Dimeric Quaternary Structure of the Prototypical Dual Specificity Phosphatase VH1

Adem C Koksal,Jonathan D Nardozzi,Gino Cingolani

doi:10.1074/jbc.m808362200

Adem C Koksal, Jonathan D Nardozzi + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m808362200

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Abstract

The Vaccinia virus H1 gene product, VH1, is a dual specificity phosphatase that down-regulates the cellular antiviral response by dephosphorylating STAT1. The crystal structure of VH1, determined at 1.32 A resolution, reveals a novel dimeric quaternary structure, which exposes two active sites spaced approximately 39 A away from each other. VH1 forms a stable dimer via an extensive domain swap of the N-terminal helix (residues 1-20). In vitro, VH1 can dephosphorylate activated STAT1, in a reaction that is competed by the nuclear transport adapter importin alpha5. Interestingly, VH1 is inactive with respect to STAT1 bound to DNA, suggesting that the viral phosphatase acts predominantly on the cytoplasmic pool of activated STAT1. We propose that the dimeric quaternary structure of VH1 is essential for specific recognition of activated STAT1, which prevents its nuclear translocation, thus blocking interferon-gamma signal transduction and antiviral response.

Highlights

IFN-␥ secreted in response to viral infection activates the Jak1 and -2 tyrosine kinase pathway, which leads to phosphorylation and activation of STAT1 [11, 12]
In the cytoplasm, phosphorylated STAT1 is recognized by importin ␣5, which together with importin ␤ mediates its rapid translocation into the cell nucleus [13, 14]
In the crystal structure of VH1, the active site is occupied by a phosphate ion, which is visible as an ϳ8␴ peak in an electron density difference map calculated with coefficient Fo Ϫ Fc (Fig. 2B)

Summary

EXPERIMENTAL PROCEDURES

Cloning and Protein Expression—The gene encoding VH1 was cloned into the NcoI and BamHI sites of the pET-14b vector (Novagen), which expresses VH1 fused to an N-terminal His tag. The gene encoding full-length STAT1 (FL-STAT1) or STAT1 lacking N-terminal residues 1–132 (⌬132-STAT1) were introduced in an engineered pMal vector containing a PreScission Protease cleavage site (plasmids pMal-PP-STAT1 and pMal-PP-⌬132STAT1). A rectangular quartz cuvette with a path length of 1 cm was used to perform the CD measurements of VH1 at a final protein concentration of 6.0 ␮M in 20 mM sodium phosphate (pH 8.0) and 100 mM NaCl. To measure the temperature-induced unfolding of dimeric VH1, we recorded variations in ellipticity at 218 nm as a function of temperature in 1 °C increments. A 10-fold molar excess of VH1 was added, and the reaction was incubated for 240 min at 37 °C, followed by SDS-PAGE and Western blot analysis as described above. The relative amount of phosphorylated versus dephosphorylated STAT1 was quantified using the NIH ImageJ software and the relative intensities of Tyr(P)701 were plotted using the program SigmaPlot

RESULTS

Data collection statistics

Core region Allowed region Generously allowed region Disallowed region

DISCUSSION