Guanylate kinase (GMPK) is a nucleoside monophosphate kinase that catalyzes the reversible phosphoryl transfer from ATP to GMP to yield ADP and GDP. In addition to phosphorylating GMP, antiviral prodrugs such as acyclovir, ganciclovir, and carbovir and anticancer prodrugs such as the thiopurines are dependent on GMPK for their activation. Hence, structural information on mammalian GMPK could play a role in the design of improved antiviral and antineoplastic agents. Here we present the structure of the mouse enzyme in an abortive complex with the nucleotides ADP and GMP, refined at 2.1 Å resolution with a final crystallographic R factor of 0.19 (R free ‫؍‬ 0.23). Guanylate kinase is a member of the nucleoside monophosphate (NMP) kinase family, a family of enzymes that despite having a low primary structure identity share a similar fold, which consists of three structurally distinct regions termed the CORE, LID, and NMP-binding regions. Previous studies on the yeast enzyme have shown that these parts move as rigid bodies upon substrate binding. It has been proposed that consecutive binding of substrates leads to "closing" of the active site bringing the NMP-binding and LID regions closer to each other and to the CORE region. Our structure, which is the first of any guanylate kinase with both substrates bound, supports this hypothesis. It also reveals the binding site of ATP and implicates arginines 44, 137, and 148 (in addition to the invariant P-loop lysine) as candidates for catalyzing the chemical step of the phosphoryl transfer.Guanylate kinase (GMPK, 1 ATP:GMP phosphotransferase, EC 2.7.4.8) is a critical enzyme for the biosynthesis of GTP and dGTP by catalyzing the phosphoryl transfer from ATP to (d)GMP resulting in ADP and (d)GDP (1, 2). GMPK also plays an important role in the recycling of the second messenger cGMP (3). In addition to these physiological roles, GMPK is essential for the activation of prodrugs used for the treatment of cancers and viral infections (4, 5). Therefore, it is medically important to elucidate its enzymatic mechanism and the structural basis for its nucleotide specificity. Our current structural understanding of this enzyme is derived from the apo-and GMP-bound structures of the yeast GMPK (6) and from analogy to other nucleoside monophosphate (NMP) kinases (7).It has been shown that the induced fit mechanism (8) plays an important role in NMP kinases, of which adenylate kinase is the best characterized (9 -11). NMP kinases catalyze phosphoryl transfer by binding both donor and acceptor nucleotides to form a ternary complex. Comparison of the crystal structures of nucleotide-free adenylate kinase to the one in which a single substrate is bound (AMP or ATP) and to the complex in which both substrates are present revealed the conformational changes that occur along the reaction coordinate: from an open unbound enzyme via a partially closed intermediate in which a single substrate is present to the fully closed form in the presence of both substrates. These substrate-induced conform...

SAP97 is a membrane cytoskeletal protein localized at the presynaptic nerve terminals of type 1 asymmetric synapses. It has been implicated in the assembly of synapses and in particular in the localization and clustering of ion channels. The C-terminal GK domain of SAP97 shares a high degree of sequence similarity with low-molecular-mass guanylate kinases. These enzymes are involved in the guanine nucleotide metabolic cycle and in the maintenance of GTP/GDP pools required for example in Ras-mediated cell signaling. It has therefore been hypothesized that SAP97 plays an essential role in cellular signaling by regulating the guanine nucleotide pools at synaptic junctions. Here, we test this hypothesis by assessing whether the GK domain in SAP97 encodes an authentic guanylate kinase. We show that the GK domain in and of itself does not encode an active guanylate kinase, that it cannot be activated by its binding partner GKAP and that flanking regions are not acting as inhibitory regulators for enzymatic activity. Thus, it would appear that the GK domain of SAP97 is not involved in the metabolism of guanine nucleotides required for signaling events.

CASK is a member of the membrane-associated guanylate kinases (MAGUK) homologs, a family of proteins that scaffold protein complexes at particular regions of the plasma membrane by utilizing multiple proteinbinding domains. The GK domain of MAGUKs, which shares high similarity in amino acid sequence with yeast guanylate kinase (yGMPK), is the least characterized MAGUK domain both in structure and function. In addition to its scaffolding function, the GK domain of hCASK has been shown to be involved in transcription regulation. Here we report the crystal structure of the GK domain of human CASK (hCASK-GK) at 1.3-Å resolution. The structure rationalizes the inability of the GK domain to catalyze phosphoryl transfer and strongly supports its new function as a protein-binding module. Comparison of the hCASK-GK structure with the available crystal structures of yGMPK provides insight into possible conformational changes that occur in hCASK upon GMP binding. These conformational changes may act to regulate hCASK-GK function in a nucleotide-dependent manner.Membrane-associated guanylate kinases (MAGUKs) 1 constitute a newly recognized class of proteins found in all animals examined to date. They act as molecular scaffolds for signaling pathway components, regulate synaptic structure and function by mediating specific interactions, and (in Drosophila) act as tumor suppressors. In addition, MAGUKs recruit molecules into localized multimolecular complexes and cluster these complexes at the plasma membrane, such as cell junctions or the apicolateral or basolateral surface, and pre-or post-synaptic sites (1-6). All MAGUK proteins share a similar multidomain structural organization that includes (from the N to C termini) one or three copies of PDZ (PSD-95/SAP-90-Dlg-Zo1) domains, followed by one Src homology 3 (SH3) domain, then followed by a guanylate kinase-like (GK) domain (3). The PDZ domain functions as a protein-protein interaction module and is typically involved in the assembly of supramolecular complexes that perform localized signaling functions at particular subcellular locations (7). Although their specific functions in MAGUKs are not very clear, SH3 domains are found in many other proteins and are recognized as modular protein-protein interaction domains with an affinity for certain proline-rich motifs (8).The GK domain in MAGUKs shares high sequence similarity with yeast guanylate kinase (yGMPK), a nucleoside monophosphate kinase that converts GMP to GDP using ATP as a phosphate donor. However, no enzymatic activity is present in MAGUK GK domains. Moreover, several protein binding partners of GK domains were found by yeast two-hybrid screens or pull-down assays (see Table III below), and this suggested that the GK domain functions as a protein-binding module. Although the GK domain cannot catalyze phosphoryl transfer, it can bind nucleotides. Binding studies have shown that the GK domain of SAP-90 binds GMP in the micromolar range and ATP in the millimolar range (9). Although a GK domain is present in all MAGUK...

Mammalian synapse-associated protein SAP97, a structural and functional homolog of Drosophila Dlg, is a membrane-associated guanylate kinase (MAGUK) that is present at pre-and postsynaptic sites as well as in epithelial cell-cell contact sites. It is a multidomain scaffolding protein that shares with other members of the MAGUK protein family a characteristic modular organization composed of three sequential protein interaction motifs known as PDZ domains, followed by an Src homology 3 (SH3) domain, and an enzymatically inactive guanylate kinase (GK)-like domain. Specific binding partners are known for each domain, and different modes of intramolecular interactions have been proposed that particularly involve the SH3 and GK domains and the so-called HOOK region located between these two domains. We identified the HOOK region as a specific site for calmodulin binding and studied the dynamics of complex formation of recombinant calmodulin and SAP97 by surface plasmon resonance spectroscopy. Binding of various SAP97 deletion constructs to immobilized calmodulin was strictly calciumdependent. From the rate constants of association and dissociation we determined an equilibrium dissociation constant K d of 122 nM for the association of calcium-saturated calmodulin and a SAP97 fragment, which encompassed the entire SH3-HOOK-GK module. Comparative structure-based sequence analysis of calmodulin binding regions from various target proteins predicts variable affinities for the interaction of calmodulin with members of the MAGUK protein family. Our findings suggest that calmodulin could regulate the intramolecular interaction between the SH3, HOOK, and GK domains of SAP97.Membrane-associated guanylate kinase homologs (MAGUKs) 1 have been implicated in the assembly of synapses and tight junctions. These are multidomain proteins consisting of one or more PDZ domains, an Src homology 3 (SH3) domain, and a guanylate kinase (GK)-like domain (for reviews, see Refs. 1-4). Rat SAP97 belongs to the MAGUK subfamily comprising Drosophila Dlg, SAP97/hDlg, SAP90/PSD-95, SAP102/NE-Dlg, and PSD93/Chapsyn110. SAP97 has earlier been reported to be presynaptic (5), but it has recently been shown also to be present at postsynaptic sites in cerebral cortex (6); it is found both at the post-synaptic density (PSD) region and in the cytoplasm of hippocampal synapses, suggesting a role for SAP97 in ionotropic glutamate receptor trafficking (7). Recently it was discovered that SAP97, via its guanylate kinase (GK)-like domain, directly regulates the function of an inwardly rectifier potassium channel (8). In addition to its crucial role as a scaffolding protein in neuronal cells, SAP97 is an essential component of the basolateral membrane cytoskeleton in a variety of epithelial cells (5).Each domain in the SAP subfamily of MAGUKs is highly conserved and has been shown to be a site of protein-protein interaction: PDZ domains bind voltage-and ligand-gated ion channels (3, 9); SH3 domains interact with proline-rich, PXXPR-like sequences (3, 4, 10) as w...

Guanylate kinase is an essential enzyme for nucleotide metabolism, phosphorylating GMP to GDP or dGMP to dGDP. The low molecular mass cytosolic forms of guanylate kinase are implicated primarily in the regulation of the supply of guanine nucleotides to cell signalling pathways. The high molecular mass and membrane-associated forms of guanylate kinase homologues, notably found in neuronal tissues, are assigned roles in cell junction organization and transmembrane regulation. Here, we describe the first plant guanylate kinase-encoding genes, AGK1 and AGK2, from Arabidopsis thaliana. The nucleotide sequences of their genomic and cDNA clones predict proteins that carry N-terminal and C-terminal extensions of the guanylate kinase-like domain. The amino acid sequences of this domain share 46±52% identity with guanylate kinases from yeast, Escherichia coli, human, mouse and Caenorhabditis elegans. Arabidopsis guanylate kinases (AGKs) exhibit a high degree of conservation of active site residues and sequence motifs in common with other nucleoside monophosphate kinases, which suggests overall structural similarity of the plant proteins. Although bacterially expressed AGK-1 is enzymatically much less active than yeast guanylate kinase, its kinase domain is shown to complement yeast GUK1 recessive lethal mutations. AGKs are expressed ubiquitously in plant tissues with highest transcriptional activity detected in roots. The identification of AGKs provides new perspectives for understanding the role of guanylate kinases in plant cell signalling pathways.