Structural basis of higher order oligomerization of KSHV inhibitor of cGAS

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) inhibitor of cyclic GMP-AMP synthase (cGAS) (KicGAS) encoded by ORF52 is a conserved major tegument protein of KSHV and the first reported viral inhibitor of cGAS. In our previous study, we found that KicGAS is highly oligomerized in solution and that oligomerization is required for its cooperative DNA binding and for inhibiting DNA-induced phase separation and activation of cGAS. However, how KicGAS oligomerizes remained unclear. Here, we present the crystal structure of KicGAS at 2.5 Å resolution, which reveals an "L"-shaped molecule with each arm of the L essentially formed by a single α helix (α1 and α2). Antiparallel dimerization of α2 helices from two KicGAS molecules leads to a unique "Z"-shaped dimer. Surprisingly, α1 is also a dimerization domain. It forms a parallel dimeric leucine zipper with the α1 from a neighboring dimer, leading to the formation of an infinite chain of KicGAS dimers. Residues involved in leucine zipper dimer formation are among the most conserved residues across ORF52 homologs of gammaherpesviruses. The self-oligomerization increases the valence and cooperativity of interaction with DNA. The resultant multivalent interaction is critical for the formation of liquid condensates with DNA and consequent sequestration of DNA from being sensed by cGAS, explaining its role in restricting cGAS activation. The structure presented here not only provides a mechanistic understanding of the function of KicGAS but also informs a molecular target for rational design of antivirals against KSHV and related viruses.

Keywords: DNA binding; KicGAS; ORF52; cGAS; higher order oligomerization.

Fig. 1.

KicGAS forms oligomers in solution. (A) KicGAS forms concentration-dependent oligomers as characterized by size exclusion chromatography; Molecular standards are marked on the profile. OD230, optical density at 230 nm. (B) Histogram envelope plot showing the sedimentation coefficient distribution from the SV-AUC experiment with KicGAS. (C) Hydrodynamic properties of major KicGAS species in solution as determined in (B).

Fig. 2.

KicGAS repeating unit is a dimer. (A) Diagrams of full-length and truncated versions of KicGAS. (B) Size exclusion chromatography profiles of KicGAS^9-104, KicGAS^9-95, and KicGAS^9-95 (L62M). OD230, optical density at 230 nm. (C) Dimer structure of KicGAS^9-95 with one chain (molA) colored in yellow and the other (molB) in blue. The secondary structure elements are labeled with chain ID as superscript. (D) Close-up view of the dimer interface mediated by α2 helix (red rectangle in (C)). Since the interface is symmetrical, only the left half is shown.

Fig. 3.

KicGAS dimer further polymerizes via α1 leucine zipper. (A) KicGAS forms α1-mediated infinite chain of dimers in the crystal lattice. Molecules A and B in each dimer are colored in different shades of yellow and blue, respectively. (B) Highly conserved hydrophobic residues mediate leucine zipper formation between α1 helices of molecule A (molA) in one dimer and molecule B (molB′) in a symmetry-related dimer. (C) Negative stain electron microscopy showing KicGAS^9-95 filaments. KicGAS^9-95 concentration: 3.4 mg/mL Scale bar: 200 nm. (D) Superposition of KSHV KicGAS tetramer (yellow and blue) with MHV-68 ORF52 tetramer (gray, PDB ID: 2OA5). Black arrows mark the movement of the two α1 helices in the structures. The sequences of the α1–α2 loops are labeled next to the loops.

Fig. 4.

Unique α1–α2 linker sequence dictates KicGAS oligomerization. (A) Alignment of the linker sequences of KicGAS homologs from gamma-2 herpesviruses. The two conserved proline residues are highlighted in cyan. RFHV, Retroperitoneal fibromatosis herpesvirus; RRV, Rhesus monkey rhadinovirus; MFRV, Macaca fuscata rhadinovirus; MnRV, Macaca nemestrina rhadinovirus 2; HVS, Herpesvirus saimiri; AtHV-3, Ateline herpesvirus 3; MHV-68, Murid herpesvirus 68. (B) Size exclusion chromatography profiles of KicGAS, the P41S/P45I mutant, and the linker switch KicGAS-LS mutant. OD230, optical density at 230 nm. (C) Fluorescence polarization binding curves of KicGAS, P41S/P45I, and KicGAS-LS with ISD45. (D) Schematic of the fusion of the N-terminal α helix of KicGAS to the VP22 core domain (α1-VP22^190-301). (E) Size exclusion chromatography profiles of α1-VP22^190-301 and VP22^174-301. (F) Fluorescence polarization binding curves of α1-VP22^190-301 and VP22^174-301 with ISD45.

Fig. 5.

Polymeric KicGAS presents multiple DNA binding sites. (A) Modeling of KicGAS^9-104 dimer juxtaposes the two R97 residues. (B) KicGAS^9-104 polymer chain in two orthogonal views. Red arrowheads point to the R97 sites. (C) Electrostatic surface of KicGAS^9-104 chain in two orthogonal views, highlighting multiple positively charged patches along the chain. Red, green, brown, and cyan arrowheads indicate various positively charged patches for DNA binding. (D) Proposed multivalent DNA binding mode of KicGAS (created with BioRender.com). Both the structured region (space-filling models) and the flexible R clusters (blue blobs connected with dashed lines) participate in DNA binding.