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. 2014 Jul 1;111(26):9609-14.
doi: 10.1073/pnas.1402448111. Epub 2014 Jun 16.

Structural studies of postentry restriction factors reveal antiparallel dimers that enable avid binding to the HIV-1 capsid lattice

David C Goldstone  1 , Philip A Walker  2 , Lesley J Calder  3 , Peter J Coombs  2 , Joshua Kirkpatrick  4 , Neil J Ball  2 , Laura Hilditch  5 , Melvyn W Yap  5 , Peter B Rosenthal  3 , Jonathan P Stoye  6 , Ian A Taylor  7
Affiliations

Structural studies of postentry restriction factors reveal antiparallel dimers that enable avid binding to the HIV-1 capsid lattice

David C Goldstone et al. Proc Natl Acad Sci U S A. .

Abstract

Restriction factors (RFs) form important components of host defenses to retroviral infection. The Fv1, Trim5α, and TrimCyp RFs contain N-terminal dimerization and C-terminal specificity domains that target assembled retroviral capsid (CA) proteins enclosing the viral core. However, the molecular detail of the interaction between RFs and their CA targets is unknown. Therefore, we have determined the crystal structure of the B-box and coiled-coil (BCC) region from Trim5α and used small-angle X-ray scattering to examine the solution structure of Trim5α BCC, the dimerization domain of Fv1 (Fv1Ntd), and the hybrid restriction factor Fv1Cyp comprising Fv1NtD fused to the HIV-1 binding protein Cyclophilin A (CypA). These data reveal that coiled-coil regions of Fv1 and Trim5α form extended antiparallel dimers. In Fv1Cyp, two CypA moieties are located at opposing ends, creating a molecule with a dumbbell appearance. In Trim5α, the B-boxes are located at either end of the coiled-coil, held in place by interactions with a helical motif from the L2 region of the opposing monomer. A comparative analysis of Fv1Cyp and CypA binding to a preformed HIV-1 CA lattice reveals how RF dimerization enhances the affinity of interaction through avidity effects. We conclude that the antiparallel organization of the NtD regions of Fv1 and Trim5α dimers correctly positions C-terminal specificity and N-terminal effector domains and facilitates stable binding to adjacent CA hexamers in viral cores.

Keywords: MLV; SAXS; X-ray crystallography; retrovirus.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
SAXS analysis of Fv1Ntd, Fv1Cyp, and RhT5 (88–296) EK/RD. (A) SEC-MALLS analysis of Fv1-Ntd, Fv1-CypA, and RhT5 (88–296) EK/RD demonstrates that all proteins are dimers in solution. (B) Concentration-corrected merged SAXS curves. (Insets) Enlargement of the Guinier region and analysis. (C) Normalized pair-distribution functions. (D) Consensus ab initio models.
Fig. 2.
Fig. 2.
Crystal structure of RhT5-T4L. (A) Cartoon representation of the RhT5-T4L structure with zinc ions shown as spheres. Residues in the L2-E region (between α4 and α5) are missing in chain A (red) and are represented as a dashed line but are present in the other monomer (chain B, blue). (B) Zn1 is coordinated by C108, D111, H125, and H128 and Zn2 is coordinated by C97, H100, C116, and C119. The B-box of one monomer packs with the α3–α4 region of the opposing monomer to form a hydrophobic core. (C) The “top” of the extended antiparallel helices has no distinct hydrophobic groove, whereas the “bottom” has a clear hydrophobic groove (green) that spans the length of the helices. (D) The L2-E region (orange sticks, labeled every 10th residue) closely follows the hydrophobic groove on the surface of the extended coil. Hydrophobicity of residues was calculated using AAindex (database code FASG890101) in PyMol where increasing hydrophobicity is proportional to the intensity of the green color.
Fig. 3.
Fig. 3.
EM and Biacore analysis of Fv1Cyp. (A) Gallery of negatively stained EM images of Fv1Cyp. Images are the sums of three different images of the same particle recorded in 0.5-μm steps (Fig. S5A), revealing thin connections between the globular domains. (B) The interaction of Fv1Cyp and monomeric CypA with a HIV-1 CA-p2 lattice immobilized on a Ni-NTA tagged lipid monolayer analyzed by Biacore. Equilibrium affinity measurements derived from sensorgram plateau values (Left) show Fv1Cyp exhibits an ∼18-fold higher affinity. Analysis of the kinetics for individual sensorgram binding curves gives comparable values for equilibrium dissociation constants (Right).
Fig. 4.
Fig. 4.
RF CA–lattice interactions. (A) A layer of CA lattice taken from 3J34. Red spheres mark the Cyp loops and the gray region highlights those that are spaced at 130–170 Å from the central Cyp loop (yellow) and accessible to bind the second CypA of Fv1Cyp. (B) The SAXS envelope of Fv1Cyp dimer is positioned with one CypA domain located on the Cyp loop of a central hexamer and the second spaced between 140–150 Å located on the Cyp loop of an adjacent CA hexamer. (C) A schematic representation of the HIV-CA lattice (green) with the Trim5-21R lattice, from ref. , is overlaid as dashed lines (black). The SAXS model for the RhT5 (88–296) EK/RD dimer shown in orange surface representation has dimensions equating to the length of a single edge of the hexamer in the Trim5-21R lattice and sufficient to span adjacent hexamers in the CA lattice. The B-boxes are shown as cyan circles at the interface between Trim5α dimers and the B30.2 domains are represented by the ovals toward the Trim5-21R twofold axis.
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