Open AccessMultinuclear magnetic resonance studies of Escherichia coli adenylate kinase in free and bound forms
Author(s) -
BurlacuMiron Simona,
Gilles AnneMarie,
Popescu Aurel,
Bârzu Octavian,
Craescu Constantin T.
Publication year - 1999
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1999.00633.x
Subject(s) - antiparallel (mathematics) , crystallography , chemistry , protein secondary structure , nuclear magnetic resonance spectroscopy , stereochemistry , adenylate kinase , chemical shift , side chain , active site , beta sheet , crystal structure , protein structure , helix (gastropod) , resonance (particle physics) , enzyme , physics , biology , ecology , biochemistry , organic chemistry , particle physics , quantum mechanics , snail , magnetic field , polymer
The crystal structure of Escherichia coli adenylate kinase (AKe) revealed three main components: a CORE domain, composed of a five‐stranded parallel β‐sheet surrounded by α‐helices, and two peripheral domains involved in covering the ATP in the active site (LID) and binding of the AMP (NMP bind ). We initiated a long‐term NMR study aiming to characterize the solution structure, binding mechanism and internal dynamics of the various domains. Using single ( 15 N) and double‐labeled ( 13 C and 15 N) samples and double‐ and triple‐resonance NMR experiments we assigned 97% of the 1 H, 13 C and 15 N backbone resonances, and proton and 13 C β resonances for more than 40% of the side chains in the free protein. Analysis of a 15 N‐labeled enzyme in complex with the bi‐substrate analogue [P 1 ,P 5 ‐bis(5′‐adenosine)‐pentaphosphate] (Ap5A) resulted in the assignment of 90% of the backbone 1 H and 15 N resonances and 42% of the side chain resonances. Based on short‐range NOEs and 1 H and 13 C secondary chemical shifts, we identified the elements of secondary structure and the topology of the β‐strands in the unliganded form. The α‐helices and the β‐strands of the parallel β‐sheet in solution have the same limits (± 1 residue) as those observed in the crystal. The first helix (α1) appears to have a frayed N‐terminal side. Significant differences relative to the crystal were noticed in the LID domain, which in solution exhibits four antiparallel β‐strands. The secondary structure of the nucleoside‐bound form, as deduced from intramolecular NOEs and the 1 H α chemical shifts, is similar to that of the free enzyme. The largest chemical shift differences allowed us to map the regions of protein–ligand contacts. 1 H/ 2 H exchange experiments performed on free and Ap5A‐bound enzymes showed a general decrease of the structural flexibility in the complex which is accompanied by a local increased flexibility on the N‐side of the parallel β‐sheet.