WHAT IS THE STRUCTURAL BASIS FOR THE COUPLING OF Ca2+ AND PEPTIDE BINDING BY CALMODULIN? S. Weigand, S. Mirzoeva, L. Shuvalova, T. Lukas, D.M. Watterson, W.F. Anderson. Mol. Pharm. & Biol. Chem., Northwestern University Medical School, Chicago, IL 60611, USA
The goal of this research is to determine the structural basis of the observed enhancement of Ca2+ binding by calmodulin (CaM) in the presence of CaM binding structures. Calmodulin is a Ca2+ modulated signal transducing protein that activates many important eukaryotic enzymes. It is known that in the presence of CaM binding structures, the Ca2+ binding affinity of CaM is enhanced (Haiech, J., et al. 1991. J. Biol. Chem. 266: 3427-31). The structural basis of this enhancement is not known. Glutamate 84 of CaM appears to be an interface contact residue in a CaM:peptide complex (Meador, W.E., et al. 1992. Science 257: 1251-5). A charge reversal mutant of CaM, E84K-CaM, has altered biological activity, but still binds peptide. However, initial data indicate that the Ca2+ binding activity of the E84K-CaM:peptide complex is diminished.
In order to address the structural basis for the Ca2+ binding enhancement, it is important to compare high resolution structures (< 2.0 Å) of CaM and E84K-CaM in different binding states. Suitable resolution structures exist of the uncomplexed CaM (Chattopadhyaya, R., et al 1992. J. Mol. Biol. 288: 1177-92, Ban, C., et al. 1993. PDB:1osa). However, a high resolution structure of CaM complexed with RS20, a peptide corresponding to the CaM recognition sequence of smooth muscle/non-muscle myosin light chain kinase, is needed. Also, the structure of E84K-CaM:RS20 complex has not yet been determined. We have collected high resolution data, and are determining the structures of both the wild type CaM:RS20 complex (1.73 Å) and the E84K-CaM:RS20 complex (1.90 Å). Once these structures are determined, we believe they will offer insight into the basis for the coupling of CaM's Ca2+ binding and peptide binding activities.
(Supported in part by NIH grants GM30861 and T32-GM08320.)