conformation of the substrate could be ascertained by modeling based on the data available for other trypsin-like enzymes with ligands present in their active sites. Thus, modeling of the consensus tetrapeptide Trp-Leu-His-Gly in the active site of SplE provides convincing clues about structural features around the S1 pocket of the enzyme responsible for its preference of His in this place. The peptide in the active site of SplE with the surrounding enzyme residues interacting with it are shown in Figure 1 (as well as Figure 4 of Stach et al., 2018Stach N. Kalinska M. Zdzalik M. Kitel R. Karim A. Serwin K. Rut W. Larsen K. Jabaiah A. Firlej M. et al.Unique substrate specificity of SpiE serine protease from Staphylococcus aureus.Structure. 2018; 26 (this issue): 572-579Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). The optimal positioning of the peptide requires only very small rearrangement of the P1 pocket, as observed in the crystal structure of apo-SplE presented by Stach et al., 2018Stach N. Kalinska M. Zdzalik M. Kitel R. Karim A. Serwin K. Rut W. Larsen K. Jabaiah A. Firlej M. et al.Unique substrate specificity of SpiE serine protease from Staphylococcus aureus.Structure. 2018; 26 (this issue): 572-579Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar. The imidazole ring of P1 His is hydrogen bonded through its ND1 atom with the enzyme histidine and through the NE2 atom with the carbonyl oxygen atom of glycine at the bottom of the S1 pocket. These interactions, as well as the presence of Val residue lining the S1 pocket, provide an effective arrangement encompassing the His side chain in the pocket. It is also possible that a Glu side chain, similar in length to His, may also create similar interactions with the same His residue, which would explain the partial specificity of SplE to this amino acid in the P1 site. All members of the Spl family of proteases differ to some extent in the structural details around their active sites from the canonical arrangement observed in other trypsin-like proteases. Stach et al., 2018Stach N. Kalinska M. Zdzalik M. Kitel R. Karim A. Serwin K. Rut W. Larsen K. Jabaiah A. Firlej M. et al.Unique substrate specificity of SpiE serine protease from Staphylococcus aureus.Structure. 2018; 26 (this issue): 572-579Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar suggest a possibility that only some specific substrates induce certain conformational changes leading to the catalytically effective arrangement of the active site. This—currently speculative—idea could explain the exceptional substrate specificity of Spl proteases, which is stricter than that of other related enzymes from different sources. However, as these hypotheses are solely based on the structure of the apoenzyme, and since it is necessary to make some rearrangements to the coordinates to accommodate modeled substrates, these suggestions need to be considered with some caution. It may be expected that the work of Stach et al., 2018Stach N. Kalinska M. Zdzalik M. Kitel R. Karim A. Serwin K. Rut W. Larsen K. Jabaiah A. Firlej M. et al.Unique substrate specificity of SpiE serine protease from Staphylococcus aureus.Structure. 2018; 26 (this issue): 572-579Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar will be pursued, particularly by investigating SplE structures in the presence of non-cleavable substrates or inhibitors, which will shed light on the structural and functional characteristics of this unusual and interesting group of proteases. Unique Substrate Specificity of SplE Serine Protease from Staphylococcus aureusStach et al.StructureMarch 8, 2018In BriefSerine proteases of the chymotrypsin family provide a textbook example of enzyme-substrate recognition. Stach et al. analyze the unique specificity of SplE protease for histidine residues and characterize the structural basis guiding this specificity. Successful rational grafting of SplE specificity onto a protease of different initial substrate preference supports the conclusions. 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