Structural determinants of limited proteolysis

Abstract

Limited or regulatory proteolysis plays a critical role in many important biological pathways like blood coagulation, cell proliferation, and apoptosis. A better understanding of mechanisms that control this process is required for discovering new proteolytic events and for developing inhibitors with potential therapeutic value. Two features that determine the susceptibility of peptide bonds to proteolysis are the sequence in the vicinity of the scissile bond and the structural context in which the bond is displayed. In this study, we assessed statistical significance and predictive power of individual structural descriptors and combination thereof for the identification of cleavage sites. The analysis was performed on a data set of >200 proteolytic events documented in CutDB for a variety of mammalian regulatory proteases and their physiological substrates with known 3D structures. The results confirmed the significance and provided a ranking within three main categories of structural features: exposure > flexibility > local interactions. Among secondary structure elements, the largest frequency of proteolytic cleavage was confirmed for loops and lower but significant frequency for helices. Limited proteolysis has lower albeit appreciable frequency of occurrence in certain types of β-strands, which is in contrast with some previous reports. Descriptors deduced directly from the amino acid sequence displayed only marginal predictive capabilities. Homology-based structural models showed a predictive performance comparable to protein substrates with experimentally established structures. Overall, this study provided a foundation for accurate automated prediction of segments of protein structure susceptible to proteolytic processing and, potentially, other post-translational modifications.

Figure 1

Examples of the mapping of proteolytic events into 3D structure of substrate along with visualization of selected structural descriptors. Values of the solvent accessibility, B-Factor, and hydrogen bonding energy were color-mapped into substrate's structure for the cases of proteolytic processing of antithrombin by thrombin [65] (a), actin by Bacteroides fragilis enterotoxin [66] (b), and human profilin by mast cell alpha-chymase [55] (c).

Figure 2

Structural importance of the subsites around the cleavage site. Three bar plots demonstrate distribution of P-values over subsites for three structural descriptors: solved accessibility (a), protrusion index (b), and packing (c). The significances of P5–P5′ subsites were estimated by t-test for the raw values of structural descriptors calculated for the solved structure dataset.

Figure 3

Estimations of the prediction capabilities of the three individual structural descriptors and the combined descriptors set, presented by ROC curves of the corresponded classifiers.

Figure 4

Probabilities of the particular type of secondary structure in the sites of limited proteolysis calculated by maximum likelihood method for solved structure (a) and structure models (b) datasets.

Figure 5

Examples of cleavage sites in β structures. (a) Two proteolytic events located at the ends of the β strands were reported to be connected with the autoactivation of lactoferrin [56]. (b) The cleavage site in the buried internal β-strand of the β-sheet of actin protein, which is located close to the N-termini, was registered both for caspase 1 and for Granzyme B proteases [57, 58]. (c) Another example of the internal β-sheet cleavage, which is located inside the β-strand and next to the N-terminal β-strand of the alpha-enolase protein [58].