Cloning, purification and structure determination of the HIV integrase-binding domain of lens epithelium-derived growth factor

Abstract

Lens epithelium-derived growth factor (LEDGF)/p75 is the dominant binding partner of HIV-1 integrase in human cells. The crystal structure of the HIV integrase-binding domain (IBD) of LEDGF has been determined in the absence of ligand. IBD was overexpressed inEscherichia coli, purified and crystallized by sitting-drop vapour diffusion. X-ray diffraction data were collected at Diamond Light Source to a resolution of 2.05 Å. The crystals belonged to space groupP21, with eight polypeptide chains in the asymmetric unit arranged as an unusual octamer composed of four domain-swapped IBD dimers. IBD exists as a mixture of monomers and dimers in concentrated solutions, but the dimers are unlikely to be biologically relevant.

Molecular Mechanism of LEDGF/p75 Dimerization

“…We initially tried to structurally characterize the LEDGF345-467 dimer using X-ray crystallography, but this effort was unsuccessful due to inherent dynamics of several regions of the construct. However, we obtained high-quality crystals for the IBD (residues 345-431) that yielded a domain-swapped dimeric structure highly similar to one published during the course of this study (Figure 4A; (Hannon et al, 2018); Table S2). The fact that we obtained reproducible results using a different protein construct indicates that swapping of helix 5 is not a crystallographic artifact, as might be inferred from an estimated dimer dissociation constant in a millimolar range (Figure 1C,D; (Hannon et al, 2018)).…”

“…However, we obtained high-quality crystals for the IBD (residues 345-431) that yielded a domain-swapped dimeric structure highly similar to one published during the course of this study (Figure 4A; (Hannon et al, 2018); Table S2). The fact that we obtained reproducible results using a different protein construct indicates that swapping of helix 5 is not a crystallographic artifact, as might be inferred from an estimated dimer dissociation constant in a millimolar range (Figure 1C,D; (Hannon et al, 2018)). The swapping of helix 5 between two monomers preserves all the key structural features of the monomer, except the straightening of the hinge region between helices 4 and 5.…”

Twenty years of research on the DFS70/LEDGF autoantibody-autoantigen system: many lessons learned but still many questions

“…5). The IBD crystal structure reveals four long α-helices arranged as a helical bundle, with a fifth short helix linking two of the other α chains [62]. IDRs typically confer conformational flexibility to transcription factors, allowing them to engage in multiple transient protein-protein and protein-nucleic acid interactions that facilitate molecular events associated with transcription, DNA repair, mRNA splicing, and signal transduction [63][64][65].…”