Calmodulin (CaM) is a ubiquitous second messenger protein that regulates a variety of structurally and functionally diverse targets in response to changes in Ca2+ 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 Ca2+/CaM, the differential affinity of these enzymes for CaM is crucial for achieving a balance in Ca2+ 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.
Bibliographical noteFunding Information:
This work was supported by the Lejwa Foundation in Biochemistry (J.M.S.) and National Institutes of Health grant K25NS047300 (M.H.C). E.Y. acknowledges the scholarship from the Dan scholarship fund. We would like to thank A. Rabinkov for help with initial SPR experiments. We are grateful to the Bio-Rad Haifa team, especially to M. Dines, S. Nimri, S. Choen, V. Bronner, and M. Tabul for their assistance with SPR experiments performed on the ProteOn XPR36 array system. We also thank M. Fromer for plotting the HSSP logo in Table 1 . In addition, we are grateful to G. Schreiber for allowing us to use equipment in his lab and for productive scientific discussions.
- computational protein design
- protein binding specificity
- protein-protein interactions