Crystal structure of the serine protease domain of Sesbania mosaic virus polyprotein and mutational analysis of residues forming the S1-binding pocket

Abstract

Sesbania mosaic virus (SeMV) polyprotein is processed by its N-terminal serine protease domain. The crystal structure of the protease domain was determined to a resolution of 2.4 A using multiple isomorphous replacement and anomalous scattering. The SeMV protease domain exhibited the characteristic trypsin fold and was found to be closer to cellular serine proteases than to other viral proteases. The residues of the S1-binding pocket, H298, T279 and N308 were mutated to alanine in the DeltaN70-Protease-VPg polyprotein, and the cis-cleavage activity was examined. The H298A and T279A mutants were inactive, while the N308A mutant was partially active, suggesting that the interactions of H298 and T279 with P1-glutamate are crucial for the E-T/S cleavage. A region of exposed aromatic amino acids, probably essential for interaction with VPg, was identified on the protease domain, and this interaction could play a major role in modulating the function of the protease.

Structural and Biochemical Analysis of Human Pathogenic Astrovirus Serine Protease at 2.0 Å Resolution

“…8c) similar to the HAstV enzyme, also recognises Glu as the P1 side chain of the substrate. 22 More important, the analysis of the HAstV protease structure (Fig. 7a) shows that the S1 site is characterised by a positive electrostatic potential, which is in perfect agreement with the idea that it recognises a negatively charged Glu and/or Asp side chain.…”

“…Remarkably, the highest levels of similarity were observed for two non-viral proteins, bacterial protease/chaperone DegS 20 (root-mean-square deviation of 2.6 Å for 161 C α atoms with 22% sequence identity) and human HtrA2 serine protease (2.6 Å for 157 C α , 22% identity). 21 The highest levels of similarity with viral proteins were observed for the serine proteases from Sesbania mosaic virus (2.9 Å for 151 C α , 18% identity) 22 and from various picornaviruses; namely, the enzymes from human pathogenic rhinovirus serotype 2 (2.6 Å for 152 C α , 19% identity), 23 hepatitis A virus 24 (2.7 Å for 158 C α , 12% identity), and foot-and-mouth disease virus (2.8 Å for 148 C α , 10% identity). 25 From the comparison between the HAstV protease and the picornavirus enzymes, it is immediately clear that the largest difference among them is the truncation in the HAstV protease structure of the loops that Alignment of 3C and 3C-like proteases.…”

“…8c). 22 Another major feature exhibited by the HAstV enzyme is the absence of the so-called β-ribbons, two long loops that connect β-strands eI and fI, and cII and dII, and define the shape of the S2 and S4 pockets (Fig. 5).…”

The Ryegrass mottle virus genome codes for a sobemovirus 3C-like serine protease and RNA-dependent RNA polymerase translated via −1 ribosomal frameshifting

“…1). The importance of H298 and T279 in substratebinding has been recently experimentally demonstrated for the E/T or E/S cleavage in SeMV serine protease [11].…”

“…The presence of alanine instead of the characteristic substrate-binding serine or cysteine caused uncertainty regarding the catalytic property of the protease domain in RGMoV [1]. Recently published SeMV serine protease crystallography data demonstrated the spatial arrangement of catalytic amino acids H, D, S, and in vitro experiments using the corresponding mutants confirmed the involvement of these AA in processing of SeMV polyproteins [10,11].…”