Substrate‐induced double sided H‐bond network as a means of domain closure in 3‐phosphoglycerate kinase

Abstract

Closure of the two domains of 3-phosphoglycerate kinase, upon substrate binding, is essential for the enzyme function. The available crystal structures cannot provide sufficient information about the mechanism of substrate assisted domain closure and about the requirement of only one or both substrates, since lattice forces may hinder the large scale domain movements. In this study the known X-ray data, obtained for the open and closed conformations, were probed by solution small-angle Xray scattering experiments. The results prove that binding of both substrates is essential for domain closure. Molecular graphical analysis, indeed, reveals formation of a double-sided H-bond network, which affects substantially the shape of the main molecular hinge at b-strand L, under the concerted action of both substrates.

A Spring-loaded Release Mechanism Regulates Domain Movement and Catalysis in Phosphoglycerate Kinase

“…Without the constraints of a crystal lattice, the protein thus adopts its fully open resting state. Scattering curves of the binary complexes, with either 3PG or ADP alone, show an R g close to that of the apoprotein implying a requirement for both substrates to bind before domain closure, in agreement with previous suggestions (11,19). Scattering curves obtained from the AlF 4 Ϫ -TSA complex have a similar R g to the crystal structure of the same complex, and ab initio modeling confirms a more closed conformation of this complex (supplemental Fig.…”

“…1). Although similar experiments have been performed previously (18,19), the experiments described here benefit from the ability to trap the fully closed conformation in solution by inhibition with aluminum fluoride (the ADP-AlF 4 Ϫ -3PG complex) and compare the curves to the crystal structure of this complex and to the various open forms from the same species. In this respect, the study provides a full explanation of domain movement during catalysis.…”

“…Therefore, the closed conformation is "primed" on substrate binding. Substrate binding may also assist in the interdomain communication and domain closure by the aid of well described interaction networks (17)(18)(19) and acts in concert with the internal dynamics of the protein. Therefore, as soon as the domains come together, the salt bridges are formed, and the closed conformation will be stabilized sufficiently for catalysis to occur.…”

“…In the closed conformation of PGK, the two domains reorient by 33°, compared with the open form, about central hinge regions to bring the substrates together. Much effort has been put into elucidating the mechanism of this hinge bending and the role that substrate binding plays in inducing the conformational changes needed for catalysis to occur (15)(16)(17)(18)(19).…”

Expression, purification, crystallization and preliminary X-ray diffraction studies of phosphoglycerate kinase from methicillin-resistantStaphylococcus aureusMRSA252

“…There is also evidence of a novel mechanism in which a synergistic combination of substrate-induced effects produces major conformational changes in the enzyme (Bernstein et al, 1997). Some of the structures of PGK solved to date by X-ray crystallography and solution small-angle X-ray scattering have provided new insights into the role of the nucleotide in domain closure (Szilá gyi et al, 2001), the hydrogen-bonding network (Varga et al, 2006) and the different interdomain interactions, together with the residues involved in the stabilization of the transition state of the phosphoryl group (Auerbach et al, 1997). Comparison of the enzyme structure in the open and closed conformations between different species reveals that conformational change reflects a change in the interactions between the different domains (Lee et al, 2006).…”