Department of Anatomy and Histology, The University of Sydney, NSW 2006 and *St. Vincent's Institute of Medical Research, 41 Victoria Pde. Fitzroy, Vic. 3065
Myosin light chain kinase (MLCK) is a serine/threonine kinase possessing the ability to recognize the structure around the phosphorylation site in its substrate. A synthetic peptide corresponding to chicken gizzard MLCK(774-807) has been examined. This sequence encompasses much of the regulatory domain of the enzyme including the calmodulin binding site at the C-terminal end and an N-terminal segment which mimics the sequence of the N-terminus of the phosphorylatable myosin light chain substrate. MLCK is activated by calmodulin binding which allows Ser19 on the myosin light chain to gain access to the active site on MLCK. The active site remains blocked by its own pseudosubstrate sequence in the absence of bound calmodulin.
In order to gain an understanding of the structural processes involved in this regulation of kinase activity by calmodulin, the structure of the pseudosubstrate domain was determined using NMR spectroscopy. Following the sequence-specific identification of all proton resonances and several proton coupling constants, the proton-proton distance and angle constraints were used in the distance geometry algorithm DIANA II to generate an ensemble of structures in solution. These were refined afterwards using a dynamic simulated annealing protocol to provide low energy solution structures. The average structure reveals that the connecting peptide comprising Leu774-Lys785 exhibits mostly a helical conformation (Asp777-Lys785) while the central portion of the pseudosubstrate segment extending from Arg790-Trp800 also forms [[alpha]]-helix. The segment connecting these regions formed by Leu786-Ser787 (corresponding to Ser1 in the myosin light chain substrate)-Lys788-Asp789 is a well-defined turn which may be involved in allowing rotation of the pseudosubstrate away from the active site following binding of calmodulin.