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We present a high-resolution mass spectrometric (MS) footprinting method enabling identification of contact amino acids in protein–protein complexes. The method is based on comparing surface topologies of a free protein versus its complex with the binding partner using differential accessibility of small chemical group selective modifying reagents. Subsequent MS analysis reveals the individual amino acids selectively shielded from modification in the protein–protein complex. The current report focuses on probing interactions between full-length HIV-1 integrase and its principal cellular partner lens epithelium-derived growth factor. This method has a generic application and is particularly attractive for studying large protein–protein interactions that are less amenable for crystallographic or NMR analysis.
Protein–protein interactions established by HIV-1 integrase (IN) within the preintegration complex (PIC) are essential for productive integration of the viral cDNA into human chromatin. Dimeric and tetrameric forms of IN have been implicated in promoting its two catalytic activities, 3′ processing and strand transfer, respectively [1–4]. IN is comprised of three-domains, the N-terminal domain, catalytic core domain (CCD), and C-terminal domain. While the atomic structures of the individual domains [5–9] and two-domain fragments [10,11] are available, the intra- and inter-protein interfaces formed by full-length multimeric IN within the catalytic complexes are for the most part unknown. Limited protein solubility at relatively low ionic strength, as well as an inherent flexibility of the three-domain protein, has likely contributed to the inability to solve the structure of the full-length HIV-1 enzyme.
Here, we present a mass spectrometric (MS) footprinting method that allows to probe in detail interactions between full-length proteins within complexes that may resist rigorous structural analysis like X-ray crystallography or nuclear magnetic spectroscopy (NMR). The application of this approach for studying nucleic acid–protein interactions consistently revealed biologically essential contact amino acid residues [12–15] and, as documented here, the method can be readily extended to examine protein–protein interfaces. MS footprinting provides a tool to build upon available high-resolution structures of protein subdomains as “pieces of the puzzle” to assemble a plausible molecular model for full-length protein–protein complexes. By example, we provide details of the analysis of HIV-1 IN with its principal cellular binding partner lens epithelium-derived growth factor (LEDGF), which is critically important for effective integration [16–21]. The solution structure of the IN binding domain (IBD) of LEDGF and its co-crystallization with the IN CCD have been reported [22,23]. However, mutagenesis studies had indicated that contacts contributing to the high affinity interaction between full-length IN and LEDGF extended beyond the inter-protein interfaces observed in the co-crystal structure of isolated domains . The MS-based approach detailed here enabled us to reveal novel inter- and intra-protein contacts important for effective formation of the IN–LEDGF complex .
The experimental strategy is depicted in Fig. 1. The method exploits differential accessibility of small chemical modifying reagents in free protein versus the protein–protein complex. To identify IN residues essential for high affinity binding with LEDGF, free IN and the IN–LEDGF complex are subjected to treatment by small covalent modifiers. Surface residues in free IN and the complex are susceptible to modification, while the interacting amino acids in the complex are shielded from modification. The concentrations of the modifying reagents are carefully optimized in preliminary experiments to ensure mild modification conditions, under which the integrity of the protein–protein complex is preserved. To recover only the LEDGF bound form of IN from the reaction mixture, we use His-tag LEDGF and tag-free IN proteins. Following the modification reactions, the IN–LEDGF complex and free LEDGF are recovered from the reaction mixture using Ni-nitrilotriacetic acid (Ni-NTA) beads (GE Healthcare). The separate control reaction, which interrogates IN protein in the absence of LEDGF, proceeds without the affinity purification step. Following separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE), the IN bands are excised and subjected to in-gel proteolysis to generate peptide fragments amenable for MS experiments. Subsequent MS analyses are conducted to compare the modification patterns of IN in its free form and in the complex with LEDGF. The modified peaks persisting in both free IN and the complex indicate accessible, surface-exposed residues, while the modified peaks observed in free IN but effectively diminished in the complex reveal the amino acids specifically shielded via the protein–protein interaction (Fig. 1).
From this step forward, treat all samples including free IN and IN–LEDGF complexes identically.
Protein-protein interactions play a vital role in numerous biological processes. Like HIV-1 IN, high-resolution structural data are available for many individual protein domains, while biologically-relevant large multi-protein complexes may be less amenable to crystallographic and NMR analysis. Our MS footprinting approach, which has unveiled important protein–protein interactions that occur during HIV-1 integration , can be readily adopted for other protein-based interaction systems. Our methodology importantly allows the examination of protein–protein interactions using limited amounts of starting materials. While amine buffers such as Tris should be avoided in the reactions with NHS-biotin, the modification buffers could include any of a wide variety of components that contribute to stability and solubility of large protein–protein complexes. Preliminary functional analyses should precede the footprinting experiments to determine the optimal modification conditions under which the integrity of the complexes is preserved. The above Lys and Arg footprinting could be augmented by probing other residues. For example, commercial reagents are available for surface topology analysis of Cys, His, Tyr, and Trp residues .
This study has been supported by NIH grants AI062520, AI077341 and CA100730 (to M.K.).