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, 435 (2), 411-20

Probing the S1 Specificity Pocket of the Aminopeptidases That Generate Antigenic Peptides

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Probing the S1 Specificity Pocket of the Aminopeptidases That Generate Antigenic Peptides

Efthalia Zervoudi et al. Biochem J.

Abstract

ERAP1 (endoplasmic reticulum aminopeptidase 1), ERAP2 and IRAP (insulin-regulated aminopeptidase) are three homologous enzymes that play critical roles in the generation of antigenic peptides. These aminopeptidases excise amino acids from N-terminally extended precursors of antigenic peptides in order to generate the correct length epitopes for binding on to MHC class I molecules. The specificity of these peptidases can affect antigenic peptide selection, but has not yet been investigated in detail. In the present study we utilized a collection of 82 fluorigenic substrates to define a detailed selectivity profile for each of the three enzymes and to probe structural and functional features of the S1 (primary specificity) pocket. Molecular modelling of the three S1 pockets reveals substrate-enzyme interactions that are critical determinants for specificity. The substrate selectivity profiles suggest that IRAP largely combines the S1 specificity of ERAP1 and ERAP2, consistent with its proposed biological function. IRAP, however, does not achieve this dual specificity by simply combining structural features of ERAP1 and ERAP2, but rather by an unique amino acid change at position 541. The results of the present study provide insights on antigenic peptide selection and may prove valuable in designing selective inhibitors or activity markers for this class of enzymes.

Figures

Figure 1
Figure 1
Selectivity profiles of ERAP1, ERAP2 and IRAP. Trimming rates were calculated for each substrate and then normalized for the best substrate for each enzyme. Error bars correspond to the standard deviation for 3–6 measurements. Substrates for which no bar is drawn failed to be hydrolyzed by the enzyme even when measured at 100μM substrate concentration. Panel A, Natural amino acid side chains in L- or D- configuration. Panel B, non-natural amino acid side chains. Panel C, reaction kinetics and specific rates for the best substrate for each enzyme (hTyr-ACC for ERAP1, Arg-ACC for ERAP2 and hArg-ACC for IRAP). Error bars indicate the standard deviation of 3 measurements.
Figure 2
Figure 2
ERAP1 and ERAP2 were mixed at 2:1 molar concentration and the selectivity profile of the mix was compared to that of IRAP using the L-substrates.
Figure 3
Figure 3
Surface representation of the S1 pocket for ERAP1, ERAP2 and IRAP colored by electrostatic potential. The best substrates for each enzyme are shown as stick models in the predicted conformations.
Figure 4
Figure 4
Key residues that define the S1 pocket. Panel A, Phe433 in ERAP1 is stacked closely with a leucine side chain of the substrate. Panel B, Met319 makes unfavorable steric interactions with the Cβ of D-leucine, leading to an altered binding conformation of the scissile peptide bond. Panel C, Simulated interactions between a 4-guanyl-phenylalanine side chain with Phe544 and Glu541 in IRAP. Panel D, The six non-conserved amino acids that define the S1 pocket of each enzyme; the predicted conformation of homo-arginine is depicted in yellow.
Figure 5
Figure 5
Selectivity profile of IRAP E541R mutation compared to wild-type protein. Data have been normalized for Leucine. Arrows highlight the most significant changes brought about by the mutation.
Figure 6
Figure 6
Michaelis-Menten kinetics for hydrolysis of the substrate R-AMC by E541R IRAP as well as the WT enzyme. The enzymatic parameters KM and Kcat are depicted for each enzyme.

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