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Peptide Recognition and Dephosphorylation by the Vaccinia VH1 Phosphatase

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Peptide Recognition and Dephosphorylation by the Vaccinia VH1 Phosphatase bioRxiv preprint doi: https://doi.org/10.1101/2020.05.26.100743; this version posted May 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. VH1 recognition of phosphorylated peptides Peptide recognition and dephosphorylation by the vaccinia VH1 phosphatase Bryan M. Zhao1*, Megan Hogan2, Michael S Lee3, Beverly K. Dyas1, and Robert G. Ulrich1* From the 1U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702, USA; 2Pfizer Pharmaceuticals, Ringaskiddy, Cork, Ireland; 3Battlefield Environment Division, U.S. CCDC Army Research Laboratory, Aberdeen Proving Ground, MD 21005, USA Running title: VH1 recognition of phosphorylated peptides *To whom correspondence should be addressed: U.S. Army Medical Research Institute of Infectious Diseases, 1425 Porter St., Frederick, MD 21702, USA; Tel.: (301)-619-9657; Fax: 301 619-8334; Email: [email protected] or [email protected] Keywords: Vaccinia H1 phosphatase; dual-specificity phosphatase; protein phosphatase; protein microarray; peptide substrates; site-specific mutagenesis; protein-protein interactions; poxvirus ______________________________________________________________________________ ABSTRACT targets for the development of inhibitors that specifically block VH1 catalytic activity and thus The VH1 protein encoded by the highly poxvirus replication. conserved H1 locus of orthopoxviruses is a dual- ____________________________________ specificity phosphatase (DUSPs) that hydrolyzes phosphate groups from phosphorylated tyrosine, INTRODUCTION serine, and threonine residues of viral and host cell proteins. Because the DUSP activities are Poxviruses (Poxviridae) are large double- required for virus replication, VH1 is a prime stranded DNA viruses that replicate exclusively target for the development of therapeutic in the cytoplasm of vertebrate and invertebrate inhibitors. However, the presentation of a host cells. Notable orthopoxviruses within the shallow catalytic site has thwarted all drug Poxviridae family include monkeypox viruses development efforts. As an alternative to direct circulating in Central Africa that have 10% case- targeting of catalytic pockets, we describe surface fatality rates (1), the variola major virus of contacts between VH1 and substrates that are smallpox that carries a 30% fatality rate within essential for full activity and provide a new non-vaccinated populations (2), posing a threat as pathway for developing inhibitors of protein- a bioterrorist weapon, and vaccinia virus strains protein interactions. Critical amino acid residues used as vaccines to control monkeypox and were manipulated by site-directed mutagenesis of smallpox infections. The orthopoxvirus genome VH1, and perturbation of peptide substrate encodes kinases B1 and F10 that phosphorylate interactions based on these mutations were six or more virus-encoded proteins (3-5), as well assessed by high-throughput assays that as a dual-specificity protein phosphatase (DUSP) employed surface plasmon resonance and VH1 that removes phosphate groups from phosphatase activities. Two positively-charged phosphorylated Ser (pSer), Thr (pThr) and Tyr residues (Lys-20 and Lys-22) and the (pTyr) residues of protein substrates (6). The hydrophobic side chain of Met-60 appear to kinases and VH1 phosphatase are essential for orient the polarity of the pTyr peptide on the VH1 viral replication (7). Significantly, surface, while additional amino acid residues that phosphorylation of tyrosine residues contained flank the catalytic site contribute to substrate within host proteins is also controlled by the recognition and productive dephosphorylation. reversible actions of cellular protein tyrosine We propose that the enzyme-substrate contact kinases (PTKs) and protein tyrosine phosphatases residues described here may serve as molecular (PTPs), including the DUSPs (8,9). These 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.26.100743; this version posted May 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. VH1 recognition of phosphorylated peptides phospho-transfer enzymes critically affect cell RESULTS proliferation, metabolism, adhesion, cell growth, and cell differentiation (10-13). VH1 was the VH1 peptide phosphatase assay first DUSP described (14), and is highly conserved within the orthopoxvirus genus The vaccinia VH1 phosphatase was expressed in (Figure 1). Active sites of VH1 phosphatases Escherichia coli as a full-length, 187-amino acid share with most DUSPs a highly conserved PTP- protein (18-kDa) and a fusion of VH1 to maltose- loop (HCXXXXXR) that harbors the catalytic binding protein (MBP)-poly His tag to increase thiol of a nucleophilic Cys residue and a general protein expression levels (21). The recombinant acid residue (Asp). Genetic repression of VH1 proteins were purified by Immobilized Metal expression leads to the production of Affinity Chromatography (IMAC) as previously noninfectious virions (7), and studies suggest that described (supplemental Figure 1) (22). The VH1 overcomes host defense mechanisms during activities of both native VH1 and MBP-VH1 infection by dephosphorylating the Signal fusion proteins were confirmed by hydrolysis of Transducer And Activator Of Transcription 1 the small molecular substrate, para- (STAT1), thereby blocking the interferon-γ nitrophenylphosphate (pNPP). We next signaling pathway (15). Because VH1 does not investigated enzymatic activity with dephosphorylate DNA bound STAT1, the viral phosphorylated peptides. Immune responses that phosphatase may act predominantly on the are mediated by STAT1 are inhibited by VH1 cytoplasmic pool of activated STAT1 (16). (15), while VH1-dependent dephosphorylation of While catalytic sites of many protein the poxvirus proteins A17 and A14 is necessary tyrosine phosphatases (PTPs) are challenging for for maturation and assembly of infection- drug discovery efforts, a strategy of targeting competent viruses (23). However, these secondary binding pockets along with active site intracellular protein substrates for VH1 were residues has resulted in significant progress in the previously identified primarily by cell-culture development of highly potent and selective assays (15,23), and little information is available inhibitors (17-19). However, the crystal structure for the in vitro activity of peptides based on the of VH1 reveals a much shallower catalytic cleft essential sequences for DUSP recognition. (6 Å) compared to other PTPs, for example, Therefore, we synthesized pTyr peptides from PTP1B (9 Å)(16,20), which is more difficult for human STAT1(hSTAT1), mouse STAT1 the design of small-molecule inhibitors. The (mSTAT1), and vaccinia A17, along with an A14 study described here examines the protein- pSer peptide from A14 (Table1), and examined in protein interactions of VH1 as a possible vitro activity of VH1 by liberation of phosphate. alternative guide for inhibitor development. By Although all four peptides were screening a library of pTyr peptides from host cell dephosphorylated by VH1, the observed reaction proteins, we identified a pTyr-peptide of EGFR rates were too slow (Figure 1B) to allow us to as a highly potent substrate and evaluated the establish a robust assay. To identify peptide optimal peptide length for VH1 catalytic activity substrates that had greater activity with VH1 (Km) in comparison with binding (KD). Amino compared to the putative endogenous cellular and acids that mapped to protein surfaces around the viral proteins, we screened a phosphorylated catalytic site of VH1 were sequentially mutated peptide substrate library that contained a to alanine to examine the effects on catalytic selection of 360 pTyr-peptide sequences that activity, peptide binding, and VH1 thermal were derived from phosphorylation sites of stability (Tm). Peptide substrate binding and human cellular proteins. A consensus amino acid dephosphorylation were evaluated en masse by sequence of the most active peptides from the using unique protein microarray assays. Finally, phosphorylated peptide library screening (Figure based on the experimental results and known 1C) included dominant negatively charged protein structures, a model of the peptide binding residues around the pTyr residue. These results footprint on VH1 was developed to guide future with vaccinia virus VH1 were consistent with development of inhibitors. previous data for VH1 from variola virus (24) that demonstrated the requirement for negatively 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.26.100743; this version posted May 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. VH1 recognition of phosphorylated peptides charged amino acid residues near the pTyr. on the catalytic activity of VH1 protein (VH1- Comparing these two viral DUSPs, there are four MBP fusion) with the small molecule substrate amino acid sequence differences between 6,8-difluoro-4-methylumbelliferyl phosphate vaccinia and variola virus VH1, as shown in (DiFMUP) and the EGFR peptide (Figure 2A). Figure 1A, while the active site is completely We hypothesized that the alanine substitutions conserved among the orthopoxviruses. Among should have a significant impact on EGFR the most active VH1 peptide substrates
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