Structure of the Autocatalytic Cysteine Protease Domain of Potyvirus Helper-component Proteinase

Bihong Guo,Jinzhong Lin,Keqiong Ye

doi:10.1074/jbc.m111.230706

Abstract

The helper-component proteinase (HC-Pro) of potyvirus is involved in polyprotein processing, aphid transmission, and suppression of antiviral RNA silencing. There is no high resolution structure reported for any part of HC-Pro, hindering mechanistic understanding of its multiple functions. We have determined the crystal structure of the cysteine protease domain of HC-Pro from turnip mosaic virus at 2.0 Å resolution. As a protease, HC-Pro only cleaves a Gly-Gly dipeptide at its own C terminus. The structure represents a postcleavage state in which the cleaved C terminus remains tightly bound at the active site cleft to prevent trans activity. The structure adopts a compact α/β-fold, which differs from papain-like cysteine proteases and shows weak similarity to nsP2 protease from Venezuelan equine encephalitis alphavirus. Nevertheless, the catalytic cysteine and histidine residues constitute an active site that is highly similar to these in papain-like and nsP2 proteases. HC-Pro recognizes a consensus sequence YXVGG around the cleavage site between the two glycine residues. The structure delineates the sequence specificity at sites P1-P4. Structural modeling and covariation analysis across the Potyviridae family suggest a tryptophan residue accounting for the glycine specificity at site P1'. Moreover, a surface of the protease domain is conserved in potyvirus but not in other genera of the Potyviridae family, likely due to extra functional constrain. The structure provides insight into the catalysis mechanism, cis-acting mode, cleavage site specificity, and other functions of the HC-Pro protease domain.

Highlights

Nent proteinase (HC-Pro)2 [5, 6], and NIa-Pro [7]
Structural Description—The proteolytic activity of the tobacco etch virus (TEV) helper-component proteinase (HC-Pro) was previously shown to reside in 155 residues within its C terminus [5]
We found that the residues universally conserved in the Potyviridae family primarily constitute the hydrophobic core of the cysteine protease domain (CPD) structure or surround the active site, which is consistent with a role in structure maintenance and catalysis

Summary

EXPERIMENTAL PROCEDURES

Protein Expression, Purification, and Crystallization—The DNA sequence encoding residues 301– 458 of TuMV HC-Pro was PCR-amplified and cloned into an engineered pET28a vector, in which the inserted protein was fused to the C terminus of a His6-SMT3 tag. The sample was diluted 10-fold with PBS buffer and passed through a HisTrap column to remove the His6-SMT3 tag. The flowthrough was concentrated and further purified in a Superdex-75 column equilibrated in 20 mM HEPES-K (pH 7.6) and 100 mM KCl. Because the protein is prone to reversible aggregation at room temperature, all purification and crystallization steps were performed at 4 °C. Crystallization was performed at 4 °C using the hangingdrop vapor diffusion method by mixing 1 ␮l of protein solution (ϳ30 mg/ml in 100 mM KCl, 10 mM DTT, and 20 mM HEPES-K (pH 7.6)) and 1 ␮l of reservoir solution (1 M (NH4)2SO4, 4% (w/v) PEG3350, and 0.1 M BisTris (pH 5.4)). Se-Met-labeled protein was crystallized in 0.1 M NaH2PO4, 0.1 M KH2PO4, 1.0 M NaCl, and 0.1 M MES (pH 6.0) at 4 °C. Structural figures were created in PyMOL [39]

RESULTS

No reflections

DISCUSSION