Modulating calmodulin binding specificity through computational protein design

Abstract

We report the computational redesign of the protein-binding interface of calmodulin (CaM), a small, ubiquitous Ca^(2+)-binding protein that is known to bind to and regulate a variety of functionally and structurally diverse proteins. The CaM binding interface was optimized to improve binding specificity towards one of its natural targets, smooth muscle myosin light chain kinase (smMLCK). The optimization was performed using optimization of rotamers by iterative techniques (ORBIT), a protein design program that utilizes a physically based force-field and the Dead-End Elimination theorem to compute sequences that are optimal for a given protein scaffold. Starting from the structure of the CaM-smMLCK complex, the program considered 10^(22) amino acid residue sequences to obtain the lowest-energy CaM sequence. The resulting eightfold mutant, CaM_8, was constructed and tested for binding to a set of seven CaM target peptides. CaM_8 displayed high binding affinity to the smMLCK peptide (1.3nM), similar to that of the wild-type protein (1.8 nM). The affinity of CaM_8 to six other target peptides was reduced, as intended, by 1.5-fold to 86-fold. Hence, CaM_8 exhibited increased binding specificity, preferring the smMLCK peptide to the other targets. Studies of this type may increase our understanding of the origins of binding specificity in protein-ligand complexes and may provide valuable information that can be used in the design of novel protein receptors and/or ligands.

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