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Modulating calmodulin binding specificity through computational protein design

Shifman, Julia M. and Mayo, Stephen L. (2002) Modulating calmodulin binding specificity through computational protein design. Journal of Molecular Biology, 323 (3). pp. 417-423. ISSN 0022-2836. doi:10.1016/S0022-2836(02)00881-1.

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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.

Item Type:Article
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Mayo, Stephen L.0000-0002-9785-5018
Additional Information:© 2002 Elsevier Science Ltd. Received 10 April 2002; revised 13 August 2002; Accepted 16 August 2002. Edited by J. Thornton. Available online 9 October 2002. This work was supported by the Howard Hughes Medical Institute, the Ralph M. Parsons Foundation, an IBM Shared University Research Grant (to S.L.M.), an NIH postdoctoral fellowship, and the Caltech Initiative in Computational Molecular Biology, awarded by the Burroughs Wellcome Fund (to J.M.S.). We thank K. Beckingham for providing a plasmid containing WT CaM and M. Ary for assistance with the manuscript.
Funding AgencyGrant Number
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Ralph M. Parsons FoundationUNSPECIFIED
IBM Shared University Research GrantUNSPECIFIED
NIH Postdoctoral FellowshipUNSPECIFIED
Caltech Initiative in Computational Molecular BiologyUNSPECIFIED
Burroughs Wellcome FundUNSPECIFIED
Subject Keywords:Substrate Specificity; Peptides; Sequence Alignment; Protein Binding; Models: Molecular; Calmodulin; Amino Acid Sequence; Myosin-Light-Chain Kinase; Binding Sites; Mutation; Crystallography: X-Ray; Protein Conformation; Computer Simulation; Protein Structure: Tertiary; Molecular Sequence Data; protein design; binding affinity; binding specificity; protein–ligand interactions
Issue or Number:3
Record Number:CaltechAUTHORS:20110928-154513858
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Official Citation:Julia M. Shifman, Stephen L. Mayo, Modulating Calmodulin Binding Specificity through Computational Protein Design, Journal of Molecular Biology, Volume 323, Issue 3, 25 October 2002, Pages 417-423, ISSN 0022-2836, 10.1016/S0022-2836(02)00881-1.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:25479
Deposited By: Tony Diaz
Deposited On:04 Oct 2011 21:00
Last Modified:09 Nov 2021 16:33

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