Identification of Phe187 as a Crucial Dimerization Determinant Facilitates Crystallization of a Monomeric Retroviral Integrase Core Domain

Abstract

Retroviral DNA integration into the host genome is mediated by nucleoprotein assemblies containing tetramers of viral integrase (IN). Whereas the fully active form of IN comprises a dimer of dimers, the molecular basis of IN multimerization has not been fully characterized. IN has consistently been crystallized in an analogous dimeric form in all crystallographic structures and experimental evidence as to the level of similarity between IN monomeric and dimeric conformations is missing because of the lack of IN monomeric structures. Here we identify Phe187 as a critical dimerization determinant of IN from feline immunodeficiency virus (FIV), a nonprimate lentivirus that causes AIDS in the natural host, and report, in addition to a canonical dimeric structure of the FIV IN core-domain, a monomeric structure revealing the preservation of the backbone structure between the two multimeric forms and suggest a role for Phe187 in "hinging" the flexible IN dimer.

The Preserved HTH-Docking Cleft of HIV-1 Integrase Is Functionally Critical

“…NTD bridging of the two protomers, within the canonical dimer, can potentially stabilize IN dimers during multimer assembly and reorganization upon DNA binding. We previously proposed that the hydrophobic core at the C-terminal tip of CCD α-5 (around F185 of HIV-1) is delicately tuned for optimal flexibility hinging the two protomers together during IN rearrangements, and a single Phe to Lys mutation at this hinge rendered IN of feline-lentivirus (FIV) monomeric (Galilee and Alian, 2014). …”

“…We exploited the distinct multimerization features, tetrameric for HIV-1 IN and dimeric for the FIV IN (Galilee and Alian, 2014), to assess the effects of domain swapping on IN multimerization using size exclusion chromatography. Swapping CTD (H-H-F, CTD of FIV), NTD (F-H-H, NTD of FIV) or both domains (F-H-F, NTD and CTD of FIV) rendered HIV-1 IN chimeras dimeric, an intrinsic feature of the FIV IN (Galilee and Alian, 2014).…”

“…We exploited the distinct multimerization features, tetrameric for HIV-1 IN and dimeric for the FIV IN (Galilee and Alian, 2014), to assess the effects of domain swapping on IN multimerization using size exclusion chromatography. Swapping CTD (H-H-F, CTD of FIV), NTD (F-H-H, NTD of FIV) or both domains (F-H-F, NTD and CTD of FIV) rendered HIV-1 IN chimeras dimeric, an intrinsic feature of the FIV IN (Galilee and Alian, 2014). Interestingly, while chimeric FIV IN containing either HIV-1 IN CTD (F-F-H) or NTD (H-F-F) remained dimeric, swapping both CTD and NTD (H-F-H) rendered FIV IN chimera tetrameric (Figure 2C, arrow) especially noticeable at higher protein concentrations (Figure 2D).…”

“…Single-site integration and 3′-processing activities of IN were assessed (1 hr in 37°C) using the previously described in vitro fluorescence assay (Merkel et al, 2009). We previously reported the inefficient in vitro strand-transfer activity of recombinant FIV IN (Galilee and Alian, 2014). …”

The Road Less Traveled: HIV's Use of Alternative Routes through Cellular Pathways

“…These intrinsic species-specific pathway preferences, which apparently hinge on variations as subtle as singleresidue mutations, emphasize the power of cross-species analysis in highlighting potentially accessible alternative routes awaiting HIV-1 exploitation. We have recently shown that recombinant FIV integrase (IN), unlike HIV-1, lacks in vitro integration activity and that a single residue mutation in the typically dimeric FIV integrase results in monomerization (25). Further characterization of these distinctions may highlight additional critical requirements for integration in FIV or bring to light divergent cellular routes accessible to monomeric integrase.…”