Structure-function relationships in Anabaena ferredoxin: correlations between X-ray crystal structures, reduction potentials, and rate constants of electron transfer to ferredoxin:NADP+ reductase for site-specific ferredoxin mutants

A combination of structural, thermodynamic, and transient kinetic data on wild-type and mutant Anabaena vegetative cell ferredoxins has been used to investigate the nature of the protein-protein interactions leading to electron transfer from reduced ferredoxin to oxidized ferredoxin:NADP+ reductase (FNR). We have determined the reduction potentials of wild-type vegetative ferredoxin, heterocyst ferredoxin, and 12 site-specific mutants at seven surface residues of vegetative ferredoxin, as well as the one- and two-electron reduction potentials of FNR, both alone and in complexes with wild-type and three mutant ferredoxins. X-ray crystallographic structure determinations have been carried out for six of the ferredoxin mutants. None of the mutants showed significant structural changes in the immediate vicinity of the [2Fe-2S] cluster, despite large decreases in electron-transfer reactivity (for E94K and S47A) and sizable increases in reduction potential (80 mV for E94K and 47 mV for S47A). Furthermore, the relatively small changes in Calpha backbone atom positions which were observed in these mutants do not correlate with the kinetic and thermodynamic properties. In sharp contrast to the S47A mutant, S47T retains electron-transfer activity, and its reduction potential is 100 mV more negative than that of the S47A mutant, implicating the importance of the hydrogen bond which exists between the side chain hydroxyl group of S47 and the side chain carboxyl oxygen of E94. Other ferredoxin mutations that alter both reduction potential and electron-transfer reactivity are E94Q, F65A, and F65I, whereas D62K, D68K, Q70K, E94D, and F65Y have reduction potentials and electron-transfer reactivity that are similar to those of wild-type ferredoxin. In electrostatic complexes with recombinant FNR, three of the kinetically impaired ferredoxin mutants, as did wild-type ferredoxin, induced large (approximately 40 mV) positive shifts in the reduction potential of the flavoprotein, thereby making electron transfer thermodynamically feasible. On the basis of these observations, we conclude that nonconservative mutations of three critical residues (S47, F65, and E94) on the surface of ferredoxin have large parallel effects on both the reduction potential and the electron-transfer reactivity of the [2Fe-2S] cluster and that the reduction potential changes are not the principal factor governing electron-transfer reactivity. Rather, the kinetic properties are most likely controlled by the specific orientations of the proteins within the transient electron-transfer complex.     

Characterization of D10S and K71E mutants of human cytosolic hsp70

To determine the effect of mutations at the nucleotide-binding site of recombinant Hsp70 on its interaction with protein and peptide substrates, point mutations were made at D10 and K71, two residues at the active site. The D10S mutation weakened both ATP and ADP binding, while the K71E mutation weakened only ATP binding. In binding experiments using Hsp70 with no bound nucleotide, the mutated Hsp70s interacted with clathrin and peptide just like the wild-type Hsp70. However, the D10 mutation completely abolished the effects of both ATP and ADP on peptide and clathrin binding. The K71 mutation also abolished the effect of ATP on substrate binding, but ADP, which still bound tightly, had its normal effect on substrate binding. In addition, the D10S and K71E mutants had greatly reduced ability to uncoat clathrin-coated vesicles at pH 7.0, bind to clathrin baskets at pH 6.0, and undergo polymerization induced by YDJ1 in the presence of ATP. We conclude, first, that nucleotides must bind strongly to Hsp70 to affect substrate binding and, second, that interaction of Hsp70 with DnaJ homologues may also require a strongly bound ATP.     

Involvement of two aspartate residues of Rubisco activase in coordination of the ATP gamma-phosphate and subunit cooperativity

Aspartate residues are involved in coordination of the nucleotide-metal of several nucleotide triphosphatases. To examine interactions between Rubisco activase and ATP, site-directed mutations were made at two species-invariant aspartate residues, D174 and D231. In the absence of the magnesium cofactor, the mutant proteins D231R, D174Q, and D174A, but not D174E, bound ATP with higher affinity than did wild-type. In the presence of Mg2+, the affinity for ATP of D231R was further increased, but was reduced with mutations at D174. Although all mutants bound ATP, only D174E aggregated in response to ATP/Mg2+ and retained partial ATPase and Rubisco activation activities. In mixing experiments, the catalytically competent D174E stimulated wild-type ATPase activity, whereas the mutants lacking ATPase activity were inhibitory to wild-type enzyme and prevented aggregation. These results are consistent with a mechanism for activase that involves ATP-binding, subunit aggregation and ATP hydrolysis as sequential steps in the catalytic mechanism. The results also indicated that precise coordination of the gamma-phosphate is required for aggregation and depends on D174 and D231. To account for the pronounced cooperativity of Rubisco activase subunits, we suggest that coordination of the ATP gamma-phosphate may involve participation of residues from adjacent subunits.     

Site-directed mutagenesis clarifies the substrate position within the three-dimensional model of the active site of herpes simplex virus type-1 thymidine kinase

Site-directed mutagenesis was used to experimentally verify the 3D model of the active site of herpes simplex virus type-1 thymidine kinase (HSV 1 TK) obtained by homology modelling. For this purpose, D215 and K317 were replaced by R and G, respectively, at homologous positions in the aciclovir-insensitive bovine herpes virus type-1 thymidine kinase (BHV 1 TK). Wild-type and mutated enzymes were expressed in Escherichia coli using a gene fusion vector and purified to homogeneity. While both mutants had the same Km value for thymidine as the recombinant wild-type enzyme (0.2 microM), Vmax was decreased to 20-25% of the original wild-type value. The recombinant wild-type enzyme was inhibited by the substrate analogue aciclovir with a Ki of 146 microM. Both mutants were able to phosphorylate aciclovir to about the same extent as the wild-type enzyme. These findings suggest that neither D215 nor K317 are directly involved in substrate binding. Therefore, a rearrangement of the 3D model is suggested, concerning the assignment of the substrate-binding site and co-substrate-binding site at the right and left side of the phosphate-binding loop, respectively.     

Relationship of conserved residues in the IMP binding site to substrate recognition and catalysis in Escherichia coli adenylosuccinate synthetase

Gln34, Gln224, Leu228, and Ser240 are conserved residues in the vicinity of bound IMP in the crystal structure of Escherichia coli adenylosuccinate synthetase. Directed mutations were carried out, and wild-type and mutant enzymes were purified to homogeneity. Circular dichroism spectroscopy indicated no difference in secondary structure between the mutants and the wild-type enzyme in the absence of substrates. Mutants L228A and S240A exhibited modest changes in their initial rate kinetics relative to the wild-type enzyme, suggesting that neither Leu228 nor Ser240 play essential roles in substrate binding or catalysis. The mutants Q224M and Q224E exhibited no significant change in KmGTP and KmASP and modest changes in KmIMP relative to the wild-type enzyme. However, kcat decreased 13-fold for the Q224M mutant and 10(4)-fold for the Q224E mutant relative to the wild-type enzyme. Furthermore, the Q224E mutant showed an optimum pH at 6.2, which is 1.5 pH units lower than that of the wild-type enzyme. Tryptophan emission fluorescence spectra of Q224M, Q224E, and wild-type proteins under denaturing conditions indicate comparable stabilities. Mutant Q34E exhibits a 60-fold decrease in kcat compared with that of the wild-type enzyme, which is attributed to the disruption of the Gln34 to Gln224 hydrogen bond observed in crystal structures. Presented here is a mechanism for the synthetase, whereby Gln224 works in concert with Asp13 to stabilize the 6-oxyanion of IMP.     

Topological and functional relationship of subunits F1-gamma and F0I-PVP(b) in the mitochondrial H+-ATP synthase

Diamide treatment of the F0F1-ATP synthase in "inside out" submitochondrial particles (ESMP) in the absence of a respiratory Delta mu H+ as well as of isolated Fo reconstituted with F1 or F1-gamma subunit results in direct disulfide cross-linking between cysteine 197 in the carboxy-terminal region of the F0I-PVP(b) subunit and cysteine 91 at the carboxyl end of a small alpha-helix of subunit F1-gamma, both located in the stalk. The F0I-PVP(b) and F1-gamma cross-linking cause dramatic enhancement of oligomycin-sensitive decay of Delta mu H+. In ESMP and MgATP particles the cross-linking is accompanied by decoupling of respiratory ATP synthesis. These effects are consistent with the view that F0I-PVP(b) and F1-gamma are components of the stator and rotor of the proposed rotary motor, respectively. The fact that the carboxy-terminal region of F0I-PVP(b) and the short alpha-helix of F1-gamma can form a direct disulfide bridge shows that these two protein domains are, at least in the resting state of the enzyme, in direct contact. In isolated F0, diamide also induces cross-linking of OSCP with another subunit of F0, but this has no significant effect on proton conduction. When ESMP are treated with diamide in the presence of Delta mu H+ generated by respiration, neither cross-linking between F0I-PVP(b) and F1-gamma subunits nor the associated effects on proton conduction and ATP synthesis is observed. Cross-linking is restored in respiring ESMP by Delta mu H+ collapsing agents as well as by DCCD or oligomycin. These observations indicate that the torque generated by Delta mu H+ decay through Fo induces a relative motion and/or a separation of the F0I-PVP(b) subunit and F1-gamma which places the single cysteine residues, present in each of the two subunits, at a distance at which they cannot be engaged in disulfide bridging.     

Functional analysis of conserved domains in the phosphotyrosyl phosphatase activator. Molecular cloning of the homologues from Drosophila melanogaster and Saccharomyces cerevisiae

Phosphotyrosyl phosphatase activator (PTPA), a 37 kDa cytosolic protein that specifically activates the phosphotyrosyl phosphatase activity of the dimeric form of PP2A, was cloned from Drosophila melanogaster and Saccharomyces cerevisiae. Sequence alignment of PTPA from yeast to human revealed highly conserved regions including the type B fragment of the putative PTPA ATP binding site. We generated PTPA deletion mutants of these conserved regions as well as point mutations within regions that were suggested to be functionally important. The recombinant proteins were expressed in E. coli and subsequently purified. Activity measurements, linked with immunological detection, revealed that most of the well-conserved regions are essential for PTPA activity. However, neither the type A fragment of the putative ATP binding site nor the cysteine-rich region, present in all but the Drosophila and yeast homologues, appeared to be essential for PTPA activity. Moreover, we observed that PTPA truncated at glycine266 behaves as a dominant negative mutant since it is inhibitory to the wild-type PTPA.     

Education for the nurse of tomorrow: a community-focused curriculum

A competitive enzyme-linked immunoadsorbent assay (ELISA) technique has been developed to facilitate quantitative analysis of the earliest step in the initiation of the extrinsic pathway of coagulation, i.e., complex formation of factor VII/VIIa with tissue factor. The ELISA measures the binding of biotinylated human plasma factor VII to relipidated recombinant human tissue factor. Quantitation of the relative affinity (expressed as IC50) of any factor VII molecular population or structural analogue for tissue factor can be determined by competitive binding. Subnanomolar concentrations of both wild-type recombinant human factor VII (rFVII) and rFVII(R152Q), a mutation at the FVII activation site, competed effectively with biotinylated plasma-derived factor VII in binding to tissue factor. In contrast, the affinity of rFVII(R79Q), a mutation in the first epidermal growth factor-like domain, was 12-fold lower. Following activation of rFVII(R79Q), its affinity for tissue factor and enzymatic activity increased 4-fold and 6-fold, respectively. For wild-type rFVII, enzymatic activity rose significantly following activation. However, its affinity for tissue factor was unchanged. We conclude that both the activation state of factor VII and the mutation of amino-acid residues within the first epidermal growth factor-like domain may alter the affinity of factor VII for tissue factor.     

Structural changes in insulin-like growth factor (IGF) I mutant proteins affecting binding kinetic rates to IGF binding protein 1 and IGF-I receptor

Ligand binding properties of five single amino acid substituted variants (V11A, D12A, Q15A, Q15E, and F16A) of human insulin-like growth factor I (IGF-I) were analyzed with respect to their binding affinities and binding kinetics to recombinant IGF binding protein 1 (IGFBP-1) and a soluble form of the IGF type I receptor (sIGF-I(R)), respectively. Side chains of the substituted residues are all predicted to be the most surface exposed in the alpha-helical portion of the B-region of the IGF-I molecule. The IGF-I variants were produced as fusion proteins to a IgG(Fc) binding protein domain, Z. Ligand binding kinetic rates were determined using BIAcore biosensor interaction analysis technology. All IGF-I variants showed altered binding affinities to both IGFBP- I and sIGF-I(R). Secondary structure content of the IGF-I variants was estimated using far-UV circular dichroism spectroscopy, followed by variable selection secondary structure calculations. The amount of calculated alpha-helicity is reduced for all the mutants, most predominantly for IGF-I(V11A) and IGF-I(F16A) proteins. Surprisingly, most of the effects of reduced binding affinities to both target proteins are attributed to lowered on-rates of binding, and these are correlated with the amount of alpha-helicity in each IGF-I variant. In addition, in some of the IGF-I variants, lowered off-rates of binding are observed. From the results, we propose that IGF-I is unusually sensitive to structural changes by surface amino acid substitutions in the B-region of the molecule. Therefore, biochemical or biological properties of amino acid substituted variants of IGF-I cannot be used in a straightforward way to dissect the direct involvement in binding of individual amino acid residues since structural changes may be involved.     

Role of electrostatic interactions in defining the potency of neurotoxin B-IV from Cerebratulus lacteus

Chemical modification implicates arginine residues of the Cerebratulus lacteus neurotoxin B-IV in biological activity. In the present study, we used site-directed mutagenesis to assess the functional contributions of each of these residues. Panels of mutants at each site have been constructed by polymerase chain reaction and recombinant proteins expressed and purified to homogeneity using an Escherichia coli expression system developed in this laboratory. All substitutions for Arg-17 (Gln, Ala, or Lys) yield proteins having undetectable levels of activity, while charge neutralizing replacement of Arg-25 (R25Q) causes a 400-fold reduction in specific toxicity. However, the R25K mutein is almost as active as natural toxin. Circular dichroism spectroscopy indicates that there are no major conformational changes in any of these muteins. These results therefore demonstrate the requirement for a guanidinium group at position 17, and a positive charge at position 25. NMR analyses (Hansen, P. E., Kem, W. R., Bieber, A. L., and Norton, R. S. (1992) Eur. J. Biochem. 210, 231-240) reveal neurotoxin B-IV to contain two antiparallel alpha-helices, which together include 57% of the sequence. Both Arg-17 and Arg-25 lie on the same face of the N-terminal helix (residues 13-26), as do the carboxyl groups of Glu-13 and Asp-21. However, charge neutralizing mutations of the latter two sites have no effects on biological activity. Arg-34, situated near the N terminus of helix 2 (residues 33-49) is also important for activity, as its replacement by Gln or Ala diminishes activity by 20- and 80-fold, respectively. However, unlike Arg-17 and Arg-25, thermal denaturation experiments suggest that R34Q may be structurally destabilized relative to wild-type B-IV.     

Structural cues involved in endoplasmic reticulum degradation of G85E and G91R mutant cystic fibrosis transmembrane conductance regulator

Abnormal folding of mutant cystic fibrosis transmembrane conductance regulator (CFTR) and subsequent degradation in the endoplasmic reticulum is the basis for most cases of cystic fibrosis. Structural differences between wild-type (WT) and mutant proteins, however, remain unknown. Here we examine the intracellular trafficking, degradation, and transmembrane topology of two mutant CFTR proteins, G85E and G91R, each of which contains an additional charged residue within the first putative transmembrane helix (TM1). In microinjected Xenopus laevis oocytes, these mutations markedly disrupted CFTR plasma membrane chloride channel activity. G85E and G91R mutants (but not a conservative mutant, G91A) failed to acquire complex N-linked carbohydrates, and were rapidly degraded before reaching the Golgi complex thus exhibiting a trafficking phenotype similar to DeltaF508 CFTR. Topologic analysis revealed that neither G85E nor G91R mutations disrupted CFTR NH2 terminus transmembrane topology. Instead, WT as well as mutant TM1 spanned the membrane in the predicted C-trans (type II) orientation, and residues 85E and 91R were localized within or adjacent to the plane of the lipid bilayer. To understand how these charged residues might provide structural cues for ER degradation, we examined the stability of WT, G85E, and G91R CFTR proteins truncated at codons 188, 393, 589, or 836 (after TM2, TM6, the first nucleotide binding domain, or the R domain, respectively). These results indicated that G85E and G91R mutations affected CFTR folding, not by gross disruption of transmembrane assembly, but rather through insertion of a charged residue within the plane of the bilayer, which in turn influenced higher order tertiary structure.     

Identification of the substrate and pseudosubstrate binding sites of phosphorylase kinase gamma-subunit

Using site-directed mutagenesis, we proposed that an autoinhibitory domain(s) is located at the C-terminal region (301-386) of the phosphorylase kinase gamma-subunit (Huang, C.-Y.F., Yuan C.-J., Livanova, N.B., and Graves, D.J. (1993) Mol. Cell. Biochem. 127/128, 7-18). Removal of the putative inhibitory domain(s) by truncation results in the generation of a constitutively active and calmodulin-independent form, gamma 1-300. To probe the structural basis of autoinhibition of gamma-subunit activity, two synthetic peptides, PhK13 (gamma 303-327) and PhK5 (gamma 343-367), corresponding to the two calmodulin-binding regions, were assayed for their ability to inhibit gamma 1-300. Competitive inhibition of gamma 1-300 by PhK13 was found versus phosphorylase b (Ki = 1.8 microM) and noncompetitive inhibition versus ATP. PhK5 showed noncompetitive inhibition with respect to both phosphorylase b and ATP. Calmodulin released the inhibition caused by both peptides. These results indicate that there are two distinct auto-inhibitory domains within the C terminus of the gamma-subunit and that these two domains overlap with the calmodulin-binding regions. Two mutant forms of gamma 1-300, E111K and E154R, were used to probe the enzyme-substrate-binding region using peptide substrate analogs corresponding to residues 9-18 of phosphorylase b (KRK11Q12ISVRGL). The data suggest that Glu111 interacts with the P-3 position of the substrate (Lys11) and Glu154 interacts with the P-2 site (Gln12). Both E111K and E154R were competitively inhibited with respect to phosphorylase b by PhK13, with 14- and 8-fold higher Ki values, respectively, than that observed with the wild-type enzyme. These data are consistent with a model for the regulation of the gamma-subunit of phosphorylase kinase in which PhK13 acts as a competitive pseudosubstrate that directly binds the substrate binding site of the gamma-subunit (Glu111 and Glu154).     

Identification of the cilium binding epitope of the Mycoplasma hyopneumoniae P97 adhesin

Mycoplasma hyopneumoniae colonizes the swine respiratory tract at the level of ciliated cells by attaching specifically to the cilium membrane. This interaction involves an adhesin called P97; the cilium binding activity of this protein was localized to the carboxy terminus, which included two repeat regions, R1 and R2 (T. Hsu, S. Artiushin, and F. C. Minion, J. Bacteriol. 179:1317-1323, 1997). To further delineate the molecular mechanisms of M. hyopneumoniae interactions with ciliated epithelium, we used a bank of transposon inserts in the cloned P97 gene to identify the site for cilium binding by testing the truncated gene products in an in vitro microtiter plate adherence assay. These studies showed that the cilium binding site was located in the AAKPV(E) repeat sequence of P97, referred to as the R1 repeat. For functional binding, at least seven AAKPV(E) repeats were required. The adherence-blocking monoclonal antibody F1B6 also recognized this region but required fewer AAKPV(E) repeats for recognition. We then constructed R1 region-lacZ gene fusions and used the resulting R1 repeat-beta-galactosidase fusion proteins in an in vitro assay to confirm the role of R1 in cilium binding. A comparison of the R1 regions of M. hyopneumoniae strains displaying variation in cilium adherence failed to identify changes that could account for the differences in adherence shown by the strains. Thus, we concluded that other proteins, in addition to P97, must be involved in cilium adherence, possibly in combination with P97.     

Mutations in the activation loop tyrosine of the oncoprotein v-Fps

Mutations were made in the activation loop tyrosine of the kinase domain of the oncoprotein v-Fps to assess the role of autophosphorylation in catalysis. Three mutant proteins, Y1073E, Y1073Q, and Y1073F, were expressed and purified as fusion proteins of glutathione-S-transferase from Escherichia coli and their catalytic properties were evaluated. Y1073E, Y1073Q, and Y1073F have k(cat) values that are reduced by 5-, 35-, and 40-fold relative to the wild-type enzyme, respectively. For all mutant enzymes, the Km values for ATP and a peptide substrate, EAEIYEAIE, are changed by 0.4-2-fold compared to the wild-type enzyme. The slopes for the plots of relative turnover versus solvent viscosity [(k(cat))eta] are 0.71 +/- 0.08, 0.10 +/- 0.06, and approximately 0 for wild type, Y1073Q, and Y1073E, respectively. These results imply that the phosphoryl transfer rate constant is reduced by 19- and 130-fold for Y1073E and Y1073Q compared to the wild-type enzyme. The dissociation constant of the substrate peptide is 1.5-2.5-fold lower for the mutants compared to wild type. The inhibition constant for EAEIFEAIE, a competitive inhibitor, is unaffected for Y1073E and raised 3-fold for Y1073Q compared to wild type. Y1073E and Y1073Q are strongly activated by free magnesium to the same extent and the apparent affinity constant for the metal is similar to that for the wild-type enzyme. The data indicate that the major role of autophosphorylation in the tyrosine kinase domain of v-Fps is to increase the rate of phosphoryl transfer without greatly affecting active-site accessibility or the local environment of the activating metal. Finally, the similar rate enhancements for phosphoryl transfer in v-Fps compared to protein kinase A [Adams et al. (1995) Biochemistry 34, 2447-2454] upon autophosphorylation suggest a conserved mechanism for communication between the activation loop and the catalytic residues of these two enzymes.     

American Board of Emergency Medicine Longitudinal Study of Emergency Physicians

Mutant adenylosuccinate lyases of Bacillus subtilis were prepared by site-directed mutagenesis with replacements for His141, previously identified by affinity labeling as being in the active site [Lee, T. T., Worby, C., Dixon, J. E., and Colman, R. F. (1997) J. Biol. Chem. 272, 458-465]. Four substitutions (A, L, E, Q) yield mutant enzyme with no detectable catalytic activity, while the H141R mutant is about 10(-)5 as active as the wild-type enzyme. Kinetic studies show, for the H141R enzyme, a Km that is only 3 times that of the wild-type enzyme. Minimal activity was also observed for mutant enzymes with replacements for His68 [Lee, T. T., Worby, C., Bao, Z. -Q., Dixon, J. E., and Colman, R. F. (1998) Biochemistry 37, 8481-8489]. Measurement of the reversible binding of radioactive adenylosuccinate by inactive mutant enzymes with substitutions at either position 68 or 141 shows that their affinities for substrate are decreased by only 10-40-fold. These results suggest that His141, like His68, plays an important role in catalysis, but not in substrate binding. Evidence is consistent with the hypothesis that His141 and His68 function, respectively, as the catalytic base and acid. Circular dichroism spectroscopy and gel filtration chromatography conducted on wild-type and all His141 and His68 mutants reveal that none of the mutant enzymes exhibits major structural changes and that all the enzymes are tetramers. Mixing inactive His141 with inactive His68 mutant enzymes leads to striking increases in catalytic activity. This complementation of mutant enzymes indicates that His141 and His68 come from different subunits to form the active site. A tetrameric structure of adenylosuccinate lyase was constructed by homology modeling based on the known structures in the fumarase superfamily, including argininosuccinate lyase, delta-crystallin, fumarase, and aspartase. The model suggests that each active site is constituted by residues from three subunits, and that His141 and His68 come from two different subunits.     

Molecular distance measurements reveal an (alpha beta)2 dimeric structure of Na+/K+-ATPase. High affinity ATP binding site and K+-activated phosphatase reside on different alpha-subunits

ATP hydrolysis by Na+/K+-ATPase proceeds via the interaction of simultaneously existing and cooperating high (E1ATP) and low (E2ATP) substrate binding sites. It is unclear whether both ATP sites reside on the same or on different catalytic alpha-subunits. To answer this question, we looked for a fluorescent label for the E2ATP site that would be suitable for distance measurements by F?rster energy transfer after affinity labeling of the E1ATP site by fluorescein 5'-isothiocyanate (FITC). Erythrosin 5'-isothiocyanate (ErITC) inactivated, in an E1ATP site-blocked enzyme (by FITC), the residual activity of the E2ATP site, namely K+-activated p-nitrophenylphosphatase in a concentration-dependent way that was ATP-protectable. The molar ratios of FITC/alpha-subunit of 0.6 and of ErITC/alpha-subunit of 0.48 indicate 2 ATP sites per (alpha beta)2 diprotomer. Measurements of F?rster energy transfer between the FITC-labeled E1ATP and the ErITC-labeled or Co(NH3)4ATP-inactivated E2ATP sites gave a distance of 6.45 +/- 0.64 nm. This distance excludes 2 ATP sites per alpha-subunit since the diameter of alpha is 4-5 nm. F?rster energy transfer between cardiac glycoside binding sites labeled with anthroylouabain and fluoresceinylethylenediamino ouabain gave a distance of 4.9 +/- 0.5 nm. Hence all data are consistent with the hypothesis that Na+/K+-ATPase in cellular membranes is an (alpha beta)2 diprotomer and works as a functional dimer (Thoenges, D., and Schoner, W. (1997) J. Biol. Chem. 272, 16315-16321).     

Interactions of the amino acid residue at position 31 of the c-Ha-Ras protein with Raf-1 and RalGDS

The Ras and Rap1A proteins can bind to the Raf and RalGDS families. Ras and Rap1A have Glu and Lys, respectively, at position 31. In the present study, we analyzed the effects of mutating the Glu at position 31 of the c-Ha-Ras protein to Asp, Ala, Arg, and Lys on the interactions with Raf-1 and RalGDS. The Ras-binding domain (RBD) of Raf-1 binds the E31R and E31K Ras mutants less tightly than the wild-type, E31A, and E31D Ras proteins; the introduction of the positively charged Lys or Arg residue at position 31 specifically impairs the binding of Ras with the Raf-1 RBD. On the other hand, the ability of the oncogenic RasG12V protein to activate Raf-1 in HEK293 cells was only partially reduced by the E31R mutation but was drastically impaired by the E31K mutation. Correspondingly, RasG12V(E31K) as well as Rap1A, but not RasG12V(E31R), exhibited abnormally tight binding with the cysteine-rich domain of Raf-1. On the other hand, the E31A, E31R, and E31K mutations, but not the E31D mutation, enhanced the RalGDS RBD-binding activity of Ras, indicating that the negative charge at position 31 of Ras is particularly unfavorable to the interaction with the RalGDS RBD. RasG12V(E31K), RasG12V(E31A), and Rap1A stimulate the RalGDS action more efficiently than the wild-type Ras in the liposome reconstitution assay. All of these results clearly show that the sharp contrast between the characteristics of Ras and Rap1A, with respect to the interactions with Raf-1 and RalGDS, depends on their residues at position 31.     

Location of the phosphate binding site within Clostridium symbiosum pyruvate phosphate dikinase

Pyruvate phosphate dikinase (PPDK) catalyzes the interconversion of ATP, Pi, and pyruvate with AMP, PPi, and PEP in three partial reactions: (1) E + ATP --> E.ATP --> E-PP.AMP, (2) E-PP.AMP + Pi --> E-PP.AMP.Pi --> E-P.AMP.PPi, and (3) E-P + pyruvate --> E-P.pyruvate --> E.PEP. The Clostridium symbiosum PPDK structure consists of N-terminal, central, and C-terminal domains. The N-terminal and central domains catalyze partial reactions 1 and 2 whereas the C-terminal and central domains catalyze partial reaction 3. The goal of the present work is to determine where on the N-terminal domain catalysis of partial reactions 1 and 2 occurs and, in particular, where the Pi binding site is located. Computer modeling studies implicated Arg337 as a key residue for Pi binding. This role was tested by site-directed mutagenesis. The R337A PPDK was shown to be impaired in catalysis of the forward (kcat 300-fold lower) and reverse (kcat 30-fold lower) full reactions. Time courses for the single turnover reactions were measured to show that catalysis of partial reaction 1 is 5-fold slower in the mutant, catalysis of the second partial reaction is 140-fold slower in the mutant, and catalysis of the third partial reaction is unaffected. With the exception of the mutation site, the crystal structure of the R337A PPDK closely resembles the structure of the wild-type protein. Thus, the altered kinetic properties observed for this mutant are attributed solely to the elimination of the interaction between substrate and the guanidinium group of the Arg337 side chain. On the basis of these findings we propose that the Pi binding site is located within the crevice of the PPDK N-terminal domain, at a site that is flanked by the ATP beta-P and the Mg2+ cofactor.     

ATPase activity and molecular chaperone function of the stress70 proteins

The codons for the amino acid residues making up the proposed ATP-binding sites of the maize (Zea mays L.) endoplasmic reticulum and tomato (Lycopersicon esculentum) cytoplasmic Stress70 proteins were deleted from their respective cDNAs. The deletions had little effect on the predicted secondary structure characteristics of the encoded proteins. Both wild-type and mutant proteins were expressed in Escherichia coli and purified to electrophoretic homogeneity. The mutant recombinant proteins did not bind to immobilized ATP columns, had no detectable ATPase activity, and were unable to function in vitro as molecular chaperones. Additionally, the inability to bind ATP was associated with changes in the oligomerization state of the Stress70 proteins.