Studies on the mechanism of catalysis of iron-sulfur cluster transfer from IscU[2Fe2S] by HscA/HscB chaperones

The HscA/HscB chaperone/cochaperone system accelerates transfer of iron-sulfur clusters from the FeS-scaffold protein IscU (IscU(2)[2Fe2S], holo-IscU) to acceptor proteins in an ATP-dependent manner. We have employed visible region circular dichroism (CD) measurements to monitor chaperone-catalyzed cluster transfer from holo-IscU to apoferredoxin and to investigate chaperone-induced changes in properties of the IscU(2)[2Fe2S] cluster. HscA-mediated acceleration of [2Fe2S] cluster transfer exhibited an absolute requirement for both HscB and ATP. A mutant form of HscA lacking ATPase activity, HscA(T212V), was unable to accelerate cluster transfer, suggesting that ATP hydrolysis and conformational changes accompanying the ATP (T-state) to ADP (R-state) transition in the HscA chaperone are required for catalysis. Addition of HscA and HscB to IscU(2)[2Fe2S] did not affect the properties of the [2Fe2S] cluster, but subsequent addition of ATP was found to cause a transient change of the visible region CD spectrum, indicating distortion of the IscU-bound cluster. The dependence of the rate of decay of the observed CD change on ATP concentration and the lack of an effect of the HscA(T212V) mutant were consistent with conformational changes in the cluster coupled to ATP hydrolysis by HscA. Experiments carried out under conditions with limiting concentrations of HscA, HscB, and ATP further showed that formation of a 1:1:1 HscA-HscB-IscU(2)[2Fe2S] complex and a single ATP hydrolysis step are sufficient to elicit the full effect of the chaperones on the [2Fe2S] cluster. These results suggest that acceleration of iron-sulfur cluster transfer involves a structural change in the IscU(2)[2Fe2S] complex during the T --> R transition of HscA accompanying ATP hydrolysis.     

HscA and HscB stimulate [2Fe-2S] cluster transfer from IscU to apoferredoxin in an ATP-dependent reaction

The role of the Azotobacter vinelandii HscA/HscB cochaperone system in ISC-mediated iron-sulfur cluster biogenesis has been investigated in vitro by using CD and EPR spectrometry to monitor the effect of HscA, HscB, MgATP, and MgADP on the time course of cluster transfer from [2Fe-2S]IscU to apo-Isc ferredoxin. CD spectra indicate that both HscB and HscA interact with [2Fe-2S]IscU and the rate of cluster transfer was stimulated more than 20-fold in the presence stoichiometric HscA and HscB and excess MgATP. No stimulation was observed in the absence of either HscB or MgATP, and cluster transfer was found to be an ATP-dependent reaction based on concomitant phosphate production and the enhanced rates of cluster transfer in the presence of KCl which is known to stimulate HscA ATPase activity. The results demonstrate a role of the ISC HscA/HscB cochaperone system in facilitating efficient [2Fe-2S] cluster transfer from the IscU scaffold protein to acceptor proteins and that [2Fe-2S] cluster transfer from IscU is an ATP-dependent process. The data are consistent with the proposed regulation of the HscA ATPase cycle by HscB and IscU [Silberg, J. J., Tapley, T. L., Hoff, K. G., and Vickery, L. E. (2004) J. Biol. Chem. 279, 53924-53931], and mechanistic proposals for coupling of the HscA ATPase cycle with cluster transfer from [2Fe-2S]IscU to apo-IscFdx are discussed.     

Contributions of the LPPVK motif of the iron-sulfur template protein IscU to interactions with the Hsc66-Hsc20 chaperone system

Hsc66 (HscA) and Hsc20 (HscB) from Escherichia coli comprise a specialized chaperone system that selectively binds the iron-sulfur cluster template protein IscU. Hsc66 interacts with peptides corresponding to a discrete region of IscU including residues 99-103 (LPPVK), and a peptide containing residues 98-106 stimulates Hsc66 ATPase activity in a manner similar to IscU. To determine the relative contributions of individual residues in the LPPVK motif to Hsc66 binding and regulation, we have carried out an alanine mutagenesis scan of this motif in the Glu98-Cys106 peptide and the IscU protein. Alanine substitutions in the Glu98-Cys106 peptide resulted in decreased ATPase stimulation (2-10-fold) because of reduced binding affinity, with peptide(P101A) eliciting <10% of the parent peptide stimulation. Alanine substitutions in the IscU protein also revealed lower activities resulting from decreased apparent binding affinity, with the greatest changes in Km observed for the Pro101 (77-fold), Val102 (4-fold), and Lys103 (15-fold) mutants. Calorimetric studies of the binding of IscU mutants to the Hsc66.ADP complex showed that the P101A and K103A mutants also exhibit decreased binding affinity for the ADP-bound state. When ATPase stimulatory activity was assayed in the presence of the co-chaperone Hsc20, each of the mutants displayed enhanced binding affinity, but the P101A and V102A mutants exhibited decreased ability to maximally simulate Hsc66 ATPase. A charge mutant containing the motif sequence of NifU, IscU(V102E), did not bind the ATP or ADP states of Hsc66 but did bind Hsc20 and weakly stimulated Hsc66 ATPase in the presence of the co-chaperone. These results indicate that residues in the LPPVK motif are important for IscU interactions with Hsc66 but not for the ability of Hsc20 to target IscU to Hsc66. The results are discussed in the context of a structural model based on the crystallographic structure of the DnaK peptide-binding domain.     

Specialized Hsp70 Chaperone (HscA) Binds Preferentially to the Disordered Form, whereas J-protein (HscB) Binds Preferentially to the Structured Form of the Iron-Sulfur Cluster Scaffold Protein (IscU)

The Escherichia coli protein IscU serves as the scaffold for Fe-S cluster assembly and the vehicle for Fe-S cluster transfer to acceptor proteins, such as apoferredoxin. IscU populates two conformational states in solution, a structured conformation (S) that resembles the conformation of the holoprotein IscU-[2Fe-2S] and a dynamically disordered conformation (D) that does not bind metal ions. NMR spectroscopic results presented here show that the specialized Hsp70 chaperone (HscA), alone or as the HscA-ADP complex, preferentially binds to and stabilizes the D-state of IscU. IscU is released when HscA binds ATP. By contrast, the J-protein HscB binds preferentially to the S-state of IscU. Consistent with these findings, we propose a mechanism in which cluster transfer is coupled to hydrolysis of ATP bound to HscA, conversion of IscU to the D-state, and release of HscB.     

Molecular chaperones HscA/Ssq1 and HscB/Jac1 and their roles in iron-sulfur protein maturation

Genetic and biochemical studies have led to the identification of several cellular pathways for the biosynthesis of iron-sulfur proteins in different organisms. The most broadly distributed and highly conserved system involves an Hsp70 chaperone and J-protein co-chaperone system that interacts with a scaffold-like protein involved in [FeS]-cluster preassembly. Specialized forms of Hsp70 and their co-chaperones have evolved in bacteria (HscA, HscB) and in certain fungi (Ssq1, Jac1), whereas most eukaryotes employ a multifunctional mitochondrial Hsp70 (mtHsp70) together with a specialized co-chaperone homologous to HscB/Jac1. HscA and Ssq1 have been shown to specifically bind to a conserved sequence present in the [FeS]-scaffold protein designated IscU in bacteria and Isu in fungi, and the crystal structure of a complex of a peptide containing the IscU recognition region bound to the HscA substrate binding domain has been determined. The interaction of IscU/Isu with HscA/Ssq1 is regulated by HscB/Jac1 which bind the scaffold protein to assist delivery to the chaperone and stabilize the chaperone-scaffold complex by enhancing chaperone ATPase activity. The crystal structure of HscB reveals that the N-terminal J-domain involved in regulation of HscA ATPase activity is similar to other J-proteins, whereas the C-terminal domain is unique and appears to mediate specific interactions with IscU. At the present time the exact function(s) of chaperone-[FeS]-scaffold interactions in iron-sulfur protein biosynthesis remain(s) to be established. In vivo and in vitro studies of yeast Ssq1 and Jac1 indicate that the chaperones are not required for [FeS]-cluster assembly on Isu. Recent in vitro studies using bacterial HscA, HscB and IscU have shown that the chaperones destabilize the IscU[FeS] complex and facilitate cluster delivery to an acceptor apo-protein consistent with a role in regulating cluster release and transfer. Additional genetic and biochemical studies are needed to extend these findings to mtHsp70 activities in higher eukaryotes.     

Molecular Chaperones HscA/Ssq1 and HscB/Jac1 and Their Roles in Iron-Sulfur Protein Maturation

ABSTRACT

Genetic and biochemical studies have led to the identification of several cellular pathways for the biosynthesis of iron-sulfur proteins in different organisms. The most broadly distributed and highly conserved system involves an Hsp70 chaperone and J-protein co-chaperone system that interacts with a scaffold-like protein involved in [FeS]-cluster preassembly. Specialized forms of Hsp70 and their co-chaperones have evolved in bacteria (HscA, HscB) and in certain fungi (Ssq1, Jac1), whereas most eukaryotes employ a multifunctional mitochondrial Hsp70 (mtHsp70) together with a specialized co-chaperone homologous to HscB/Jac1. HscA and Ssq1 have been shown to specifically bind to a conserved sequence present in the [FeS]-scaffold protein designated IscU in bacteria and Isu in fungi, and the crystal structure of a complex of a peptide containing the IscU recognition region bound to the HscA substrate binding domain has been determined. The interaction of IscU/Isu with HscA/Ssq1 is regulated by HscB/Jac1 which bind the scaffold protein to assist delivery to the chaperone and stabilize the chaperone-scaffold complex by enhancing chaperone ATPase activity. The crystal structure of HscB reveals that the N-terminal J-domain involved in regulation of HscA ATPase activity is similar to other J-proteins, whereas the C-terminal domain is unique and appears to mediate specific interactions with IscU. At the present time the exact function(s) of chaperone-[FeS]-scaffold interactions in iron-sulfur protein biosynthesis remain(s) to be established. In vivo and in vitro studies of yeast Ssq1 and Jac1 indicate that the chaperones are not required for [FeS]-cluster assembly on Isu. Recent in vitro studies using bacterial HscA, HscB and IscU have shown that the chaperones destabilize the IscU[FeS] complex and facilitate cluster delivery to an acceptor apo-protein consistent with a role in regulating cluster release and transfer. Additional genetic and biochemical studies are needed to extend these findings to mtHsp70 activities in higher eukaryotes.     

Solution structure of the iron-sulfur cluster cochaperone HscB and its binding surface for the iron-sulfur assembly scaffold protein IscU

The interaction between IscU and HscB is critical for successful assembly of iron-sulfur clusters. NMR experiments were performed on HscB to investigate which of its residues might be part of the IscU binding surface. Residual dipolar couplings ( (1) D HN and (1) D CalphaHalpha) indicated that the crystal structure of HscB [Cupp-Vickery, J. R., and Vickery, L. E. (2000) Crystal structure of Hsc20, a J-type cochaperone from Escherichia coli, J. Mol. Biol. 304, 835-845] faithfully represents its solution state. NMR relaxation rates ( (15)N R 1, R 2) and (1)H- (15)N heteronuclear NOE values indicated that HscB is rigid along its entire backbone except for three short regions which exhibit flexibility on a fast time scale. Changes in the NMR spectrum of HscB upon addition of IscU mapped to the J-domain/C-domain interface, the interdomain linker, and the C-domain. Sequence conservation is low in the interface and in the linker, and NMR changes observed for these residues likely result from indirect effects of IscU binding. NMR changes observed in the conserved patch of residues in the C-domain (L92, M93, L96, E97, E100, E104, and F153) were suggestive of a direct interaction with IscU. To test this, we replaced several of these residues with alanine and assayed for the ability of HscB to interact with IscU and to stimulate HscA ATPase activity. HscB(L92A,M93A,F153A) and HscB(E97A,E100A,E104A) both showed decreased binding affinity for IscU; the (L92A,M93A,F153A) substitution also strongly perturbed the allosteric interaction within the HscA.IscU.HscB ternary complex. We propose that the conserved patch in the C-domain of HscB is the principal binding site for IscU.     

Facilitated transfer of IscU-[2Fe2S] clusters by chaperone-mediated ligand exchange

The scaffold protein IscU and molecular chaperones HscA and HscB play central roles in biological assembly of iron-sulfur clusters and maturation of iron-sulfur proteins. However, the structure of IscU-FeS complexes and the molecular mechanism whereby the chaperones facilitate cluster transfer to acceptor proteins are not well understood. We have prepared amino acid substitution mutants of Escherichia coli IscU in which potential ligands to the FeS cluster (Cys-37, Cys-63, His-105, and Cys-106) were individually replaced with alanine. The properties of the IscU-FeS complexes formed were investigated by measuring both their ability to transfer preformed FeS clusters to apo-ferredoxin and the activity of the IscU proteins in catalyzing cluster assembly on apo-ferredoxin using inorganic iron with inorganic sulfide or with IscS and cysteine as a sulfur source. The ability of the HscA/HscB chaperone system to accelerate ATP-dependent cluster transfer from each IscU substitution mutant to apo-ferredoxin was also determined. All of the mutants formed FeS complexes with a stoichiometry similar to the wild-type holo-protein, i.e., IscU(2)[2Fe2S], raising the possibility that different cluster ligation states may occur during iron-sulfur protein maturation. Spectroscopic properties of the mutants and the kinetics of transfer of performed IscU-FeS clusters to apo-ferredoxin indicate that the most stable form of holo-IscU involves iron coordination by Cys-63 and Cys-106. Results of studies on the ability of mutants to catalyze formation of holo-ferredoxin using iron and different sulfur sources were consistent with proposed roles for Cys-63 and Cys-106 in FeS cluster binding and also indicated an essential role for Cys-106 in sulfide transfer to IscU from IscS. Measurements of the ability of the chaperones HscA and HscB to facilitate cluster transfer from holo-IscU to apo-ferredoxin showed that only IscU(H105A) behaved similarly to wild-type IscU in exhibiting ATP-dependent stimulation of cluster transfer. IscU(C63A) and IscU(C106A) displayed elevated rates of cluster transfer in the ±ATP whereas IscU(C37A) exhibited low rates of cluster transfer ±ATP. In interpreting these findings, we propose that IscU(2)[2Fe2S] is able undergo structural isomerization to yield conformers having different cysteine residues bound to the cluster. On the basis of the crystal structure of HscA complexed with an IscU-derived peptide, we propose that the chaperone binds and stabilizes an isomer of IscU(2)[2Fe2S] in which the cluster is bound by cysteine residues 37 and 63 and that the [2Fe2S] cluster, being held less tightly than that coordinated by Cys-63 and Cys-106 in free IscU(2)[2Fe2S], is more readily transferred to acceptor proteins such as apo-ferredoxin.     

Network of protein-protein interactions among iron-sulfur cluster assembly proteins in Escherichia coli

The assembly of iron-sulfur (Fe-S) clusters is mediated by complex machinery which, in Escherichia coli, is encoded by the iscRSUA-hscBA-fdx-ORF3 gene cluster. Here, we demonstrate the network of protein-protein interactions among the components involved in the machinery. We have constructed (His)(6)-tagged versions of the components and identified their interacting partners that were co-purified from E. coli extracts with a Ni-affinity column. Direct associations of the defined pair of proteins were further examined in yeast cells using the two-hybrid system. In accord with the previous in vitro binding and kinetic experiments, interactions were observed for the combinations of IscS and IscU, IscU and HscB, IscU and HscA, and HscB and HscA. In addition, we have identified previously unreported interactions between IscS and Fdx, IscS and ORF3, IscA and HscA, and HscA and Fdx. We also found, by site-directed mutational analysis combined with the two-hybrid system, that two cysteine residues in IscU are essential for binding with HscB but not with IscS. Despite the complex network of interactions in various combinations of components, heteromultimeric complexes were not observed in our experiments except for the putative oligomeric form of IscU-IscS-ORF3. Thus, the sequential association and dissociation among the IscS, IscU, IscA, HscB, HscA, Fdx, and ORF3 proteins may be a critical process in the assembly of Fe-S clusters.     

Preferential substrate binding orientation by the molecular chaperone HscA

HscA, a specialized bacterial hsp70-class chaperone, interacts with the iron-sulfur cluster assembly protein IscU by recognizing a conserved LPPVK sequence motif at positions 99-103. We have used a site-directed fluorescence labeling and quenching strategy to determine whether HscA binds to IscU in a preferred orientation. HscA was selectively labeled on opposite sides of the substrate binding domain with the fluorescent probe bimane, and the ability of LPPVK-containing peptides having tryptophan at the N or C terminus to quench bimane fluorescence was measured. Quenching was highly dependent on the position of tryptophan in the peptide and the location of bimane on HscA implying a strong directional preference for peptide binding. Similar experiments showed that full-length IscU binds in the same orientation as IscU-derived peptides and that binding orientation is unaffected by the co-chaperone HscB. The preferred orientation of the HscA-IscU complex is the reverse of that previously described for peptide complexes of Escherichia coli DnaK and rat Hsc70 substrate binding domain fragments establishing that hsp70 isoforms can bind peptide/polypeptide substrates in different orientations.     

Kinetic characterization of the ATPase cycle of the molecular chaperone Hsc66 from Escherichia coli

Hsc66 from Escherichia coli is a constitutively expressed hsp70 class molecular chaperone whose activity is coupled to ATP binding and hydrolysis. To better understand the mechanism and regulation of Hsc66, we investigated the kinetics of ATP hydrolysis and the interactions of Hsc66 with nucleotides. Steady-state experiments revealed that Hsc66 has a low affinity for ATP (K(m)(ATP) = 12.7 microM) compared with other hsp70 chaperones. The kinetics of nucleotide binding were determined by analyzing changes in the Hsc66 absorbance spectrum using stopped-flow methods at 23 degrees C. ATP binding results in a rapid, biphasic increase of Hsc66 absorbance at 280 nm; this is interpreted as arising from a two-step process in which ATP binding (k(a)(ATP) = 4.2 x 10(4) M(-1) s(-1), k(d)(ATP) = 1.1 s(-1)) is followed by a slow conformational change (k(conf) = 0. 1 s(-1)). Under single turnover conditions, the ATP-induced transition decays exponentially with a rate (k(decay) = 0.0013 s(-1)) similar to that observed in both steady-state and single turnover ATP hydrolysis experiments (k(hyd) = 0.0014 s(-1)). ADP binding to Hsc66 results in a monophasic transition in the absence (k(a)(ADP) = 7 x 10(5) M(-1) s(-1), k(d)(ADP) = 60 s(-1)) and presence of physiological levels of inorganic phosphate (k(a)(ADP(P(i)) = 0.28 x 10(5) M(-1) s(-1), k(d)(ADP(P(i)) = 9.1 s(-1)). These results indicate that ATP hydrolysis is the rate-limiting step under steady-state conditions and is >10(3)-fold slower than the rate of ADP/ATP exchange. Thus, in contrast to DnaK and eukaryotic forms of hsp70 that have been characterized to date, the R if T equilibrium balance for Hsc66 is shifted in favor of the low peptide affinity T state, and regulation of the reaction cycle is expected to occur at the ATP hydrolysis step rather than at nucleotide exchange.     

Kinetic mechanism of human myosin IIIA

Myosin IIIA is specifically expressed in photoreceptors and cochlea and is important for the phototransduction and hearing processes. In addition, myosin IIIA contains a unique N-terminal kinase domain and C-terminal tail actin-binding motif. We examined the kinetic properties of baculovirus expressed human myosin IIIA containing the kinase, motor, and two IQ domains. The maximum actin-activated ATPase rate is relatively slow (k(cat) = 0.77 +/- 0.08 s(-1)), and high actin concentrations are required to fully activate the ATPase rate (K(ATPase) = 34 +/- 11 microm). However, actin co-sedimentation assays suggest that myosin III has a relatively high steady-state affinity for actin in the presence of ATP (K(actin) approximately 7 microm). The rate of ATP binding to the motor domain is quite slow both in the presence and absence of actin (K(1)k(+2) = 0.020 and 0.001 microm(-1).s(-1), respectively). The rate of actin-activated phosphate release is more than 100-fold faster (85 s(-1)) than the k(cat), whereas ADP release in the presence of actin follows a two-step mechanism (7.0 and 0.6 s(-1)). Thus, our data suggest a transition between two actomyosin-ADP states is the rate-limiting step in the actomyosin III ATPase cycle. Our data also suggest the myosin III motor spends a large fraction of its cycle in an actomyosin ADP state that has an intermediate affinity for actin (K(d) approximately 5 microm). The long lived actomyosin-ADP state may be important for the ability of myosin III to function as a cellular transporter and actin cross-linker in the actin bundles of sensory cells.     

Structural determinants of HscA peptide-binding specificity

The Hsp70-class molecular chaperone HscA interacts specifically with a conserved (99)LPPVK(103) motif of the iron-sulfur cluster scaffold protein IscU. We used a cellulose-bound peptide array to perform single-site saturation substitution of peptide residues corresponding to Glu(98)-Ile(104) of IscU to determine positional amino acid requirements for recognition by HscA. Two mutant chaperone forms, HscA(F426A) with a DnaK-like arch structure and HscA(M433V) with a DnaK-like substrate-binding pocket, were also studied. Wild-type HscA and HscA(F426A) exhibited a strict preference for proline in the central peptide position (ELPPVKI), whereas HscA(M433V) bound a peptide containing a Pro-->Leu substitution at this location (ELPLVKI). Contributions of Phe(426) and Met(433) to HscA peptide specificity were further tested in solution using a fluorescence-based peptide-binding assay. Bimane-labeled HscA and HscA(F426A) bound ELPPVKI peptides with higher affinity than leucine-substituted peptides, whereas HscA(M433V) favored binding of ELPLVKI peptides. Fluorescence-binding studies were also carried out with derivatives of the peptide NRLLLTG, a model substrate for DnaK. HscA and HscA(F426A) bound NRLLLTG peptides weakly, whereas HscA(M433V) bound NRLLLTG peptides with higher affinity than IscU-derived peptides ELPPVKI and ELPLVKI. These results suggest that the specificity of HscA for the LPPVK recognition sequence is determined in part by steric obstruction of the hydrophobic binding pocket by Met(433) and that substitution with the Val(433) sidechain imparts a broader, more DnaK-like, substrate recognition pattern.     

The ATPase cycle of the mitochondrial Hsp90 analog Trap1

Hsp90 is an ATP-dependent molecular chaperone whose mechanism is not yet understood in detail. Here, we present the first ATPase cycle for the mitochondrial member of the Hsp90 family called Trap1 (tumor necrosis factor receptor-associated protein 1). Using biochemical, thermodynamic, and rapid kinetic methods we dissected the kinetics of the nucleotide-regulated rearrangements between the open and the closed conformations. Surprisingly, upon ATP binding, Trap1 shifts predominantly to the closed conformation (70%), but, unlike cytosolic Hsp90 from yeast, this process is rather slow at 0.076 s(-1). Because reopening (0.034 s(-1)) is about ten times faster than hydrolysis (k(hyd) = 0.0039 s(-1)), which is the rate-limiting step, Trap1 is not able to commit ATP to hydrolysis. The proposed ATPase cycle was further scrutinized by a global fitting procedure that utilizes all relevant experimental data simultaneously. This analysis corroborates our model of a two-step binding mechanism of ATP followed by irreversible ATP hydrolysis and a one-step product (ADP) release.     

Biochemical characterization of the human RAD51 protein. II. Adenosine nucleotide binding and competition

RecA mediated homologous recombination requires cooperative ATP binding and hydrolysis to assume and maintain an active, extended DNA-protein (nucleoprotein) filament. Human RAD51 protein (hRAD51) lacks the magnitude of ATP-induced cooperativity and catalytic efficiency displayed by RecA. Here, we examined hRAD51 binding and ATPase inhibition pattern by ADP and ATP/adenosine 5'-O-(thiotriphosphate) (ATPgammaS). hRAD51 fully saturates with ATP/ATPgammaS regardless of DNA cofactor (K(D) approximately 5 microm; 1 ATP/1 hRAD51). The binding of ADP to hRAD51 appeared bimodal. The first mode was identical to ATP/ATPgammaS binding (K(app1) approximately 3 microm; 1 ADP/1 hRAD51), while a second mode occurred at elevated ADP concentrations (K(app2) > or = 125 microm; >1 ADP/1 hRAD51). We could detect ADP --> ATP exchange in the high affinity ADP binding mode (K(app1)) but not the low affinity binding mode (K(app2)). At low ATP concentrations (<0.3 mm), ADP and ATPgammaS competitively inhibit the hRAD51 ATPase (K(m)((app)) > K(m)). However, at high ATP (>0.3 mm), the hRAD51 ATPase was stimulated by concentrations of ATPgammaS that were 20-fold above the K(D). Ammonium sulfate plus spermidine decreased the affinity of hRAD51 for ADP substantially ( approximately 10-fold) and ATP modestly ( approximately 3-fold). Our results suggest that ATP binding is not rate-limiting but that the inability to sustain an active nucleoprotein filament probably restricts the hRAD51 ATPase.     

Mechanism of nucleotide binding to actomyosin VI: evidence for allosteric head-head communication

We have examined the kinetics of nucleotide binding to actomyosin VI by monitoring the fluorescence of pyrene-labeled actin filaments. ATP binds single-headed myosin VI following a two-step reaction mechanism with formation of a low affinity collision complex (1/K(1)' = 5.6 mm) followed by isomerization (k(+2)' = 176 s-1) to a state with weak actin affinity. The rates and affinity for ADP binding were measured by kinetic competition with ATP. This approach allows a broader range of ADP concentrations to be examined than with fluorescent nucleotide analogs, permitting the identification and characterization of transiently populated intermediates in the pathway. ADP binding to actomyosin VI, as with ATP binding, occurs via a two-step mechanism. The association rate constant for ADP binding is approximately five times greater than for ATP binding because of a higher affinity in the collision complex (1/K(5b)' = 2.2 mm) and faster isomerization rate constant (k(+5a)' = 366 s(-1)). By equilibrium titration, both heads of a myosin VI dimer bind actin strongly in rigor and with bound ADP. In the presence of ATP, conditions that favor processive stepping, myosin VI does not dwell with both heads strongly bound to actin, indicating that the second head inhibits strong binding of the lead head to actin. With both heads bound strongly, ATP binding is accelerated 2.5-fold, and ADP binding is accelerated >10-fold without affecting the rate of ADP release. We conclude that the heads of myosin VI communicate allosterically and accelerate nucleotide binding, but not dissociation, when both are bound strongly to actin.     

Effect of phosphorylation on the binding of smooth muscle heavy meromyosin X ADP to actin

Relaxation of both smooth and skeletal muscles appears to be caused primarily by inhibition of the step associated with Pi release in the actomyosin ATPase cycle, rather than by a block in the binding of the myosin X ATP and myosin X ADP X Pi complexes to actin. In skeletal muscle, troponin-tropomyosin not only causes marked inhibition of Pi release, but it also markedly inhibits the binding of myosin subfragment-1 X ADP to actin, raising the possibility that the two phenomena are coupled in some way. In the present study we determined whether phosphorylation of smooth muscle heavy meromyosin (HMM) also affects both the binding of HMM X ADP to actin and the Pi release step. This was done by having phosphorylated and unphosphorylated HMM X ADP compete for sites on F-actin. At mu = 30 mM, phosphorylation increased the affinity of the HMM molecule for actin about 12-fold and at mu = 170 mM, there was less than a 3-fold increase in the affinity of HMM. If phosphorylation affects the binding of each head of HMM to the same extent, then phosphorylation caused about a 4- and 2-fold increase in the affinity of each head of HMM for actin at mu = 30 and 170 mM, respectively. In contrast, at both ionic strengths, phosphorylation caused more than 100-fold actin activation of the ATPase activity of smooth muscle HMM. Therefore, the marked activation of Pi release in the acto X HMM ATPase cycle upon phosphorylation of HMM is not accompanied by a comparable increase in the affinity of HMM X ADP for actin. We have also found that phosphorylation increases by only 4-fold the rate of Pi release from HMM alone. These results suggest that in smooth muscle, phosphorylation accelerates the step associated with the release of Pi both in the forward and the reverse direction without correspondingly affecting the binding of myosin X ADP to actin.     

Modulation of the Kinesin ATPase Cycle by Neck Linker Docking and Microtubule Binding

Kinesin motor proteins use an ATP hydrolysis cycle to perform various functions in eukaryotic cells. Many questions remain about how the kinesin mechanochemical ATPase cycle is fine-tuned for specific work outputs. In this study, we use isothermal titration calorimetry and stopped-flow fluorometry to determine and analyze the thermodynamics of the human kinesin-5 (Eg5/KSP) ATPase cycle. In the absence of microtubules, the binding interactions of kinesin-5 with both ADP product and ATP substrate involve significant enthalpic gains coupled to smaller entropic penalties. However, when the wild-type enzyme is titrated with a non-hydrolyzable ATP analog or the enzyme is mutated such that it is able to bind but not hydrolyze ATP, substrate binding is 10-fold weaker than ADP binding because of a greater entropic penalty due to the structural rearrangements of switch 1, switch 2, and loop L5 on ATP binding. We propose that these rearrangements are reversed upon ATP hydrolysis and phosphate release. In addition, experiments on a truncated kinesin-5 construct reveal that upon nucleotide binding, both the N-terminal cover strand and the neck linker interact to modulate kinesin-5 nucleotide affinity. Moreover, interactions with microtubules significantly weaken the affinity of kinesin-5 for ADP without altering the affinity of the enzyme for ATP in the absence of ATP hydrolysis. Together, these results define the energy landscape of a kinesin ATPase cycle in the absence and presence of microtubules and shed light on the role of molecular motor mechanochemistry in cellular microtubule dynamics.     

ATPase domain of Hsp70 exhibits intrinsic ATP-ADP exchange activity

The chaperone activity of Hsp70 in protein folding and its conformational switching are regulated through the hydrolysis of ATP and the ATP-ADP exchange cycle. It was reported that, in the presence of physiological concentrations of ATP (approximately 5 mM) and ADP (approximately 0.5 mM), Hsp70 catalyzes ATP-ADP exchange through transfer of gamma-phosphate between ATP and ADP, via an autophosphorylated intermediate, whereas it only catalyzes the hydrolysis of ATP in the absence of ADP. To clarify the functional domain of the ATP-ADP exchange activity of Hsp70, we isolated the 44-kD ATPase domain of Hsp70 after limited proteolysis with alpha-chymotrypsin (EC 3.4.21.1). The possibility of ATP-ADP exchange activity of a contaminating nucleoside diphosphate kinase (EC 2.7.4.6) was monitored throughout the experiments. The purified 44-kD ATPase domain exhibited intrinsic ATP-ADP exchange by catalyzing the transfer of gamma-phosphate between ATP and ADP with acid-stable autophosphorylation at Thr204.     

The kinetic mechanism of kinesin

The chemical kinetic mechanism of kinesin (K) is considered by using a consensus scheme incorporating biochemically defined open, closed and trapped states. In the absence of microtubules, the dominant species is a trapped K*ADP state, which is defined by its ultra-slow release of ADP (off rate, k(off) approximately 0.002 s(-1)) and weak microtubule binding (dissociation constant, K(d) approximately 10-20 microM). Once bound, this trapped state equilibrates with a strongly binding open state that rapidly releases ADP (k(off) approximately 300 s(-1)). After ADP release, Mg*ATP binds (on rate, k(on) approximately 2 microM(-1)s(-1)) driving formation of a closed state that is defined by hydrolysis competence and by strong binding to microtubules. Hydrolysis (k(hyd) approximately 100-300 s(-1)) and phosphate release (k(off)>100 s(-1)) both occur in this microtubule-bound closed state. Phosphate release acts as a gate that controls reversion to the trapped K*ADP state, which detaches from the microtubule, completing the cycle.