Structural analysis of monkeypox virus H1 dual phosphatase as a pan-poxvirus target

Structural analysis of monkeypox virus H1 dual phosphatase as a pan-poxvirus target

In a recent study published in bioRxiv * Preprint server: Researchers determined that the crystal structure of monkeypox virus (MPXV) dual-specific H1 phosphatase (DSP) at 1.8 Å resolution is critical for virus replication, making it an attractive antiviral target.

Lernen: Kristallstruktur der Affenpocken-H1-Phosphatase, einem antiviralen Wirkstoffziel. Bildnachweis: Die Fuchswerkstatt/Shutterstock

This news article was a review of a preliminary scientific report that had not been peer-reviewed at the time of publication. Since its initial publication, the scientific report has now been peer-reviewed and accepted for publication in an academic journal. Links to the preliminary and peer-reviewed reports can be found in the Sources section at the end of this article. View sources

The number of MPX cases continues to increase hourly worldwide, justifying the need for the development of effective antiviral therapeutics and vaccines to improve global preparedness for MPX and contain viral infections. H1 is essential for MPXV replication as it dephosphorylates proteins such as A14, F18, A17 and signal transducer and activator of transcription 1 (STAT1) and inhibits interferon (IFN) signaling. Importantly, HI is conserved among poxviruses and can therefore be targeted for the development of broad antiviral agents.

About the study

In the present study, researchers examined the H1 crystal structure at 1.8 Å resolution to improve the understanding of MPXV H1-catalyzed dephosphorylation and to develop a precise model for the development of novel antiviral drugs.

The deoxyribonucleic acid (DNA) encoding H1 of the current 2022 MPXV outbreak MPXV_USA_2022_MA001 isolate was synthesized and cloned into an expression plasmid vector, which was verified by sequencing. H1 was expressed in Escherichia coli BL21 (or DE3) cells and H1-expressing bacteria were lysed, after which the protein was subjected to chromatographic analysis.

H1 was crystallized using the sitting drop vapor diffusion technique and the crystals were subjected to X-ray diffraction analysis. The H1 structure was determined using the molecular replacement technique with the vaccinia virus H1 structure as a search model. A structural comparison with human protein tyrosine phosphatases (PTP)/DSP phosphatases was then carried out.

MPXV-H1 coordinates were uploaded to the Dali server and searched for similarly structured proteins in the PDB (protein database) using the PDB50 subset. As a result, structures of 30 phosphatases with significant structural similarity were identified (Z-scores >13). Among them, two human phosphatases were crystallized as dimers: human dual-specificity phosphatase (hDSP)-5 and 27, and their dimerization modes were compared.

Results

MPXV H1 comprised 171 residues with well-matched electron densities, one asymmetric H1 molecule, and two symmetrically aligned H1 molecules, forming butterfly-shaped and domain-swapped dimers. The entire H1 structure consisted of six and four alpha helices (α) and beta strands (β), respectively, with a β sheet arranged on the sides between helices α2 and α3 to α6.

The two active sites were located at a distance of 39 Å near the C-terminals of the terminal β-strand. Each active site comprised a cysteine ​​(Cys)-arginine (Arg)-aspartic acid (Asp) triad. In each active site, the conserved Arg and Cys residues were located in the phosphate ion binding loop between the α4 and β4 residues. The Arg116 residue of the loop captured phosphate ions whose guanidinium group interacted with two PO (phosphate-oxygen) molecules connected by hydrogen bonds.

The Arg116 residue ensured efficient substrate orientation and binding. The N-terminal α1 helices of each protomer were exchanged to mediate H1 dimerizations. The α1 helices of a single H1 protomer formed a bundled structure with three α4 to α6 helices of the corresponding promoters involved in pairing. At the base of the catalytic pocket, the Cys110 residue attacked phosphorus atoms during dephosphorylation, resulting in the transient formation of an enzyme-phosphate intermediate, which was hydrolyzed to regenerate inorganic phosphate and enzyme.

The sulfur atom of the Cys110 residue was positioned parallel to the PO bond, which corresponds to the bond formed upon regeneration of the enzyme. The Asp79 residue was involved in coordination with the water molecule and acted as an acid, facilitating the formation and hydrolysis of the intermediate. Thus, the H1 structure indicated the last catalytic step before product release.

The surface buried between two promoters was 1000 Å2 apart and was stabilized by hydrophobic and hydrophilic interactions. The serine residues (Ser)14 and threonine residues (Thr)15 in α1 showed hydrogen bonds with histidine residues (His)143 and tyrosine residues (Tyr)142/lysine residues (Lys)159 of the corresponding H1 protomer, respectively. In contrast, residues Tyr7, leucine (Leu)11 and Leu12 participated in hydrophobic bonds with residues methionine (Met)135, Leu139, Lys159, isoleucine (Ile)163, valine (Val)167 and Ile168 from the H1 pairing.

The Leu136, Leu139, and Met135 residues in α5 were opposed by symmetry-associated dimer residues to extend the hydrophobic binding interface. The α1 residue was stabilized using hydrophobic interactions and intramolecular hydrogen bonds between the α1 and α5 residues. The H1 dimer was confirmed in chromatographic analysis, indicating that the dimers represented functional states.

The MPXV H1 crystal structure revealed two hotspots for the development of novel antiviral drugs. The dimer contact site is a hotspot unique to PTP/DSP molecules. Inhibition of H1 dimerization could potentially inhibit the dimerization and dephosphorylation of the activated STAT1 phosphorus-tyrosine homodimer. The second hotspot is the active site, which, although located around the phosphate ion binding loops with comparable backbone structures, has different side chains and therefore the substrate specificity may be altered, paving the way for the development of antiviral drugs.

Overall, the study results revealed the high-resolution H1 crystal structure, which provides a solid foundation for further mechanistic analysis and development of novel antiviral drugs against emerging viral pathogens.

This news article was a review of a preliminary scientific report that had not been peer-reviewed at the time of publication. Since its initial publication, the scientific report has now been peer-reviewed and accepted for publication in an academic journal. Links to the preliminary and peer-reviewed reports can be found in the Sources section at the end of this article. View sources

References:

• Vorläufiger wissenschaftlicher Bericht.
  Wen Cui et al. (2022). Kristallstruktur der Affenpocken-H1-Phosphatase, einem antiviralen Wirkstoffziel. bioRxiv. doi: https://doi.org/10.1101/2022.09.30.510410 https://www.biorxiv.org/content/10.1101/2022.09.30.510410v1
• Von Experten begutachteter und veröffentlichter wissenschaftlicher Bericht.
  Cui, Wen, Haojun Huang, Yinkai Duan, Zhi Luo, Haofeng Wang, Tenan Zhang, Henry C Nguyen, et al. 2022. „Kristallstruktur der Monkeypox H1-Phosphatase, einem antiviralen Wirkstoffziel.“ Protein & Zelle, November. https://doi.org/10.1093/procel/pwac051. https://academic.oup.com/proteincell/advance-article/doi/10.1093/procel/pwac051/6805938.

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