Domain Closure in Adenylate Kinase

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Abstract

The method of time-resolved dynamic nonradiative excitation energy transfer (ET) was used to analyze the proposed domain closure in adenylate kinase (AK). A highly active mutant of Escherichia coli AK, (C77S, V169W, A55C)-AK, was prepared, in which the solvent- accessible residues valine 169 and alanine 55 were replaced by tryptophan (the donor of excitation energy) and cysteine, respectively. The latter was subsequently labeled with either 5- or 4-acetamidosalicylic acid (the acceptor). From the comparative analysis of AK crystal structures [Schulz, G.E., Müller, C.W., & Diederichs, K. (1990) J. Mol. Biol. 213, 627-630] (apo-AK,AK.AMP complex and AK.AP5A [P1,P5-di(adenosine-5') pentaphosphate] complex), "sequential formation" of the pseudoternary AK.AP5A complex is followed by two- step domain closure. The domain closure reduces interdomain distances in a two-step manner. Specifically, the distance between C alpha-atoms of the residues 169 and 55 (numbers correspond to those of E. coli AK) is decreased from 23.6 A in the apo-enzyme to 16.2 A upon the formation of the AK.AMP complex and to 12.3 A upon the further formation of the pseudoternary AK.AP5A complex. Time-resolved dynamic nonradiative excitation energy transfer was measured for the following ligand forms of the labeled derivative of the mutant enzyme: the apo-enzyme, the enzyme-MgATP complex, the enzyme.AMP complex, and the enzyme.AP5A "ternary" complex. The transfer efficiencies, which were determined in these experiments, were approximately 7.5%, 22%, 33%, and 65%, respectively. Global analyses of the time resolved ET experiments with the same ligand forms yielded intermolecular distance distributions with corresponding means of 31, 23, 19, and 12 A and full widths at half- maximum of 29, 24, 14, and 11 A. The data confirmed the proposed stepwise manner of the domain closure of the enzyme and revealed the presence of multiple conformations of E. coli AK in solution.

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