Site-specific proteolysis by the enzymatic cleavage of small linear sequence motifs is a key posttranslational modification involved in physiology and disease. The ability to robustly and rapidly predict protease–substrate specificity would also enable targeted proteolytic cleavage by designed proteases. Current methods for predicting protease specificity are limited to sequence pattern recognition in experimentally derived cleavage data obtained for libraries of potential substrates and generated separately for each protease variant. We reasoned that a more semantically rich and robust model of protease specificity could be developed by incorporating the energetics of molecular interactions between protease and substrates into machine learning workflows. We present Protein Graph Convolutional Network (PGCN), which develops a physically grounded, structure-based molecular interaction graph representation that describes molecular topology and interaction energetics to predict enzyme specificity. We show that PGCN accurately predicts the specificity landscapes of several variants of two model proteases. Node and edge ablation tests identified key graph elements for specificity prediction, some of which are consistent with known biochemical constraints for protease:substrate recognition. We used a pretrained PGCN model to guide the design of protease libraries for cleaving two noncanonical substrates, and found good agreement with experimental cleavage results. Importantly, the model can accurately assess designs featuring diversity at positions not present in the training data. The described methodology should enable the structure-based prediction of specificity landscapes of a wide variety of proteases and the construction of tailor-made protease editors for site-selectively and irreversibly modifying chosen target proteins.
Data-driven supervised learning of a viral protease specificity landscape from deep sequencing and molecular simulations
Biophysical interactions between proteins and peptides are key determinants of molecular recognition specificity landscapes. However, an understanding of how molecular structure and residue-level energetics at protein−peptide interfaces shape these landscapes remains elusive. We combine information from yeast-based library screening, next-generation sequencing, and structure-based modeling in a supervised machine learning approach to report the comprehensive sequence−energetics−function mapping of the specificity landscape of the hepatitis C virus (HCV) NS3/4A protease, whose function—site-specific cleavages of the viral polyprotein—is a key determinant of viral fitness. We screened a library of substrates in which five residue positions were randomized and measured cleavability of ∼30,000 substrates (∼1% of the library) using yeast display and fluorescence-activated cell sorting followed by deep sequencing. Structure-based models of a subset of experimentally derived sequences were used in a supervised learning procedure to train a support vector machine to predict the cleavability of 3.2 million substrate variants by the HCV protease. The resulting landscape allows identification of previously unidentified HCV protease substrates, and graph-theoretic analyses reveal extensive clustering of cleavable and uncleavable motifs in sequence space. Specificity landscapes of known drug-resistant variants are similarly clustered. The described approach should enable the elucidation and redesign of specificity landscapes of a wide variety of proteases, including human-origin enzymes. Our results also suggest a possible role for residue-level energetics in shaping plateau-like functional landscapes predicted from viral quasispecies theory.
more » « less- Award ID(s):
- 1716623
- NSF-PAR ID:
- 10082246
- Publisher / Repository:
- Proceedings of the National Academy of Sciences
- Date Published:
- Journal Name:
- Proceedings of the National Academy of Sciences
- Volume:
- 116
- Issue:
- 1
- ISSN:
- 0027-8424
- Page Range / eLocation ID:
- p. 168-176
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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