Phosphagen Kinase Family Research Articles

The Horizontally Transferred Arginine Kinase in Myxococcus Xanthus Has Evolved Roles in Multiple Physiological Processes

Arginine kinase (AK) is a member of the phosphagen kinase family that is involved in energy homeostasis in a range of animal and protozoan species. However, recent studies have shown that some bacterial species have acquired AKs through horizontal gene transfer (HGT), raising the question as to what role they may play in bacterial systems. A recent study with one of these bacterial species, the soil bacteria Myxococcus xanthus, knocked out the endogenous AK to reveal several phenotypes, including diminished recovery from certain stressors and an unexpected role in the sporulation response induced by starvation conditions characteristic to the myxoccocales family. We have expanded upon these initial observations, identifying another novel phenotype in which the deletion strain is unexpectedly no longer recognized as kin by the non‐deletion strain. To further investigate how this AK has evolved in this system, we asked whether a heterologous AK could rescue the deletion strain. Our results will help us determine if the myxococcus AK has uniquely evolved after transmission and acquired novel traits not seen in other AKs.

The Influence of Identified Key Residues on Catalytic Activity and Substrate Specificity of Taurocyamine Kinases from Arenicola brasiliensis, An Enzyme in the Phosphagen Kinases Family

Tauracyamine kinases (TKs) are part of the phosphagen kinase family which act as an energy buffering mechanism in some invertebrates and unicellular organisms by catalyzing the transfer of phosphate from ATP to guanidino containing compounds, such as arginine, creatine and tauracycamine. TKs utilize tauroayamine as their primary substrate but many TKs exhibit promiscuity in substrate specificity and can also phosprylate glycocyamine or lumbricine. Multiple amino acid residues have been identified as key residues for substrate binding and/or specificity in the phosphagen kinases; specifically, amino acids residue 62‐72 have been identified as key residues within the guanidino substrate (GS) loop that play an important role in substrate binding and preference. Previous work has shown that a single substitution in this loop can ‘flip’ substrate specificity from tauracyamine to glycocyamine although with a significant drop in activity. Using site‐directed mutagenesis we have created additional substitutions in the GS loop to see if they can improve overall activity for the enzyme while still maintaining a preference for glycocyamine. We will be describing our kinetic analysis (KM, kcat, and kcat/KM) of each of the variants and the wildtype TKs to help determine whether the residues mutated are critical in the determining the enzyme’s preference and overall activity with the two substrates, glycocyamine and tauracyamine. This may lead to insight into how different substrate preferences may have evolved.

Arginine kinase from Haemonchus contortus decreased the proliferation and increased the apoptosis of goat PBMCs in vitro

BackgroundArginine kinase (AK), an important member of phosphagen kinase family has been extensively studied in various vertebrates and invertebrates. Immunologically, AKs are important constituents of different body parts, involved in various biological and cellular functions, and considered as immune-modulator and effector for pro-inflammatory cytokines. However, immunoregulatory changes of host cells triggered by AK protein of Haemonchus contortus, a parasitic nematode of ruminants, are still unknown. The current study was focused on cloning and characterisation of Hc-AK, and its regulatory effects on cytokines level, cell migration, cell proliferation, nitric oxide production and apoptosis of goat peripheral blood mononuclear cells (PBMCs) were observed.MethodsThe full-length sequence of the Hc-AK gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and sub-cloned into the prokaryotic expression vector pET-32a. The biochemical characteristics of recombinant protein Hc-AK, which was purified by affinity chromatography, were performed based on the enzymatic assay. Binding of rHc-AK with PBMCs was confirmed by immunofluorescence assay (IFA). Immunohistochemical analysis was used to detect localisation of Hc-AK within adult worms sections. The immunoregulatory effects of rHc-AK on cytokine secretions, cell proliferation, cell migration, nitric oxide production and apoptosis were determined by co-incubation of rHc-AK with goat PBMCs.ResultsThe full-length ORF (1080 bp) of the Hc-AK gene was successfully cloned, and His-tagged AK protein was expressed in the Escherichia coli strain BL21. The recombinant protein of Hc-AK (rHc-AK) was about 58.5 kDa together with the fused vector protein of 18 kDa. The biochemical assay showed that the protein encoded by the Hc-ak exhibited enzymatic activity. Western blot analysis confirmed that the rHc-AK was recognised by the sera from rat (rat-antiHc-AK). The IFA results showed that rHc-AK could bind on the surface of goat PBMCs. Immunohistochemically, Hc-AK was localised at the inner and outer membrane as well as in the gut region of adult worms. The binding of rHc-AK to host cells increased the levels of IL-4, IL-10, IL-17, IFN-γ, nitric oxide (NO) production and cell apoptosis of goat PBMCs, whereas, TGF-β1 levels, cell proliferation and PBMCs migration were significantly decreased in a dose dependent manner.ConclusionsOur findings suggested that rHc-AK is an important excretory and secretory (ES) protein involved in host immune responses and exhibit distinct immunomodulatory properties during interaction with goat PBMCs.

The Michaelis Complex of Arginine Kinase Samples the Transition State at a Frequency That Matches the Catalytic Rate.

Arginine kinase (AK), which is a member of the phosphagen kinase family, serves as a model system for studying the structural and dynamic determinants of biomolecular enzyme catalysis of all major states involved of the enzymatic cycle. These states are the apo state (substrate free), the Michaelis complex analogue AK:Arg:Mg·AMPPNP (MCA), a product complex analogue AK:pAIE:Mg·ADP (PCA), and the transition state analogue AK:Arg:Mg·ADP:NO3- (TSA). The conformational dynamics of these states have been studied by NMR relaxation dispersion measurements of the methyl groups of the Ile, Leu, and Val residues at two static magnetic fields. Although all states undergo significant amounts of μs-ms time scale dynamics, only the MCA samples a dominant excited state that resembles the TSA, as evidenced by the strong correlation between the relaxation dispersion derived chemical shift differences Δω and the equilibrium chemical shift differences Δδ of these states. The average lifetime of the MCA is 36 ms and the free energy difference to the TSA-like form is 8.5 kJ/mol. It is shown that the conformational energy landscape of the Michaelis complex analogue is shaped in a way that at room temperature it channels passage to the transition state, thereby determining the rate-limiting step of the phosphorylation reaction of arginine. Conversely, relaxation dispersion experiments of the TSA reveal that it samples the structures of the Michaelis complex analogue or the apo state as its dominant excited state. This reciprocal behavior shows that the free energy of the TSA, with all ligands bound, is lower by only about 8.9 kJ/mol than that of the Michaelis or apo complex conformations with the TSA ligands present.

Shrimp arginine kinase being a binding protein of WSSV envelope protein VP31

Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31 (WSV340/WSSV396), an envelope protein of white spot syndrome virus (WSSV), contains an Arg-Gly-Asp (RGD) peptide domain known as a cellular attachment site. At present, the process of VP31 interacting with shrimp host cells has not been explored. Therefore, the VP31 gene was cloned into pET30a (+), expressed in Escherichia coli strain BL21 and purified with immobilized metal ion affinity chromatography. Four gill cellular proteins of shrimp (Fenneropenaeus chinensis) were pulled down by an affinity column coupled with recombinant VP31 (rVP31), and the amino acid sequences were identified with MALDI-TOF/TOF mass spectrometry. Hemocyanin, beta-actin, arginine kinase (AK), and an unknown protein were suggested as the putative VP31 receptor proteins. SDS-PAGE showed that AK is the predominant binding protein of VP31. An i n vitro binding activity experiment indicated that recombinant AK’s (rAK) binding activity with rVP31 is comparable to that with the same amount of WSSV. These results suggested that AK, as a member of the phosphagen kinase family, plays a role in WSSV infection. This is the first evidence showing that AK is a binding protein of VP31. Further studies on this topic will elucidate WSSV infection mechanism in the future.

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

The taurocyamine kinase from the blood fluke Schistosoma mansoni (SmTK) belongs to the phosphagen kinase (PK) family and catalyzes the reversible Mg(2+)-dependent transfer of a phosphoryl group between ATP and taurocyamine. SmTK is derived from gene duplication, as are all known trematode TKs. Our crystallographic study of SmTK reveals the first atomic structure of both a TK and a PK with a bilobal structure. The two unliganded lobes present a canonical open conformation and interact via their respective C- and N-terminal domains at a helix-mediated interface. This spatial arrangement differs from that observed in true dimeric PKs, in which both N-terminal domains make contact. Our structures of SmTK complexed with taurocyamine or l-arginine compounds explain the mechanism by which an arginine residue of the phosphagen specificity loop is crucial for substrate specificity. An SmTK crystal was soaked with the dead end transition state analog (TSA) components taurocyamine-NO3 (2-)-MgADP. One SmTK monomer was observed with two bound TSAs and an asymmetric conformation, with the first lobe semiclosed and the second closed. However, isothermal titration calorimetry and enzyme kinetics experiments showed that the two lobes function independently. A small angle x-ray scattering model of SmTK-TSA in solution with two closed active sites was generated.

Characterization of arginine kinase from the caenogastropod Semisulcospira libertina, an intermediate host of Paragonimus westermani

Arginine kinase (AK) belongs to the phosphagen kinase (PK) family of enzymes that have a significant role in the maintenance of energy homeostasis. In this study, cDNA of Semisulcospira libertina AK was determined, cloned and the recombinant protein was expressed as fusion protein with maltose-binding protein. The protein has a theoretical molecular mass of 39,412 Da and an estimated isoelectric point (p I) of 6.37. The recombinant enzyme showed high affinity and significant activity only for the substrate L-arginine (Km ¼ 0.53 mM; Vmax ¼ 61.30 mmol/min mg protein). Phylogenetic analyses showed that S. libertina AK clusters with gastropod and cephalopod AKs. Comparison of gene structure showed that all intron positions of S. libertina AK are shared with molluscan AKs. Certain intron positions are also shared with trematode TK and sipunculid HTK. Semisulcospira libertina AK can be utilized in the control of paragonimiasis since it is absent in mammalian hosts.

Characterization of a putative oomycete taurocyamine kinase: Implications for the evolution of the phosphagen kinase family

Phosphagen kinases (PKs) are known to be distributed throughout the animal kingdom, but have recently been discovered in some protozoan and bacterial species. Within animal species, these enzymes play a critical role in energy homeostasis by catalyzing the reversible transfer of a high-energy phosphoryl group from Mg⋅ATP to an acceptor molecule containing a guanidinium group. In this work, a putative PK gene was identified in the oomycete Phytophthora sojae that was predicted, based on sequence homology, to encode a multimeric hypotaurocyamine kinase. The recombinant P. sojae enzyme was purified and shown to catalyze taurocyamine phosphorylation efficiently (kcat/KM (taurocyamine) = 2 × 10(5) M(-1) s(-1)) and glycocyamine phosphorylation only weakly (kcat/KM (glycocyamine) = 2 × 10(2) M(-1) s(-1)), but lacked any observable kinase activity with the more ubiquitous guanidinium substrates, creatine or arginine. Additionally, the enzyme was observed to be dimeric but lacked cooperativity between the subunits in forming a transition state analog complex. These results suggest that protozoan PKs may exhibit more diversity in substrate specificity than was previously thought.

Mutation of residue arginine 330 of arginine kinase results in the generation of the oxidized form more susceptible

Arginine kinase (AK), a crucial enzyme for the energy metabolism of invertebrates, catalyzes the reversible phosphorylation of arginine by Mg2+ATP to form phosphoarginine and Mg2+ADP. Arginine 330 (R330), not involved in the catalysis of phosphoryl transfer, is a residue highly conserved in the phosphagen kinase family. In order to investigate the role of R330 in AK, it was replaced by lysine (R330K). Non-reduced SDS-PAGE analysis suggested that wild type AK (Wt-AK) and R330K existed in two forms, the reduced form (R-AK or R-R330K) and the oxidized form (O-AK or O-R330K), whereas O-R330K was more susceptible to generate than O-AK. Subsequently, an intramolecular disulfide bond in O-R330K was demonstrated to be formed between Cys201 and Cys271 by site-directed mutagenesis. Biochemical analysis revealed that conformational changes of R330K were concomitant with the sharp decline of catalytic activity. These results were further confirmed by structure modeling of AK and R330K. Therefore, it can be concluded that R330 residue plays an important role in the structural stability and activity of AK.

Crystal structures of arginine kinase in complex with ADP, nitrate, and various phosphagen analogs

Arginine kinase catalyzes the reversible transfer of a phosphoryl group between ATP and l-arginine and is a monomeric homolog of the human enzyme creatine kinase. Arginine and creatine kinases belongs to the phosphagen kinase family of enzymes, which consists of eight known members, each of which is specific for its own phosphagen. Here, the source of phosphagen specificity in arginine kinase is investigated through the use of phosphagen analogs. Crystal structures have been determined for Limulus polyphemus arginine kinase with one of four arginine analogs bound in a transition state analog complex: l-ornithine, l-citrulline, imino-l-ornithine, and d-arginine. In all complexes, the enzyme achieves a closed conformation very similar to that of the cognate transition state analog complex, but differences are observed in the configurations of bound ligands. Arginine kinase exhibits no detectable activity towards ornithine, citrulline, or imino-l-ornithine, and only trace activity towards d-arginine. The crystal structures presented here demonstrate that phosphagen specificity is derived neither from a lock-and-key mechanism nor a modulation of induced-fit conformational changes, but potentially from subtle distortions in bound substrate configurations.

Variable intron/exon structure in the oligochaete lombricine kinase gene

Lombricine kinase is an annelid enzyme that belongs to the phosphagen kinase family of which creatine kinase and arginine kinase are the typical representatives. The enzymes play important roles in the cellular energy metabolism of animals. Biochemical, physiological and molecular information with respect to lombricine kinase is limited compared to other phosphagen kinases. This study presents data on the cDNA sequences of lombricine kinase from two smaller oligochaetes, Enchytraeus sp. and Stylaria sp. The deduced amino acid sequences are analyzed and compared with other selected phosphagen kinases. The intron/exon structure of the lombricine kinase gene was determined for these two species as well as two additional oligochaetes, Lumbriculus variegatus and Tubifex tubifex, and compared with available data for annelid phosphagen kinases. The data indicate the existence of a variable organization of the proposed 8-intron/9-exon gene structure. The results provide further insights in the evolution and position of these enzymes within the phosphagen kinase family.

Characterization of a hypotaurocyamine kinase from the protozoan, Phytophthora sojae, and its implications on the evolution of substrate specificity in the phosphagen kinase family

Phosphagen kinases (PK) are a family of enzymes that catalyze the reversible transfer of a high‐energy gamma phosphoryl group from ATP to a guanidino acceptor molecule. A novel hypotaurocyamine kinase (HTK) from the protozoan oomycete Phytophthora sojae (PsPK) was studied to determine substrate specificity, oligomerization state, and cooperativity. Isothermal titration calorimetry was used to determine the kinetic constants of PsPK reacting with taurocyamine and, less strongly, glycocyamine by titrating the guanidino substrate at varying concentrations into a solution containing the HTK and MgATP. The Km for taurocyamine and MgATP was found to be 0.59 mM and 3.78 mM, respectively, with a kcat of 34.66 s−1, while the Km for glycocyamine and MgATP was found to be 5.1 mM and 5.1 mM, respectively, with a kcat of 3.2 s−1. Size exclusion chromatography indicates PsPK is an oligomer and isothermal titration calorimetry suggests no cooperativity among subunits. Our results coupled with findings in Monosiga suggest oligomerization evolved first in the protozoa and that cooperativity sometime later. The data also indicate there is diverse substrate specificity in protozoan PKs previously thought to exist only in annelida. This project was made possible by the support of USDA grant (USDA 2011‐68004‐30104) and the Howard Hughes Medical Institute Undergraduate Science Education Program.

Spatial buffering of ATP by arginine kinase may be critical for Myxococcus xanthus development

Previous work suggested M. xanthus contains an arginine kinase (ark) gene that was incorporated into its genome by horizontal gene transfer. This protein is a member of the phosphagen kinase family, which regulates the concentration of the energy‐carrier molecule ATP by catalyzing the reversible transfer a high‐energy phosphate from ATP to another molecule, in this case arginine. In response to starvation, Myxococcus xanthus cells aggregate to form mounds called fruiting bodies containing resistant spores that can survive until conditions improve. When ark is knocked out in M. xanthus the developmental pathway does not proceed normally. However, when M. xanthus cells with ark knocked‐out are exposed to osmotic stress during development, development is partially restored. The goal of this study is to determine whether this enzyme has evolved a novel function that makes it important for development. To test the effect of AK activity, I will insert genes for AKs of different activity into the M. xanthus DNA. These enzymes will provide AK activity, but not any novel functions that the M. xanthus AK may have. I will then observe whether development proceeds normally in these new strains. I am currently confirming the production of the plasmid that will be used to re‐insert the M. xanthus AK into its genome. This research is supported by the College of Wooster Biology department and Copeland Funding.

Immunolocalization of arginine kinase (AK) in Toxocara canis, Toxocara vitulorum, and Ascaris lumbricoides

Arginine kinase (AK) is a member of the phosphagen kinase family. AK plays a major role in cellular energy metabolism in invertebrates including nematodes. In the present study, we performed the direct immunofluorescence test to determine the immunolocalization of AK in different stages of the life cycle (eggs, larvae, and adult worms) of Toxocara canis, Toxocara vitulorum, and Ascaris lumbricoides. Our results indicated variable levels of expression of AK in different stages. Moreover, strong fluorescence was observed in cleaving eggs than in dormant eggs. The highest activity of the enzyme was observed in the fully developed eggs. This may be due to high expression of AK in embryonic development, which is associated with increased energy demand due to cleavage and cellular differentiation. Surprisingly, expression of AK is significantly higher in the middle part and posterior end compared to anterior end of the larvae. In addition, AK is highly concentrated in cellular and metabolically active parts of the body such as hypodermis, muscle, intestine, ovaries, oviducts, and uterus, while it is absent in noncellular areas like cuticle. The present study revealed the presence of AK in T. canis, A. lumbricoides, and T. vitulorum and that it plays a major role in energy metabolism of these nematodes. Interestingly, antiserum was prepared against the recombinant T. canis AK and reacts with the native AKs of T. canis, A. lumbricoides, and T. vitulorum. AK levels could vary in relation to maximum potential rates of ATP turnover, oxidative capacity, and energy output. Further studies on subcellular localization of AK in these important helminths provide new information for researchers to develop effective anthelmintics against the parasites of veterinary and of public health importance.

Characterization of the Phytophthora sojae hypotaurocyamine kinase suggests early evolution of quaternary structure in the phosphagen kinase family

We investigated the substrate specificity and kinetic constants of a Phytophthora sojae hypotaurocyamine kinase (HTK) to better understand the evolution of the phosphagen kinase (PK) protein family. The substrate specificity and kinetic constants were determined using Isothermal Titration Calorimetry by titrating arginine, creatine, glycocyamine, or taurocyamine at varying concentrations in to a solution containing the HTK and MgATP. The protein had strong reactivity with taurocyamine and weak reactivity with glycocyamine, while it showed no reactivity with either arginine or creatine. The Km for taurocyamine and MgATP were found to be 0.59mM and 3.78mM respectively. The kcat was found to be 34.66s−1. Based on our phylogenetic analysis and the ability for the P. sojae HTK to react with glycocyamine, we hypothesized that the dimeric PKs evolved from an ancestral kinase that may have had some promiscuity in substrate specificity. Using protein sequences of known dimeric PKs, we tested this hypothesis by constructing ancestral sequences using the web‐based program ANCESCON. This project was made possible by the support of the Howard Hughes Medical Institute and the Henry J. Copeland Fund.

The Structure of Lombricine Kinase: IMPLICATIONS FOR PHOSPHAGEN KINASE CONFORMATIONAL CHANGES

Lombricine kinase is a member of the phosphagen kinase family and a homolog of creatine and arginine kinases, enzymes responsible for buffering cellular ATP levels. Structures of lombricine kinase from the marine worm Urechis caupo were determined by x-ray crystallography. One form was crystallized as a nucleotide complex, and the other was substrate-free. The two structures are similar to each other and more similar to the substrate-free forms of homologs than to the substrate-bound forms of the other phosphagen kinases. Active site specificity loop 309-317, which is disordered in substrate-free structures of homologs and is known from the NMR of arginine kinase to be inherently dynamic, is resolved in both lombricine kinase structures, providing an improved basis for understanding the loop dynamics. Phosphagen kinases undergo a segmented closing on substrate binding, but the lombricine kinase ADP complex is in the open form more typical of substrate-free homologs. Through a comparison with prior complexes of intermediate structure, a correlation was revealed between the overall enzyme conformation and the substrate interactions of His(178). Comparative modeling provides a rationale for the more relaxed specificity of these kinases, of which the natural substrates are among the largest of the phosphagen substrates.

Arginine Kinase: Joint Crystallographic and NMR RDC Analyses Link Substrate-Associated Motions to Intrinsic Flexibility

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.

CDNA identification, comparison and phylogenetic aspects of lombricine kinase from two oligochaete species

Creatine kinase and arginine kinase are the typical representatives of an eight-member phosphagen kinase family, which play important roles in the cellular energy metabolism of animals. The phylum Annelida underwent a series of evolutionary processes that resulted in rapid divergence and radiation of these enzymes, producing the greatest diversity of the phosphagen kinases within this phylum. Lombricine kinase (EC 2.7.3.5) is one of such enzymes and sequence information is rather limited compared to other phosphagen kinases. This study presents data on the cDNA sequences of lombricine kinase from two oligochaete species, the California blackworm (Lumbriculus variegatus) and the sludge worm (Tubifex tubifex). The deduced amino acid sequences are analyzed and compared with other selected phosphagen kinases, including two additional lombricine kinase sequences extracted from DNA databases and provide further insights in the evolution and position of these enzymes within the phosphagen kinase family. The data confirms the presence of a deleted region within the flexible loop (the GS region) of all six examined lombricine kinases. A phylogenetic analysis of these six lombricine kinases clearly positions the enzymes together in a small subcluster within the larger creatine kinase (EC 2.7.3.2) clade.

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

Glycocyamine kinase (GK), a member of the phosphagen kinase family, catalyzes the Mg(2+)-dependent reversible phosphoryl group transfer of the N-phosphoryl group of phosphoglycocyamine 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, alpha and beta, derived from a common pre-mRNA by mutually exclusive N-terminal alternative exons. The N-terminal exon of GKbeta encodes a peptide that is different in sequence and is 16 amino acids longer than that encoded by the N-terminal exon of GKalpha. The crystal structures of recombinant GKalphabeta and GKbetabeta from Namalycastis sp. were determined at 2.6 and 2.4 A resolution, respectively. In addition, the structure of the GKbetabeta was determined at 2.3 A resolution in complex with a transition state analogue, Mg(2+)-ADP-NO(3)(-)-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 GKalphabeta are structurally defined, and the longer N-terminus of the beta subunit is anchored at the dimer interface. In GKbetabeta 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 GKalphabeta 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 alpha or solely beta 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 analogue, both subunits of GK undergo the same closure motion that clasps the transition state analogue, in contrast to the transition state analogue complexes of CK, where the corresponding transition state analogue 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 dimer, 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.

Creatine kinases: a cornerstone for structural research in the phosphagen kinase family

In their recently published manuscript, Wu et al. (1) solved a 1.75 A structure of a dimeric arginine kinase (AK) and propose a mechanism for negative cooperativity in dimeric phosphagen kinases (PK) in general. The authors correctly cite that such AK dimers probably evolved from another PK family member, a dimeric creatine kinase (CK)-like ancestor (2), that CKs are the most studied family members for their multiple implications in human pathologies, and that their results have repercussions for the understanding of CK function. Unfortunately, some fundamental results concerning the structure/function relationship of CKs have been omitted. Pertinent to this work is the historically first X-ray structure of a PK proper, that is the large cuboidal octamer of sarcomeric mitochondrial CK with bound ADP (3), whose coordinates have been used to solve many subsequent PK structures by molecular replacement, as well as the first X-ray structures of two other CK isoforms. These include octameric ubiquitous mitochondrial CK (4) and dimeric human brain BB-CK (5), solved at 1.41 A, the highest resolution obtained so far within the PK family. These structures allowed already 13 years ago to conclude on separated active sites within the CK homooligomers and to examine the monomer/monomer interface. Even before, in 1994, the importance of the N-termini in CK protomer communication has been shown by mutational analysis (6), similar to what is presented by Wu et al. (1). Cooperativity between CK monomers within a functional CK dimer was demonstrated (7) and the conformational change in CK upon substrate (TSAC) binding from an open to a closed conformation elaborated by in-solution X-ray scattering (8). Recent reviews on these results are available (9–11). REFERENCES