Movatterモバイル変換

[0]ホーム
URL:

Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
Thehttps:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log inShow account info
Access keysNCBI HomepageMyNCBI HomepageMain ContentMain Navigation
pubmed logo
Advanced Clipboard
User Guide

Full text links

Wiley full text link Wiley Free PMC article
Full text links

Actions

Share

.2010 Nov;19(11):2240-51.
doi: 10.1002/pro.506.

Structural determinants of tobacco vein mottling virus protease substrate specificity

Affiliations

Structural determinants of tobacco vein mottling virus protease substrate specificity

Ping Sun et al. Protein Sci.2010 Nov.

Abstract

Tobacco vein mottling virus (TVMV) is a member of the Potyviridae, one of the largest families of plant viruses. The TVMV genome is translated into a single large polyprotein that is subsequently processed by three virally encoded proteases. Seven of the nine cleavage events are carried out by the NIa protease. Its homolog from the tobacco etch virus (TEV) is a widely used reagent for the removal of affinity tags from recombinant proteins. Although TVMV protease is a close relative of TEV protease, they exhibit distinct sequence specificities. We report here the crystal structure of a catalytically inactive mutant TVMV protease (K65A/K67A/C151A) in complex with a canonical peptide substrate (Ac-RETVRFQSD) at 1.7-Å resolution. As observed in several crystal structures of TEV protease, the C-terminus (∼20 residues) of TVMV protease is disordered. Unexpectedly, although deleting the disordered residues from TEV protease reduces its catalytic activity by ∼10-fold, an analogous truncation mutant of TVMV protease is significantly more active. Comparison of the structures of TEV and TVMV protease in complex with their respective canonical substrate peptides reveals that the S3 and S4 pockets are mainly responsible for the differing substrate specificities. The structure of TVMV protease suggests that it is less tolerant of variation at the P1' position than TEV protease. This conjecture was confirmed experimentally by determining kinetic parameters k(cat) and K(m) for a series of oligopeptide substrates. Also, as predicted by the cocrystal structure, we confirm that substitutions in the P6 position are more readily tolerated by TVMV than TEV protease.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Three-dimensional structure of TVMV protease. (A) Overall structure of TVMV protease in one asymmetric unit. Chain A (1–217, purple) and Chain B (3–216, green) bound to peptide substrates Chain C (residues −6 to 1, cyan) and Chain D (residues −6 to 2, red), respectively. The N- and C-termini are labeled with the letters N and C. The catalytic triad residues are shown in ball-and-stick representation. (B) Crystal structure of TEV protease (PDB ID:1LVM) viewed from the same perspective. Chains A to E are colored in pink, blue, green, pale pink, and salmon, respectively. (C) Stereoview of the peptide substrate (Chain D, yellow) bound to inactive TVMV protease (Chain B, green). The catalytic triad residues [H46, D81, and A151 (normally C151 in wild-type TVMV protease)] are shown in ball-and-stick representation. The peptide substrate is also displayed in a ball-and-stick format and covered by an omit map contoured at 1.0σ. The composite omit map was calculated at 1.7 Å resolution by CNS, with an omit rations of 7.5%.
Figure 2
Figure 2
Comparison between TVMV and TEV proteases. (A) Stereoview of superposition of the crystal structures of TVMV (Chain B, gray) and TEV (PDB ID:1LVB, Chain A, magenta) proteases with their peptide substrates. Secondary structure elements are labeled according to the TVMV structure. Canonical peptide substrates bound to TVMV and TEV proteases are shown in green and yellow, respectively. The N- and C-termini of the proteases are labeled in blue and red, respectively. The side chains of the mutated catalytic residue (C151A in TVMV protease) from both structures are shown as cyan spheres. (B) Sequence alignment of TVMV (gray) and TEV (magenta) proteases. The secondary structures were determined by the results from iMolTalk server (http://i.moltalk.org/). β-strands and α-helices are numbered and 310-helices are labeled A–C. Identical residues are shaded in cyan. Two surface entropy reduction mutation sites (K65&K67) are shaded in yellow. The active-site cysteine residues are shaded in salmon. The self-cleavage site within TEV protease is indicated by the yellow arrow. The C-termini of available crystal structure models of TEV protease (PDB ID:1LVB and1LVM) are denoted by the brown arrow. The C-terminus of the truncated TVMV protease (TVMV1–217 protease) investigated in this study is indicated by the green arrow. (C) Surface representation of TVMV (left) and TEV (right) proteases viewed at the same angle.
Figure 3
Figure 3
Comparison between the P6 positions of TVMV and TEV protease (PDB ID:1LVB, Chain A) substrates. (A, B) P6 Glu binds to the surface of its corresponding protease, viewed at the same angle. (C, D) Hydrogen-bond interactions between P6 Glu and TVMV protease (C) and TEV protease (D). Residues are shown in ball-and-stick representation. The hydrogen bonds are shown as dashed lines colored red. Residues from TVMV and TEV protease are colored in purple and green, respectively. The P6 substrate residues for TVMV and TEV proteases are colored cyan and yellow, respectively.
Figure 4
Figure 4
Comparison of the S1′ and S3 subsites in TVMV and TEV proteases. Left and right panels refer to the TVMV and TEV protease, respectively. (A, B) Surface representation of the S3 subsites. P3 residues are colored cyan (TVMV) and yellow (TEV). (C, D) Hydrogen-bond interaction at the S3 subsites. Residues from TVMV and TEV protease are colored gray and magenta, respectively. (E, F) Surface representation of the S1′ pockets. P1′ Ser residues of the peptide substrates are colored green (TVMV) and yellow (TEV). (G, H) Hydrogen-bond interactions in the S1′ pockets. Residues are shown in ball-and-stick representation. The hydrogen bonds are shown as dashed lines colored red. Residues from TVMV and TEV proteases are colored yellow and cyan, respectively. P1′ Ser residues of the peptide substrates are colored in green (TVMV) and magenta (TEV).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ryan MD, Flint M. Virus-encoded proteinases of the picornavirus super-group. J Gen Virol. 1997;78(Part 4):699–723. - PubMed
    1. Stanway G. Structure, function and evolution of picornaviruses. J Gen Virol. 1990;71(Part 11):2483–2501. - PubMed
    1. Domier LL, Franklin KM, Shahabuddin M, Hellmann GM, Overmeyer JH, Hiremath ST, Siaw MF, Lomonossoff GP, Shaw JG, Rhoads RE. The nucleotide sequence of tobacco vein mottling virus RNA. Nucleic Acids Res. 1986;14:5417–5430. - PMC - PubMed
    1. Hellmann GM, Shaw JG, Rhoads RE. In vitro analysis of tobacco vein mottling virus NIa cistron: evidence for a virus-encoded protease. Virology. 1988;163:554–562. - PubMed
    1. Seipelt J, Guarne A, Bergmann E, James M, Sommergruber W, Fita I, Skern T. The structures of picornaviral proteinases. Virus Res. 1999;62:159–168. - PubMed

Publication types

MeSH terms

Substances

Related information

Grants and funding

LinkOut - more resources

Full text links
Wiley full text link Wiley Free PMC article
Cite
Send To

NCBI Literature Resources

MeSHPMCBookshelfDisclaimer

The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited.

[8]ページ先頭
©2009-2025 Movatter.jp