TY - JOUR
T1 - Arginine kinase
T2 - Joint crystallographic and NMR RDC analyses link substrate-associated motions to intrinsic flexibility
AU - Niu, Xiaogang
AU - Bruschweiler-Li, Lei
AU - Davulcu, Omar
AU - Skalicky, Jack J.
AU - Brüschweiler, Rafael
AU - Chapman, Michael S.
N1 - Funding Information:
Crystallographic data were collected with the assistance of Mohammad Yousef, Shawn Clark, and T. Somasundaram. We thank Dr. Charles. D. Schwieters for help with using Xplor-NIH. This work was supported by National Institutes of Health–National Institute of General Medical Sciences grant R01GM077643 (M.S.C.). The NMR RDC experiments were conducted at the National High Magnetic Field Laboratory supported by cooperative agreement DMR 0654118 between the National Science Foundation and the State of Florida.
PY - 2011/1/14
Y1 - 2011/1/14
N2 - The phosphagen kinase family, including creatine and arginine kinases (AKs), catalyzes the reversible transfer of a "high-energy" phosphate between ATP and a phosphoguanidino substrate. They have become a model for the study of both substrate-induced conformational change and intrinsic protein dynamics. Prior crystallographic studies indicated large substrate-induced domain rotations, but differences among a recent set of AK structures were interpreted as a plastic deformation. Here, the structure of Limulus substrate-free AK is refined against high-resolution crystallographic data and compared quantitatively with NMR chemical shifts and residual dipolar couplings (RDCs). This demonstrates the feasibility of this type of RDC analysis of proteins that are large by NMR standards (42 kDa) and illuminates the solution structure, free from crystal-packing constraints. Detailed comparison of the 1.7 Å resolution substrate-free crystal structure against the 1.7 Å transition-state analog complex shows large substrate-induced domain motions that can be broken down into movements of smaller quasi-rigid bodies. The solution-state structure of substrate-free AK is most consistent with an equilibrium of substrate-free and substrate-bound structures, with the substrate-free form dominating, but with varying displacements of the quasi-rigid groups. Rigid-group rotations evident from the crystal structures are about axes previously associated with intrinsic millisecond dynamics using NMR relaxation dispersion. Thus, "substrate-induced" motions are along modes that are intrinsically flexible in the substrate-free enzyme and likely involve some degree of conformational selection.
AB - The phosphagen kinase family, including creatine and arginine kinases (AKs), catalyzes the reversible transfer of a "high-energy" phosphate between ATP and a phosphoguanidino substrate. They have become a model for the study of both substrate-induced conformational change and intrinsic protein dynamics. Prior crystallographic studies indicated large substrate-induced domain rotations, but differences among a recent set of AK structures were interpreted as a plastic deformation. Here, the structure of Limulus substrate-free AK is refined against high-resolution crystallographic data and compared quantitatively with NMR chemical shifts and residual dipolar couplings (RDCs). This demonstrates the feasibility of this type of RDC analysis of proteins that are large by NMR standards (42 kDa) and illuminates the solution structure, free from crystal-packing constraints. Detailed comparison of the 1.7 Å resolution substrate-free crystal structure against the 1.7 Å transition-state analog complex shows large substrate-induced domain motions that can be broken down into movements of smaller quasi-rigid bodies. The solution-state structure of substrate-free AK is most consistent with an equilibrium of substrate-free and substrate-bound structures, with the substrate-free form dominating, but with varying displacements of the quasi-rigid groups. Rigid-group rotations evident from the crystal structures are about axes previously associated with intrinsic millisecond dynamics using NMR relaxation dispersion. Thus, "substrate-induced" motions are along modes that are intrinsically flexible in the substrate-free enzyme and likely involve some degree of conformational selection.
KW - conformational change
KW - conformational selection
KW - induced fit
KW - protein dynamics
KW - residual dipolar coupling
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U2 - 10.1016/j.jmb.2010.11.007
DO - 10.1016/j.jmb.2010.11.007
M3 - Article
C2 - 21075117
AN - SCOPUS:78650909033
SN - 0022-2836
VL - 405
SP - 479
EP - 496
JO - Journal of molecular biology
JF - Journal of molecular biology
IS - 2
ER -