Structural biology of retroviral DNA integration

Abstract

Three-dimensional macromolecular structures shed critical light on biological mechanism and facilitate development of small molecule inhibitors. Clinical success of raltegravir, a potent inhibitor of HIV-1 integrase, demonstrated the utility of this viral DNA recombinase as an antiviral target. A variety of partial integrase structures reported in the past 16 years have been instrumental and very informative to the field. Nonetheless, because integrase protein fragments are unable to functionally engage the viral DNA substrate critical for strand transfer inhibitor binding, the early structures did little to materially impact drug development efforts. However, recent results based on prototype foamy virus integrase have fully reversed this trend, as a number of X-ray crystal structures of active integrase-DNA complexes revealed key mechanistic details and moreover established the foundation of HIV-1 integrase strand transfer inhibitor action. In this review we discuss the landmarks in the progress of integrase structural biology during the past 17 years.

Fig. 1

Domain organization and secondary structural elements of representative IN proteins. Interdomain linker and C-terminal tail region lengths are indicated above lines; the terminal 38 residues of the ASLV Gag-Pol precursor (parentheses) is cleaved during virus morphogenesis to yield the mature 286-residue IN, though small amounts of the 323-residue protein are detected in virions (Alexander et al., 1987). Secondary structural elements (line, alpha helix; arrow, beta strand) from the HIV-1 intasome model (Krishnan et al., 2010) and ASLV CCD-CTD (Yang et al., 2000) and PFV intasome (Hare et al., 2010a) crystal structures shown underneath each drawing are numbered according to domain (NED, N-terminal extension domain; NTD, N-terminal domain; CCD, catalytic core domain; CTD, C-terminal domain). Domains are color-coded for simplicity; residues conserved among retroviral INs are coded based on chemical property: electronegative, red; electropositive, blue; sulfur-containing, yellow. Proteins alignments based on start of NTD sequences (Jaskolski et al., 2009).

Fig. 2

Structures of individual HIV-1 IN domains. (A) X-ray crystal structure (PDB code 1THG) highlighting the common CCD dimer and active site residues Asp64 and Asp116 (red sticks); the third member of the DDE catalytic triad, Glu152, was undecipherable in this initial structure. Only one of two Lys185 solubility-enhancing substitutions (blue stick) is visible in this orientation. Dashed lines indicate disordered gaps in polypeptide chains. (B) CTD NMR structure (PDB code 1IHV) revealed a dimer, with each monomer folded into a five-stranded beta barrel. (C) HIV-1 NTD monomer from PDB code 1WJC. Highlighted are Zn-coordinating residues His12, His16, Cys 40, and Cys43 as well as the bound Zn²⁺ ion (grey sphere). Visualized IN residue termini are indicated in panels B and C.

Fig. 3

Two-domain X-ray crystal structures revealed variable CCD-CTD arrangements among retroviral INs. (A) The HIV-1 structure (PDB code 1EX4) unveiled CTDs extending from each monomer within the canonical CCD dimer. Arg199 side chains are indicated by blue stick. (B) SIV structure (PDB code 1C6V) showing a single CTD in closer proximity to its CCD dimer as compared to the HIV-1 structure in panel A. (C) The ASLV 2-domain structure revealed two CTDs off kilter from the 2-fold CCD dimer symmetry axis. The drawing is scaled for similar CCD dimer sizes, each positioned as in Fig. 2A. Red sticks indicate catalytic DDE triads; dashed lines, disordered gaps.

Fig. 4

Intermolecular NTD-CCD interactions among different multidomain IN crystal structures. (A) The dimer of dimers observed in the HIV-1 IN NTD-CCD crystallographic asymmetric unit (PDB code 1K6Y) (Wang et al., 2001). Inner monomers are painted green and cyan; outer monomer (yellow) interactions are intradimer with partners connected via canonical CCD interfaces. Interdimer NTC (green)–CCD (cyan) contact residues are shown in the lower panel, with salt bridges between ionic side chains highlighted by red dashes. Interactions for other highlighted residues are mediated through polypeptide backbone atoms. (B) The MVV IN NTDCCD structure from PDB code 3HPH (Hare et al., 2009a), oriented and labeled as in panel A. LEDGF IBDs were omitted for clarity. (C) The Mn-bound PFV intasome (PDB code 3OYA) (Hare et al., 2010a; Hare et al., 2010b) oriented 45° to the right and 90° into the page with respect to panels A and B to highlight interactions between the green NTD and cyan CCD. DNA molecules were omitted for clarity. Side chains of DDE catalytic triads are shown as red sticks in upper panels; secondary elements in lower panels based on Figure 1; grey spheres, Zn atoms.

Fig. 5

Crystal structure of the active PFV intasome. (A) The crystallographic asymmetric unit housed a dimer of IN bound to a single DNA molecule (PDB code 3L2Q) (Hare et al., 2010a). The non-transferred DNA strand is painted orange whereas the transferred strand terminating in dA_OH is magenta. (B) The intasome structure, formed by duplication, C2 symmetry rotation, and merger of the panel A structure, with the second DNA-bound monomer painted cyan. Other labeling as in Figures 1 and 3.

Fig. 6

Wild type and S217H mutant intasome structures in committed and drug-bound forms elucidate the mechanism of INSTI action and basis for HIV-1 Q148H/R/K drug resistance. (A) Mn-bound active site (PDB code 3OY9) revealed the coordination of two metal ions by the DDE catalytic triad (see main text for additional details). (B) RAL (yellow scaffold) binding to an induced fit pocket formed through interactions with coordinated metal ion (Mg²⁺) and the penultimate C16/G4 bp of the vDNA end ejects the terminal adenine nucleotide (A17) and its affiliated 3′-OH nucleophile from the IN active site (PDB code 3OYA). (C) Superposition of wild-type intasome in committed (grey trace; same view as panel A) and MK-2048 (magenta backbone) bound (blue trace; PDB code 3OYB) conformations. (D) Comparison of MK-2048-bound (PDB code 3OYL; blue trace) and unbound S127H mutant (grey trace; PDB code 3OYK) intasome structures revealed significant active site conformational changes (alterations in side chain positions indicated by arrows) elicited by drug binding. Other labeling is same as in Figures 4 and 5.

Fig. 7

PFV IN TCC and STC crystal structures elucidate the mechanism of DNA strand transfer. (A) The PFV IN TCC structure highlighted the highly bent conformation of bound tDNA (magenta and black strands), with the arrow indicating the spacing between scissile phosphodiester bonds (PDB code 3OS1). Red sticks, DDE side chains. (B) The mechanism of DNA strand transfer elucidated by PFV IN CDC, TCC, and STC overlays (PDB codes 3OY9, 3OS1, and 3OS0, respectively). The dotted line represents the path taken by the adenosine 3′-OH nucleophile during DNA strand transfer; the curved arrow highlights the rotation around the deoxyribose bond that ejects the newly formed vDNA-tDNA phosphodiester bond from the enzyme active site. Color codes are as follows: magenta vDNA, CDC; grey tDNA, TCC; cyan vDNA and black tDNA, STC; green IN protein and DDE side chains, CDC; purple IN ribbon and cyan DDE side chains, STC; grey metal ions, Mn²⁺ in CDC; black metal ion, position A in STC.