Computational design of calmodulin mutants with up to 900-fold increase in binding specificity.
Calmodulin (CaM) is a ubiquitous second messenger protein that regulates a variety of structurally and functionally diverse targets in response to changes in Ca(2+) concentration. CaM-dependent protein kinase II (CaMKII) and calcineurin (CaN) are the prominent CaM targets that play an opposing role in many cellular functions including synaptic regulation. Since CaMKII and CaN compete for the available Ca(2+)/CaM, the differential affinity of these enzymes for CaM is crucial for achieving a balance in Ca(2+) signaling. We used the computational protein design approach to modify CaM binding specificity for these two targets. Starting from the X-ray structure of CaM in complex with the CaM-binding domain of CaMKII, we optimized CaM interactions with CaMKII by introducing mutations into the CaM sequence. CaM optimization was performed with a protein design program, ORBIT, using a modified energy function that emphasized intermolecular interactions in the sequence selection procedure. Several CaM variants were experimentally constructed and tested for binding to the CaMKII and CaN peptides using the surface plasmon resonance technique. Most of our CaM mutants demonstrated small increase in affinity for the CaMKII peptide and substantial decrease in affinity for the CaN peptide compared to that of wild-type CaM. Our best CaM design exhibited an about 900-fold increase in binding specificity towards the CaMKII peptide, becoming the highest specificity switch achieved in any protein-protein interface through the computational protein design approach. Our results show that computational redesign of protein-protein interfaces becomes a reliable method for altering protein binding affinity and specificity.
Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.
Eliyahu
E
Yosef
Regina
R
Politi
Mee H
MH
Choi
Julia M
JM
Shifman
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding
D002147:Calmodulin; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D002118:Calcium