Alternative views of functional protein binding epitopes obtained by combinatorial shotgun scanning mutagenesis

Abstract

Combinatorial shotgun scanning mutagenesis was used to analyze two large, related protein binding sites to assess the specificity and importance of individual side chain contributions to binding affinity. The strategy allowed for cost-effective generation of a plethora of functional data. The ease of the technology promoted comprehensive investigations, in which the classic alanine-scanning approach was expanded with two additional strategies, serine- and homolog-scanning. Binding of human growth hormone (hGH) to the hGH receptor served as the model system. The entire high affinity receptor-binding sites (site 1) of wild-type hGH (hGHwt) and of an affinity-improved variant (hGHv) were investigated and the results were compared. The contributions that 35 residue positions make to binding were assessed on each hormone molecule by both serine- and homolog-scanning. The hormone molecules were displayed on the surfaces of bacteriophage, and the 35 positions were randomized simultaneously to allow equal starting frequencies of the wild-type residue and either serine or a homologous mutation in separate libraries. Functional selections for binding to the hGH receptor shifted the relative wild-type/mutant frequencies at each position to an extent characteristic of the functional importance of the side chain. Functional epitope maps were created and compared to previous maps obtained by alanine-scanning. Comparisons between the different scans provide insights into the affinity maturation process that produced hGHv. The serine and homolog-scanning results expand upon and complement the alanine-scanning results and provide additional data on the robustness of the high affinity receptor-binding site of hGH.

Figure 1.

ΔΔG_mut-wt values measuring the effects of mutations on site 1 binding affinity for the hGHR. Data in (A) are for hGH_wt and those in (B) are for hGH_v. Data are shown for homolog (red bars), serine (white bars), or alanine (green bars) substitutions. The homolog- and serine-scanning data are from Table 2, alanine-scanning data for hGH_wt are from Cunningham and Wells (1993), and shotgun alanine-scanning data for hGH_v are from Pal et al. (2003). Asterisks (*) indicate positions for which alanine-scanning data are not available. The hGH_wt and hGH_v residues are denoted by the single-letter amino acid code followed by their position number in the sequence. The letter following the position number denotes the homolog substitution used at each position.

Figure 2.

The effects of mutations on site 1 binding affinity for the hGHR mapped onto the structure of hGH_wt (A–C) or hGH_v (D–F). The maps show the effects of substitution by homologous residues (A,D), serine (B,E), or alanine (C,F). The residues are colored according to the ΔΔG_mut-wt values shown in Table 2 and Figure 1 ▶, as follows: cyan< −0.4 kcal/mol; −0.4 kcal/mol ≤ green<0.4 kcal/mol; 0.4 kcal/ mol ≤ orange<1.0 kcal/mol; red ≥ 1.0 kcal/mol; gray, untested. The structures were derived from the 2:1 hGHR-ECD:hGH complexes for hGH_wt (de Vos et al. 1992) and hGH_v (Schiffer et al. 2002), and were rendered as molecular surfaces using Pymol (DeLano Scientific).