A nucleosome interaction module is required for normal function of Arabidopsis thaliana BRAHMA

The BRAHMA (BRM) gene encodes the SNF2-type ATPase of the putative Arabidopsis thaliana SWI/SNF chromatin remodelling complex. This family of ATPases is characterized by the presence of a conserved catalytic domain and an arrangement of auxiliary domains, whose functions in the remodelling activity remains unclear. Here, we characterize, at the molecular and functional level, the carboxy-terminal part of Arabidopsis BRM. We have found three DNA-binding regions that bind various free DNA and nucleosomal probes with different specificity. One of these regions contains an AT-hook motif. The carboxy terminus also contains a bromodomain able to bind histones H3 and H4. We propose that this array of domains constitute a nucleosome interaction module that helps BRM to interact with its substrate. We also characterize an Arabidopsis mutant that expresses a BRM protein lacking the last 454 amino acid residues (BRM-DeltaC), encompassing the bromodomain and two of the three DNA-binding activities identified. This mutant displays an intermediate phenotype between those of the wild-type and a null allele mutant, suggesting that the nucleosome interaction module is required for the normal function of BRM but it is not essential for the remodelling activity of BRM-containing SWI/SNF complexes.     

Dissection of the contribution of individual domains to the ATPase mechanism of Hsp90

Hsp90 is a dimeric, ATP-regulated molecular chaperone. Its ATPase cycle involves the N-terminal ATP binding domain (amino acids (aa) 1-272) and, in addition, to some extent the middle domain (aa 273-528) and the C-terminal dimerization domain (aa 529-709). To analyze the contribution of the different domains and the oligomeric state on the progression of the ATPase cycle of yeast Hsp90, we created deletion constructs lacking either the C-terminal or both the C-terminal and the middle domain. To test the effect of dimerization on the ATPase activity of the different constructs, we introduced a Cys residue at the C-terminal ends of the constructs, which allowed covalent dimerization. We show that all monomeric constructs tested exhibit reduced ATPase activity and a decreased affinity for ATP in comparison with wild type Hsp90. The covalently linked dimers lacking only the C-terminal domain hydrolyze ATP as efficiently as the wild type protein. Furthermore, this construct is able to trap the ATP molecule similar to the full-length protein. This demonstrates that in the ATPase cycle, the C-terminal domain can be replaced by a cystine bridge. In contrast, the ATPase activity of the artificially linked N-terminal domains remains very low and bound ATP is not trapped. Taken together, we show that both the dimerization of the N-terminal domains and the association of the N-terminal with the middle domain are important for the efficiency of the ATPase cycle. These reactions are synergistic and require Hsp90 to be in the dimeric state.     

Construction and analysis of hybrid Escherichia coli-Bacillus subtilis dnaK genes

The highly conserved DnaK chaperones consist of an N-terminal ATPase domain, a central substrate-binding domain, and a C-terminal domain whose function is not known. Since Bacillus subtilis dnaK was not able to complement an Escherichia coli dnaK null mutant, we performed domain element swap experiments to identify the regions responsible for this finding. It turned out that the B. subtilis DnaK protein needed approximately normal amounts of the cochaperone DnaJ to be functional in E. coli. The ATPase domain and the substrate-binding domain form a species-specific functional unit, while the C-terminal domains, although less conserved, are exchangeable. Deletion of the C-terminal domain in E. coli DnaK affected neither complementation of growth at high temperatures nor propagation of phage lambda but abolished degradation of sigma32.     

ATPase activity of the heat shock protein hsp72 is dispensable for its effects on dephosphorylation of stress kinase JNK and on heat-induced apoptosis

A major inducible heat shock protein, Hsp72, has previously been found to stimulate dephosphorylation (inactivation) of stress kinase JNK in heat-shocked cells and protect them from apoptosis. Using Rat-1 fibroblasts with constitutive expression of a human Hsp72 or its deletion mutant lacking an ATPase domain (C-terminal fragment (CTF)), we tested whether ATPase activity of Hsp72 is necessary for these effects. We found that expression of CTF markedly increased, similarly to the intact protein, JNK dephosphorylation in heat-shocked cells. As a result, JNK inactivation following heat shock occurred much faster in cells expressing either full-length or mutant Hsp72 than in parental cells and this was accompanied by suppression of heat-induced apoptosis. Thus, protein refolding activity of Hsp72 appears to be dispensable for its effect on JNK inactivation and apoptosis.     

Structural and functional studies of FkpA from Escherichia coli, a cis/trans peptidyl-prolyl isomerase with chaperone activity

The protein FkpA from the periplasm of Escherichia coli exhibits both cis/trans peptidyl-prolyl isomerase (PPIase) and chaperone activities. The crystal structure of the protein has been determined in three different forms: as the full-length native molecule, as a truncated form lacking the last 21 residues, and as the same truncated form in complex with the immunosuppressant ligand, FK506. FkpA is a dimeric molecule in which the 245-residue subunit is divided into two domains. The N-terminal domain includes three helices that are interlaced with those of the other subunit to provide all inter-subunit contacts maintaining the dimeric species. The C-terminal domain, which belongs to the FK506-binding protein (FKBP) family, binds the FK506 ligand. The overall form of the dimer is V-shaped, and the different crystal structures reveal a flexibility in the relative orientation of the two C-terminal domains located at the extremities of the V. The deletion mutant FkpNL, comprising the N-terminal domain only, exists in solution as a mixture of monomeric and dimeric species, and exhibits chaperone activity. By contrast, a deletion mutant comprising the C-terminal domain only is monomeric, and although it shows PPIase activity, it is devoid of chaperone function. These results suggest that the chaperone and catalytic activities reside in the N and C-terminal domains, respectively. Accordingly, the observed mobility of the C-terminal domains of the dimeric molecule could effectively adapt these two independent folding functions of FkpA to polypeptide substrates.     

Two domains of superfamily I helicases may exist as separate proteins.

DNA and RNA helicases of superfamily I are characterized by seven conserved motifs. The five N-terminal motifs are separated from the two C-terminal ones by a spacer that is highly variable in both sequence and length, suggesting the existence of two distinct domains. Using computer methods for protein sequence analysis, we show that PhoH, an ATP-binding protein that is conserved in Escherichia coli and Mycobacterium leprae, is homologous to the putative N-terminal domain of the helicases, whereas the putative E. coli protein YjhR is homologous to the C-terminal domain. These findings suggest that the N-and C-terminal domains of superfamily I helicases have distinct activities, with only the N-terminal domain having the ATPase activity. It is speculated that PhoH and YjhR have evolved from helicases through deletion of the portions of the helicase genes coding for the C- and N-terminal domain, respectively.     

The chaperone function of hsp70 is required for protection against stress-induced apoptosis

Cellular stress can trigger a process of self-destruction known as apoptosis. Cells can also respond to stress by adaptive changes that increase their ability to tolerate normally lethal conditions. Expression of the major heat-inducible protein hsp70 protects cells from heat-induced apoptosis. hsp70 has been reported to act in some situations upstream or downstream of caspase activation, and its protective effects have been said to be either dependent on or independent of its ability to inhibit JNK activation. Purified hsp70 has been shown to block procaspase processing in vitro but is unable to inhibit the activity of active caspase 3. Since some aspects of hsp70 function can occur in the absence of its chaperone activity, we examined whether hsp70 lacking its ATPase domain or the C-terminal EEVD sequence that is essential for peptide binding was required for the prevention of apoptosis. We generated stable cell lines with tetracycline-regulated expression of hsp70, hsc70, and chaperone-defective hsp70 mutants lacking the ATPase domain or the C-terminal EEVD sequence or containing AAAA in place of EEVD. Overexpression of hsp70 or hsc70 protected cells from heat shock-induced cell death by preventing the processing of procaspases 9 and 3. This required the chaperone function of hsp70 since hsp70 mutant proteins did not prevent procaspase processing or provide protection from apoptosis. JNK activation was inhibited by both hsp70 and hsc70 and by each of the hsp70 domain mutant proteins. The chaperoning activity of hsp70 is therefore not required for inhibition of JNK activation, and JNK inhibition was not sufficient for the prevention of apoptosis. Release of cytochrome c from mitochondria was inhibited in cells expressing full-length hsp70 but not in cells expressing the protein with ATPase deleted. Together with the recently identified ability of hsp70 to inhibit cytochrome c-mediated procaspase 9 processing in vitro, these data demonstrate that hsp70 can affect the apoptotic pathway at the levels of both cytochrome c release and initiator caspase activation and that the chaperone function of hsp70 is required for these effects.     

UvrD2 is essential in Mycobacterium tuberculosis, but its helicase activity is not required

UvrD is an SF1 family helicase involved in DNA repair that is widely conserved in bacteria. Mycobacterium tuberculosis has two annotated UvrD homologues; here we investigate the role of UvrD2. The uvrD2 gene at its native locus could be knocked out only in the presence of a second copy of the gene, demonstrating that uvrD2 is essential. Analysis of the putative protein domain structure of UvrD2 shows a distinctive domain architecture, with an extended C terminus containing an HRDC domain normally found in SF2 family helicases and a linking domain carrying a tetracysteine motif. Truncated constructs lacking the C-terminal domains of UvrD2 were able to compensate for the loss of the chromosomal copy, showing that these C-terminal domains are not essential. Although UvrD2 is a functional helicase, a mutant form of the protein lacking helicase activity was able to permit deletion of uvrD2 at its native locus. However, a mutant protein unable to hydrolyze ATP or translocate along DNA was not able to compensate for lack of the wild-type protein. Therefore, we concluded that the essential role played by UvrD2 is unlikely to involve its DNA unwinding activity and is more likely to involve DNA translocation and, possibly, protein displacement.     

Roles of the glycogen-binding domain and Snf4 in glucose inhibition of SNF1 protein kinase

The SNF1/AMP-activated protein kinase (AMPK) family is required for adaptation to metabolic stress and energy homeostasis. The gamma subunit of AMPK binds AMP and ATP, and mutations that affect binding cause human disease. We have here addressed the role of the Snf4 (gamma) subunit in regulating SNF1 protein kinase in response to glucose availability in Saccharomyces cerevisiae. Previous studies of mutant cells lacking Snf4 suggested that Snf4 counteracts autoinhibition by the C-terminal sequence of the Snf1 catalytic subunit but is dispensable for glucose regulation, and AMP does not activate SNF1 in vitro. We first introduced substitutions at sites that, in AMPK, contribute to nucleotide binding and regulation. Mutations at several sites relieved glucose inhibition of SNF1, as judged by catalytic activity, phosphorylation of the activation-loop Thr-210, and growth assays, although analogs of the severe human mutations R531G/Q had little effect. We further showed that alterations of Snf4 residues that interact with the glycogen-binding domain (GBD) of the beta subunit strongly relieved glucose inhibition. Finally, substitutions in the GBD of the Gal83 beta subunit that are predicted to disrupt interactions with Snf4 and also complete deletion of the GBD similarly relieved glucose inhibition of SNF1. Analysis of mutant cells lacking glycogen synthase showed that regulation of SNF1 is normal in the absence of glycogen. These findings reveal novel roles for Snf4 and the GBD in regulation of SNF1.     

A family of proteins containing a conserved domain that mediates interaction with the yeast SNF1 protein kinase complex.

The SNF1 protein kinase is required for the regulatory response to glucose starvation in Saccharomyces cerevisiae. SNF1 is a protein serine/threonine kinase that has been widely conserved in both plants and mammals. Previously, we identified SIP1 and SIP2 as proteins that interact with SNF1 in vivo by the two-hybrid system. We have cloned the SIP2 gene and the encoded protein is homologous to SIP1 and to GAL83, which affects glucose repression of the GAL genes. We show that SIP2 and GAL83, like SIP1, co-immunoprecipitate with SNF1 and are phosphorylated in vitro. An 80 amino acid sequence, designated the ASC domain, is highly conserved at the C-termini of all three proteins. We show that this small domain can mediate protein-protein interaction with the SNF1 kinase complex. Thus, SIP1, SIP2 and GAL83 define a family of homologous proteins that are tightly associated with the SNF1 kinase, probably in alternative forms of the complex. Genetic evidence suggests that the three proteins have distinct, but related, functions in the SNF1 pathway, and deletion of GAL83 dramatically reduces SNF1 activity in immune complex assays. We propose that SIP1, SIP2 and GAL83 act as adaptors that promote the activity of SNF1 towards specific targets.     

Two independent mechanisms down-regulate the intrinsic SecA ATPase activity

SecA initiates protein translocation by interacting with ATP, preprotein, and the SecYEG membrane components. Under such conditions, it undergoes a conformational change characterized as membrane insertion, which is then followed by hydrolysis of ATP, enabling the release of the preprotein and deinsertion of SecA itself for the next cycle of reactions. Without ongoing translocation, the ATPase activity of SecA is kept very low. Previously, it was shown that the C-terminal 34-kDa domain of SecA interacts with the N-terminal 68-kDa ATPase domain to down-regulate the ATPase. Here, we show, using a deregulated SecA mutant, that the intrinsic ATPase activity is subject to dual inhibitory mechanisms. Thus, the proposed second ATP-binding domain down-regulates the ATPase activity executed by the primary ATPase domain. This regulation, within the N-terminal ATPase domain, operates independently of the C-terminal domain-mediated regulation. The absence of both the mechanisms resulted in a 50-fold elevation of translocation-uncoupled ATP hydrolysis.     

The plant cyclin-dependent kinase inhibitor ICK1 has distinct functional domains for in vivo kinase inhibition,protein instability and nuclear localization

Interactor/inhibitor 1 of Cdc2 kinase (ICK1) from Arabidopsis thaliana is the first plant cyclin-dependent kinase (CDK) inhibitor, and overexpression of ICK1 inhibits CDK activity, cell division and plant growth in transgenic plants. In this study, ICK1 and deletion mutants were expressed either alone or as green fluorescent protein (GFP) fusion proteins in transgenic Arabidopsis plants. Deletion of the C-terminal 15 or 29 amino acids greatly reduced or completely abolished the effects of ICK1 on the transgenic plants, and recombinant proteins lacking the C-terminal residues lost the ability to bind to CDK complex and the kinase inhibition activity, demonstrating the role of the conserved C-terminal domain in in vivo kinase inhibition. In contrast, the mutant ICK1DeltaN108 with the N-terminal 108 residues deleted had much stronger effects on plants than the full-length ICK1. Analyses demonstrated that this effect was not because of an enhanced ability of ICK1DeltaN108 protein to inhibit CDK activity, but a result of a much higher level of ICK1DeltaN108 protein in the plants, indicating that the N-terminal domain contains a sequence or element increasing protein instability in vivo. Furthermore, GFP-ICK1 protein was restricted to the nuclei in roots of transgenic plants, even with the C-terminal or the N-terminal domain deleted, suggesting that a sequence in the central domain of ICK1 is responsible for nuclear localization. These results provide mechanistic understanding about the function and regulation of this cell cycle regulator in plants.     

Inhibition of human NTPDase 2 by modification of an intramembrane cysteine by p-chloromercuriphenylsulfonate and oxidative cross-linking of the transmembrane domains

Human NTPDase 2 is a cell surface integral membrane glycoprotein that is anchored to the membranes by two transmembrane domains while the bulk of the protein containing the active site faces the extracellular milieu. It contains 10 conserved cysteine residues in the extracellular domain that are involved in disulfide bond formation and one free cysteine residue, C26, which is located in the N-terminal transmembrane domain. The human NTPDase 2 activity is inactivated by membrane perturbation that disrupts interaction of the transmembrane domains and is inhibited by p-chloromercuriphenylsulfonate (pCMPS), a sulfhydryl reagent. In this report, we show that C26 is the target of pCMPS modification, since a mutant in which C26 was replaced with a serine was no longer inhibited by pCMPS. Mutants in which cysteine residues are placed in the C-terminal transmembrane domain near the extracellular surface were still modified by pCMPS, but the degree of inhibition of their ATPase activity was lower than that of the wild-type enzyme. Thus, loss of the ATPase activity of human NTPDase 2 in the presence of pCMPS probably results from the disturbance of both transmembrane domain interaction and its active site. Inhibition of human NTPDase 2 activity by pCMPS and membrane perturbation is attenuated when the enzyme is cross-linked by glutaraldehyde. On the other hand, NTPDase 2 dimers formed from oxidative cross-linking of the wild-type enzyme and mutants containing a single cysteine residue in the C-terminal transmembrane domain displayed reduced ATPase activity. A similar reduction in activity was also obtained upon intramolecular disulfide formation in mutants that contain a cysteine residue in each of the two transmembrane domains. These results indicate that the mobility of the transmembrane helices is necessary for maximal catalysis.