Biochemical and Biophysical Research Communications
Crystal structures of arginine kinase in complex with ADP, nitrate, and various phosphagen analogs
Highlights
► X-ray structures of arginine kinase bound to four phosphagen analogs are presented. ► The structures are of the transition state analog complex, with ADP and nitrate. ► The protein conformation with analogs bound is similar to the cognate complex. ► Analog configurations, however, differ from that of bound l-arginine. ► Substrate binding synergy may have an inter-substrate component.
Introduction
Arginine kinase, a homolog of the human enzyme creatine kinase, is a member of the phosphagen kinase family of enzymes which buffer cellular ATP levels in cells with high and/or fluctuating energy demands. To this end, these enzymes catalyze reversible phosphoryl transfer between ATP and their respective phosphagens, substrates with guanidinium groups to and from which the phosphoryl group is transferred. Through these enzyme-catalyzed reactions, cells are capable of maintaining a constant ATP/ADP ratio, thus retaining a high free energy of ATP hydrolysis which can be used to fuel a number of cellular processes [1].
Phosphagen substrates vary by organism, but all have a guanidino group that is reversibly phosphorylated. Crystal structures for several homologous phosphagen kinases have been determined, including arginine kinase (AK), creatine kinase (CK), glycocyamine kinase (GK), and lombricine kinase (LK) in substrate-free “open” forms [2], [3], [4], [5], [6], [7]. AK, CK and GK structures have also been determined as transition state analog (TSA) complexes, composed of the phosphagen, MgADP, and nitrate [2], [4], [8], [9], [10]. In these closed-conformation complexes, the planar nitrate group mimics the phosphoryl group being transferred. Comparison of open- and closed-form structures reveals a conserved set of conformational changes on substrate binding that may mediate substrate alignment, position catalytically important residues, and/or exclude bulk solvent from the enzyme’s active site, potentially preventing the wasteful hydrolysis of ATP [8], [11], [12], [13]. NMR has demonstrated that a subset of the conformational changes observed crystallographically occur intrinsically in substrate-free arginine kinase and that they are likely turnover-limiting [14], [15], [16].
The structure of the arginine kinase TSA complex, solved at a resolution of 1.2 Å, provides a precise and unperturbed view of substrate alignment [9]. Substrates were aligned within 4 degrees of optimal for in-line phosphoryl transfer [9], [10]. Two glutamates, E225 and E314, were shown to interact with the substrate guanidinium. Mutation of these glutamates reduces activity to 0.5–1.7% of wild type (wt), respectively, but does not eliminate activity. Structures of E225Q and E314D mutants as TSA complexes showed subtle distortions in the precise alignment of substrates [17]. The TSA complex structure of another active site mutant, C271A, exhibits 15° distortions of the phosphagen and nucleotide attack angles, although loss of catalysis has been thought to be an electrostatic/stereoelectronic effect of losing a thiolate [18]. The structure of the non-cognate complex of wtAK with ADP and creatine, a phosphagen for which AK has no detectable activity, suggests that the lack of activity may result from AK’s inability to position precisely this smaller phosphagen [19]. Taken together, a number of observations can be interpreted to indicate that substrate alignment plays an important role in rate enhancement and/or substrate specificity.
The role of substrate alignment is further investigated here through structural studies of AK with a set of phosphagen analogs. Crystal structures are determined for arginine kinase from the Atlantic horseshoe crab Limulus polyphemus with one of four phosphagen analogs bound in a TSA-like complex with nitrate and MgADP. No enzymatic activity is observed for three of the analogs: l-ornithine, imino-l-ornithine, and l-citrulline; and trace activity is detected for d-arginine. The enzyme structures are very similar to the native TSA complex, but significant distortions are observed in substrate configurations [9], [10]. The crystal structures show the non-cognate substrate analogs bound at high occupancy, i.e. they are not excluded in a lock-and-key fashion, so that it appears that activity and substrate specificity are mediated through appropriate substrate alignment.
Section snippets
Protein expression, purification, and crystallization
Limulus polyphemus arginine kinase was expressed and purified as previously described [20]. Initial crystals were grown at room temperature by hanging drop vapor diffusion. Purified arginine kinase at 30 mg/ml was mixed with substrate analog and crystallant solutions in 4:4:1 ratio. The crystallant solution contained 26% (w/v) PEG 6000, 50 mM HEPES, and 100 mM MgCl2 at a pH of 8.0. The substrate analog solution contained 25 mM MgCl2, 20 mM K–ADP, 250 mM NaNO3, 25 mM NaN3, 5 mM DTT, and phosphagen
Crystallographic structure determination
Crystal structures were successfully determined for four complexes of arginine kinase bound each of the phosphagen analogs l-citrulline, l-ornithine, iminoethyl-l-ornithine, and d-arginine. Crystallographic and refinement statistics are shown in Table 1. The unit cells of all phosphagen analog complexes except d-arginine are very similar to that of the transition state analog (TSA) complex of arginine kinase [9], [10]. In fact, molecular replacement was only necessary for the iminoethyl-l
Acknowledgments
T. Somasundaram is thanked for his help in X-ray data collection and processing, as is W. Ross Ellington for helpful discussions. The work was supported in part by NIH R01 GM077643 (MSC).
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