Structural basis for an exceptionally strong preference for asparagine residue at the S2 subsite of Stenotrophomonas maltophilia dipeptidyl peptidase 7

The emergence of drug-resistant bacteria has become a major problem worldwide. Bacterial dipeptidyl peptidases 7 and 11 (DPP7s and DPP11s), belonging to the family-S46 peptidases, are important enzymes for bacterial growth and are not present in mammals. Therefore, specific inhibitors for these peptidases are promising as potential antibiotics. While the molecular mechanisms underlining strict specificity at the S1 subsite of S46 peptidases have been well studied, those of relatively broad preference at the S2 subsite of these peptidases are unknown. In this study, we performed structural and biochemical analyses on DPP7 from Stenotrophomonas maltophilia (SmDPP7). SmDPP7 showed preference for the accommodation of hydrophobic amino acids at the S2 subsite in general, but as an exception, also for asparagine, a hydrophilic amino acid. Structural analyses of SmDPP7 revealed that this exceptional preference to asparagine is caused by a hydrogen bonding network at the bottom of the S2 subsite. The residues in the S2 subsite are well conserved among S46 peptidases as compared with those in the S1 subsite. We expect that our findings will contribute toward the development of a universal inhibitor of S46 peptidases.

A pentagonal hydrogen-bond network facilitates an exceptional preference for asparagine in the S2 subsite of Stenotrophomonas maltophilia dipeptidyl peptidase 7

The emergence of drug-resistant bacteria has become a major problem worldwide. Bacterial dipeptidyl peptidases 7 and 11 (DPP7s and DPP11s), belonging to the family-S46 peptidases, are important enzymes for bacterial growth and are not present in mammals. Therefore, specific inhibitors for these peptidases are promising as potential antibiotics. While the molecular mechanisms underlining strict substrate specificity at the S1 subsite of S46 peptidases have been well studied, those of relatively broad substrate preference at the S2 subsite of these peptidases are unknown. In this study, we performed structural and biochemical analyses on DPP7 from Stenotrophomonas maltophilia (SmDPP7). SmDPP7 showed substrate preference for hydrophobic amino acids at the S2 subsite in general, but as an exception, also for asparagine, a hydrophilic amino acid. Structural analyses of SmDPP7 revealed that this exceptional preference to asparagine is caused by a hydrogen bonding network at the bottom of the S2 subsite. The residues in the S2 subsite are well conserved among S46 peptidases as compared with those in the S1 subsite. We expect that our findings will contribute toward the development of a universal inhibitor of S46 peptidases. (184 words / 200 words)

Structural insight into the catalytic mechanism and inhibitor binding of aminopeptidase A

Aminopeptidase A (APA) is a membrane-bound monozinc aminopeptidase. In the brain, APA generates angiotensin III which exerts a tonic stimulatory effect on the control of blood pressure (BP) in hypertensive animals. The oral administration of RB150 renamed firibastat by WHO, an APA inhibitor prodrug, targeting only the S1 subsite, decreases BP in hypertensive patients from various ethnic origins. To identify new families of potent and selective APA inhibitors, we explored the organization of the APA active site, especially the S2′ subsite. By molecular modeling, docking, molecular dynamics simulations and site-directed mutagenesis, we revealed that Arg368 and Arg386, in the S2′ subsite of human APA established various types of interactions in major part with the P2′ residue but also with the P1′ residue of APA inhibitors, required for their nanomolar inhibitory potency. We also demonstrated an important role for Arg368 in APA catalysis, in maintaining the structural integrity of the GAMEN motif, a conserved sequence involved in exopeptidase specificity and optimal positioning of the substrate in monozinc aminopeptidases. This arginine together with the GAMEN motif are key players for the catalytic mechanism of these enzymes.

Structural basis for the inhibition of SARS-CoV-2 main protease by antineoplastic drug Carmofur

The antineoplastic drug Carmofur was shown to inhibit SARS-CoV-2 main protease (Mpro). Here the X-ray crystal structure of Mpro in complex with Carmofur reveals that the carbonyl reactive group of Carmofur is covalently bound to catalytic Cys145, whereas its fatty acid tail occupies the hydrophobic S2 subsite. Carmofur inhibits viral replication in cells (EC50 = 24.30 μM) and it is a promising lead compound to develop new antiviral treatment for COVID-19.

HIV-1 Protease Uses Bi-Specific S2/S2’ Subsites To Optimize Cleavage of Two Classes of Target Sites

Retroviral proteases (PR) have a unique specificity that allows cleavage of sites with or without a P1’ proline. A P1’ proline is required at the MA/CA cleavage site due to its role in a post-cleavage conformational change in the capsid protein. However, the HIV-1 PR prefers to have large hydrophobic amino acids flanking the scissile bond, suggesting PR recognizes two different classes of substrate sequences. We analyzed the cleavage rate of over 150 iterations of six different HIV-1 cleavage sites to explore rate determinants of cleavage. We found that cleavage rates are strongly influenced by the two amino acids flanking the amino acids at the scissile bond (P2-P1/P1’-P2’), with two complementary sets of rules. When P1’ is proline, the P2 side chain interacts with a polar region in the S2 subsite of the PR, while the P2’ amino acid interacts with a hydrophobic region of the S2’ subsite. When P1’ is not proline, the orientations of the P2 and P2’ side chains with respect to the scissile bond are reversed; P2 residues interact with a hydrophobic face of the S2 subsite while the P2’ amino acid usually engages hydrophilic amino acids in the S2’ subsite. These results reveal that the HIV-1 PR has evolved bi-functional S2 and S2’ subsites to accommodate the steric effects imposed by a P1’ proline on the orientation of P2 and P2’ substrate side chains. These results also suggest a new strategy for inhibitor design to engage the multiple specificities in these subsites.

Effect of D168V mutation in NS3/4A HCV protease on susceptibilities of faldaprevir and danoprevir

Disrupted hydrogen bonding network in the extended S2 subsite lead to faldaprevir and danoprevir resistances.

Aspartate 496 from the subsite S2 drives specificity of human dipeptidyl peptidase III

Human dipeptidyl peptidase III (hDPP III) is a member of the M49 metallopeptidase family, which is involved in intracellular protein catabolism and oxidative stress response. To investigate the structural basis of hDPP III preference for diarginyl arylamide, using site-directed mutagenesis, we altered its S2 subsite to mimic the counterpart in yeast enzyme. Kinetic studies revealed that the single mutant D496G lost selectivity due to the increase of the Km value. The D496G, but not S504G, showed significantly decreased binding of peptides with N-terminal arginine, and of tynorphin. The results obtained identify Asp496 as an important determinant of human DPP III substrate specificity.