N-terminal Nucleotide-binding Domain Research Articles

Phenylalanine-508 mediates a cytoplasmic–membrane domain contact in the CFTR 3D structure crucial to assembly and channel function

Deletion of phenylalanine-508 (Phe-508) from the N-terminal nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ATP-binding cassette (ABC) transporter family, disrupts both its folding and function and causes most cystic fibrosis. Most mutant nascent chains do not pass quality control in the ER, and those that do remain thermally unstable, only partially functional, and are rapidly endocytosed and degraded. Although the lack of the Phe-508 peptide backbone diminishes the NBD1 folding yield, the absence of the aromatic side chain is primarily responsible for defective CFTR assembly and channel gating. However, the site of interdomain contact by the side chain is unknown as is the high-resolution 3D structure of the complete protein. Here we present a 3D structure of CFTR, constructed by molecular modeling and supported biochemically, in which Phe-508 mediates a tertiary interaction between the surface of NBD1 and a cytoplasmic loop (CL4) in the C-terminal membrane-spanning domain (MSD2). This crucial cytoplasmic membrane interface, which is dynamically involved in regulation of channel gating, explains the known sensitivity of CFTR assembly to many disease-associated mutations in CL4 as well as NBD1 and provides a sharply focused target for small molecules to treat CF. In addition to identifying a key intramolecular site to be repaired therapeutically, our findings advance understanding of CFTR structure and function and provide a platform for focused biochemical studies of other features of this unique ABC ion channel.

Diminished Self-Chaperoning Activity of the ΔF508 Mutant of CFTR Results in Protein Misfolding

The absence of a functional ATP Binding Cassette (ABC) protein called the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) from apical membranes of epithelial cells is responsible for cystic fibrosis (CF). Over 90% of CF patients carry at least one mutant allele with deletion of phenylalanine at position 508 located in the N-terminal nucleotide binding domain (NBD1). Biochemical and cell biological studies show that the ΔF508 mutant exhibits inefficient biosynthetic maturation and susceptibility to degradation probably due to misfolding of NBD1 and the resultant misassembly of other domains. However, little is known about the direct effect of the Phe508 deletion on the NBD1 folding, which is essential for rational design strategies of cystic fibrosis treatment. Here we show that the deletion of Phe508 alters the folding dynamics and kinetics of NBD1, thus possibly affecting the assembly of the complete CFTR. Using molecular dynamics simulations, we find that meta-stable intermediate states appearing on wild type and mutant folding pathways are populated differently and that their kinetic accessibilities are distinct. The structural basis of the increased misfolding propensity of the ΔF508 NBD1 mutant is the perturbation of interactions in residue pairs Q493/P574 and F575/F578 found in loop S7-H6. As a proof-of-principle that the S7-H6 loop conformation can modulate the folding kinetics of NBD1, we virtually design rescue mutations in the identified critical interactions to force the S7-H6 loop into the wild type conformation. Two redesigned NBD1-ΔF508 variants exhibited significantly higher folding probabilities than the original NBD1-ΔF508, thereby partially rescuing folding ability of the NBD1-ΔF508 mutant. We propose that these observed defects in folding kinetics of mutant NBD1 may also be modulated by structures separate from the 508 site. The identified structural determinants of increased misfolding propensity of NBD1-ΔF508 are essential information in correcting this pathogenic mutant.

Sorting Nexin 9 Interacts with Dynamin 1 and N-WASP and Coordinates Synaptic Vesicle Endocytosis

Sorting nexin 9 (SNX9) is a member of the sorting nexin family of proteins, each of which contains a characteristic Phox homology domain. SNX9 is widely expressed and plays a role in clathrin-mediated endocytosis, but it is not known if it is present in neuronal cells. We report that SNX9 is expressed in the presynaptic compartment of cultured hippocampal neurons, where it binds to dynamin-1 and N-WASP. Overexpression of full-length SNX9 or a C-terminal truncated version caused severe defects in synaptic vesicle endocytosis during, as well as after, stimulation. Knockdown of SNX9 with short interfering RNA also reduced synaptic vesicle endocytosis, and the W39A mutation of SNX9 abolished the inhibitory effect of SNX9 on endocytosis. Rescue experiments showed that most of the effect of SNX9 on endocytosis results from its interaction with dynamin 1, although its interaction with N-WASP contributes in some degree. We further showed that SNX9 dimerizes through its C-terminal domain, suggesting that it may interact simultaneously with dynamin 1 and N-WASP. We propose that SNX9 interacts with dynamin-1 and N-WASP in presynaptic terminals, where it links actin dynamics and synaptic vesicle endocytosis.

Staphylococcus aureus ArcR Controls Expression of the Arginine Deiminase Operon

We identified a single open reading frame that is strongly similar to ArcR, a member of the Crp/Fnr family of bacterial transcriptional regulators, in all sequenced Staphylococcus aureus genomes. The arcR gene encoding ArcR forms an operon with the arginine deiminase (ADI) pathway genes arcABDC that enable the utilization of arginine as a source of energy for growth under anaerobic conditions. In this report, we show that under anaerobic conditions, S. aureus growth is subject to glucose catabolic repression and is enhanced by arginine. Likewise, glucose and arginine have reciprocal effects on the transcription of the arcABDCR genes. Furthermore, we show using a mutant deleted for arcR that the transcription of the arc operon under anaerobic conditions depends strictly on a functional ArcR. These findings are supported by proteome analyses, which showed that under anaerobic conditions the expression of the ADI catabolic proteins depends on ArcR. Bioinformatic analysis of S. aureus ArcR predicts an N-terminal nucleotide binding domain and a C-terminal helix-turn-helix DNA binding motif. ArcR binds to a conserved Crp-like sequence motif, TGTGA-N(6)-TCACA, present in the arc promoter region and thereby activates the expression of the ADI pathway genes. Crp-like sequence motifs were also found in the regulatory regions of some 30 other S. aureus genes mostly encoding anaerobic enzymatic systems, virulence factors, and regulatory systems. ArcR was tested and found to bind to the regulatory regions of four such genes, adh1, lctE, srrAB, and lukM. In one case, for lctE, encoding l-lactate dehydrogenase, ArcR was able to bind only in the presence of cyclic AMP. These observations suggest that ArcR is likely to play an important role in the expression of numerous genes required for anaerobic growth.

Crystal structure of the C-terminal three-helix bundle subdomain of C. elegans Hsp70

Hsp70 chaperones are composed of two domains; the 40kDa N-terminal nucleotide-binding domain (NDB) and the 30kDa C-terminal substrate-binding domain (SBD). Structures of the SBD from Escherichia coli homologues DnaK and HscA show it can be further divided into an 18kDa β-sandwich subdomain, which forms the hydrophobic binding pocket, and a 10kDa C-terminal three-helix bundle that forms a lid over the binding pocket. Across prokaryotes and eukaryotes, the NBD and β-sandwich subdomain are well conserved in both sequence and structure. The C-terminal subdomain is, however, more evolutionary variable and the only eukaryotic structure from rat Hsc70 revealed a diverged helix–loop–helix fold. We have solved the crystal structure of the C-terminal 10kDa subdomain from Caenorhabditis elegans Hsp70 which forms a helical-bundle similar to the prokaryotic homologues. This provides the first confirmation of the structural conservation of this subdomain in eukaryotes. Comparison with the rat structure reveals a domain-swap dimerisation mechanism; however, the C. elegans subdomain exists exclusively as a monomer in solution in agreement with the hypothesis that regions out with the C-terminal subdomain are necessary for Hsp70 self-association.

X-ray Crystallographic and Steady State Fluorescence Characterization of the Protein Dynamics of Yeast Polyadenylate Polymerase

Polyadenylate polymerase (PAP) catalyzes the synthesis of poly(A) tails on the 3′-end of pre-mRNA. PAP is composed of three domains: an N-terminal nucleotide-binding domain (homologous to the palm domain of DNA and RNA polymerases), a middle domain (containing other conserved, catalytically important residues), and a unique C-terminal domain (involved in protein–protein interactions required for 3′-end formation). Previous X-ray crystallographic studies have shown that the domains are arranged in a V-shape such that they form a central cleft with the active site located at the base of the cleft at the interface between the N-terminal and middle domains. In the previous studies, the nucleotides were bound directly to the N-terminal domain and exhibited a conspicuous lack of adenine-specific interactions that would constitute nucleotide recognition. Furthermore, it was postulated that base-specific contacts with residues in the middle domain could occur either as a result of a change in the conformation of the nucleotide or domain movement. To address these issues and to better characterize the structural basis of substrate recognition and catalysis, we report two new crystal structures of yeast PAP. A comparison of these structures reveals that the N-terminal and C-terminal domains of PAP move independently as rigid bodies along two well defined axes of rotation. Modeling of the nucleotide into the most closed state allows us to deduce specific nucleotide interactions involving residues in the middle domain (K215, Y224 and N226) that are proposed to be involved in substrate binding and specificity. To further investigate the nature of PAP domain flexibility, 2-aminopurine labeled molecular probes were employed in steady state fluorescence and acrylamide quenching experiments. The results suggest that the closed domain conformation is stabilized upon recognition of the correct subtrate, MgATP, in an enzyme-substrate ternary complex. The implications of these results on the enzyme mechanism of PAP and the possible role for domain motion in an induced fit mechanism are discussed.

Subcellular trafficking of the yeast plasma membrane ABC transporter, Pdr5, is impaired by a mutation in the N‐terminal nucleotide‐binding fold

The plasma membrane ATP-binding cassette (ABC) transporter, Pdr5p, mediates resistance to many different xenobiotic compounds in yeast. We have isolated several mutated forms that fail to confer resistance to cycloheximide and itraconazole. Here, we examined two variants, the expression of which was abnormally low when cells reach the stationary phase of growth. The Pdr5(1157) variant lacked the C-terminal transmembrane domain due to the presence of a nonsense mutation at codon 1158. The second variant, Pdr5(L183P), contained a Leu183Pro substitution close to the Walker A motif in the N-terminal nucleotide-binding domain. This substitution impaired UTPase activity as well as protein stability. The Pdr5(L183P) variant induced the unfolded protein response and was targeted to the proteasome for degradation. Fluorescence microscopy showed that the highly unstable Pdr5(L183P) was mislocalized to endoplasmic reticulum (ER)-associated compartments, whereas the truncated Pdr5(1157) protein was retained in the ER. When threonine 363 (located in the first nucleotide-binding domain, close to the Walker B motif) in Pdr5(L183P) was replaced with isoleucine, this double mutant conferred partial drug resistance. These results suggest that Pdr5p requires a properly folded nucleotide-binding domain for trafficking to the plasma membrane.

Conserved Asp327 of Walker B Motif in the N-Terminal Nucleotide Binding Domain (NBD-1) of Cdr1p ofCandida albicansHas Acquired a New Role in ATP Hydrolysis

The Walker A and B motifs of nucleotide binding domains (NBDs) of Cdr1p though almost identical to all ABC transporters, has unique substitutions. We have shown in the past that Trp326 of Walker B and Cys193 of Walker A motifs of N-terminal NBD of Cdr1p have distinct roles in ATP binding and hydrolysis, respectively. In the present study, we have examined the role of a well conserved Asp327 in the Walker B motif of the N-terminal NBD, which is preceded (Trp326) and followed (Asn328) by atypical amino acid substitutions and compared it with its equivalent well conserved Asp1026 of the C-terminal NBD of Cdr1p. We observed that the removal of the negative charge by D327N, D327A, D1026N, D1026A, and D327N/D1026N substitutions, resulted in Cdr1p mutant variants that were severely impaired in ATPase activity and drug efflux. Importantly, all of the mutant variants showed characteristics similar to those of the wild type with respect to cell surface expression and photoaffinity drug analogue [125I] IAAP and [3H] azidopine labeling. Although the Cdr1p D327N mutant variant showed comparable binding with [alpha-32P] 8-azido ATP, Cdr1p D1026N and Cdr1p D327N/D1026N mutant variants were crippled in nucleotide binding. That the two conserved carboxylate residues Asp327 and Asp1026 are functionally different was further evident from the pH profile of ATPase activity. The Cdr1p D327N mutant variant showed approximately 40% enhancement of its residual ATPase activity at acidic pH, whereas no such pH effect was seen with the Cdr1p D1026N mutant variant. Our experimental data suggest that Asp327 of N-terminal NBD has acquired a new role to act as a catalytic base in ATP hydrolysis, a role normally conserved for Glu present adjacent to the conserved Asp in the Walker B motif of all the non-fungal transporters.

Chimeras of the ABC drug transporter Cdr1p reveal functional indispensability of transmembrane domains and nucleotide-binding domains, but transmembrane segment 12 is replaceable with the corresponding homologous region of the non-drug transporter Cdr3p

The molecular basis of the broad substrate recognition and the transport of substrates by Cdr1p, a major drug efflux protein of Candida albicans, is not well understood. To investigate the role of transmembrane domains and nucleotide-binding domains (NBDs) of Cdr1p in drug transport, two sets of protein chimeras were constructed: one set between homologous regions of Cdr1p and the non-drug transporter Cdr3p, and another set consisting of Cdr1p variants comprising either two N- or two C-terminal NBDs of Cdr1p. The replacement of either the N- or the C-terminal half of Cdr1p by the homologous segments of Cdr3p resulted in non-functional recombinant strains expressing chimeric proteins. The results suggest that the chimeric protein could not reach the plasma membrane, probably because of misfolding and subsequent cellular trafficking problems, or the rapid degradation of the chimeras. As an exception, the replacement of transmembrane segment 12 (TMS12) of Cdr1p by the corresponding region of Cdr3p resulted in a functional chimera which displayed unaltered affinity for all the tested substrates. The variant protein comprising either two N-terminal or two C-terminal NBDs of Cdr1p also resulted in non-functional recombinant strains. However, the N-terminal NBD variant, which also showed poor cell surface localization, could be rescued to cell surface, if cells were grown in the presence of drug substrates. The rescued chimera remained non-functional, as was evident from impaired ATPase and efflux activities. Taken together, the results suggest that the two NBDs of Cdr1p are asymmetric and non-exchangeable and that the drug efflux by Cdr1p involves complex interactions between the two halves of the protein.

Modulation of Chaperone Function and Cochaperone Interaction by Novobiocin in the C-terminal Domain of Hsp90: EVIDENCE THAT COUMARIN ANTIBIOTICS DISRUPT Hsp90 DIMERIZATION

The C-terminal domain of Hsp90 displays independent chaperone activity, mediates dimerization, and contains the MEEVD motif essential for interaction with tetratricopeptide repeat-containing immunophilin cochaperones assembled in mature steroid receptor complexes. An alpha-helical region, upstream of the MEEVD peptide, helps form the dimerization interface and includes a hydrophobic microdomain that contributes to the Hsp90 interaction with the immunophilin cochaperones and corresponds to the binding site for novobiocin, a coumarin-related Hsp90 inhibitor. Mutation of selected residues within the hydrophobic microdomain significantly impacted the chaperone function of a recombinant C-terminal Hsp90 fragment and novobiocin inhibited wild-type chaperone activity. Prior incubation of the Hsp90 fragment with novobiocin led to a direct blockade of immunophilin cochaperone binding. However, the drug had little influence on the pre-formed Hsp90-immunophilin complex, suggesting that bound cochaperones mask the novobiocin-binding site. We observed a differential effect of the drug on Hsp90-immunophilin interaction, suggesting that the immunophilins make distinct contacts within the C-terminal domain to specifically modulate Hsp90 function. Novobiocin also precluded the interaction of full-length Hsp90 with the p50(cdc37) cochaperone, which targets the N-terminal nucleotide-binding domain, and is prevalent in Hsp90 complexes with protein kinase substrates. Novobiocin therefore acts locally and allosterically to induce conformational changes within multiple regions of the Hsp90 protein. We provide evidence that coumermycin A1, a coumarin structurally related to novobiocin, interferes with dimerization of the Hsp90 C-terminal domain. Coumarin-based inhibitors then may antagonize Hsp90 function by inducing a conformation favoring separation of the C-terminal domains and release of substrate.

ATP hydrolysis is required to reset the ATP-binding cassette dimer into the resting-state conformation

ATP-binding cassette (ABC) transporters couple ATP binding and hydrolysis to the movement of substances across the membrane; conformational changes clearly play an important role in the transporter mechanism. Previously, we have shown that a dimer of MalK, the ATPase subunit of the maltose transporter from Escherichia coli, undergoes a tweezers-like motion in a transport cycle. The MalK monomer consists of an N-terminal nucleotide binding domain and a C-terminal regulatory domain. The two nucleotide-binding domains in a dimer are either open or closed, depending on whether ATP is present, while the regulatory domains maintain contacts to hold the dimer together. In this work, the structure of MalK in a posthydrolysis state is presented, obtained by cocrystallizing MalK with ATP-Mg(2+). ATP was hydrolyzed in the crystallization drop, and ADP-Mg(2+) was found in the resulting crystal structure. In contrast to the ATP-bound form where two ATP molecules are buried in a closed interface between the nucleotide-binding domains, the two nucleotide-binding domains of the ADP-bound form are open, indicating that ADP, unlike ATP, cannot stabilize the closed form. This conclusion is further supported by oligomerization studies of MalK in solution. At low protein concentrations, ATP promotes dimerization of MalK, whereas ADP does not. The structures of dimeric MalK in the nucleotide-free, ATP-bound, and ADP-bound forms provide a framework for understanding the nature of the conformational changes that occur in an ATP-binding cassette transporter hydrolysis cycle, as well as how conformational changes in MalK are coupled to solute transport.

Structural Basis of Interdomain Communication in the Hsc70 Chaperone

Hsp70 family proteins are highly conserved chaperones involved in protein folding, degradation, targeting and translocation, and protein complex remodeling. They are comprised of an N-terminal nucleotide binding domain (NBD) and a C-terminal protein substrate binding domain (SBD). ATP binding to the NBD alters SBD conformation and substrate binding kinetics, but an understanding of the mechanism of interdomain communication has been hampered by the lack of a crystal structure of an intact chaperone. We report here the 2.6 angstroms structure of a functionally intact bovine Hsc70 (bHsc70) and a mutational analysis of the observed interdomain interface and the immediately adjacent interdomain linker. This analysis identifies interdomain interactions critical for chaperone function and supports an allosteric mechanism in which the interdomain linker invades and disrupts the interdomain interface when ATP binds.

Attempts to characterize the NBD heterodimer of MRP1: transient complex formation involves Gly771 of the ABC signature sequence but does not enhance the intrinsic ATPase activity

MRP1 (multidrug-resistance-associated protein 1; also known as ABCC1) is a member of the human ABC (ATP-binding cassette) transporter superfamily that confers cell resistance to chemotherapeutic agents. Considering the structural and functional similarities to the other ABC proteins, the interaction between its two NBDs (nucleotide-binding domains), NBD1 (N-terminal NBD) and NBD2 (C-terminal NBD), is proposed to be essential for the regulation of the ATP-binding/ATP-hydrolysis cycle of MRP1. We were interested in the ability of recombinant NBD1 and NBD2 to interact with each other and to influence ATPase activity. We purified NBD1 (Asn642-Ser871) and NBD2 (Ser1286-Val1531) as soluble monomers under native conditions. We measured extremely low intrinsic ATPase activity of NBD1 (10(-5) s(-1)) and NBD2 (6x10(-6) s(-1)) and no increase in the ATP-hydrolysis rate could be detected in an NBD1+NBD2 mixture, with concentrations up to 200 microM. Despite the fact that both monomers bind ATP, no stable NBD1.NBD2 heterodimer could be isolated by gel-filtration chromatography or native-PAGE, but we observed some significant modifications of the heteronuclear single-quantum correlation NMR spectrum of 15N-NBD1 in the presence of NBD2. This apparent NBD1.NBD2 interaction only occurred in the presence of Mg2+ and ATP. Partial sequential assignment of the NBD1 backbone resonances shows that residue Gly771 of the LSGGQ sequence is involved in NBD1.NBD2 complex formation. This is the first NMR observation of a direct interaction between the ABC signature and the opposite NBD. Our study also reveals that the NBD1.NBD2 heterodimer of MRP1 is a transient complex. This labile interaction is not sufficient to induce an ATPase co-operativity of the NBDs and suggests that other structures are required for the ATPase activation mechanism.

Functional Characterization of N-Terminal Nucleotide Binding Domain (NBD-1) of a Major ABC Drug Transporter Cdr1p of Candida albicans: Uncommon but Conserved Trp326 of Walker B Is Important for ATP Binding

Using purified N-terminal NBD (NBD-512) domain of Cdr1p, a major multidrug extrusion pump of human pathogenic yeast Candida albicans, we show the relevance of the unique positioning of an atypical Trp326 residue. Similar to Cys193 in Walker A, Trp326 in the Walker B motif of Cdr1p is also a conserved feature of other fungal ATP Binding Cassette (ABC) transporters. By employing fluorescence spectroscopy, chemical modification, and site-directed mutagenesis, we demonstrate that of the five Trp residues in the NBD-512 domain, Trp326 alone is important for nucleotide binding and subsequent conformational changes within the domain. Furthermore, mutation of Trp326 to Ala results in an increased K(M) without appreciably affecting V(max) of ATPase activity. Thus, Trp326 in NBD-512 appears to be important for nucleotide binding and not for its hydrolysis. Additionally, the role of Trp326 in ATP binding is independent of the presence of the adjacent well-conserved Asp327 residue which, like Cys193, has a catalytic role in ATP hydrolysis. Considering that Trp326 of Cdr1p is a typical feature of fungal transporters alone, our study suggests that these ABC transporters may reflect mechanistic differences with regard to nucleotide binding and hydrolysis as compared to their counterparts of non-fungal origin.

The Molecular Chaperone, ClpA, Has a Single High Affinity Peptide Binding Site per Hexamer

Substrate recognition by Clp chaperones is dependent on interactions with motifs composed of specific peptide sequences. We studied the binding of short motif-bearing peptides to ClpA, the chaperone component of the ATP-dependent ClpAP protease of Escherichia coli in the presence of ATPgammaS and Mg2+ at pH 7.5. Binding was measured by isothermal titration calorimetry (ITC) using the peptide, AANDENYALAA, which corresponds to the SsrA degradation motif found at the C terminus of abnormal nascent polypeptides in vivo. One SsrA peptide was bound per hexamer of ClpA with an association constant (K(A)) of 5 x 10(6) m(-1). Binding was also assayed by changes in fluorescence of an N-terminal dansylated SsrA peptide, which bound with the same stoichiometry of one per ClpA hexamer (K(A) approximately 1 x 10(7) m(-1)). Similar results were obtained when ATP was substituted for ATPgammaS at 6 degrees C. Two additional peptides, derived from the phage P1 RepA protein and the E. coli HemA protein, which bear different substrate motifs, were competitive inhibitors of SsrA binding and bound to ClpA hexamers with K(A)' > 3 x 10(7) m(-1). DNS-SsrA bound with only slightly reduced affinity to deletion mutants of ClpA missing either the N-terminal domain or the C-terminal nucleotide-binding domain, indicating that the binding site for SsrA lies within the N-terminal nucleotide-binding domain. Because only one protein at a time can be unfolded and translocated by ClpA hexamers, restricting the number of peptides initially bound should avoid nonproductive binding of substrates and aggregation of partially processed proteins.

NMR Study of Nucleotide-induced Changes in the Nucleotide Binding Domain of Thermus thermophilus Hsp70 Chaperone DnaK: IMPLICATIONS FOR THE ALLOSTERIC MECHANISM

We present an NMR investigation of the nucleotide-dependent conformational properties of a 44-kDa nucleotide binding domain (NBD) of an Hsp70 protein. Conformational changes driven by ATP binding and hydrolysis in the N-terminal NBD are believed to allosterically regulate substrate affinity in the C-terminal substrate binding domain. Several crystal structures of Hsc70 NBDs in different nucleotide states have, however, not shown significant structural differences. We have previously reported the NMR assignments of the backbone resonances of the NBD of the bacterial Hsp70 homologue Thermus thermophilus DnaK in the ADP-bound state. In this study we show, by assigning the NBD with the ATP/transition state analogue, ADP.AlFx, bound, that it closely mimics the ATP-bound state. Chemical shift difference mapping of the two nucleotide states identified differences in a cluster of residues at the interface between subdomains 1A and 1B. Further analysis of the spectra revealed that the ATP state exhibited a single conformation, whereas the ADP state was in slow conformational exchange between a form similar to the ATP state and another state unique to the ADP-bound form. A model is proposed of the allosteric mechanism based on the nucleotide state altering the balance of a dynamic equilibrium between the open and closed states. The observed chemical shift perturbations were concentrated in an area close to a previously described J-domain binding channel, confirming the importance of that region in the allosteric mechanism.

Structural basis for recruitment of the ATPase activator Aha1 to the Hsp90 chaperone machinery.

Hsp90 is a molecular chaperone essential for the activation and assembly of many key eukaryotic signalling and regulatory proteins. Hsp90 is assisted and regulated by co-chaperones that participate in an ordered series of dynamic multiprotein complexes, linked to Hsp90s conformationally coupled ATPase cycle. The co-chaperones Aha1 and Hch1 bind to Hsp90 and stimulate its ATPase activity. Biochemical analysis shows that this activity is dependent on the N-terminal domain of Aha1, which interacts with the central segment of Hsp90. The structural basis for this interaction is revealed by the crystal structure of the N-terminal domain (1-153) of Aha1 (equivalent to the whole of Hch1) in complex with the middle segment of Hsp90 (273-530). Structural analysis and mutagenesis show that binding of N-Aha1 promotes a conformational switch in the middle-segment catalytic loop (370-390) of Hsp90 that releases the catalytic Arg 380 and enables its interaction with ATP in the N-terminal nucleotide-binding domain of the chaperone.

The Mechanism of Hsp90 Regulation by the Protein Kinase-Specific Cochaperone p50 cdc37

Recruitment of protein kinase clients to the Hsp90 chaperone involves the cochaperone p50 cdc37 acting as a scaffold, binding protein kinases via its N-terminal domain and Hsp90 via its C-terminal region. p50 cdc37 also has a regulatory activity, arresting Hsp90's ATPase cycle during client-protein loading. We have localized the binding site for p50 cdc37 to the N-terminal nucleotide binding domain of Hsp90 and determined the crystal structure of the Hsp90–p50 cdc37 core complex. Dimeric p50 cdc37 binds to surfaces of the Hsp90 N-domain implicated in ATP-dependent N-terminal dimerization and association with the middle segment of the chaperone. This interaction fixes the lid segment in an open conformation, inserts an arginine side chain into the ATP binding pocket to disable catalysis, and prevents trans-activating interaction of the N domains.

A Tweezers-like Motion of the ATP-Binding Cassette Dimer in an ABC Transport Cycle

The ATPase components of ATP binding cassette (ABC) transporters power the transporters by binding and hydrolyzing ATP. Major conformational changes of an ATPase are revealed by crystal structures of MalK, the ATPase subunit of the maltose transporter from Escherichia coli, in three different dimeric configurations. While other nucleotide binding domains or subunits display low affinity for each other in the absence of the transmembrane segments, the MalK dimer is stabilized through interactions of the additional C-terminal domains. In the two nucleotide-free structures, the N-terminal nucleotide binding domains are separated to differing degrees, and the dimer is maintained through contacts of the C-terminal regulatory domains. In the ATP-bound form, the nucleotide binding domains make contact and two ATPs lie buried along the dimer interface. The two nucleotide binding domains of the dimer open and close like a pair of tweezers, suggesting a regulatory mechanism for ATPase activity that may be tightly coupled to translocation.

Purification and characterization of the N-terminal nucleotide binding domain of an ABC drug transporter of Candida albicans: uncommon cysteine 193 of Walker A is critical for ATP hydrolysis.

The Candida drug resistance protein Cdr1p (approximately 170 kDa) is a member of ATP binding cassette (ABC) superfamily of drug transporters, characterized by the presence of 2 nucleotide binding domains (NBD) and 12 transmembrane segments (TMS). NBDs of these transporters are the hub of ATP hydrolysis activity, and their sequence contains a conserved Walker A motif (GxxGxGKS/T). Mutations of the lysine residue within this motif abrogate the ability of NBDs to hydrolyze ATP. Interestingly, the sequence alignments of Cdr1p NBDs with other bacterial and eukaryotic transporters reveal that its N-terminal NBD contains an unusual Walker A sequence (GRPGAGCST), as the invariant lysine is replaced by a cysteine. In an attempt to understand the significance of this uncommon positioning of cysteine within the Walker A motif, we for the first time have purified and characterized the N-terminal NBD (encompassing first N-terminal 512 amino acids) of Cdr1p as well as its C193A mutant protein. The purified NBD-512 protein could exist as an independent functional general ribonucleoside triphosphatase with strong divalent cation dependence. It exhibited ATPase activity with an apparent K(m) in the 0.8-1.0 mM range and V(max) in the range of 147-160 nmol min(-)(1) (mg of protein)(-)(1). NBD-512-associated ATPase activity was also sensitive to inhibitors such as vanadate, azide, and NEM. The Mut-NBD-512 protein (C193A) showed a severe impairment in its ability to hydrolyze ATP (95%); however, no significant effect on ATP (TNP-ATP) binding was observed. Our results show that C193 is critical for N-terminal NBD-mediated ATP hydrolysis and represents a unique feature distinguishing the ATP-dependent functionality of the ABC transporters of fungi from those found in bacteria and other eukaryotes.