Dear Editor,
Since its appearance in May 2022, monkeypox has spread to >100 countries and afflicted tens of thousands of people. Individuals infected with monkeypox present a fever, an extensive characteristic rash, and usually swollen lymph nodes (
Chatterjee et al., 2022). The number of confirmed cases worldwide continues to grow at a rapid rate, but the treatment to this highly infectious viral disease is still very limited. Identification of new targeted-therapies will be crucial to control of this emerging public health threat.
The monkeypox virus is an enveloped double-stranded DNA virus that belongs to the
Orthopoxvirus genus of the
Poxviridae family (
Isidro et al., 2022). It has a very large genome (~200 kb) and codes around 200 proteins (
Kugelman et al., 2014). Poxviruses express a dual specific phosphatase (H1) that de-phosphorylates signal transducer and activator of transcription 1 (STAT1) and blocks interferon signal transduction (
Najarro et al., 2001;
Mann et al., 2008). H1 is conserved in poxviruses and is essential for virus replication in cell culture (
Liu et al., 1995). Inhibiting H1 expression results in greatly reduced infectivity. About 200 copies of H1 are packaged into newly formed viral particles and function in the early stage of viral infection (
Liu et al., 1995). H1 has also been suggested to de-phosphorylate monkeypox proteins F18, A14, and A17 (
Liu et al., 1995;
Derrien et al., 1999;
Traktman et al., 2000). Due to the importance of H1 in modulating interferon-signaling and viral replication, it serves as an attractive anti-poxvirus drug target.
To understand the mechanism for monkeypox H1 catalyzed dephosphorylation and provide an accurate structural model for drug discovery, we determined a crystal structure of H1 to 1.8 Å resolution (Fig. 1 and Table S1). Monkeypox H1 has 171 amino acid residues and the refined model includes residues 2–171 with well-fitting electron density. There is one H1 molecule in an asymmetric unit. Two H1 molecules are related by crystallographic symmetry and form a domain swapped dimer, which resembles a butterfly (Fig. 1A). The overall structure is composed of six α helices and four β strands. A four-stranded β-sheet is sandwiched by helices α2 and α3–α6 on either side. The active site is located near the C-terminus of the last β-strand. The two active sites are ~39 Å apart (Fig. 1A). There is a phosphate ion captured at each active site, representing the final stage of catalysis before the product is released.
The N-terminal helix α1 from each protomer interchanges to mediate H1 dimerization. The α1 from one H1 protomer forms a four-helix bundle with three helices α4−α6 of the pairing protomer. The buried surface area between Protomer A and B is ~1000 Å2, which is stabilized by both hydrophilic and hydrophobic interactions. Residues Ser14 and Thr15 in α1 form hydrogen bonds with His143 and Tyr142/Lys159 of the other H1 protomer, respectively; whereas Tyr7, Leu11, and Leu12 participate in hydrophobic interactions with Met135, Leu139, Lys159, Ile163, Val167, and Ile168 from the pairing H1 molecule (Fig. 1B, left panel). In addition, three residues in α5, Met135, Leu136, and Leu139, face the symmetry related residues in the dimer to expand the hydrophobic interface (Fig. 1B, middle panel). α1 is also stabilized by intramolecular hydrogen bonds and hydrophobic interactions between residues of α1 and α5 (Fig. 1B, right panel). Size exclusion chromatography confirms the H1 dimer in solution, suggesting that dimerization represents its functional state (Fig. 1C).
The H1 active site consists of a Cys-Arg-Asp catalytic triad (Fig. 2A). The conserved Cys and Arg residues are in a loop between β4 and α4 (109HCVAGVNRS117), which is also known as the phosphate-binding loop. The arginine residue (Arg116) of this loop captures the phosphate ion, whose guanidinium group interacts with two phosphate oxygens through two hydrogen bonds with the distances of 2.9 and 3.0 Å, respectively (Fig. 2B). This important arginine residue guarantees efficient binding and orientation of the substrate. At the bottom of the catalytic pocket (Fig. 2B), the conserved Cys110 attacks the phosphorous atom during the de-phosphorylation reaction, resulting in a transient enzyme-phosphate intermediate. This intermediate is then hydrolyzed to generate inorganic phosphate and the regenerated enzyme. The sulfur atom of Cys110 is positioned in line with a phosphorous-oxygen (P-O) bond which corresponds to the one formed during the enzyme regeneration step (Fig. 2B). Asp79 is responsible for coordinating the water molecule, which is also hydrogen bonded to oxygen from the phosphate group (Fig. 2B). This residue functions as a general acid, facilitating both the formation of the enzyme-phosphate intermediate and its hydrolysis. Thus, the crystal structure represents the final step of catalysis before the product is released.
The high-resolution crystal structure of monkeypox H1 reveals at least two hot spots for drug discovery. The first hot spot is the dimer interface, which is unique among the members in the protein tyrosine phosphatase (PTP)/dual-specificity phosphatase (DSP) family (Fig. 1A and 1D). Blocking H1 dimerization may potentially inhibit its ability to dimerize and dephosphorylate the phosphor-tyrosine in activated STAT1, which is also a homodimer (
Wenta et al., 2008;
Jeong et al., 2014). In addition, the active site is another potential target for inhibition. Although the active sites of all phosphatases in PTP/DSP family are built around a phosphate-binding loop (with a sequence HCX
5R(S/T)) and have a similar main chain structure, the side chains around the active center are different (
Yuvaniyama et al., 1996;
Tao and Tong, 2007;
Jeong et al., 2014) (Fig. 2C), which affects their own substrate specificity and may allow the development of specific inhibitors.
In conclusion, we report a high-resolution crystal structure of the monkeypox H1 phosphatase that lays a solid foundation for its mechanistic study and the discovery of antiviral compounds against this emerging pathogen.
©The Author(s) 2022. Published by Oxford University Press on behalf of Higher Education Press.
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