Research Article: Crystal structure of a soluble fragment of poliovirus 2CATPase

Date Published: September 19, 2018

Publisher: Public Library of Science

Author(s): Hongxin Guan, Juan Tian, Chu Zhang, Bo Qin, Sheng Cui, Nuria Verdaguer.

http://doi.org/10.1371/journal.ppat.1007304

Abstract

Poliovirus (PV) 2CATPase is the most studied 2C protein in the Picornaviridae family. It is involved in RNA replication, encapsidation and uncoating and many inhibitors have been found that target PV 2CATPase. Despite numerous investigations to characterize its functions, a high-resolution structure of PV 2C has not yet been determined. We report here the crystal structure of a soluble fragment of PV 2CATPase to 2.55Å, containing an ATPase domain, a zinc finger and a C-terminal helical domain but missing the N-terminal domain. The ATPase domain shares the common structural features with EV71 2C and other Superfamily 3 helicases. The C-terminal cysteine-rich motif folds into a CCCC type zinc finger in which four cysteine ligands and several auxiliary residues assist in zinc binding. By comparing with the known zinc finger fold groups, we found the zinc finger of 2C proteins belong to a new fold group, which we denote the “Enterovirus 2C-like” group. The C-terminus of PV 2CATPase forms an amphipathic helix that occupies a hydrophobic pocket located on an adjacent PV 2CATPase in the crystal lattice. The C-terminus mediated PV 2C-2C interaction promotes self-oligomerization, most likely hexamerization, which is fundamental to the ATPase activity of 2C. The zinc finger is the most structurally diverse feature in 2C proteins. Available structural and virological data suggest that the zinc finger of 2C might confer the specificity of interaction with other proteins. We built a hexameric ring model of PV 2CATPase and visualized the previously identified functional motifs and drug-resistant sites, thus providing a structure framework for antiviral drug development.

Partial Text

Poliovirus (PV) is the pathogen of poliomyelitis. PV infection can directly result in damage of motor neurons and cause neural lesions [1]. Since the launch of Global Polio Eradication Initiative (http://polioeradication.org/) by the World Health Assembly, the number of poliomyelitis cases have been significantly reduced. The incidence of paralytic polio in 1988 was 1,000 children per day, and this number decreased to 400 per day in 2013[2]. Only three endemic countries remain today. Nevertheless, obstacles to global polio eradication remain. To overcome the last hurdles in the endgame phase, effective anti-PV drugs are critical in controlling transmission of vaccine-derived polioviruses (VDPVs) and in treating patients with chronic infection or personnel casually exposed to PV [3]. Further, to minimize poliomyelitis risk in the “post-polio” era, the National Research Council of the Unite States concluded that the development of antiviral drugs would be important, and possibly essential [4].

To gain atomic insight into poliovirus 2CATPase, we carried out a crystallographic study. We first expressed a N-terminal maltose-binding protein (MBP) tagged full-length 2CATPase from human poliovirus 1 (strain Mahoney, GenBank: KU866422.1); however, the MBP tagged protein did not yield crystals. Removal of MBP tag by proteinase cleavage only led to precipitation of PV 2CATPase. Therefore, we employed a similar strategy as used in expressing the soluble fragment of EV71 2C [21]. Firstly, we removed the N-terminal 115 residues of PV 2CATPase, (PV-2C-ΔN). This fragment lacks the N-terminal membrane binding domain but retains a complete ATPase domain, a zinc binding site and a C-terminal helical domain. PV-2C-ΔN was soluble, but still failed to crystallize. Considering that surface-entropy reduction may favor crystallization [45], we predicted a set of surface residues charges based on EV71 2C structure [21]. We then introduced alanine substitutions systematically to these residues with an aim of minimizing surface charging. We finally obtained crystals of PV-2C-ΔN bearing mutations E207A, K209A and R149A (PV-2C-ΔN-3Mut). None of these mutations is located within conserved ATPase motifs, zinc binding site or the C-terminal helical domain.

Comparing the available high-resolution structures of enterovirus 2C proteins (PV 2C, EV-C species and EV71 2C, EV-A species), we identified both common and individual structural features. (i) The zinc finger is the most structurally distinct site in PV 2CATPase and EV71 2C. While PC 2CATPase has a CCCC type zinc finger, EV71 2C has only three cysteine (lacking the PCS2) for zinc coordination. Eliminating the PCS2 of PV 2CATPase resulted in temperature-sensitive phenotypes and encapsidation defects [11,49]. We added the PCS2 to the zinc finger of EV71 2C in order to convert it to a CCCC type zinc finger similar to PV 2CATPase zinc finger, but it failed to improve EV71 infectivity. Zinc fingers are ubiquitous small motifs that function as binding module for nucleic acids or proteins, etc.[50]. It was demonstrated that the C-terminal cysteine-rich site of PV 2CATPase, the zinc finger in our structure, is required for morphogenesis[49]. Therefore, the significant difference in sequence and structure of the zinc finger we observed here may underlie the specificity of 2C protein and determine what process it may involve. The cysteine-rich motif is one of the least conserved regions in Picornaviridae 2C (Fig 2E). In fact, the 2C of the foot-and-mouth disease virus (FMDV) does not even have a cysteine-rich motif between ATPase and C-terminal helical domains. So, this region of FMDV 2C might fold into a structure completely different from the zinc finger but it may still function as protein binding module with the distinct specificity. (ii) The hexameric ring models of PV 2CATPase and EV71 2C show that the ATPase active site formed between 2C subunits has nearly identical geometry and the catalytic residues (Walker A & B, Motif C and R finger) identified by structural and biochemical characterizations are invariant. (iii) The C-terminus amphipathic helix mediated self-oligomerization is common in enterovirus 2C. Analogous to EV71 2C, PV 2CATPase undergoes self-oligomerization both in crystalline state and in solution via PBD-pocket interaction. Although residues constituting the PBD and the pocket are not strictly conserved in PV 2CATPase and EV71 2C, the “knob-into-hole” interaction between PBD-pocket is identical. The PBD residues of PV 2CATPase include C323, M324, L327 and F328, whereas the PBD of EV71 2C contains residues T323, I324, L327 and F328. All PBD residues are hydrophobic, in which L327 and F328 are invariant. The strict conservation of L327 and F328 highlights their essential role in 2C activity. We showed that mutations L327 and F328 abrogated the ATPase activity and homo-oligomerization of both PV 2CATPase and EV71 2C helicases (Figs 4 and 5), and these mutations could halt EV71 infection [21]. The dimension of the hydrophobic pocket of PV 2CATPase is 17Å long, 12 Å wide and 7Å deep, which is similar to the pocket size of EV71 2C. We calculated the solvent accessible surface area (SASA) of the pocket of PV 2CATPase as 918 Å2 containing 13 residues, the SASA of the pocket of EV71 2C is 846 Å2 containing 14 residues.

 

Source:

http://doi.org/10.1371/journal.ppat.1007304

 

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