Quantitative Analysis of the Substrate Specificity of Human Rhinovirus 3C Protease and Exploration of Its Substrate Recognition Mechanisms

Abstract

Human rhinovirus 3C protease (HRV 3C-P) is a high-value commercial cysteine protease that could specifically recognize the short peptide sequence of LEVLFQ↓GP. In here, a strategy based on our previous Yeast Endoplasmic Reticulum Sequestration Screening (YESS) approach was developed in Saccharomyces cerevisiae, a model microorganism, to fully characterize the substrate specificity of a typical human virus protease, HRV 3C-P, in a quantitative and fast manner. Our results demonstrated that HRV 3C-P had very high specificity at P1 and P1′ positions, only recognizing Gln/Glu at the P1 position and Gly/Ala/Cys/Ser at the P1′ position, respectively. Comparably, it exhibited efficient recognition of most residues at the P2′ position, except Trp. Further biochemical characterization through site mutagenesis, enzyme structural modeling, and comparison with other 3C proteases indicated that the S1 pocket of HRV 3C-P was constituted by neutral and basic amino acids, in which His160 and Thr141 specifically interacted with Gln or Glu residues at the substrate P1 position. Additionally, the stringent S1′ pocket determined its unique property of only accommodating residues without or with short side chains. Based on our characterization, LEVLFQ↓GM was identified as a more favorable substrate than the original LEVLFQ↓GP at high temperature, which might be caused by the conversion of random coils to β-turns in HRV 3C-P along with the temperature increase. Our studies prompted a further understanding of the substrate specificity and recognition mechanism of HRV 3C-P. Besides, the YESS-PSSC combined with the enzyme modeling strategy in this study provides a general strategy for deciphering the substrate specificities of proteases.

Diverse viral proteases activate the NLRP1 inflammasome

“…We first compiled complete enterovirus polyprotein sequences from the Viral Pathogen Resource (ViPR) database (Pickett et al, 2012) and extracted and concatenated sequences for each of the cleavage sites within the polyproteins ( Figure 1A and 1B, Supplementary files 1 and 2). After removing redundant sequences, we used the MEME Suite (Bailey et al, 2009) to create the following 3C pro cleavage motif: [A/XXQGXXX (where denotes a hydrophobic residue and X denotes any amino acid), which is broadly consistent with previous studies that have experimentally profiled the substrate specificity of enterovirus 3C pros (Blom et al, 1996;Fan et al, 2020;Jagdeo et al, 2018;O'Donoghue et al, 2012) ( Figure 1C).…”

Diverse viral proteases activate the NLRP1 inflammasome

“…We first compiled complete enterovirus polyprotein sequences from the Viral Pathogen Resource (ViPR) database (Pickett et al, 2012) and extracted and concatenated sequences for each of the cleavage sites within the polyproteins (Figure 1A and 1B, Supplementary files 1 and 2). After removing redundant sequences, we used the MEME Suite (Bailey et al, 2009) to create the following 3C pro cleavage motif: [A/ΦXXQGXXX (where Φ denotes a hydrophobic residue and X denotes any amino acid), which is broadly consistent with previous studies (Blom et al, 1996; Fan et al, 2020; Jagdeo et al, 2018; O’Donoghue et al, 2012) (Figure 1C).…”

YESS 2.0, a Tunable Platform for Enzyme Evolution, Yields Highly Active TEV Protease Variants

“…The structure modeling was performed according to a previously published procedure. 34 The wild-type TEV protease structure (PDB: 1LVB) and peptide substrate, ENLYFQS, were derived from the protein data bank. 35 The structure of eTEV was derived from simulation using the online ITASSER program (https:// zhanglab.ccmb.med.umich.edu/ITASSER/).…”

“…The structure modeling was performed according to a previously published procedure . The wild-type TEV protease structure (PDB: 1LVB) and peptide substrate, ENLYFQS, were derived from the protein data bank .…”