Rapid mapping of protein functional epitopes by combinatorial alanine scanning

Abstract

A combinatorial alanine-scanning strategy was used to determine simultaneously the functional contributions of 19 side chains buried at the interface between human growth hormone and the extracellular domain of its receptor. A phage-displayed protein library was constructed in which the 19 side chains were preferentially allowed to vary only as the wild type or alanine. The library pool was subjected to binding selections to isolate functional clones, and DNA sequencing was used to determine the alanine/wild-type ratio at each varied position. This ratio was used to calculate the effect of each alanine substitution as a change in free energy relative to that of wild type. Only seven side chains contribute significantly to the binding interaction, and these conserved residues form a compact cluster in the human growth hormone tertiary structure. The results were in excellent agreement with free energy data previously determined by conventional alanine-scanning mutagenesis and suggest that this technology should be useful for analyzing functional epitopes in proteins.

Figures

Figure 1

Scheme for hGH shotgun scanning. The hGH gene was fused to M13 gene-8, and an hGH library was constructed with 19 mutated positions on three noncontiguous stretches of primary sequence. The library was then phage displayed, resulting in phage particles with hGH variants (blue ovals) displayed on their surface and the cognate hGH genes encapsulated within their coats. For simplicity, we show only two scanned side chains. Mutated positions initially had an approximately even distribution of wt and alanine. After selection for binding to an immobilized ligand (e.g., hGHbp shown in yellow), enrichment for wt side chains was observed for residues that contribute favorably to binding (red side chain). No selection for the wt amino acid was observed for residues that do not contribute to the binding interaction (blue side chain).

Figure 2

Shotgun scan of hGH site 1 for binding to the hGHbp. The three-dimensional structure of hGH (23) is shown in space-filling mode. The 19 shotgun-scanned residues are colored, with blue denoting ΔΔGmut-wt < 1.0 kcal/mol and red denoting ΔΔGmut-wt > 1.0 kcal/mol (Table 2). The residues shown in red constitute the functional epitope of hGH site 1. The figure was produced with grasp software (30).

Figure 3

Correlation between the change in the free energy of binding (ΔΔGmut-wt) calculated for alanine mutants relative to wt for hGH site 1 binding to hGHbp, as determined by shotgun scanning (y axis) or conventional alanine-scanning mutagenesis (x axis). Each point is labeled with the corresponding hGH residue number. The shotgun scanning data are from Table 2, whereas the alanine-scanning data are from Cunningham and Wells (7). The least squares linear fit of the data is shown, with the corresponding equation and R value given at the top left.