NEUTRON-SCATTERING STUDIES REVEAL FURTHER DETAILS OF THE CA2+ CALMODULIN-DEPENDENT ACTIVATION MECHANISM OF MYOSIN LIGHT-CHAIN KINASE/

Previously, we utilized small-angle X-ray scattering and neutron scatt ering with contrast variation to obtain the first low-resolution struc ture of 4Ca(2+). calmodulin (CaM) complexed with a functional enzyme, an enzymatically active truncation mutant of skeletal muscle myosin li ght chain kinase (MLCK). These experiments showed that, upon binding t o MLCK, CaM undergoes a conformational collapse identical to that obse rved when CaM binds to the isolated peptide corresponding to the CaM b inding sequence of MLCK. CaM thereby was shown to release the inhibiti on of the kinase by inducing a significant movement of its CaM binding and autoinhibitory sequences away from the surface of the catalytic c ore [Krueger, J. K., Olah, G. A., Rokop, S. E., Zhi, G., Stull, J. T., and Trewhella, J. (1997) Biochemistry 36, 6017-6023]. We report here similar scattering experiments on the CaM MLCK complex with the additi on of substrates; a nonhydrolyzable analogue of adenosine-triphosphate , AMPPNP, and a peptide substrate for MLCK, a phosphorylation sequence from myosin regulatory light chain (pRLC). These substrates are shown to induce an overall compaction of the complex. The separation of the centers-of-mass of the CaM and MLCK components is shortened (by simil ar to 12 Angstrom), thus bringing CaM closer to the catalytic site com pared to the complex without substrates. In addition, there appears to be a reorientation of CaM with respect to the kinase upon substrate b inding that results in interactions between the N-terminal sequence of CaM and the kinase that were not observed in the complex without subs trates. Finally, the kinase itself becomes more compact in the CaM MLC K pRLC AMPPNP complex compared to the complex without substrates. This observed compaction of MLCK upon substrate binding is similar to that arising from the closure of the catalytic cleft in cAMP-dependent pro tein kinase upon binding pseudosubstrate.