Structural Basis for the Mechanism and Substrate Specificity of Glycocyamine Kinase, a Phosphagen Kinase Family Member^,

Abstract

Glycocyamine kinase (GK), a member of the phosphagen kinase family, catalyzes the Mg2+-dependent reversible phosphoryl group transfer of the N-phosphoryl group of phospho glycocyamine to ADP to yield glycocyamine and ATP. This reaction helps to maintain the energy homeostasis of the cell in some multicelullar organisms that encounter high and variable energy turnover. GK from the marine worm Namalycastis sp. is heterodimeric, with two homologous polypeptide chains, α and β, derived from a common pre mRNA by mutually exclusive N-terminal alternative exons. The N-terminal exon of GKβ encodes a peptide that is different in sequence and is sixteen amino acids longer than that encoded by the N-terminal exon of GKα. The crystal structures of recombinant GKαβ and GKββ from Namalycastis sp. were determined at 2.6 Å and 2.4 Å resolution, respectively. In addition, the structure of the GKββ was determined at 2.3 Å resolution in complex with a transition state analog, Mg2+-ADP-NO3--glycocyamine. Consistent with the sequence homology, the GK subunits adopt the same overall fold as that of other phosphagen kinases of known structure (the homodimeric creatine kinase (CK) and the monomeric arginine kinase (AK)). As with CK, the GK N-termini mediate the dimer interface. In both heterodimeric and homodimeric GK forms, the conformations of the two N-termini are asymmetric and the asymmetry is different than that reported previously for the homodimeric CKs from several organisms. The entire polypeptide chains of GKαβ are structurally defined and the longer N-terminus of the β subunit is anchored at the dimer interface. In GKββ the 24 N-terminal residues of one subunit and 11 N-terminal residues of the second subunit are disordered. This observation is consistent with a proposal that the GKαβ amino acids involved in the interface formation were optimized once a heterodimer emerged as the physiological form of the enzyme. As a consequence, the homodimer interface (either solely α or solely β chains) has been corrupted. In the unbound state, GK exhibits an open conformation analogous to that observed with ligand-free CK or AK. Upon binding the transition state analog, both subunits of GK undergo the same closure motion that clasps the transition state analog, in contrast to the transition state analog complexes of CK, where the corresponding transition state analog occupies only one subunit, which undergoes domain closure. The active site environments of the GK, CK and AK at the bound states reveal the structural determinants of substrate specificity. Despite the equivalent binding in both active sites of the GK heterodimer, the conformational asymmetry of the N-termini is retained. Thus, the coupling between the structural asymmetry and negative cooperativity previously proposed for CK is not supported in the case of GK.

The Substrate-free and -bound Crystal Structures of the Duplicated Taurocyamine Kinase from the Human Parasite Schistosoma mansoni

“…Similar structural features were reported for a dimeric AK co-crystallized with the pretransition state analog arginine-AMPPNP, which achieved a closed liganded form in a single monomer (5). However, a dimeric GK with an asymmetric structure in the substrate-free form and a doubly closed structure in the GK-TSA complex have also recently been described (27), ruling out the hypothesis of a systematic coupling between asymmetrical unliganded dimers and catalytically interdependent monomers. In addition, several experiments performed on muscle CK did not provide any evidence for a functional asymmetry of the two identical subunits of the enzyme (30,31,36,37).…”

“…All known PK structures display a conserved topology, with an N-terminal ␣-helix bundle domain connected to a large ␣ϩ␤ C-terminal domain, as observed in the crystal structures of CKs and mitochondrial creatine kinases (9, 10, 14 -19), AKs (5, 20 -26), GK (27), and lombricine kinase (28). In the substrate-free enzyme, the active site is open and stretches as a groove on the concave face of the structure.…”

“…The C-terminal domain is mainly involved in nucleotide binding and has a consequent inward bending motion when the PK is complexed with an abortive transition state analog (TSA) consisting of the guanidino substrate, the MgADP product, and a trigonal planar nitrate anion that mimics the transferred phosphoryl group (20). The crystal structures of CK-, AK-, and GK-TSA as well as NMR and mass spectrometry studies show that this structural rearrangement is accompanied by the stabilization of a C-terminal flexible loop that shields the active site from the solvent (14,19,20,23,24,27,30,31). The PK-TSA structure is closed, and the bound compounds are locked in proper alignment for the in-line phosphoryl transfer (23,26,(32)(33)(34)).…”

The Structure of Lombricine Kinase

“…Analogously, crystals of multimeric CK have been reported with substrates bound to some subunits but not others (21,60,61). (Such asymmetry was cited as supporting negative cooperativity (61,62), but this argument has been undercut by a glycocyamine kinase structure with both subunits in the closed substrate-bound configuration (19).) Lombricine was not available, and the concentration of the alternative substrate, taurocyamine, limited by solubility to 3ϫ K m , may not have been sufficient to obtain TSAC crystals, even though LpAK has been crystallized with a wide variety of weakly binding substrate analogs (15).…”

“…One set (LpAK loop 59 -64) and Urechis caupo lombricine kinase (UcLK) loop [53][54][55][56][57][58] is located in the small N-terminal ␣-helical domain and facilitates binding of the phosphagen substrate through backbone hydrogen bonds to the carboxylate of the substrate. With loop length inversely correlated to substrate size, the mechanism of the small domain specificity loop has been rationalized in terms of lock-and-key steric hindrance (15,(17)(18)(19).…”