Analysis of ATP binding to CheA containing tryptophan substitutions near the active site

Signal transduction in the chemotaxis system of Escherichia coli involves an autophosphorylating protein histidine kinase, CheA. At the active site of CheA, phenylalanine residues 455 and 459 occupy positions near the ATP-binding pocket, immediately adjacent to one of the hinge regions of a loop that undergoes an ATP-induced conformational change ("lid closure") that has been characterized previously in X-ray crystal structures [Bilwes et al. (2001) Nat. Struct. Biol. 8, 353-360]. We generated versions of CheA carrying F455W and F459W replacements and investigated whether the fluorescence properties of the introduced tryptophan side chains were affected by nucleotide binding in a manner that would provide a signal for investigating the dynamics of active site events, such as ATP binding and lid closure. Our results indicate that CheA(F455W) is useful in this regard, but CheA(F459W) is not. CheA(F455W) retained full catalytic activity and exhibited easily monitored fluorescence changes upon binding nucleotide: we observed a 25-30% decrease in CheA(F455W) fluorescence emission intensity at 330 nm upon binding ATP in the absence of Mg(2+); in the presence of Mg(2+), the emission spectrum of the CheA(F455W):ATP complex was red-shifted by 5 nm and exhibited an increased intensity (approximately 20% higher at 345 nm relative to that of uncomplexed CheA(F455W)). Different fluorescence changes were observed when two ATP analogues (ADPNP and ADPCP) were used instead of ATP and when Mn(2+) or Ca(2+) was used in place of Mg(2+). We exploited the fluorescence changes induced by Mg(2+)-ATP to explore the kinetics and mechanism of nucleotide binding by CheA(F455W). In the absence of Mg(2+), binding appears to involve a simple one-step equilibrium (k(assn) = 0.7 microM(-1) s(-1) and k(dissn) = 270 s(-1) at 4 degrees C). In the presence of Mg(2+), the binding mechanism involves at least two steps: (i) rapid, relatively weak binding followed by (ii) a rapid, reversible step (k(forward) = 300 s(-1) and k(reverse) =15 s(-1) at 4 degrees C) that enhanced the overall affinity of the complex and generated an increase in W455 fluorescence. This second step could reflect a conformational change at the CheA active site, such as lid closure. These results provide the first insight into the dynamics of nucleotide binding and substrate-induced conformational changes at the active site of a protein histidine kinase.     

Probing the nucleotide binding domain of the osmoregulator EnvZ using fluorescent nucleotide derivatives

EnvZ is a histidine protein kinase important for osmoregulation in bacteria. While structural data are available for this enzyme, the nucleotide binding pocket is not well characterized. The ATP binding domain (EnvZB) was expressed, and its ability to bind nucleotide derivatives was assessed using equilbrium and stopped-flow fluorescence spectroscopy. The fluorescence emission of the trinitrophenyl derivatives, TNP-ATP and TNP-ADP, increase upon binding to EnvZB. The fluorescence enhancements were quantitatively abolished in the presence of excess ADP, indicating that the fluorescent probes occupy the nucleotide binding pocket. Both TNP-ATP and TNP-ADP bind to EnvZB with high affinity (K(d) = 2-3 microM). The TNP moiety attached to the ribose ring does not impede access of the fluorescent nucleotide into the binding pocket. The association rate constant for TNP-ADP is 7 microM(-1) s(-1), a value consistent with those for natural nucleotides and the eucaryotic protein kinases. Using competition experiments, it was found that ATP and ADP bind 30- and 150-fold more poorly, respectively, than the corresponding TNP-derivatized forms. Surprisingly, the physiological metal Mg(2+) is not required for ADP binding and only enhances ATP affinity by 3-fold. Although portions of the nucleotide pocket are disordered, the recombinant enzyme is highly stable, unfolding only at temperatures in excess of 70 degrees C. The unusually high affinity of the TNP derivatives compared to the natural nucleotides suggests that hydrophobic substitutions on the ribose ring enforce an altered binding mode that may be exploited for drug design strategies.     

Hexameric structures of the archaeal secretion ATPase GspE and implications for a universal secretion mechanism

The secretion superfamily ATPases are conserved motors in key microbial membrane transport and filament assembly machineries, including bacterial type II and IV secretion, type IV pilus assembly, natural competence, and archaeal flagellae assembly. We report here crystal structures and small angle X-ray scattering (SAXS) solution analyses of the Archaeoglobus fulgidus secretion superfamily ATPase, afGspE. AfGspE structures in complex with ATP analogue AMP-PNP and Mg(2+) reveal for the first time, alternating open and closed subunit conformations within a hexameric ring. The closed-form active site with bound Mg(2+) evidently reveals the catalytically active conformation. Furthermore, nucleotide binding results and SAXS analyses of ADP, ATPgammaS, ADP-Vi, and AMP-PNP-bound states in solution showed that asymmetric assembly involves ADP binding, but clamped closed conformations depend on both ATP gamma-phosphate and Mg(2+) plus the conserved motifs, arginine fingers, and subdomains of the secretion ATPase superfamily. Moreover, protruding N-terminal domain shifts caused by the closed conformation suggest a unified piston-like, push-pull mechanism for ATP hydrolysis-dependent conformational changes, suitable to drive diverse microbial secretion and assembly processes by a universal mechanism.     

Substrate binding-induced alteration of nucleotide binding site properties of chloroplast coupling factor 1

Two adenine nucleotide binding sites of chloroplast coupling factor 1 (CF1) were shown previously to switch their properties after exposure of the enzyme to Mg2(+)-ATP or Ca2(+)-ATP (Shapiro, A. B., and McCarty, R. E. (1988) J. Biol. Chem. 263, 14160-14165). The change in binding properties was monitored by fluorescence resonance energy transfer between Lucifer Yellow vinyl sulfone covalently bound to one alpha subunit and trinitrophenyl-ATP (TNP-ATP) tightly bound to nucleotide binding site 1. When the nucleotide binding properties of sites 1 and 3 switch during catalysis, site 3, which is nearer Lucifer Yellow than site 1, switches its nucleotide binding properties with site 1, allowing TNP-ATP to become tightly bound near Lucifer Yellow. The smaller separation allows energy transfer to occur, resulting in decreased Lucifer Yellow fluorescence. In this paper, we show that adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) bound to CF1 and caused nucleotide binding sites 1 and 3 to switch properties, but was not hydrolyzed. Using AMP-PNP, we also found that relaxation of the properties of the sites to the precatalysis state after removal of substrate occurred in the absence of hydrolysis of the last bound nucleotide. When Mg2+ was omitted during exposure of CF1 to ATP, there was very little hydrolysis or nucleotide site switching. When Mg2+ was added to a very low concentration which was more than stoichiometric with CF1, however, site switching occurred at its maximal level with virtually no increase in ATP hydrolysis. These results support a model in which binding of substrate Mg2(+)-ATP, not hydrolysis, causes the putative catalytic sites to switch properties, in agreement with the alternating site catalytic cooperativity hypothesis (Boyer, P. D. (1989) FASEB J. 3, 2164-2178). TNP-ATP, the fluorescence acceptor, did not cause nucleotide site switching when incubated with CF1 in the presence of EDTA to eliminate free Mg2+. Two possible additional nucleotide binding sites were detected, in addition to the three well characterized sites. At least one of these sites was close to the Lucifer Yellow site, judging by the amount of energy transfer caused by partial occupancy with TNP-ATP.     

An open conformation of switch I revealed by the crystal structure of a Mg2+-free form of RHOA complexed with GDP. Implications for the GDP/GTP exchange mechanism

Mg(2+) ions are essential for guanosine triphosphatase (GTPase) activity and play key roles in guanine nucleotide binding and preserving the structural integrity of GTP-binding proteins. We determined the crystal structure of a small GTPase RHOA complexed with GDP in the absence of Mg(2+) at 2.0-A resolution. Elimination of a Mg(2+) ion induces significant conformational changes in the switch I region that opens up the nucleotide-binding site. Similar structural changes have been observed in the switch regions of Ha-Ras bound to its guanine nucleotide exchange factor, Sos. This RHOA-GDP structure reveals an important regulatory role for Mg(2+) and suggests that guanine nucleotide exchange factor may utilize this feature of switch I to produce an open conformation in GDP/GTP exchange.     

Characterization of the N-terminal domain of NrtC, the ATP-binding subunit of ABC-type nitrate transporter of the cyanobacterium Phormidium laminosum

The N-terminal domain of NrtC, the ATP-binding subunit of nitrate/nitrite ABC-transporter in the cyanobacterium Phormidium laminosum, has been expressed in Escherichia coli as a histidine-tagged fusion protein (His(6)NrtC1). Binding of ATP to the pure His(6)NrtC1 was characterized using the nucleotide analogue TNP-ATP [2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate]. Fluorescence assays showed that His(6)NrtC1 specifically binds Mg(2+) TNP-ATP with high affinity, binding being dependent on protein concentration. The presence of ATP prevents the covalent modification of His(6)NrtC1 by fluorescein 5'-isothiocyanate (FITC), suggesting that this probe reacts at the nucleotide-binding site of NrtC. The active form of the truncated NrtC is a dimer that shows high affinity for TNP-ATP (K(d)=0.76+/-0.1 microM). Evidence for the presence of two nucleotide-binding sites per dimer protein is given. Our results indicate that nucleotide binding is strongly dependent on the dimerization of NrtC and that the N-terminal domain of the protein contains the binding site for ATP. No ATPase activity catalyzed in vitro by the truncated subunit was detected.     

Slow interaction of 5'-adenylyl-beta,gamma-imidodiphosphate with Escherichia coli DNA gyrase. Evidence for cooperativity in nucleotide binding.

We have examined the kinetics of interaction between Escherichia coli DNA gyrase and the nonhydrolyzable ATP analog 5'-adenylyl-beta,gamma-imidodiphosphate (ADPNP) in the presence and absence of ATP. In the absence of ATP, [alpha-32P]ADPNP binds extremely slowly to gyrase, with an apparent second-order rate constant (k1) of 120 M-1 min-1. Similarly, the limited negative supercoiling of closed-circular DNA caused by ADPNP binding is slow, requiring at least 2 h to reach completion in the presence of 100 microM ADPNP. A very slow but detectable rate of dissociation of ADPNP from gyrase was measured, with a rate constant of 3.5 x 10(-4) min-1. The calculated dissociation constant for ADPNP is thus 2.9 microM. ADPNP is a potent competitive inhibitor of ATP-dependent DNA supercoiling. Inhibition is established much more rapidly than can be accounted for by the slow rate of ADPNP binding in the absence of ATP. We have found that ATP can accelerate the rate of [32P]ADPNP binding by more than 15-fold (k1 = 1,850 M-1 min-1). The ATP-promoted rate enhancement requires the presence of DNA; in the absence of DNA, ATP has no effect on the rate of binding. Relaxed closed-circular, nicked-circular, and linear pBR322 DNA are all equally effective cofactors for ATP-stimulated binding of ADPNP. After a short lag, the presence of ATP also greatly speeds up ADPNP dissociation from gyrase bound initially to closed-circular DNA, with the restoration of DNA supercoiling activity. This effect is not observed in the presence of nicked-circular or linear DNA, suggesting that ADPNP dissociates more rapidly from gyrase bound to supercoiled DNA. The results of ADPNP binding provide evidence for cooperative interactions between the nucleotide binding sites. To account for these data, a model is proposed for the interaction of nucleotides at the two ATP binding sites on DNA gyrase.     

Crystal structure of phosphoserine phosphatase from Methanococcus jannaschii, a hyperthermophile, at 1.8 A resolution

BACKGROUND: D-Serine is a co-agonist of the N-methyl-D-aspartate subtype of glutamate receptors, a major neurotransmitter receptor family in mammalian nervous systems. D-Serine is converted from L-serine, 90% of which is the product of the enzyme phosphoserine phosphatase (PSP). PSP from M. jannaschii (MJ) shares significant sequence homology with human PSP. PSPs and P-type ATPases are members of the haloacid dehalogenase (HAD)-like hydrolase family, and all members share three conserved sequence motifs. PSP and P-type ATPases utilize a common mechanism that involves Mg(2+)-dependent phosphorylation and autodephosphorylation at an aspartyl side chain in the active site. The strong resemblance in sequence and mechanism implies structural similarity among these enzymes. RESULTS: The PSP crystal structure resembles the NAD(P) binding Rossmann fold with a large insertion of a four-helix-bundle domain and a beta hairpin. Three known conserved sequence motifs are arranged next to each other in space and outline the active site. A phosphate and a magnesium ion are bound to the active site. The active site is within a closed environment between the core alpha/beta domain and the four-helix-bundle domain. CONCLUSIONS: The crystal structure of MJ PSP was determined at 1.8 A resolution. Critical residues were assigned based on the active site structure and ligand binding geometry. The PSP structure is in a closed conformation that may resemble the phosphoserine bound state or the state after autodephosphorylation. Compared to a P-type ATPase (Ca(2+)-ATPase) structure, which is in an open state, this PSP structure appears also to be a good model for the closed conformation of P-type ATPase.     

Poliovirus RNA-dependent RNA polymerase (3Dpol): pre-steady-state kinetic analysis of ribonucleotide incorporation in the presence of Mn2+

The use of Mn(2+) as the divalent cation cofactor in polymerase-catalyzed reactions instead of Mg(2+) often diminishes the stringency of substrate selection and incorporation fidelity. We have solved the complete kinetic mechanism for single nucleotide incorporation catalyzed by the RNA-dependent RNA polymerase from poliovirus (3D(pol)) in the presence of Mn(2+). The steps employed during a single cycle of nucleotide incorporation are identical to those employed in the presence of Mg(2+) and include a conformational-change step after nucleotide binding to achieve catalytic competence of the polymerase-primer/template-nucleotide complex. In the presence of Mn(2+), the conformational-change step is the primary determinant of enzyme specificity, phosphoryl transfer appears as the sole rate-limiting step for nucleotide incorporation, and the rate of phosphoryl transfer is the same for all nucleotides: correct and incorrect. Because phosphoryl transfer is the rate-limiting step in the presence of Mn(2+), it was possible to determine that the maximal phosphorothioate effect in this system is in the range of 8-11. This information permitted further interrogation of the nucleotide-selection process in the presence of Mg(2+), highlighting the capacity of this cation to permit the enzyme to use the phosphoryl-transfer step for nucleotide selection. The inability of Mn(2+) to support a reduction in the efficiency of phosphoryl transfer when incorrect substrates are employed is the primary explanation for the loss of fidelity observed in the presence of this cofactor. We propose that the conformational change involves reorientation of the triphosphate moiety of the bound nucleotide into a conformation that permits binding of the second metal ion required for catalysis. In the presence of Mg(2+), this conformation requires interactions with the enzyme that permit a reduction in catalytic efficiency to occur during an attempt to incorporate an incorrect nucleotide. Adventitious interactions in the cofactor-binding site with bound Mn(2+) may diminish fidelity by compensating for interaction losses used to modulate catalytic efficiency when incorrect nucleotides are bound in the presence of Mg(2+).     

Nucleotide binding to pig muscle 3-phosphoglycerate kinase in the crystal and in solution: relationship between substrate antagonism and interdomain communication.

Binding constants for the nucleotide substrates were determined in two different crystalline forms of pig muscle 3-phosphoglycerate kinase (PGK): the binary complex with 3-phosphoglycerate (3-PG) in which the two domains are in an open conformation (Harlos, Vas, and Blake (1992) Proteins, 12, 133-144) and the ternary complex with 3-PG and the Mg salt of the ATP analogue, beta,gamma-methyleneadenosine-5'-triphosphate (AMP-PCP), the structure of which is under resolution. Competitive titrations have been performed in the presence of the chromophoric analogue of ATP, 2'3'-O-(2,4,6-trinitrophenyl)ATP (TNP-ATP), similar to those previously carried out in solution, where a weakening of the binding of the nucleotide substrates in the presence of the other substrate, 3-PG, has been observed (Vas, Merli, and Rossi (1994) Biochem. J. 301, 885-891). Here the K(d) values for MgADP were found to be 0.096 +/- 0.021 and 0.045 +/- 0.016 mM, respectively, for the crystals of the binary and ternary complexes. Both K(d) values are significantly smaller than the one obtained in solution in the presence of 3-PG (0.38 +/- 0.05 mM) and are close to the values determined in solution in the absence of 3-PG (0.06 +/- 0.01 mM). Thus, the "substrate antagonism" observed in solution is not present in either of the investigated crystal forms. Further nucleotide binding studies with the solubilized enzyme have shown that 3-PG has no effect on ADP (Mg(2+)-free) binding (K(d) = 0.34 +/- 0.05 mM), while it weakens MgADP binding. Thus, 3-PG abolishes the strengthening effect of the Mg(2+) ion on the binding of ADP. This phenomenon is apparently due to the interaction between the carboxyl group of 3-PG and the protein, since the carboxyl-lacking analogue glycerol-3-phosphate has no detectable effect on MgADP binding. Comparison of the crystallographic data of different PGK binary (with either 3-PG or MgADP) and ternary (with both 3-PG and MgADP) complexes, having open and closed conformations, respectively, provides a possible structural explanation of the substrate antagonism. We suggest that the specific interaction between the 3-PG carboxylic group and a conserved arginine side chain is changed during domain closure, and, through interdomain communication, this change may be transmitted to the site in which Mg(2+) binds the ADP phosphates. This effect is abolished in the crystals of pig muscle PGK, in which lattice forces stabilize the open domain conformation.     

The role of Mg2+ cofactor in the guanine nucleotide exchange and GTP hydrolysis reactions of Rho family GTP-binding proteins

The biological activities of Rho family GTPases are controlled by their guanine nucleotide binding states in cells. Here we have investigated the role of Mg(2+) cofactor in the guanine nucleotide binding and hydrolysis processes of the Rho family members, Cdc42, Rac1, and RhoA. Differing from Ras and Rab proteins, which require Mg(2+) for GDP and GTP binding, the Rho GTPases bind the nucleotides in the presence or absence of Mg(2+) similarly, with dissociation constants in the submicromolar concentration. The presence of Mg(2+), however, resulted in a marked decrease in the intrinsic dissociation rates of the nucleotides. The catalytic activity of the guanine nucleotide exchange factors (GEFs) appeared to be negatively regulated by free Mg(2+), and GEF binding to Rho GTPase resulted in a 10-fold decrease in affinity for Mg(2+), suggesting that one role of GEF is to displace bound Mg(2+) from the Rho proteins. The GDP dissociation rates of the GTPases could be further stimulated by GEF upon removal of bound Mg(2+), indicating that the GEF-catalyzed nucleotide exchange involves a Mg(2+)-independent as well as a Mg(2+)-dependent mechanism. Although Mg(2+) is not absolutely required for GTP hydrolysis by the Rho GTPases, the divalent ion apparently participates in the GTPase reaction, since the intrinsic GTP hydrolysis rates were enhanced 4-10-fold upon binding to Mg(2+), and k(cat) values of the Rho GTPase-activating protein (RhoGAP)-catalyzed reactions were significantly increased when Mg(2+) was present. Furthermore, the p50RhoGAP specificity for Cdc42 was lost in the absence of Mg(2+) cofactor. These studies directly demonstrate a role of Mg(2+) in regulating the kinetics of nucleotide binding and hydrolysis and in the GEF- and GAP-catalyzed reactions of Rho family GTPases. The results suggest that GEF facilitates nucleotide exchange by destabilizing both bound nucleotide and Mg(2+), whereas RhoGAP utilizes the Mg(2+) cofactor to achieve high catalytic efficiency and specificity.     

Active site mutations in CheA, the signal-transducing protein kinase of the chemotaxis system in Escherichia coli.

We investigated the functional roles of putative active site residues in Escherichia coli CheA by generating nine site-directed mutants, purifying the mutant proteins, and quantifying the effects of those mutations on autokinase activity and binding affinity for ATP. We designed these mutations to alter key positions in sequence motifs conserved in the protein histidine kinase family, including the N box (H376 and N380), the G1 box (D420 and G422), the F box (F455 and F459), the G2 box (G470, G472, and G474), and the "GT block" (T499), a motif identified by comparison of CheA to members of the GHL family of ATPases. Four of the mutant CheA proteins exhibited no detectable autokinase activity (Kin(-)). Of these, three (N380D, D420N, and G422A) exhibited moderate decreases in their affinities for ATP in the presence or absence of Mg(2+). The other Kin(-) mutant (G470A/G472A/G474A) exhibited wild-type affinity for ATP in the absence of Mg(2+), but reduced affinity (relative to that of wild-type CheA) in the presence of Mg(2+). The other five mutants (Kin(+)) autophosphorylated at rates slower than that exhibited by wild-type CheA. Of these, three mutants (H376Q, D420E, and F455Y/F459Y) exhibited severely reduced k(cat) values, but preserved K(M)(ATP) and K(d)(ATP) values close to those of wild-type CheA. Two mutants (T499S and T499A) exhibited only small effects on k(cat) and K(M)(ATP). Overall, these results suggest that conserved residues in the N box, G1 box, G2 box, and F box contribute to the ATP binding site and autokinase active site in CheA, while the GT block makes little, if any, contribution. We discuss the effects of specific mutations in relation to the three-dimensional structure of CheA and to binding interactions that contribute to the stability of the complex between CheA and Mg(2+)-bound ATP in both the ground state and the transition state for the CheA autophosphorylation reaction.     

DNA binding mediates conformational changes and metal ion coordination in the active site of PcrA helicase.

Based upon the crystal structures of PcrA helicase, we have made and characterised mutations in a number of conserved helicase signature motifs around the ATPase active site. We have also determined structures of complexes of wild-type PcrA with ADPNP and of a mutant PcrA complexed with ADPNP and Mn2+. The kinetic and structural data define roles for a number of different residues in and around the ATP binding site. More importantly, our results also show that there are two functionally distinct conformations of ATP in the active site. In one conformation, ATP is hydrolysed poorly whereas in the other (activated) conformation, ATP is hydrolysed much more rapidly. We propose a mechanism to explain how the stimulation of ATPase activity afforded by binding of single-stranded DNA stabilises the activated conformation favouring Mg2+binding and a consequent repositioning of the gamma-phosphate group which promotes ATP hydrolysis. A part of the associated conformational change in the protein forces the side-chain of K37 to vacate the Mg2+binding site, allowing the cation to bind and interact with ATP.     

The role of magnesium ions in β-galactosidase-catalysed hydrolyses. Studies on charge and shape of the β-galactopyranosyl-binding site

1. beta-d-Galactopyranosyl trimethylammonium bromide is a competitive inhibitor of beta-galactosidase, K(i)=1.4+/-0.2mm at 25 degrees C. 2. Tetramethylammonium bromide is not an inhibitor (K(i)>0.2m). 3. The kinetics of deactivation of Mg(2+)-saturated, and of inhibitor-and Mg(2+)-saturated, enzyme in 10mm-EDTA are similar. 4. The apparent K(i) for the glycosylammonium salt is approx. 2.2mm in the absence of Mg(2+). 5. It is therefore concluded that Mg(2+) and the inhibitor bind independently, and that the Mg(2+) does not act as an electrophilic catalyst. 6. Complexant fluorescence measurements indicate binding of 1 Mg(2+) ion per 135000-dalton protomer. 7. This stoicheiometry is confirmed by equilibrium dialysis. 8. 1,6-Anhydrogalactopyranose is neither a substrate (k(cat.)/K(m)< 3x10(-2)m(-1).S(-1)) nor an inhibitor (K(i)>0.2m). 9. Considerations of conformations available to the cationic inhibitor and to the anhydrogalactose indicate that the substrate is bound with the pyranose ring in a conformation not greatly different from the normal chair (C1) conformation.     

Activation and inhibition of photoreceptor guanylyl cyclase by guanylyl cyclase activating protein 1 (GCAP-1): the functional role of Mg2+/Ca2+ exchange in EF-hand domains

Guanylyl cyclase activating protein 1 (GCAP-1), a Ca(2+)/Mg(2+) sensor protein that accelerates retinal guanylyl cyclase (RetGC) in the light and decelerates it in the dark, is inactive in cation-free form. Binding of Mg(2+) in EF-hands 2 and 3 was essential for RetGC activation in the conditions mimicking light adaptation. Mg(2+) binding in EF-hand 2 affected the conformation of a neighboring non-metal binding domain, EF-hand-1, and increased GCAP-1 affinity for RetGC nearly 40-fold compared with the metal-free EF-hand 2. Mg(2+) binding in EF-hand 3 increased GCAP-1 affinity for RetGC 5-fold and its maximal RetGC stimulation 2-fold. Mg(2+) binding in EF-hand 4 affected neither GCAP-1 affinity for RetGC, nor RetGC activation. Inactivation of Ca(2+) binding in EF-hand 4 was sufficient to render GCAP-1 a constitutive activator of RetGC, whereas the EF-hand 3 role in Ca(2+)-dependent deceleration of RetGC was likely to be through the neighboring EF-hand 4. Inactivation of Ca(2+) binding in EF-hand 2 affected cooperativity of RetGC inhibition by Ca(2+), but did not prevent the inhibition. We conclude that 1) Mg(2+) binding in EF-hands 2 and 3, but not EF-hand 4, is essential for the ability of GCAP-1 to activate RetGC in the light; 2) Mg(2+) or Ca(2+) binding in EF-hand 3 and especially in EF-hand 2 is required for high-affinity interaction with the cyclase and affects the conformation of the neighboring EF-hand 1, a domain required for targeting RetGC; and 3) RetGC inhibition is likely to be primarily caused by Ca(2+) binding in EF-hand 4.     

Membrane adenosine triphosphatase of Micrococcus lysodeikticus: effect of millimolar Mg2+ in modulating the properties of the membrane-bound enzyme

The latency of Micrococcus lysodeikticus membrane-bound Mg(2+)-adenosine triphosphatase (ATPase) is expressed by the ratio of its activity assayed in the presence of trypsin ("total") versus the activity assayed in absence of the protease ("basal"). By isolating membranes in the presence of variable concentrations of Mg(2+) (50 mM, 10 mM, or none) and by washing them with different Mg(2+)- and ethylenediaminetetraacetic acid-containing tris(hydroxymethyl)aminomethane-hydrochloride buffers (pH 7.5), we showed that the enzyme latency was dependent on the environmental concentration of this divalent metal ion. Mg(2+) bound to at least two classes of sites. The binding of Mg(2+) to low-affinity sites (saturation at approximately 40 mM external Mg(2+)) induced a high basal ATPase activity, whereas its binding to medium-affinity sites (saturation at about 2 mM Mg(2+)) correlated with low basal activity and a very high stimulation by trypsin. Membranes with tightly bound Mg(2+) (high affinity?) revealed an intermediate behavior for the latency of M. lysodeikticus ATPase. The Mg(2+)/Ca(2+) antagonism as activators of the membrane ATPase was not directly related to Mg(2+) binding by the membranes. The efficiency of the ATPase release from M. lysodeikticus membrane by 3 mM tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.5) was inversely proportional to the concentration of external and/or bound Mg(2+). Deoxycholate (DOC) (1%) solubilized the ATPase from all types of membrane. All the soluble ATPases behaved as Ca(2+)-ATPases, but the DOC-soluble fractions showed degrees of latency like those of the original membranes. The DOC-soluble ATPase preparation revealed a vesicular structure and complex protein patterns by sodium dodecyl sulfate gel electrophoresis. We propose that ATPase latency is modulated via a Mg(2+)-ATPase-membrane complex.     

PurT-encoded glycinamide ribonucleotide transformylase. Accommodation of adenosine nucleotide analogs within the active site

PurT-encoded glycinamide ribonucleotide transformylase, or PurT transformylase, functions in purine biosynthesis by catalyzing the formylation of glycinamide ribonucleotide through a catalytic mechanism requiring Mg(2+)ATP and formate. From previous x-ray diffraction analyses, it has been demonstrated that PurT transformylase from Escherichia coli belongs to the ATP-grasp superfamily of enzymes, which are characterized by three structural motifs referred to as the A-, B-, and C-domains. In all of the ATP-grasp enzymes studied to date, the adenosine nucleotide ligands are invariably wedged between the B- and C-domains, and in some cases, such as biotin carboxylase and carbamoyl phosphate synthetase, the B-domains move significantly upon nucleotide binding. Here we present a systematic and high-resolution structural investigation of PurT transformylase complexed with various adenosine nucleotides or nucleotide analogs including Mg(2+)ATP, Mg(2+)-5'-adenylylimidodiphosphate, Mg(2+)-beta,gamma-methyleneadenosine 5'-triphosphate, Mg(2+)ATPgammaS, or Mg(2+)ADP. Taken together, these studies indicate that the conformation of the so-called "T-loop," delineated by Lys-155 to Gln-165, is highly sensitive to the chemical identity of the nucleotide situated in the binding pocket. This sensitivity to nucleotide identity is in sharp contrast to that observed for the "P-loop"-containing enzymes, in which the conformation of the binding motif is virtually unchanged in the presence or absence of nucleotides.     

Nucleotide binding to DNA gyrase causes loss of DNA wrap

DNA gyrase negatively supercoils DNA in a reaction coupled to the binding and hydrolysis of ATP. Limited supercoiling can be achieved in the presence of the non-hydrolysable ATP analogue, 5'-adenylyl beta,gamma-imidodiphosphate (ADPNP). In order to negatively supercoil DNA, gyrase must wrap a length of DNA around itself in a positive sense. In previous work, the effect of ADPNP on the gyrase-DNA interaction has been assessed but has produced conflicting results; the aim of this work was to resolve this conflict. We have probed the wrapping of DNA around gyrase in the presence and in the absence of ADPNP using direct observation by atomic force microscopy (AFM). We confirm that gyrase indeed generates a significant curvature in DNA in the absence of nucleotide and we show that the addition of ADPNP leads to a complete loss of wrap. These results have been corroborated using a DNA relaxation assay involving topoisomerase I. We have re-analysed previous hydroxyl-radical footprinting and crystallography data, and highlight the fact that the gyrase-DNA complex is surprisingly asymmetric in the absence of nucleotide but is symmetric in the presence of ADPNP. We suggest a revised model for the conformation of DNA bound to the enzyme that is fully consistent with these AFM data, in which a closed loop of DNA is stabilised by the enzyme in the absence of ADPNP and is lost in the presence of nucleotide.     

X-ray Structures of Magnesium and Manganese Complexes with the N-Terminal Domain of Calmodulin: Insights into the Mechanism and Specificity of Metal Ion Binding to an EF-Hand

Calmodulin (CaM), a member of the EF-hand superfamily, regulates many aspects of cell function by responding specifically to micromolar concentrations of Ca(2+) in the presence of an ～1000-fold higher concentration of cellular Mg(2+). To explain the structural basis of metal ion binding specificity, we have determined the X-ray structures of the N-terminal domain of calmodulin (N-CaM) in complexes with Mg(2+), Mn(2+), and Zn(2+). In contrast to Ca(2+), which induces domain opening in CaM, octahedrally coordinated Mg(2+) and Mn(2+) stabilize the closed-domain, apo-like conformation, while tetrahedrally coordinated Zn(2+) ions bind at the protein surface and do not compete with Ca(2+). The relative positions of bound Mg(2+) and Mn(2+) within the EF-hand loops are similar to those of Ca(2+); however, the Glu side chain at position 12 of the loop, whose bidentate interaction with Ca(2+) is critical for domain opening, does not bind directly to either Mn(2+) or Mg(2+), and the vacant ligand position is occupied by a water molecule. We conclude that this critical interaction is prevented by specific stereochemical constraints imposed on the ligands by the EF-hand β-scaffold. The structures suggest that Mg(2+) contributes to the switching off of calmodulin activity and possibly other EF-hand proteins at the resting levels of Ca(2+). The Mg(2+)-bound N-CaM structure also provides a unique view of a transiently bound hydrated metal ion and suggests a role for the hydration water in the metal-induced conformational change.