Hydrophile scanning as a complement to alanine scanning for exploring and manipulating protein-protein recognition: application to the Bim BH3 domain

Abstract

Alanine scanning has been widely employed as a method of identifying side chains that play important roles in protein-protein and protein-peptide interactions. Here we show how an analogous and complementary technique, hydrophile scanning, can provide additional insight on such interactions. Mutation of a wild-type residue to alanine removes most of the side-chain atoms, and the effect of this removal is typically interpreted to indicate contribution of the deleted side chain to the stability of the complex. Hydrophile scanning involves systematic mutation of wild-type residues to a cationic or anionic residue (lysine or glutamic acid, in this case). We find that the results of these mutations provide insights on interactions between polypeptide surfaces that are complementary to the information obtained via alanine scanning. We have applied this technique to a peptide that corresponds to the BH3 domain of the pro-apoptotic protein Bim. The wild-type Bim BH3 domain binds strongly to the anti-apoptotic proteins Bcl-x(L) and Mcl-1. Combining information from the alanine, lysine, and glutamic acid scans has enabled us to identify Bim BH3 domain mutants containing only two or three sequence changes that bind very selectively either to Bcl-x(L) or Mcl-1. Our findings suggest that hydrophile scanning may prove to be a broadly useful tool for revealing sources of protein-protein recognition and for engineering selectivity into natural sequences.

Figure 1.

(A–C) Normalized binding of the Bim (86-103) and mutant peptides to Mcl-1 and Bcl-x_L plotted on an inverse and logarithmic scale. Normalization consists of division of the K_i of the mutant by the K_i of the wild-type Bim (86-103) sequence. Dashed line signifies the K_i of the wild-type sequence. Broken bar indicates binding below the detectable limit. “*” Denotes aggregation of peptide as evidenced in assay; “X” indicates mutation not made at this residue due to similarity to the wild-type residue; “+” equals K_i(mutant)/K_i (Bim 86-103) > 100.

Figure 2.

(A,B) Inhibition constant (K_i) of the Bim (86-103) (white bar) and mutant peptides to Mcl-1 and Bcl-x_L plotted on an inverse and logarithmic scale. Dashed line signifies the K_i of the wild-type sequence. Broken bar indicates binding below the detectable limit. “*” Denotes aggregation of peptide as evidenced in assay; “+” indicates K_i ≥ 10,000 nM. Absence of symbol or bar indicates that the mutation was not made at this residue due to similarity to the wild-type residue.

Figure 3.

Circular dichroism of Bim^BH3 and mutant peptides in 10 mM sodium phosphate buffer (pH 7.0). Bim (86-103) is shown in white squares, peptide 1 is shown in gray triangles, and peptide 2 is shown in crosses.