NMR Dynamics Investigation of Ligand-Induced Changes of Main and Side-Chain Arginine N−H’s in Human Phosphomevalonate Kinase

Abstract

Studies of the dynamical properties of proteins using NMR is an emerging field, which has largely been limited to backbone N-H or sidechain methyl motions. It is often, but not always true that changes to dynamic state of the backbone will reflect changes in the sidechains. The NMR techniques for quantifying fast timescale motion involve measuring the longitudinal (R 1 ) and transverse (R 2 ) relaxation rates, as well as the heteronuclear NOE for each amino acid backbone N-H (or sidechain C-H bond vector) in the protein. 1-3 , 10 These values can then be used to calculate the generalized order parameter (S 2 ), which is a measure of protein flexibility.We have recently reported on the ligand induced structural and dynamical changes of human phosphomevalonate kinase (PMK).4 PMK is the fifth enzyme in the mevalonate pathway in humans, and is involved in steroid biosynthesis.5 , 6 PMK catalyzes phosphoryl transfer from ATP to mevalonate 5-phosphate (M5P) to form ADP and mevalonate 5-diphosphate. To permit phosphoryl transfer, the substrates are brought close together, resulting in a significant and repulsive buildup of negative charge. To facilitate this difficult task, PMK contains 17 arginines ( Figure 1) and 8 lysines, with many in the active site to help neutralize the negative charge on the phosphates. The most important arginines, based on site-directed mutagenesis studies, are R18, R48, R73, R84, R110, R111, R141. 7,8 Here we describe the use of NMR dynamics methods to characterize changes to the mobility of arginine sidechains upon ligand binding. NMR dynamics methods, recently applied to arginine sidechains, 10-13 allow study of the role that arginine sidechains play in ligand binding and catalysis. Our studies provide surprising insights into ligand effects on arginine sidechains that are remote from the active site, perhaps due to long-range coulombic attraction.daniel.sem@mu.edu. Supporting Information Available: Detailed procedures and additional NMR spectra. This material is available free of charge via the Internet at http://pubs.acs.org Phosphomevalonate kinase (PMK) catalyzes phosphoryl transfer from ATP to mevalonate 5-phosphate (M5P), on the pathway to synthesizing cholesterol and other isoprenoids. To permit this reaction, its substrates must be brought proximal, which would result in a significant and repulsive buildup of negative charge. To facilitate this difficult task, PMK contains 17 arginines and 8 lysines. But, how this charge neutralization and binding is achieved, from a structural and dynamics perspective, is not known. More broadly, the role of arginine sidechain dynamics in binding charged substrates has not been experimentally defined for any protein, to date. Herein we report a characterization of changes to the dynamical state of the arginine sidechains in PMK, due to binding its highly charged substrates, ATP and M5P. These studies have been facilitated by the use of arginine-selective labeling, to eliminate spectral overlap. Modelfree analysis indicates that while substrate bi...

Enzymes of the mevalonate pathway of isoprenoid biosynthesis

“…R48M, R111M, R130M, R141M) has facilitated NMR assignments of arginine side chains. Dynamics studies on wild type PMK and these PMK arginine mutants [82] indicate that substrate binding to PMK correlates with a transition from a flexible to a more rigid protein. The observation extends to the arginine side chains, including several that are somewhat remote from the active site.…”

Mapping temperature-dependent conformational change in the voltage-sensing domain of an engineered heat-activated K + channel

“…We use established NMR methods for specific detection of the -N e H e resonance of the arginine side chains (32), which fall in a unique region of 1 H-15 N TROSY-HSQC spectra (Fig. 2).…”

Rapid identification of protein–protein interfaces for the construction of a complex model based on multiple unassigned signals by using time-sharing NMR measurements

“…Specific stable isotope labeling with amino acids has been utilized in various aspects of biomolecular NMR analyses (Crespi et al, 1968;Kameda et al, 2009;Markley et al, 1968;Muchmore et al, 1989;Olson et al, 2010;Staunton et al, 2006;Tugarinov et al, 2006). In this study, in order to obtain the information about several amino acid types simultaneously, we used a unique combination of a single 15 N-labeled amino acid and several amino acids 13 C-labeled on specific atoms.…”