Fig 1.
HIV-1 Integrase and ALLINI Structure.
A. Domain Map of HIV-1 Integrase. B. The preclinical lead ALLINI BI-224436. Chemical features common to the ALLINI class are highlighted, including a carboxylic acid moiety (salmon), a tert-butoxy moiety (green), and a large hydrophobic appendage at the R4 position (blue). C. Structure of HIV-1 IN CCD in complex with LEDGF/p75 Integrase Binding Domain (IBD, purple, PDB 2B4J). Shown below is the structure of HIV-1 IN CCD (blue) in complex with the ALLINI GSK1264 (PDB 5HOT). D. The IN-ALLINI structure reveals an open linear polymer configuration within the crystal lattice that underlies drug-induced aggregation. Outlined in black is the minimal ternary complex describing the ALLINI-induced protein-protein interaction between CCD and CTD. E. Cartoon Schematic of the CCD•CTD domain interactions stabilized by ALLINIs and a branched polymer model for drug-induced aggregation of HIV-1 IN.
Table 1.
Data collection and refinement statisticsa.
Fig 2.
2.93 Å X-ray Crystal Structure of CCDF185K•BI-224436•CTD.
Orthogonal views of the 2:2:2 ternary complex. The CCD is colored in dark blue and the CTD is colored in light blue. The BI-224436 molecule is shown in yellow. Disordered linkers within the CCD and CTD not observed within the electron density are shown as dotted lines.
Fig 3.
Evidence for asymmetry in the minimal ternary complex.
A. B-factor analysis. A color key depicts the overall B-factor of the atomic structure. B. Representative sigma A-weighted 2Fo-Fc electron density, contoured at 1σ at the first (left) and second (right) ALLINI binding sites. In the second site, side chain electron density is lost or diminished at key interfacial interactions including Lys-266, Arg-228, and Thr-124. C. Analysis of the surface of the ALLINI binding site using CASTp reveals at large binding pocket volume at ALLINI site 2 (971 A3) when compared to ALLINI site 1 (781 A3).
Fig 4.
Crystal Lattice Packing at the C-terminal domain of HIV-1 IN.
A. Crystal lattice packing. Shown is the lattice packing observed when crystallographic symmetry operations are applied. CCD dimer is shown in blue, the canonical CTD dimer in dark blue, the alternative CTD dimer in cyan, and the drug BI-224436 in yellow. Two homomeric CTD interactions are denoted (canonical and alternative), along with their position relative to ALLINI binding sites 1 and 2. B. Shown in orthogonal views are the canonical (light blue) and alternative CTD dimers (dark blue) observed. In both dimers, the interface involves the β2, β3, and β4 sheets and is predominantly hydrophobic, with the most significant contributions from residues Val-240, Leu-242, Trp-243, and Ile-257. In the canonical dimer, loop residues between the N-terminus and β2 are disordered and depicted as a dotted grey line.
Fig 5.
CCD interactions with BI-224436 within the minimal ternary complex.
A. Shown is a view of the site I BI-224436 binding site on the CCD, with the CTD omitted for clarity. Helices α3 and α1 from one monomer subunit and α5 and α4 from another monomer subunit are shown cradling the drug (yellow). B. Electrostatic surface of ALLINI-bound CCD, with the CTD omitted for clarity. Red denotes electropositive surfaces and blue denotes electronegative surfaces. C. Known ex vivo resistance mutation sites are shown in orange on the same ALLINI-bound CCD view. D. Conservation analysis, using the 2019 HIV IN collection of sequences at the Los Alamos database that includes all subtypes (4,326 sequences) of HIV-1 IN, as well as subtype M from HIV-1/SIVcpz (2,471 sequences). Alignments were prepared and used in the calculation of sequence conservation scores using the entropy-based method from AL2CO [63] program implemented in the UCSF ChimeraX [64]. All Figs were created using the program UCSF ChimeraX. Regions of high conservation are shown in red and regions of low conservation are shown in blue.
Fig 6.
CTD interactions within the minimal ternary complex.
A. Shown is the ternary complex, with CCD monomer chains colored in blue and cyan, BI-224436 in yellow, and the CTD colored in white. are Trp-235, Lys-266, and Ile-268 interactions with BI-224436 in ALLINI site 1. B. Hydrogen bonding interactions between Lys-266 from the CTD, the carboxylic acid moiety of BI-224436, and Glu-170, His-171, and Thr-174 from the CCD are highlighted. C. Shown is a bound molecule of ethylene glycol (EDO) within ALLINI binding site 1, created by the observed conformational change in Trp131, where bridging hydrogen bonds with Tyr-226 and Asp-270 are observed. D. Interactions between Tyr-226 and Ala-128, a common ex vivo resistance mutation site, on the α3 helice is shown. E. A distal interdomain cation-π interaction network between Trp-131, Arg-224, and Asp 270 is shown. Bound behind the indole plane of the Trp-131 side chain is a well-ordered molecule of EDO, precluding further stabilizing cation- π interactions by Lys-127 and Lys-136. E. An aromatic π-π network between Tyr-271 and a cluster of aromatics at the C-terminal base of the α3 helice in the CCD dimer is shown, including Trp-132, Phe-180, and Lys-185. In wild type HIV IN-1, position 185 is occupied by a phenylalanine.
Fig 7.
A generalized model for ALLINI Binding.
A. Using available isolated CCD•ALLINI structures in the Protein Data Bank, models of ALLINI class members in the minimal ternary complex structure were created to provide a generalized model for IN-ALLINI binding. Shown on the right are the chemical structures of the ALLINI modelled, with shared features highlighted: carboxylic acid moiety (orange), tert-butoxy moiety (green), large hydrophobic moiety (lavender), and an CTD-interacting moiety (purple). The following models of the ternary complex are shown: BI-224436 (yellow), LEDGIN-6 (salmon, PDB 3LPT [36]), BI-D (green, PDB 4ID1 [47]), and GSK1264 (blue, PDB 4OJR [11]). B. Modeling of GSK002 binding by superposition of the available CCD-only structure (PDB 5HRN [11]) with the ternary complex structure. Highlighted is a clash between the diflourobenzyl moiety at R6 with the CTD residue Trp-235. C. Modelling of STP0404 binding by superposition of the available CCD-only structure (PDB 7KE0 [80]) with the ternary complex structure. Highlighted is a clash between the 1-methylpyrazole moiety at R6 with the CTD residue Trp-235. D. Modeling of KF116 binding. A predicted clash between its distal ornament and Trp 235 is shown.