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  • Reparixin mg At the top of the S subsite cylinder are

    2024-07-09

    At the top of the S1 subsite cylinder are two “cap” residues: E572 and M1034 [13]. Atomic structures of PfA-M1 have revealed important clues into potential roles for the cap residues in substrate selection. In the PfA-M1:bestatin co-crystal structure ([13]; Fig. 1), the P1 phenyl ring occupies the S1 subsite. M1034 is positioned to make stabilizing van der Waals contacts with the phenyl ring. Notably, the E572 sidechain swings away from the S1 subsite, presumably to avoid situating a polar group in a non-polar environment. Strikingly different conformations of the two cap residues are observed when a positively-charged group occupies the S1 subsite. In the co-crystal structure of the hydrolysis product arginine in the active site of the PfA-M1 mutant V459P, the E572 sidechain carboxylate points into the S1 subsite and forms a salt bridge with the Arg guanidinyl group, while the M1034 sidechain adopts two conformations, both of which orient it Reparixin mg away from the S1 subsite [23]. These observations suggest a “complementarity” model for the contributions of the two cap residues to substrate selection: large, non-polar P1 sidechains engage M1034, whereas the positively-charged P1 sidechains of Arg and Lys are stabilized by electrostatic interaction with E572. Conformational flexibility of the cap residue sidechains is a key attribute of this model as it allows for the mitigation of unfavorable interactions. Here, we have undertaken a mutagenic study to define the contributions of the S1 subsite cap residues E572 and M1034 to substrate selection and catalytic efficiency. The two residues were mutated to alanine, both independently and together. In addition, both cap residues were mutated to those in Escherichia coli PepN (EcPepN), a distantly-related M1-family aminopeptidase, yielding the double mutant E572N/M1034Q. Analysis of the E572N/M1034Q mutant was undertaken to assess the context dependence of cap residue function. We predicted that the E572N/M1034Q mutant would retain high catalytic efficiency with substrates having an Arg or Lys P1 residue, as the cap residues of EcPepN (N373, Q821) have been shown to directly interact with P1-sidechains of Lys (N373 only) and Arg (N373 and Q821) [24]. Effects of the mutations on S1 subsite preferences were assessed by determining the steady-state kinetic parameters for catalysis of hydrolysis of a series of model dipeptide substrates. To inform future inhibitor development efforts, we also determined the effects of cap residue mutations on the binding affinity of the aminopeptidase inhibitor bestatin.
    Materials and methods
    Results
    Discussion The view emerging from these studies is that the two PfA-M1 cap residues E572 and M1034 contribute to efficient catalysis of peptide hydrolysis by stabilizing the binding of chemically diverse P1 sidechains in the S1 subsite. This effect was most pronounced for substrates with basic and large aromatic sidechains. The stabilizing effect of E572 on the binding of substrates with positively-charged P1 sidechain guanidinyl and amino groups (Arg and Lys, respectively) is likely due to the formation of a salt bridge, such as that previously observed in the PfA-M1(V459P): Arg co-crystal structure [23]. In contrast, the stabilization of non-polar P1 sidechains likely occurs through two possible mechanisms: direct van der Waals interactions with the sidechain of M1034A and/or exclusion of water from the top of the S1 subsite (i.e., the hydrophobic effect). The effect of the M1034A mutation was greatest for the substrate with the largest non-polar P1 sidechain (Trp-Ala); this may reflect the ability of the M1034 sidechain to form more extensive interactions with the bulky Trp sidechain as compared to the smaller Leu and Met sidechains. At the same time, the results did not accord with the simple “complementarity” model described in the Introduction. For example, in the case of Trp-Ala and the E572A mutant, a statistically significant increase in Km and decreases in kcat and kcat/Km were observed. One possible explanation is that the indole NH forms a hydrogen bond with the E572 sidechain. While we can’t rule this out, we note that in the EcPepN:Trp co-crystal structure, the indole NH forms a hydrogen bond to a water molecule and does not directly interact with the cap residues [24]. Another example is the increase in bestatin Ki in the E572A mutant. Available structural evidence [13] indicates that the E572 sidechain does not interact directly with bestatin. One possible explanation for these findings is that water molecules fill the space vacated by the E572A mutation, altering the electrostatic properties of the S1 subsite. More generally, the bestatin data indicate that productive interactions with the cap residues can substantially increase the binding affinity of inhibitors. Strategies to develop potent PfA-M1 inhibitors should take into consideration the benefits of occupying the S1 subsite such that E572 and/or M1034 are engaged in inhibitor binding.