Abstract
Members of the same protease family show different substrate specificity, even if they share identical folds, depending on the physiological processes they are part of. Here, we investigate the key factors for subpocket and global specificity of factor Xa, elastase, and granzyme B which despite all being serine proteases and sharing the chymotrypsin-fold show distinct substrate specificity profiles. We determined subpocket interaction potentials with GRID for static X-ray structures and an in silico generated ensemble of conformations. Subpocket interaction potentials determined for static X-ray structures turned out to be insufficient to explain serine protease specificity for all subpockets. Therefore, we generated conformational ensembles using molecular dynamics simulations. We identified representative binding site conformations using distance-based hierarchical agglomerative clustering and determined subpocket interaction potentials for each representative conformation of the binding site. Considering the differences in subpocket interaction potentials for these representative conformations as well as their abundance allowed us to quantitatively explain subpocket specificity for the nonprime side for all three example proteases on a molecular level. The methods to identify key regions determining subpocket specificity introduced in this study are directly applicable to other serine proteases, and the results provide starting points for new strategies in rational drug design.
Highlights
Proteases are enzymes that catalyze the cleavage of peptide bonds and are important in numerous fundamental cellular processes
Comparison of subpocket interaction potentials determined from the X-ray structure and weighted mean of subpocket interaction potentials calculated from representative cluster conformations showed a high dependence of subpocket interaction potentials on the investigated conformation
While subpocket interaction potentials determined from the X-ray structure showed little to no correlation to specificity quantified as cleavage entropy, subpocket interaction potentials determined as weighted mean from representative cluster conformations show high correlation to specificity in the case of elastase and Factor Xa (fXa) and minor correlation in the case of granzyme B
Summary
Proteases are enzymes that catalyze the cleavage of peptide bonds and are important in numerous fundamental cellular processes. To elucidate the molecular determinants of serine protease substrate recognition, we chose three serine proteases with chymotrypsin-fold adopting different biological functions and showing distinct specificity profiles. In the S4 pocket nonpolar amino acids such as Ile, Ala, and Phe are preferred. The S2′−S4′ pockets all prefer nonpolar amino acids, with the S2′ pocket showing a preference for Val according to MEROPS data. Elastases are a group of proteases which cleave the important connective tissue protein elastin.[11] The here investigated porcine pancreatic elastase is structurally similar to human leukocyte elastase[12] and preferentially cleaves C-terminal amino acids with small alkyl side chains such as Ile, Val, and Ala.[13] Because elastases can destroy connective tissue proteins and Received: October 30, 2015 Revised: December 28, 2015 Published: December 28, 2015
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