ATP-Binding site of annexin VI characterized by photochemical release of nucleotide and infrared difference spectroscopy.

Structural changes induced by nucleotide binding to porcine liver annexin VI (AnxVI) were probed by reaction-induced difference spectroscopy (RIDS). Photorelease of the nucleotide from ATP[Et(PhNO2)] produced RIDS of AnxVI characterized by reproducible changes in the amide I region. The magnitude of the infrared change was comparable to RIDS of other ATP-binding proteins, such as Ca(2+)-ATPase and creatine and arginine kinases. Analysis of RIDS revealed the existence of ATP-binding site(s) (K(d) < 1 microM) within the AnxVI molecule, comprising five to six amino acid residues located in the C-terminal portion of the protein molecule. The binding stoichiometry of ATP:AnxVI was determined as 1:1 (mol/mol). ATP, in the presence of Ca2+, induced changes in protein secondary structure reflected by a 5% decrease in alpha-helix content of the protein in favor of unordered structure. Such changes may influence the affinity of AnxVI for Ca2+ and modulate its interaction with membranes.     

A role for CBS domain 2 in trafficking of chloride channel CLC-5

CLC-5 is a member of the CLC family of voltage-gated chloride channels. Mutations disrupting CLC-5 lead to Dent's disease, an X-linked renal tubular disorder, characterised by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis, and renal stones. Sequence analysis of CLC-5 reveals a 746 amino acid protein with an intracellular amino-terminus, transmembrane spanning domains, and two CBS domains within its intracellular carboxy-terminus. CBS domains have been implicated in intracellular targetting and trafficking as well as protein-protein interactions. We investigate subcellular localisation of three naturally occurring CLC-5 mutants which all lead to a truncated protein, disrupting the second CBS domain. These mutants are unable to traffic normally to acidic endosomes but are retained in perinuclear compartments, colocalising with the Golgi complex. This is the first identification of the cellular pathogenesis of CBS domain mutations of CLC-5.     

Introducing Wilson disease mutations into the zinc-transporting P-type ATPase of Escherichia coli. The mutation P634L in the 'hinge' motif (GDGXNDXP) perturbs the formation of the E2P state.

ZntA, a bacterial zinc-transporting P-type ATPase, is homologous to two human ATPases mutated in Menkes and Wilson diseases. To explore the roles of the bacterial ATPase residues homologous to those involved in the human diseases, we have introduced several point mutations into ZntA. The mutants P401L, D628A and P634L correspond to the Wilson disease mutations P992L, D1267A and P1273L, respectively. The mutations D628A and P634L are located in the C-terminal part of the phosphorylation domain in the so-called hinge motif conserved in all P-type ATPases. P401L resides near the N-terminal portion of the phosphorylation domain whereas the mutations H475Q and P476L affect the heavy metal ATPase-specific HP motif in the nucleotide binding domain. All mutants show reduced ATPase activity corresponding 0-37% of the wild-type activity. The mutants P401L, H475Q and P476L are poorly phosphorylated by both ATP and P(i). Their dephosphorylation rates are slow. The D628A mutant is inactive and cannot be phosphorylated at all. In contrast, the mutant P634L six residues apart in the same domain shows normal phosphorylation by ATP. However, phosphorylation by P(i) is almost absent. In the absence of added ADP the P634L mutant dephosphorylates much more slowly than the wild-type, whereas in the presence of ADP the dephosphorylation rate is faster than that of the wild-type. We conclude that the mutation P634L affects the conversion between the states E1P and E2P so that the mutant favors the E1 or E1P state.     

Deconstructing nucleotide binding activity of the Mdm2 RING domain

Mdm2, a central negative regulator of the p53 tumor suppressor, possesses a Really Interesting New Gene (RING) domain within its C-terminus. In addition to E3 ubiquitin ligase activity, the Mdm2 RING preferentially binds adenine base nucleotides, and such binding leads to a conformational change in the Mdm2 C-terminus. Here, we present further biochemical analysis of the nucleotide–Mdm2 interaction. We have found that MdmX, an Mdm2 family member with high sequence homology, binds adenine nucleotides with similar affinity and specificity as Mdm2, suggesting that residues involved in nucleotide binding may be conserved between the two proteins and adenosine triphosphate (ATP) binding may have similar functional consequences for both Mdm family members. By generating and testing a series of proteins with deletions and substitution mutations within the Mdm2 RING, we mapped the specific adenine nucleotide binding region of Mdm2 to residues 429–484, encompassing the minimal RING domain. Using a series of ATP derivatives, we demonstrate that phosphate coordination by the Mdm2 P-loop contributes to, but is not primarily responsible for, ATP binding. Additionally, we have identified the 2′ and 3′ hydroxyls of the ribose and the C6 amino group of the adenine base moiety as being essential for binding.     

Role of the ATP-Binding Domain of the Human Papillomavirus Type 11 E1 Helicase in E2-Dependent Binding to the Origin

Replication of the genome of human papillomaviruses (HPV) is initiated by the recruitment of the viral E1 helicase to the origin of DNA replication by the viral E2 protein, which binds specifically to the origin. We determined, for HPV type 11 (HPV-11), that the C-terminal 296 amino acids of E1 are sufficient for interaction with the transactivation domain of E2 in the yeast two-hybrid system and in vitro. This region of E1 encompasses the ATP-binding domain. Here we have examined the role of this ATP-binding domain, and of ATP, on E2-dependent binding of E1 to the origin. Several amino acid substitutions in the phosphate-binding loop (P loop), which is implicated in binding the triphosphate moiety of ATP, abolished E2 binding, indicating that the structural integrity of this domain is essential for the interaction. The structural constraints imposed on the E1 P loop may differ between HPV-11 and bovine papillomavirus type 1 (BPV-1), since the P479S substitution that inactivates BPV-1 E1 is tolerated in the HPV-11 enzyme. Other substitutions in the E1 P loop, or in two other conserved motifs of the ATP-binding domain, were tolerated, indicating that ATP binding is not essential for interaction with E2. Nevertheless, ATP-Mg stimulated the E2-dependent binding of E1 to the origin in vitro. This stimulation was maximal at the physiological temperature (37 degrees C) and did not require ATP hydrolysis. In contrast, ATP-Mg did not stimulate the E2-dependent binding to the origin of an E1 protein containing only the C-terminal domain (353 to 649) or that of mutant E1 proteins with alterations in the DNA-binding domain. These results are discussed in light of a model in which the E1 ATP-binding domain is required for formation of the E2-binding surface and can, upon the binding of ATP, facilitate and/or stabilize the interaction of E1 with the origin.     

Site-directed mutagenesis of the GTP-binding domain of beta-tubulin.

Tubulin binds guanine nucleotides with high affinity and specificity. GTP, an allosteric effector of microtubule assembly, requires Mg2+ for its interaction with beta-tubulin and binds as the MgGTP complex. In contrast, GDP binding does not require Mg2+. The structural basis for this difference is not understood but may be of fundamental importance for microtubule assembly. We investigated the interaction of beta-tubulin with guanine nucleotides using site-directed mutagenesis. Acidic amino acid residues have been shown to interact with nucleotide in numerous nucleotide-binding proteins. In this study, we mutated seven highly conserved aspartic acid residues and one highly conserved glutamic acid residue in the putative GTP-binding domain of beta-tubulin (N-terminal 300 amino acids) to asparagine and glutamine, respectively. The mutants were synthesized in vitro using rabbit reticulocyte lysates, and their affinities for nucleotide determined by an h.p.l.c.-based assay. Our results indicate that the mutations can be placed in six separate categories on the basis of their effects on nucleotide binding. These categories range from having no effect on nucleotide binding to a mutation that apparently abolishes nucleotide binding. One mutation at Asp224 reduced the affinity of beta-tubulin for GTP in the presence but not in the absence of Mg2+. The specific effect of this mutation on nucleotide binding is consistent with an interaction of this amino acid with the Mg2+ moiety of MgGTP. This residue is in a region sharing sequence homology with the putative Mg2+ site in myosin and other ATP-binding proteins. As a result, tubulin belongs to a distinct class of GTP-binding proteins which may be evolutionarily related to the ATP-binding proteins.     

Site-directed mutagenesis of conserved charged amino acid residues in ClpB from Escherichia coli

ClpB is a member of a multichaperone system in Escherichia coli (with DnaK, DnaJ, and GrpE) that reactivates strongly aggregated proteins. The sequence of ClpB contains two ATP-binding domains, each containing Walker consensus motifs. The N- and C-terminal sequence regions of ClpB do not contain known functional motifs. In this study, we performed site-directed mutagenesis of selected charged residues within the Walker A motifs (Lys212 and Lys611) and the C-terminal region of ClpB (Asp797, Arg815, Arg819, and Glu826). We found that the mutations K212T, K611T, D797A, R815A, R819A, and E826A did not significantly affect the secondary structure of ClpB. The mutation of the N-terminal ATP-binding site (K212T), but not of the C-terminal ATP-binding site (K611T), and two mutations within the C-terminal domain (R815A and R819A) inhibited the self-association of ClpB in the absence of nucleotides. The defects in self-association of these mutants were also observed in the presence of ATP and ADP. The four mutants K212T, K611T, R815A, and R819A showed an inhibition of chaperone activity, which correlated with their low ATPase activity in the presence of casein. Our results indicate that positively charged amino acids that are located along the intersubunit interface (this includes Lys212 in the Walker A motif of the N-terminal ATP-binding domain as well as Arg815 and Arg819 in the C-terminal domain) participate in intersubunit salt bridges and stabilize the ClpB oligomer. Interestingly, we have identified a conserved residue within the C-terminal domain (Arg819) which does not participate directly in nucleotide binding but is essential for the chaperone activity of ClpB.     

Mechanism of flexibility control for ATP access of hepatitis C virus NS3 helicase

Hepatitis C virus (HCV) NS3 helicase couples adenosine triphosphate (ATP) binding and hydrolysis to polynucleotide unwinding. Understanding the regulation mechanism of ATP binding will facilitate targeting of the ATP-binding site for potential therapeutic development for hepatitis C. T324, an amino acid residue connecting domains 1 and 2 of NS3 helicase, has been suggested as part of a flexible hinge for opening of the ATP-binding cleft, although the detailed mechanism remains largely unclear. We used computational simulation to examine the mutational effect of T324 on the dynamics of the ATP-binding site. A mutant model, T324A, of the NS3 helicase apo structure was created and energy was minimized. Molecular dynamics simulation was conducted for both wild type and the T324A mutant apo structures to compare their differences. For the mutant structure, histogram analysis of pairwise distances between residues in domains 1 and 2 (E291-Q460, K210-R464 and R467-T212) showed that separation between the two domains was reduced by ～10% and the standard deviation by ～33%. Root mean square fluctuation (RMSF) analysis demonstrated that residues in close proximity to residue 324 have at least 30% RMSF value reductions in the mutant structure. Solvent RMSF analysis showed that more water molecules were trapped near D290 and H293 in domain 1 to form an extensive interaction network constraining cleft opening. We also demonstrated that the T324A mutation established a new atomic interaction with V331, revealing that an atomic interaction cascade from T324 to residues in domains 1 and 2 controls the flexibility of the ATP-binding cleft.     

Insight into Pleiotropic Drug Resistance ATP-binding Cassette Pump Drug Transport through Mutagenesis of Cdr1p Transmembrane Domains

The fungal ATP-binding cassette (ABC) transporter Cdr1 protein (Cdr1p), responsible for clinically significant drug resistance, is composed of two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs). We have probed the nature of the drug binding pocket by performing systematic mutagenesis of the primary sequences of the 12 transmembrane segments (TMSs) found in the TMDs. All mutated proteins were expressed equally well and localized properly at the plasma membrane in the heterologous host Saccharomyces cerevisiae, but some variants differed significantly in efflux activity, substrate specificity, and coupled ATPase activity. Replacement of the majority of the amino acid residues with alanine or glycine yielded neutral mutations, but about 42% of the variants lost resistance to drug efflux substrates completely or selectively. A predicted three-dimensional homology model shows that all the TMSs, apart from TMS4 and TMS10, interact directly with the drug-binding cavity in both the open and closed Cdr1p conformations. However, TMS4 and TMS10 mutations can also induce total or selective drug susceptibility. Functional data and homology modeling assisted identification of critical amino acids within a drug-binding cavity that, upon mutation, abolished resistance to all drugs tested singly or in combinations. The open and closed Cdr1p models enabled the identification of amino acid residues that bordered a drug-binding cavity dominated by hydrophobic residues. The disposition of TMD residues with differential effects on drug binding and transport are consistent with a large polyspecific drug binding pocket in this yeast multidrug transporter.     

Identification of amino acid residues modified by two ATP analogs in Bacillus subtilis glutamine synthetase.

Bacillus subtilis glutamine synthetase was modified by two ATP analogs, 5'-p-fluorosulfonylbenzoyladenosine (FSBA) and 8-azidoadenosine 5'-triphosphate (8-N3-ATP), each one containing either Mg2+ or Mn2+. The FSBA labeled peptide was monitored by measuring the characteristic absorbance of the 4-carboxybenzenesulfonyl (CBS) part at 243 nm. The 8-N3ATP photolabeled peptide could also be monitored by measuring its absorption at 310 nm. A single CBS-labeled tryptic peptide was obtained, spanning residues 89-91 from the N-terminal of the subunit polypeptide chain, and sequence analysis by Edman degradation revealed that CBS-arginine was at position 91. The amino acids photolabeled by 8-N3ATP at the ATP-binding site in B. subtilis GS were His-186, His-187, and Trp-424. These results suggested that these four amino acids constitute an ATP-binding active site located at the interface between two subunits. The region surrounding Trp-424, which varies among different prokaryotic enzymes, was considered to be involved in a catalytic or regulatory role in B. subtilis GS. Since the same amino acids were labeled when B. subtilis GS was modified with FSBA or 8-N3ATP in the presence of Mn2+ or Mg2+, no conformational difference between B. subtilis GS binding Mn(2+)-ATP and that binding Mg(2+)-ATP was detected by affinity labeling with ATP analogs.     

Two distinct ATP-binding domains are needed to promote protein export by Escherichia coli SecA ATPase

Six putative ATP-binding motifs of SecA protein were altered by oligonucleotide-directed mutagenesis to try to define the ATP-binding regions of this multifunctional protein. The effects of the mutations were analysed by genetic and biochemical assays. The results show that SecA contains two essential ATP-binding domains. One domain is responsible for high-affinity ATP binding and contains motifs AO and BO, located at amino acid residues 102-109 and 198-210, respectively. A second domain is responsible for low-affinity ATP binding and contains motifs A3 and a predicted B motif located at amino acid residues 503-511 and 631-653, respectively. The ATP-binding properties of both domains were essential for SecA-dependent translocation ATPase and in vitro protein translocation activities. The significance of these findings for the mechanism of SecA-dependent protein translocation is discussed.     

Rat liver mitochondrial ATP synthase. Effects of mutations in the glycine-rich region of a beta subunit peptide on its interaction with adenine nucleotides

The beta subunit of the rat liver mitochondrial ATP synthase contains a glycine-rich amino acid sequence implicated in binding nucleotides by its similarity to a sequence found in many other nucleotide-binding proteins. A C-terminal three-quarter-length rat liver beta subunit fragment (Glu122 through Ser479), containing this homology region, interacts with adenine nucleotides (Garboczi, D.N., Hullihen, J.H., and Pedersen, P.L. (1988) J. Biol. Chem. 263, 15694-15698). Here we directly test the involvement of the glycine-rich region in nucleotide binding by altering its amino acid sequence through mutation or deletion. Twenty-one mutations within the glycine-rich region of the beta subunit cDNA were randomly generated. Wild-type and mutant beta subunit proteins were purified from overexpressing Escherichia coli strains. The mutant proteins were screened for changes in their interaction with 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), a fluorescent nucleotide analog. Only one mutant protein bearing two amino acid changes (Gly153----Val, Gly156----Arg) exhibited a fluorescence enhancement higher than that of the wild-type "control." Further analysis of this protein revealed a lower affinity for TNP-ATP (Kd = 10 microM) compared with wild type (Kd = 5 microM). In addition, a mutant from which amino acids Gly149-Lys214 had been deleted was prepared. This mutant protein, which lacks the entire glycine-rich region, also displayed a marked reduction in affinity for TNP-ATP (Kd greater than 60 microM). Prior addition of 0.5 mM ATP significantly reduced the binding of TNP-ATP to both the double and deletion mutants. The altered interaction of nucleotide with both glycine-rich region mutants points to the involvement of this region in the binding site. Further, this work shows that a beta subunit protein that lacks the glycine-rich homology region can still interact with nucleotide, indicating that one or more additional regions of this subunit contribute to the nucleotide binding site.     

Eight amino acids form the ATP recognition site of Na(+)/K(+)-ATPase

Point mutations of a part of the H(4)-H(5) loop (Leu(354)-Ile(604)) of Na(+)/K(+)-ATPase have been used to study the ATP and TNP-ATP binding affinities. Besides the previously reported amino acid residues Lys(480), Lys(501), Gly(502), and Cys(549), we have found four more amino acid residues, viz., Glu(446), Phe(475), Gln(482), and Phe(548), completing the ATP-binding pocket of Na(+)/K(+)-ATPase. Moreover, mutation of Arg(423) has also resulted in a large decrease in the extent of ATP binding. This residue, localized outside the binding pocket, seems to play a key role in supporting the proper structure and shape of the binding site, probably due to formation of a hydrogen bond with Glu(472). On the other hand, only some minor effects were caused by mutations of Ile(417), Asn(422), Ser(445), and Glu(505).     

The last 10 amino acid residues beyond the hydrophobic motif are critical for the catalytic competence and function of protein kinase Calpha

The segment C-terminal to the hydrophobic motif at the V5 domain of protein kinase C (PKC) is the least conserved both in length and in amino acid identity among all PKC isozymes. By generating serial truncation mutants followed by biochemical and functional analyses, we show here that the very C terminus of PKCalpha is critical in conferring the full catalytic competence to the kinase and for transducing signals in cells. Deletion of one C-terminal amino acid residue caused the loss of approximately 60% of the catalytic activity of the mutant PKCalpha, whereas deletion of 10 C-terminal amino acid residues abrogated the catalytic activity of PKCalpha in immune complex kinase assays. The PKCalpha C-terminal truncation mutants were found to lose their ability to activate mitogen-activated protein kinase, to rescue apoptosis induced by the inhibition of endogenous PKC in COS cells, and to augment melatonin-stimulated neurite outgrowth. Furthermore, molecular dynamics simulations revealed that the deletion of 1 or 10 C-terminal residues results in the deformation of the V5 domain and the ATP-binding pocket, respectively. Finally, PKCalpha immunoprecipitated using an antibody against its C terminus had only marginal catalytic activity compared with that of the PKCalpha immunoprecipitated by an antibody against its N terminus. Therefore, the very C-terminal tail of PKCalpha is a novel determinant of the catalytic activity of PKC and a promising target for selective modulation of PKCalpha function. Molecules that bind preferentially to the very C terminus of distinct PKC isozymes and suppress their catalytic activity may constitute a new class of selective inhibitors of PKC.     

Comparative analysis of the ATP-binding sites of Hsp90 by nucleotide affinity cleavage: a distinct nucleotide specificity of the C-terminal ATP-binding site.

The 90-kDa heat shock protein (Hsp90) is a molecular chaperone that assists both in ATP-independent sequestration of damaged proteins, and in ATP-dependent folding of numerous targets, such as nuclear hormone receptors and protein kinases. Recent work from our lab and others has established the existence of a second, C-terminal nucleotide binding site besides the well characterized N-terminal, geldanamycin-sensitive ATP-binding site. The cryptic C-terminal site becomes open only after the occupancy of the N-terminal site. Our present work demonstrates the applicability of the oxidative nucleotide affinity cleavage in the site-specific characterization of nucleotide binding proteins. We performed a systematic analysis of the nucleotide binding specificity of the Hsp90 nucleotide binding sites. N-terminal binding is specific to adenosine nucleotides with an intact adenine ring. Nicotinamide adenine dinucleotides and diadenosine polyphosphate alarmones are specific N-terminal nucleotides. The C-terminal binding site is much more unspecific-it interacts with both purine and pirimidine nucleotides. Efficient binding to the C-terminal site requires both charged residues and a larger hydrophobic moiety. GTP and UTP are specific C-terminal nucleotides. 2',3'-O-(2,4,6-trinitrophenyl)-nucleotides (TNP-ATP, TNP-GTP) and pyrophosphate access the C-terminal binding site without the need for an occupied N-terminal site. Our data provide additional evidence for the dynamic domain-domain interactions of Hsp90, give hints for the design of novel types of specific Hsp90 inhibitors, and raise the possibility that besides ATP, other small molecules might also interact with the C-terminal nucleotide binding site in vivo.     

Biochemical properties of a minimal functional domain with ATP-binding activity of the NTPase/helicase of hepatitis C virus.

The RNA-stimulated nucleoside triphosphatase (NTPase) and helicase of hepatitis C virus (HCV) consists of three domains with highly conserved NTP binding motifs located in the first domain. The ATP-binding domain was obtained by limited proteolysis of a greater fragment of the HCV polyprotein, and it was purified to homogenity by column chromatography. The identity of the domain, comprising amino acids 1203 to 1364 of the HCV polyprotein, was confirmed by N- and C-terminal sequencing and by its capability to bind 5'-fluorosulfonylbenzoyladenosine (FSBA). The analyses of the kinetics of ATP binding revealed a single class of binding site with the Kd of 43.6 microM. The binding is saturable and dependent on Mn2+ or Mg2+ ions. Poly(A) and poly(dA) show interesting properties as regulators of the ATP-binding capacity of the domain. Polynucleotides bind to the domain and enhance its affinity for ATP. In addition, ATP enhances the affinity of the domain for the polynucleotides. Different compounds, which are known to interact with nucleotide binding sites of various classes of enzymes, were tested for their ability to inhibit the binding of ATP to the domain. Of the compounds tested, two agents behaved as inhibitors: paclitaxel, which inhibits the ATP binding competitively (IC50 = 22 microM), and trifluoperazine, which inhibits the ATP binding by a noncompetitive mechanism (IC50 = 98 microM). Kinetic experiments with the NTPase/helicase indicate that both compounds inhibit the NTPase activity of the holoenzyme by interacting with its ATP-binding domain.     

Sequence requirements of the ATP-binding site within the C-terminal nucleotide-binding domain of mouse P-glycoprotein: structure-activity relationships for flavonoid binding

Sequence requirements of the ATP-binding site within the C-terminal nucleotide-binding domain (NBD2) of mouse P-glycoprotein were investigated by using two recombinantly expressed soluble proteins of different lengths and photoactive ATP analogues, 8-azidoadenosine triphosphate (8N(3)-ATP) and 2',3',4'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N(3)-ATP). The two proteins, Thr(1044)-Thr(1224) (NBD2(short)) and Lys(1025)-Ser(1276) (NBD2(long)), both incorporated the four consensus sequences of ABC (ATP-binding cassette) transporters, Walker A and B motifs, the Q-loop, and the ABC signature, while differing in N-terminal and C-terminal extensions. Radioactive photolabeling of both proteins was characterized by hyperbolic dependence on nucleotide concentration and high-affinity binding with K(0.5)(8N(3)-ATP) = 36-37 microM and K(0.5)(TNP-8N(3)-ATP) = 0.8-2.6 microM and was maximal at acidic pH. Photolabeling was strongly inhibited by TNP-ATP (K(D) = 0.1-5 microM) and ATP (K(D) = 0.5-2.7 mM). Since flavonoids display bifunctional interactions at the ATP-binding site and a vicinal steroid-interacting hydrophobic sequence [Conseil, G., Baubichon-Cortay, H., Dayan, G., Jault, J.-M., Barron, D., and Di Pietro, A. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9831-9836], a series of 30 flavonoids from different classes were investigated for structure-activity relationships toward binding to the ATP site, monitored by protection against photolabeling. The 3-OH and aromaticity of conjugated rings A and C appeared important, whereas opening of ring C abolished the binding in all but one case. It can be concluded that the benzopyrone portion of the flavonoids binds at the adenyl site and the phenyl ring B at the ribosyl site. The Walker A and B motifs, intervening sequences, and small segments on both sides are sufficient to constitute the ATP site.     

Protein modeling combined with spectroscopic techniques: an attractive quick alternative to obtain structural information

Beside of the protein crystallography or NMR, another attractive option in protein structure analysis has recently appeared: computer modeling of the protein structure based on homology and similarity with proteins of already known structures. We have used the combination of computer modeling with spectroscopic techniques, such as steady-state or time-resolved fluorescence spectroscopy, and with molecular biology techniques. This method could provide useful structural information in the cases where crystal or NMR structure is not available. Molecular modeling of the ATP site within the H4-H5-loop revealed eight amino acids residues, namely besides the previously reported amino acids Asp443, Lys480, Lys501, Gly502 and Arg544, also Glu446, Phe475 and Gln482, which form the complete ATP recognition site. Moreover, we have proved that a hydrogen bond between Arg423 and Glu472 supports the connection of two opposite halves of the ATP-binding pocket. Similarly, the conserved residue Pro489 is important for the proper interaction of the third and fourth beta-strands, which both contain residues that take part in the ATP-binding. Alternatively, molecular dynamics simulation combined with dynamic fluorescence spectroscopy revealed that 14-3-3 zeta C-terminal stretch is directly involved in the interaction of 14-3-3 protein with the ligand. Phosphorylation at Thr232 induces a conformational change of the C-terminus, which is presumably responsible for observed inhibition of binding abilities. Phosphorylation at Thr232 induces more extended conformation of 14-3-3zeta C-terminal stretch and changes its interaction with the rest of the 14-3-3 molecule. This could explain negative regulatory effect of phosphorylation at Thr232 on 14-3-3 binding properties.     

TmrB protein, responsible for tunicamycin resistance of Bacillus subtilis, is a novel ATP-binding membrane protein.

tmrB is the gene responsible for tunicamycin resistance in Bacillus subtilis. It is predicted that an increase in tmrB gene expression makes B. subtilis tunicamycin resistant. To examine the tmrB gene product, we produced the tmrB gene product in Escherichia coli by using the tac promoter. TmrB protein was found not only in the cytoplasm fraction but also in the membrane fraction. Although TmrB protein is entirely hydrophilic and has no hydrophobic stretch of amino acids sufficient to span the membrane, its C-terminal 18 amino acids could form an amphiphilic alpha-helix. Breaking this potential alpha-helix by introducing proline residues or a stop codon into this region caused the release of this membrane-bound protein into the cytoplasmic fraction, indicating that the C-terminal 18 residues were essential for membrane binding. On the other hand, TmrB protein has an ATP-binding consensus sequence in the N-terminal region. We have tested whether this sequence actually has the ability to bind ATP by photoaffinity cross-linking with azido-[alpha-32P]ATP. Wild-type protein bound azido-ATP well, but mutants with substitutions in the consensus amino acids were unable to bind azido-ATP. These C-terminal or N-terminal mutant genes were unable to confer tunicamycin resistance on B. subtilis in a multicopy state. It is concluded that TmrB protein is a novel ATP-binding protein which is anchored to the membrane with its C-terminal amphiphilic alpha-helix.     

Kar3Vik1 Mechanochemistry Is Inhibited by Mutation or Deletion of the C Terminus of the Vik1 Subunit

Force production by kinesins has been linked to structural rearrangements of the N and C termini of their motor domain upon nucleotide binding. In recent crystal structures, the Kar3-associated protein Vik1 shows unexpected homology to these conformational states even though it lacks a nucleotide-binding site. This conservation infers a degree of commonality in the function of the N- and C-terminal regions during the mechanochemical cycle of all kinesins and kinesin-related proteins. We tested this inference by examining the functional effects on Kar3Vik1 of mutating or deleting residues in Vik1 that are involved in stabilizing the C terminus against the core and N terminus of the Vik1 motor homology domain (MHD). Point mutations at two moderately conserved residues near the Vik1 C terminus impaired microtubule gliding and microtubule-stimulated ATP turnover by Kar3Vik1. Deletion of the seven C-terminal residues inhibited Kar3Vik1 motility much more drastically. Interestingly, none of the point mutants seemed to perturb the ability of Kar3Vik1 to bind microtubules, whereas the C-terminal truncation mutant did. Molecular dynamics simulations of these C-terminal mutants showed distinct root mean square fluctuations in the N-terminal region of the Vik1 MHD that connects it to Kar3. Here, the degree of motion in the N-terminal portion of Vik1 highly correlated with that in the C terminus. These observations suggest that the N and C termini of the Vik1 MHD form a discrete folding motif that is part of a communication pathway to the nucleotide-binding site of Kar3.