Conformational fluctuations play a central role in enzymatic catalysis. However, it is not clear how the rates and the coordination of the motions affect the different catalytic steps. Here, we used NMR spectroscopy to analyze the conformational fluctuations of the catalytic subunit of the cAMP-dependent protein kinase (PKA-C), a ubiquitous enzyme involved in a myriad of cell signaling events. We found that the wild-type enzyme undergoes synchronous motions involving several structural elements located in the small lobe of the kinase, which is responsible for nucleotide binding and release. In contrast, a mutation (Y204A) located far from the active site desynchronizes the opening and closing of the active cleft without changing the enzyme's structure, rendering it catalytically inefficient. Since the opening and closing motions govern the rate-determining product release, we conclude that optimal and coherent conformational fluctuations are necessary for efficient turnover of protein kinases.
Bibliographical noteFunding Information:
The NMR experiments were acquired at the Minnesota NMR Center and at the University of Colorado. Many thanks to Dr. G. Armstrong for the NMR experiments carried out in Colorado, Dr. A. Kornev for assistance with visualization of the community maps, and Professor L.E. Kay and Professor D. Korznev for providing the CPMG-fit software. This work is supported by the NIH (GM 100301).