Abstract
We determined the size and shape of full-length avian sarcoma virus (ASV) integrase (IN) monomers and dimers in solution using small angle x-ray scattering. The low resolution data obtained establish constraints for the relative arrangements of the three component domains in both forms. Domain organization within the small angle x-ray envelopes was determined by combining available atomic resolution data for individual domains with results from cross-linking coupled with mass spectrometry. The full-length dimer architecture so revealed is unequivocally different from that proposed from x-ray crystallographic analyses of two-domain fragments, in which interactions between the catalytic core domains play a prominent role. Core-core interactions are detected only in cross-linked IN tetramers and are required for concerted integration. The solution dimer is stabilized by C-terminal domain (CTD-CTD) interactions and by interactions of the N-terminal domain in one subunit with the core and CTD in the second subunit. These results suggest a pathway for formation of functional IN-DNA complexes that has not previously been considered and possible strategies for preventing such assembly.
Highlights
Retroviral integrase (IN)3 catalyzes the insertion of viral DNA into the DNA of the infected host cell
As retroviral INs are likely to share architectures for active complexes [1], we have focused our studies on avian sarcoma virus (ASV) IN [2], which is more soluble than HIV IN and, as we show here, suitable for structural analyses
We report the use of small angle x-ray scattering (SAXS) and biochemical cross-linking analyses to determine the architecture of full-length ASV IN monomers and dimers, in the absence of DNA substrates
Summary
Light Scattering Analysis—Measurements were made with a Protein Solutions DynaPro temperature-controlled microsampler. The apparent molecular mass (MW-I) was calculated from the static light scattering measurements (at least 300 acquisitions per protein sample) using the DynaPro software. Several qmax cutoff values were sampled in the range of 0.3– 0.9, with the standard final processing using a qmax of 0.4 These produced dummy atom output files that were used to generate the final envelopes with the Situs software [44]. To test for uniqueness, 10 shape reconstructions were performed with the program GASBOR for wild type ASV IN dimers and monomers (supplemental Fig. S1). The results showed a high degree of stability and convergence in the shape modeling for both monomer (normalized spatial discrepancies (NSDs) of 1.0 to 1.19) and dimer (NSDs of 1.0 to 1.34) envelopes, with ranges comparable with those reported in other SAXS analyses [45].
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