The crystal structures of Chikungunya and Venezuelan equine encephalitis virus nsP3 macro domains define a conserved adenosine binding pocket

Abstract

Macro domains (also called "X domains") constitute a protein module family present in all kingdoms of life, including viruses of the Coronaviridae and Togaviridae families. Crystal structures of the macro domain from the Chikungunya virus (an "Old World" alphavirus) and the Venezuelan equine encephalitis virus (a "New World" alphavirus) were determined at resolutions of 1.65 and 2.30 A, respectively. These domains are active as adenosine di-phosphoribose 1''-phosphate phosphatases. Both the Chikungunya and the Venezuelan equine encephalitis virus macro domains are ADP-ribose binding modules, as revealed by structural and functional analysis. A single aspartic acid conserved through all macro domains is responsible for the specific binding of the adenine base. Sequence-unspecific binding to long, negatively charged polymers such as poly(ADP-ribose), DNA, and RNA is observed and attributed to positively charged patches outside of the active site pocket, as judged by mutagenesis and binding studies. The crystal structure of the Chikungunya virus macro domain with an RNA trimer shows a binding mode utilizing the same adenine-binding pocket as ADP-ribose, but avoiding the ADP-ribose 1''-phosphate phosphatase active site. This leaves the AMP binding site as the sole common feature in all macro domains.

FIG. 1.

Structures of the macro domains from the alphaviruses CHIKV and VEEV. (A) Representation of CHIKV and VEEV macro domains in a purple-to-red gradient (from N terminus to C terminus). Secondary structure elements are labeled on the CHIKV macro domain. (B) Superposition of representations of the CHIKV, VEEV, SARS-CoV, and E. coli macro domains, colored, respectively, in dark blue, cyan, purple, and white. (C) Electrostatic surface potential presented between −4 and 4 kT/e for the CHIKV and VEEV macro domains. The potential was generated using the PDB2PQR server (8) and Adaptative Poisson-Boltzmann Solver (APBS) plug-in in Pymol (http://www.pymol.org). (D) Representation of the superposition between the macro domains from CHIKV (apo form in dark blue), VEEV (apo form in cyan and complexed with ADP-ribose in light orange). The two main divergences between the three structures are indicated and circled. (E) Sequence alignment of the macro domains studied. They belong to the genus Alphavirus, except for SARS-CoV. Residues in red boxes are strictly conserved, while those in yellow boxes are conserved by at least four out of seven viruses aligned. Secondary structure elements from the viral macro domain crystal structures obtained are represented above the alignment.

FIG. 2.

ADP-ribose binding. (A) Thermal denaturation shift at a given ligand concentration (2 mM) for the CHIKV macro domain. T_m is the melting temperature of the protein in the presence of the ligand, and T_o is the melting temperature of the protein alone. (B) ADP-ribose titration using the thermal denaturation shift assay on CHIKV (wild type and D10A mutant), VEEV, and SARS-CoV macro domains. (C) ITC assay of ADP-ribose binding to the CHIKV macro domain. (Upper panel) ITC raw data of ADP-ribose binding to the CHIKV and VEEV macro domains. (Lower panel) ADP-ribose binding isotherm derived from raw data. (D) Same experiment as in panel C but using the VEEV macro domain.

FIG. 3.

Structural basis for ADP-ribose binding. (A) ADP-ribose binding site of the CHIKV macro domain. On the left side is a representation of the CHIKV macro domain with helices, strands, and loops colored, respectively, in red, yellow, and green. ADP-ribose is displayed in sticks with carbons in yellow, oxygens in red, nitrogens in blue, and phosphorus in orange. The Fo − Fc difference map, contoured at 3σ, was calculated at a 1.80-Å resolution from a model in which the ligand was omitted. On the right side is an electrostatic surface representation of the CHIKV macro domain in complex with ADP-ribose. The electrostatic potential is shown between −8 and 8 kT/e and has been generated as in Fig. 1C. The ADP-ribose molecule is shown as on the left side of panel A. (B) The ADP-ribose binding site presented with the same orientation as in panel A. The CHIKV macro domain in complex with ADP-ribose is shown in the upper part, the VEEV macro domain in complex with a Bicine molecule originating from the buffer is presented in the middle part, and the VEEV macro domain in complex with ADP-ribose is shown in the lower part. ADP-ribose and Bicine are shown in cyan. Residues interacting with ADP-ribose in the CHIKV macro domain are shown in blue, and mutated residues are indicated with an asterisk. Corresponding residues in VEEV are colored in blue. Hydrogen bonds between the ligand and the protein are shown in black dotted lines. The loops containing residues 30 to 37 are shown in yellow in CHIKV and VEEV macro domains: their conformations diverged particularly between residues 32 and 35.

FIG. 4.

Detection by TLC of ADP-ribose 1″-P, the product of the ADP-ribose phosphatase activity. Control lanes (ADP-ribose 1″-phosphate and ADP-ribose) and reaction lanes with corresponding enzymes and D10A, N24A, and Y114A mutants are indicated. The EEEV lane corresponds to the purified EEEV macro domain designed and produced in the same manner as the CHIKV macro domain. The yeast macro domain protein Poa1p serves as a positive control.

FIG. 5.

Slot blot PAR binding assay of macro domains from CHIKV, VEEV, Sindbis virus, SARS-CoV, and SFV. BSA was used as a negative control. PAR was synthezised using PARP-1 and [³²P]NAD⁺, diluted, and used as a probe to label immobilized proteins (2,000 to 0.98 pmol) blotted onto a nitrocellulose membrane as described in Materials and Methods.

FIG. 6.

RNA binding assays. (A) Slot blot RNA binding assay with wild-type and mutant CHIKV macro domains using the 5′-³²P-labeled RNA AAAAAAAAAGCUACC as a probe to label immobilized proteins. The amount of proteins blotted onto the membrane ranges from 2,000 to 0.98 pmol, as indicated. (B) Surface representation of the CHIKV macro domain in complex with the RNA AAA. Proposed nt 1, 2, and 3 (I, II, and III, respectively) are circled. The electrostatic potential is shown between −6 and 6 kT/e and has been generated as in Fig. 1C. (C) RNA binding site. RNA is shown in green and interacting residues in blue, with oxygens in red. H-bonds are indicated with dotted lines.