Lens-epithelium-derived-growth factor (LEDGF/p75) is an essential host protein for integration of HIV-1 DNA into host genome. Earlier alanine scanning mutational analysis has revealed that residues I365, D366 and F406 in the integrase binding domain (IBD) of p75 are critical for interaction with HIV-1 integrase (IN), while K364, V408 have intermediate effect and residues N367, L368, R405, K407 show wild type binding with IN. To gain insight into contribution of side chains of L368 and V408 that are adjacent to critical residues I365 and F406, respectively, site directed mutation of these residues to Ile/Leu, Met and Phe has been performed and characterized in this study. In contrast to alanine substitution, L368F mutation showed a ∼25% decrease, while V408L and V408F showed wild type binding, to IN. Docking analysis of I365, D366 and F406 mutants of IBD with IN predicts that interaction between residue M178(IN) and I365(IBD) might lead to an encounter complex formation. Accordingly, M178I mutant of IN failed to interact with IBD. Interestingly, a L368F/V408F double mutant of IBD restored binding to M178I mutant of IN, indicating that altered hydrophobicity in the inter helical loops of IBD might make I365 more accessible for interaction with IN. © 2017 Elsevier Inc.