ATP Binding Activity Research Articles

Crystal Structures of the Mnk2 Kinase Domain Reveal an Inhibitory Conformation and a Zinc Binding Site

Human mitogen-activated protein kinases (MAPK)-interacting kinases 1 and 2 (Mnk1 and Mnk2) target the translational machinery by phosphorylation of the eukaryotic initiation factor 4E (eIF4E). Here, we present the 2.1 A crystal structure of a nonphosphorylated Mnk2 fragment that encompasses the kinase domain. The results show Mnk-specific features such as a zinc binding motif and an atypical open conformation of the activation segment. In addition, the ATP binding pocket contains an Asp-Phe-Asp (DFD) in place of the canonical magnesium binding Asp-Phe-Gly (DFG) motif. The phenylalanine of this motif sticks into the ATP binding pocket and blocks ATP binding as observed with inhibitor bound and, thus, inactive p38 kinase. Replacement of the DFD by the canonical DFG motif affects the conformation of Mnk2, but not ATP binding and kinase activity. The results suggest that the ATP binding pocket and the activation segment of Mnk2 require conformational switches to provide kinase activity.

Universal Stress Proteins inEscherichia coli

Universal Stress Proteins in<i>Escherichia coli</i>

Chemical ablation of androgen receptor in prostate cancer cells by the histone deacetylase inhibitor LAQ824

Androgen receptor plays a critical role in the development of primary as well as advanced hormone-refractory prostate cancer. Therefore, ablation of androgen receptor from prostate cancer cells is an interesting concept for developing a new therapy not only for androgen-dependent prostate cancer but also for metastatic hormone-refractory prostate cancer, for which there is no effective treatment available. We report here that LAQ824, a cinnamyl hydroxamatic acid histone deacetylase inhibitor currently in human clinical trials, effectively depleted androgen receptor in prostate cancer cells at nanomolar concentrations. LAQ824 seemed capable of depleting both the mutant and wild-type androgen receptors in either androgen-dependent and androgen-independent prostate cancer cells. Although LAQ824 may exert its effect through multiple mechanisms, several lines of evidence suggest that inactivation of the heat shock protein-90 (Hsp90) molecular chaperone is involved in LAQ824-induced androgen receptor depletion. Besides androgen receptor, LAQ824 reduced the level of Hsp90 client proteins HER-2 (ErbB2), Akt/PKB, and Raf-1 in LNCaP cells. Another Hsp90 inhibitor, 17-allyamino-17-demethoxygeldanamycin (17-AAG), also induced androgen receptor diminution. LAQ824 induced Hsp90 acetylation in LNCaP cells, which resulted in inhibition of its ATP-binding activity, dissociation of Hsp90-androgen receptor complex, and proteasome-mediated degradation of androgen receptor. Consequently, LAQ824 blocked androgen-induced prostate-specific antigen production in LNCaP cells. LAQ824 effectively inhibited cell proliferation and induced apoptosis of these prostate cancer cells. These results reveal that LAQ824 is a potent agent for depletion of androgen receptor and a potential new drug for prostate cancer.

Adenosine 5′-Triphosphate-Dependent Vitamin D Sterol Binding to Heat Shock Protein-70 Chaperones

Chaperone proteins in the heat shock protein-70 family possess endogenous ATP binding and ATPase activity and interact with intracellular protein substrates in an ATP-dependent manner; the hydrolysis of ATP to ADP results in an increase in the affinity of the chaperone for protein substrates. Heat shock protein-70s can also specifically interact with 25-hydroxylated vitamin D metabolites. Using constitutively expressed heat shock protein-70 (hsc70) as chaperone, here we demonstrate that vitamin D metabolite binding to hsc70 is also ATP dependent. Transient overexpression of an hsc70-green fluorescent protein chimeric construct in primate kidney cells resulted in a 6-fold increase in specific, extractable 25-hydroxyvitamin D(3) binding. When ATPase capability of hsc70 was disabled, this increase was completely blocked. In solution, the binding of 25-hydroxylated vitamin D metabolites to hsc70 was significantly increased (P < 0.01) in the presence of ATP and a nonmetabolizable ATP analog. The ATP-directed increase in specific binding resulted from an increase in the abundance of relatively high-affinity hormone-binding sites (K(d), approximately 0.24 nM). These results suggest that ATP hydrolysis to ADP would favor the release of vitamin D from a donor hsc70 molecule at a time when an hsc70-bound acceptor protein substrate is anchored to the chaperone with relative avidity. We theorize that the endogenous ATPase activity of hsc70 promotes the transfer of vitamin D sterols to other intracellular vitamin D binding proteins, such as the vitamin D receptor and vitamin D hydroxylases, to which hsc70 is known to bind.

Coat proteins of two filamentous plant viruses display NTPase activity in vitro

Coat proteins (CPs) of plant viruses are involved in different stages of the viral life cycle such as virion assembly, replication, movement, vector transmission, and regulation of host defense responses. Here, we report that the CPs of two filamentous RNA viruses, potato virus X (PVX, Potexvirus) and potato virus A (PVA, Potyvirus) exhibit an enzyme activity. The CP isolated from PVX virions possesses ATP-binding and ATPase activities. Recombinant PVX and PVA CPs produced in Escherichia coli show Mg 2+-dependent ATPase and UTPase activities inhibited by antibodies against virus particles. Deletion of the C-terminal regions of these proteins diminishes their ATPase activity.

Hsp90 and environmental impacts on epigenetic states: a model for the trans-generational effects of diethylstibesterol on uterine development and cancer

Hsp90 is a chaperone for over 100 'client proteins' in the cell, most of which are involved in signaling pathways. For example, Hsp90 maintains several nuclear hormone receptors, such as the estrogen receptor (ER), as agonist-receptive monomers in the cytoplasm. In the presence of agonist, Hsp90 dissociates and the receptors dimerize, enter the nucleus and ultimately activate transcription of the target genes. Increasing evidence suggests that Hsp90 also has a role in modifying the chromatin conformation of many genes. For example, Hsp90 has recently been shown to increase the activity of the histone H3 lysine-4 methyltransferase SMYD3, which activates the chromatin of target genes. Further evidence for chromatin-remodeling functions is that Hsp90 acts as a capacitor for morphological evolution by masking epigenetic variation. Release of the capacitor function of Hsp90, such as by environmental stress or by drugs that inhibit the ATP-binding activity of Hsp90, exposes previously hidden morphological phenotypes in the next generation and for several generations thereafter. The chromatin-modifying phenotypes of Hsp90 have striking similarities to the trans-generational effects of the ER agonist diethylstilbesterol (DES). Prenatal and perinatal exposure to DES increases the predisposition to uterine developmental abnormalities and cancer in the daughters and granddaughters of exposed pregnant mice. In this review, we propose that trans-generational epigenetic phenomena involving Hsp90 and DES are related and that chromatin-mediated WNT signaling modifications are required. This model suggests that inhibitors of Hsp90, WNT signaling and chromatin-remodeling enzymes might function as anticancer agents by interfering with epigenetic reprogramming and canalization in cancer stem cells.

Rapid enrichment and analysis of yeast phosphoproteins using affinity chromatography, 2D‐PAGE and peptide mass fingerprinting

A combination of affinity purification, 2D-PAGE and peptide mass fingerprinting was employed to study the phosphoprotein complement of Saccharomyces cerevisiae. Protein extracts were first passed through a phosphoprotein affinity column, and the phosphoprotein-enriched eluate fractions were then separated on 2D gels and visualized by staining with SYPRO Ruby. Proteins were excised from the gels and identified by peptide mass fingerprinting; 11/13 protein spots identified from a gel of the phosphoprotein-enriched fraction had prior published evidence indicating that they were phosphoproteins. Additional experiments using a specific stain for phosphoproteins, prior incubation of the protein extract with alkaline phosphatase and blotting with monoclonal antibodies to phosphothreonine, phosphoserine and phosphotyrosine demonstrated that the phosphoprotein affinity column was an effective method for enriching phosphoproteins. Further validating the method, growth of yeast in the presence of sorbic acid resulted in altered phosphorylation of 17 proteins, 13 of which had prior published evidence that they were phosphoproteins or had ATP binding activity.

Functional Interactions Between Nucleotide Binding Domains and Leukotriene C4Binding Sites of Multidrug Resistance Protein 1 (ABCC1)

Multidrug resistance protein 1 (MRP1) is a member of the "C" branch of the ATP-binding cassette transporter superfamily. The NH(2)-proximal nucleotide-binding domain (NBD1) of MRP1 differs functionally from its COOH-proximal domain (NBD2). NBD1 displays intrinsic high-affinity ATP binding and little ATPase activity. In contrast, ATP binding to NBD2 is strongly dependent on nucleotide binding by NBD1, and NBD2 is more hydrolytically active. We have demonstrated that occupancy of NBD2 by ATP or ADP markedly decreased substrate binding by MRP1. We have further explored the relationship between nucleotide and substrate binding by examining the effects of various ATP analogs and ADP trapping, as well as mutations in conserved functional elements in the NBDs, on the ability of MRP1 to bind the photoactivatable, high-affinity substrate cysteinyl leukotriene C(4) (LTC(4))(.) Overall, the results support a model in which occupancy of both NBD1 and NBD2 by ATP results in the formation of a low-affinity conformation of the protein. However, nonhydrolyzable ATP analogs (beta,gamma-imidoadenosine 5'-triphosphate and adenylylmethylene diphosphonate) failed to substitute for ATP or adenosine 5'-O-(thiotriphosphate) (ATPgammaS) in decreasing LTC(4) photolabeling. Furthermore, mutations of the signature sequence in either NBD that had no apparent effect on azido-ATP binding abrogated the formation of a low-affinity substrate binding state in the presence of ATP or ATPgammaS. We suggest that the effect of these mutations, and possibly the failure of some ATP analogs to decrease LTC(4) binding, may be attributable to an inability to elicit a conformational change in the NBDs that involves interactions between the signature sequence and the gamma-phosphate of the bound nucleotide.

Cadmium inhibits mismatch repair by blocking the ATPase activity of the MSH2-MSH6 complex

Cadmium (Cd2+) is a known carcinogen that inactivates the DNA mismatch repair (MMR) pathway. In this study, we have tested the effect of Cd2+ exposure on the enzymatic activity of the mismatch binding complex MSH2–MSH6. Our results indicate that Cd2+ is highly inhibitory to the ATP binding and hydrolysis activities of MSH2–MSH6, and less inhibitory to its DNA mismatch binding activity. The inhibition of the ATPase activity appears to be dose and exposure time dependent. However, the inhibition of the ATPase activity by Cd2+ is prevented by cysteine and histidine, suggesting that these residues are essential for the ATPase activity and are targeted by Cd2+. A comparison of the mechanism of inhibition with N-ethyl maleimide, a sulfhydryl group inhibitor, indicates that this inhibition does not occur through direct inactivation of sulfhydryl groups. Zinc (Zn2+) does not overcome the direct inhibitory effect of Cd2+ on the MSH2–MSH6 ATPase activity in vitro. However, the increase in the mutator phenotype of yeast cells exposed to Cd2+ was prevented by excess Zn2+, probably by blocking the entry of Cd2+ into the cell. We conclude that the inhibition of MMR by Cd2+ is through the inactivation of the ATPase activity of the MSH2–MSH6 heterodimer, resulting in a dominant negative effect and causing a mutator phenotype.

Distinct roles for the Saccharomyces cerevisiae mismatch repair proteins in heteroduplex rejection, mismatch repair and nonhomologous tail removal.

The Saccharomyces cerevisiae mismatch repair (MMR) protein MSH6 and the SGS1 helicase were recently shown to play similarly important roles in preventing recombination between divergent DNA sequences in a single-strand annealing (SSA) assay. In contrast, MMR factors such as Mlh1p, Pms1p, and Exo1p were shown to not be required or to play only minimal roles. In this study we tested mutations that disrupt Sgs1p helicase activity, Msh2p-Msh6p mismatch recognition, and ATP binding and hydrolysis activities for their effect on preventing recombination between divergent DNA sequences (heteroduplex rejection) during SSA. The results support a model in which the Msh proteins act with Sgs1p to unwind DNA recombination intermediates containing mismatches. Importantly, msh2 mutants that displayed separation-of-function phenotypes with respect to nonhomologous tail removal during SSA and heteroduplex rejection were characterized. These studies suggest that nonhomologous tail removal is a separate function of Msh proteins that is likely to involve a distinct DNA binding activity. The involvement of Sgs1p in heteroduplex rejection but not nonhomologous tail removal further illustrates that subsets of MMR proteins collaborate with factors in different DNA repair pathways to maintain genome stability.

Impaired Recycling of Apolipoprotein E4 Is Associated with Intracellular Cholesterol Accumulation

After internalization of triglyceride-rich lipoproteins (TRL) in hepatoma cells, TRL particles are immediately disintegrated in the early endosomal compartment. This involves the targeting of lipids and apoprotein B along the degradative pathway and the recycling of TRL-derived apoE through recycling endosomes. Re-secretion of apoE is accompanied by the concomitant association of apoE and cellular cholesterol with high-density lipoproteins (HDL). Since epidemiological data showed that apoE3 and apoE4 have differential effects on HDL metabolism, we investigated whether the intracellular processing of TRL-derived apoE4 differs from apoE3-TRL. In this study, we demonstrated by radioactive and immunofluorescence uptake experiments that cell-surface binding and internalization of TRL-derived apoE4 are increased compared with apoE3 in hepatoma cells. Pulse-chase experiments revealed that HDL-induced recycling, but not disintegration and degradation, of apoE4-enriched TRL is strongly reduced in these cells. Furthermore, impaired HDL-induced apoE4 recycling is associated with reduced cholesterol efflux. Studies performed in Tangier fibroblasts showed that apoE recycling does not depend on ATP-binding cassette transporter A1 activity. These studies provide initial evidence that impaired recycling of apoE4 could interfere with intracellular cholesterol transport and contribute to the pathophysiological lipoprotein profile observed in apoE4 homozygotes.

A CDC6-like Factor from the Archaea Sulfolobus solfataricus Promotes Binding of the Mini-chromosome Maintenance Complex to DNA

The archaeal replication apparatus appears to be a simplified version of the eukaryotic one with fewer polypeptides and simpler protein complexes. Herein, we report evidence that a Cdc6-like factor from the hyperthermophilic crenarchaea Sulfolobus solfataricus stimulates binding of the homohexameric MCM-like complex to bubble- and fork-containing DNA oligonucleotides that mimic early replication intermediates. This function does not require the Cdc6 ATP and DNA binding activities. These findings may provide important clues to understanding how the DNA replication initiation process has evolved in the more complex eukaryotic organisms.

Communication between Esigma(54) , promoter DNA and the conserved threonine residue in the GAFTGA motif of the PspF sigma-dependent activator during transcription activation.

Conversion of Esigma(54) closed promoter complexes to open promoter complexes requires specialized activators which are members of the AAA (ATPases Associated with various cellular Activities) protein family. The ATP binding and hydrolysis activity of Esigma(54) activators is used in an energy coupling reaction to remodel the Esigma(54) closed promoter complex and to overcome the sigma(54)-imposed block on open complex formation. The remodelling target for the AAA activator within the Esigma(54) closed complex includes a complex interface contributed to by Region I of sigma(54), core RNA polymerase and a promoter DNA fork junction structure, comprising the Esigma(54) regulatory centre. One sigma(54) binding surface on Esigma(54) activators is a conserved sequence known as the GAFTGA motif. Here, we present a detailed characterization of the interaction between Region I of sigma(54) and the Escherichia coli AAA sigma(54) activator Phage shock protein F. Using Esigma(54) promoter complexes that mimic different conformations adopted by the DNA during open complex formation, we investigated the contribution of the conserved threonine residue in the GAFTGA motif to transcription activation. Our results suggest that the organization of the Esigma(54) regulatory centre, and in particular the conformation adopted by the sigma(54) Region I and the DNA fork junction structure during open complex formation, is communicated to the AAA activator via the conserved T residue of the GAFTGA motif.

RNA helicase A interacts with nuclear factor kappaB p65 and functions as a transcriptional coactivator.

RNA helicase A (RHA), a member of DNA and RNA helicase family containing ATPase activity, is involved in many steps of gene expression such as transcription and mRNA export. RHA has been reported to bind directly to the transcriptional coactivator, CREB-binding protein, and the tumor suppressor protein, BRCA1, and links them to RNA Polymerase II holoenzyme complex. Using yeast two-hybrid screening, we have identified RHA as an interacting molecule of the p65 subunit of nuclear factor kappaB (NF-kappaB). The interaction between p65 and RHA was confirmed by glutathione-S transferase pull-down assay in vitro, and by co-immunoprecipitation assay in vivo. In transient transfection assays, RHA enhanced NF-kappaB dependent reporter gene expression induced by p65, tumor necrosis factor-alpha, or NF-kappaB inducing kinase. The mutant form of RHA lacking ATP-binding activity inhibited NF-kappaB dependent reporter gene expression induced by these activators. Moreover, depletion of RHA using short interfering RNA reduced the NF-kappaB dependent transactivation. These data suggest that RHA is an essential component of the transactivation complex by mediating the transcriptional activity of NF-kappaB.

Adenosine nucleotides and the regulation of GRP94-client protein interactions.

The molecular chaperone heat shock protein 90 (Hsp90) serves essential roles in the regulation of signaling protein function, trafficking, and turnover. Hsp90 function is intimately linked to intrinsic ATP binding and hydrolysis activities, the latter of which is under the regulatory control of accessory factors. Glucose-regulated protein of 94 kDa (GRP94), the endoplasmic reticulum Hsp90, is highly homologous to cytosolic Hsp90. However, neither accessory factors nor adenosine nucleotides have been clearly implicated in the regulation of GRP94-client protein interactions. In the current study, the structural and regulatory consequences of adenosine nucleotide binding to GRP94 were investigated. We report that apo-GRP94 undergoes a time- and temperature-dependent tertiary conformational change that exposes a site(s) of protein-protein interaction; ATP, ADP, and radicicol markedly suppress this conformational change. In concert with these findings, ATP and ADP act identically to suppress GRP94 homooligomerization, as well as both local and global conformational activity. To identify a role(s) for ATP or ADP in the regulation of GRP94-client protein interactions, immunoglobulin (Ig) heavy chain folding intermediates containing bound GRP94 and immunoglobulin binding protein (BiP) were isolated from myeloma cells, and the effects of adenosine nucleotides on chaperone-Ig heavy chain interactions were examined. Whereas ATP elicited efficient release of BiP from both wild-type and mutant Ig heavy chain intermediates, GRP94 remained in stable association with Ig heavy chains in the presence of ATP or ADP. On the basis of these data, we propose that structural maturation of the client protein substrate, rather than ATP binding or hydrolysis, serves as the primary signal for dissociation of GRP94-client protein complexes.

Identification of a Conserved Sequence Motif That Promotes Cdc37 and Cyclin D1 Binding to Cdk4

Cdc37 is a molecular chaperone that is important for the stability and activity of several protein kinases, including Cdk4 and Raf1. We first determined, using in vitro assays, that Cdc37 binds to the amino-terminal lobe of Cdk4. Subsequent mutagenesis revealed that Gly-15 (G15A) and Gly-18 (G18A) were critical for Cdc37-Cdk4 complex formation. Gly-15 and Gly-18 of Cdk4 are within the conserved Gly-X-Gly-X-X-Gly motif that is required for ATP binding to the kinase. Mutation of either glycine at the equivalent positions of Raf1 (G358A and G361A) also inhibited Cdc37 binding to Raf1. Replacing another conserved residue critical for ATP binding and kinase activity, Lys-35 (K35A), reduced Cdc37-Cdk4 complex formation but to a lesser extent. The interaction of Cdk4 with Cdc37 in vitro was not sensitive to changes in ATP levels. Cell-based assays indicated that Cdk4(G15A) and Cdk4(G18A) were present at the same level as wild type Cdk4. Equivalent amounts of p16 bound to Cdk4(G15A) and Cdk4(G18A) relative to wild type Cdk4, suggesting that Cdk4(G15A) and Cdk4(G18A) adopt significant tertiary structure. However, in contrast to wild type Cdk4, Cdk4(G15A), and Cdk4(G18A) had greatly reduced binding of cyclin D1, Cdc37, and Hsp90. Importantly, overexpression of Cdc37 not only stimulated cyclin D1 binding to wild type Cdk4 but also restored its binding to Cdk4(G15A). Under the same conditions, p16 binding to wild type Cdk4 was suppressed. Our findings show that the interaction of Cdc37 with its client protein kinases requires amino acid residues within a motif that is present in many protein kinases.

Ribonucleopeptides: functional RNA-peptide complexes.

Selection of functional RNAs from randomized pool of RNA molecules successfully affords RNA aptamers that specifically bind to small molecules, and that have catalytic activities. Recent structural analyses of the ribosomal RNA complex suggest that the RNA-protein complex would be a new structural candidate for the design of tailor-made receptors and enzymes. We have designed an ATP binding domain that consists of an RNA subunit and a peptide subunit by means of structure-based design approach and successive in vitro selection method. The RNA subunit is designed to consist of two functional domains; an ATP binding domain with 20 randomized nucleotides and an adjacent stem region that serves as a binding site for the RNA-binding peptide. The randomized nucleotide region was placed next to the HIV-1 Rev response element to enable the formation of "ribonucleopeptide" pools in the presence of the Rev peptide. In vitro selection of RNA oligonucleotides from the randomized pool afforded a ribonucleopeptide receptor specific for ATP. The ATP-binding ribonucleopeptide did not share the known consensus nucleotide sequence for ATP aptamers, and completely lost its ATP-binding ability in the absence of the Rev peptide. The ATP-binding activity of the ribonucleopeptide was increased by a substitution of the N-terminal amino acid of the Rev peptide. These results demonstrate that the peptide stabilizes the functional structure of RNA and suggest that amino acids outside the RNA binding region of the peptide participate in the ATP binding. Our approach would provide a new strategy for the design of tailor-made ribonucleopeptide receptors.

Biological functions of the cytoplasmic TGBp1 inclusions of bamboo mosaic potexvirus

The TGBp1 protein encoded by the first open reading frame of the triple-gene-block (TGB) of bamboo mosaic potexvirus (BaMV) plays important roles in virus movement; one of them is to shape the viral RNA before its being transported from cell to cell. However, TGBp1 mainly forms cytoplasmic inclusions which are devoid of the RNA-binding activity. With the purified TGBp1 inclusions of BaMV, we analyzed the possibility of dissociation of functional TGBp1 from the inclusions. Our data showed that the cytoplasmic inclusions were able to dissociate continuously functional TGBp1, which possessed ATP-binding, ATPase, and RNA-binding activities. Moreover, the dissociation of TGBp1 was significantly enhanced by the presence of viral RNA and capsid protein (CP), consistent with the finding that TGBp1 are able to form ribonucleoprotein complex with viral RNA and CP. Taken together, these results support the idea that the cytoplasmic TGBp1 inclusions are active pools of TGBp1, upon which viral RNA can be shaped into a transferable form.

Functional dissection of the transmembrane domains of the transporter associated with antigen processing (TAP).

The transporter associated with antigen processing (TAP1/2) translocates cytosolic peptides of proteasomal degradation into the endoplasmic reticulum (ER) lumen. A peptide-loading complex of tapasin, major histocompatibility complex class I, and several auxiliary factors is assembled at the transporter to optimize antigen display to cytotoxic T-lymphocytes at the cell surface. The heterodimeric TAP complex has unique N-terminal domains in addition to a 6 + 6-transmembrane segment core common to most ABC transporters. Here we provide direct evidence that this core TAP complex is sufficient for (i) ER targeting, (ii) heterodimeric assembly within the ER membrane, (iii) peptide binding, (iv) peptide transport, and (v) specific inhibition by the herpes simplex virus protein ICP47 and the human cytomegalovirus protein US6. We show for the first time that the translocation pore of the transporter is composed of the predicted TM-(5-10) of TAP1 and TM-(4-9) of TAP2. Moreover, we demonstrate that the N-terminal domains of TAP1 and TAP2 are essential for recruitment of tapasin, consequently mediating assembly of the macromolecular peptide-loading complex.

Structure of the N-terminal Domain of GRP94: BASIS FOR LIGAND SPECIFICITY AND REGULATION

GRP94, the endoplasmic reticulum (ER) paralog of the chaperone Hsp90, plays an essential role in the structural maturation or secretion of a subset of proteins destined for transport to the cell surface, such as the Toll-like receptors 2 and 4, and IgG, respectively. GRP94 differs from cytoplasmic Hsp90 by exhibiting very weak ATP binding and hydrolysis activity. GRP94 also binds selectively to a series of substituted adenosine analogs. The high resolution crystal structures at 1.75-2.1 A of the N-terminal and adjacent charged domains of GRP94 in complex with N-ethylcarboxamidoadenosine, radicicol, and 2-chlorodideoxyadenosine reveals a structural mechanism for ligand discrimination among hsp90 family members. The structures also identify a putative subdomain that may act as a ligand-responsive switch. The residues of the charged region fold into a disordered loop whose termini are ordered and continue the twisted beta sheet that forms the structural core of the N-domain. This continuation of the beta sheet past the charged domain suggests a structural basis for the association of the N-terminal and middle domains of the full-length chaperone.