Substrate recognition by the ClpA chaperone component of ClpAP protease

ClpA, a member of the Clp/Hsp100 ATPase family, is a molecular chaperone and regulatory component of ClpAP protease. We explored the mechanism of protein recognition by ClpA using a high affinity substrate, RepA, which is activated for DNA binding by ClpA and degraded by ClpAP. By characterizing RepA derivatives with N- or C-terminal deletions, we found that the N-terminal portion of RepA is required for recognition. More precisely, RepA derivatives lacking the N-terminal 5 or 10 amino acids are degraded by ClpAP at a rate similar to full-length RepA, whereas RepA derivatives lacking 15 or 20 amino acids are degraded much more slowly. Thus, ClpA recognizes an N-terminal signal in RepA beginning in the vicinity of amino acids 10-15. Moreover, peptides corresponding to RepA amino acids 4-13 and 1-15 inhibit interactions between ClpA and RepA. We constructed fusions of RepA and green fluorescent protein, a protein not recognized by ClpA, and found that the N-terminal 15 amino acids of RepA are sufficient to target the fusion protein for degradation by ClpAP. However, fusion proteins containing 46 or 70 N-terminal amino acids of RepA are degraded more efficiently in vitro and are noticeably stabilized in vivo in clpADelta and clpPDelta strains compared with wild type.     

Concurrent chaperone and protease activities of ClpAP and the requirement for the N-terminal ClpA ATP binding site for chaperone activity.

ClpA, a member of the Clp/Hsp100 family of ATPases, is both an ATP-dependent molecular chaperone and the regulatory component of ClpAP protease. We demonstrate that chaperone and protease activities occur concurrently in ClpAP complexes during a single round of RepA binding to ClpAP and ATP-dependent release. This result was substantiated with a ClpA mutant, ClpA(K220V), carrying an amino acid substitution in the N-terminal ATP binding site. ClpA(K220V) is unable to activate RepA, but the presence of ClpP or chemically inactivated ClpP restores its ability to activate RepA. The presence of ClpP simultaneously facilitates degradation of RepA. ClpP must remain bound to ClpA(K220V) for these effects, indicating that both chaperone and proteolytic activities of the mutant complex occur concurrently. ClpA(K220V) itself is able to form stable complexes with RepA in the presence of a poorly hydrolyzed ATP analog, adenosine 5'-O-(thiotriphosphate), and to release RepA upon exchange of adenosine 5'-O-(thiotriphosphate) with ATP. However, the released RepA is inactive in DNA binding, indicating that the N-terminal ATP binding site is essential for the chaperone activity of ClpA. Taken together, these results suggest that substrates bound to the complex of the proteolytic and ATPase components can be partitioned between release/reactivation and translocation/degradation.     

Crystal structure of ClpA,an Hsp100 chaperone and regulator of ClpAP protease

Escherichia coli ClpA, an Hsp100/Clp chaperone and an integral component of the ATP-dependent ClpAP protease, participates in regulatory protein degradation and the dissolution and degradation of protein aggregates. The crystal structure of the ClpA subunit reveals an N-terminal domain with pseudo-twofold symmetry and two AAA(+) modules (D1 and D2) each consisting of a large and a small sub-domain with ADP bound in the sub-domain junction. The N-terminal domain interacts with the D1 domain in a manner similar to adaptor-binding domains of other AAA(+) proteins. D1 and D2 are connected head-to-tail consistent with a cooperative and vectorial translocation of protein substrates. In a planar hexamer model of ClpA, built by assembling ClpA D1 and D2 into homohexameric rings of known structures of AAA(+) modules, the differences in D1-D1 and D2-D2 interfaces correlate with their respective contributions to hexamer stability and ATPase activity.     

ClpA and ClpP remain associated during multiple rounds of ATP-dependent protein degradation by ClpAP protease.

The Escherichia coli ClpA and ClpP proteins form a complex, ClpAP, that catalyzes ATP-dependent degradation of proteins. Formation of stable ClpA hexamers and stable ClpAP complexes requires binding of ATP or nonhydrolyzable ATP analogues to ClpA. To understand the order of events during substrate binding, unfolding, and degradation by ClpAP, it is essential to know the oligomeric state of the enzyme during multiple catalytic cycles. Using inactive forms of ClpA or ClpP as traps for dissociated species, we measured the rates of dissociation of ClpA hexamers or ClpAP complexes. When ATP was saturating, the rate constant for dissociation of ClpA hexamers was 0.032 min(-1) (t(1/2) of 22 min) at 37 degrees C, and dissociation of ClpP from the ClpAP complexes occurred with a rate constant of 0. 092 min(-1) (t(1/2) of 7.5 min). Because the k(cat) for casein degradation is approximately 10 min(-1), these results indicate that tens of molecules of casein can be turned over by the ClpAP complex before significant dissociation occurs. Mutations in the N-terminal ATP binding site led to faster rates of ClpA and ClpAP dissociation, whereas mutations in the C-terminal ATP binding site, which cause significant decreases in ATPase activity, led to lower rates of dissociation of ClpA and ClpAP complexes. Dissociation rates for wild-type and first domain mutants of ClpA were faster at low nucleotide concentrations. The t(1/2) for dissociation of ClpAP complexes in the presence of nonhydrolyzable analogues was >/=30 min. Thus, ATP binding stabilizes the oligomeric state of ClpA, and cycles of ATP hydrolysis affect the dynamics of oligomer interaction. However, since the k(cat) for ATP hydrolysis is approximately 140 min(-1), ClpA and the ClpAP complex remain associated during hundreds of rounds of ATP hydrolysis. Our results indicate that the ClpAP complex is the functional form of the protease and as such engages in multiple rounds of interaction with substrate proteins, degradation, and release of peptide products without dissociation.     

Roles of the N-domains of the ClpA unfoldase in binding substrate proteins and in stable complex formation with the ClpP protease

The hexameric cylindrical Hsp100 chaperone ClpA mediates ATP-dependent unfolding and translocation of recognized substrate proteins into the coaxially associated serine protease ClpP. Each subunit of ClpA is composed of an N-terminal domain of approximately 150 amino acids at the top of the cylinder followed by two AAA+ domains. In earlier studies, deletion of the N-domain was shown to have no effect on the rate of unfolding of substrate proteins bearing a C-terminal ssrA tag, but it did reduce the rate of degradation of these proteins (Lo, J. H., Baker, T. A., and Sauer, R. T. (2001) Protein Sci. 10, 551-559; Singh, S. K., Rozycki, J., Ortega, J., Ishikawa, T., Lo, J., Steven, A. C., and Maurizi, M. R. (2001) J. Biol. Chem. 276, 29420-29429). Here we demonstrate, using both fluorescence resonance energy transfer to measure the arrival of substrate at ClpP and competition between wild-type and an inactive mutant form of ClpP, that this effect on degradation is caused by diminished stability of the ClpA-ClpP complex during translocation and proteolysis, effectively disrupting the targeting of unfolded substrates to the protease. We have also examined two larger ssrA-tagged substrates, CFP-GFP-ssrA and luciferase-ssrA, and observed different behaviors. CFP-GFP-ssrA is not efficiently unfolded by the truncated chaperone whereas luciferase-ssrA is, suggesting that the former requires interaction with the N-domains, likely via the body of the protein, to stabilize its binding. Thus, the N-domains play a key allosteric role in complex formation with ClpP and may also have a critical role in recognizing certain tag elements and binding some substrate proteins.     

Structural analysis of the adaptor protein ClpS in complex with the N-terminal domain of ClpA

In Escherichia coli, protein degradation is performed by several proteolytic machines, including ClpAP. Generally, the substrate specificity of these machines is determined by chaperone components, such as ClpA. In some cases, however, the specificity is modified by adaptor proteins, such as ClpS. Here we report the 2.5 A resolution crystal structure of ClpS in complex with the N-terminal domain of ClpA. Using mutagenesis, we demonstrate that two contact residues (Glu79 and Lys 84) are essential not only for ClpAS complex formation but also for ClpAPS-mediated substrate degradation. The corresponding residues are absent in the chaperone ClpB, providing a structural rationale for the unique specificity shown by ClpS despite the high overall similarity between ClpA and ClpB. To determine the location of ClpS within the ClpA hexamer, we modeled the N-terminal domain of ClpA onto a structurally defined, homologous AAA+ protein. From this model, we proposed a molecular mechanism to explain the ClpS-mediated switch in ClpA substrate specificity.     

The anti-adhesive activity of thrombospondin is mediated by the N-terminal domain of cell surface calreticulin

Thrombospondin (TSP) induces reorganization of the actin cytoskeleton and restructuring of focal adhesions through binding of amino acids (aa) 17-35 (hep I peptide) of thrombospondin to a cell surface form of calreticulin (CRT). In this report we provide further evidence for the involvement of calreticulin in thrombospondin signaling and characterize thrombospondin-calreticulin interactions. Wild type but not crt(-/-) cells respond to hep I/TSP. Responsiveness can be restored by incubation of cells with exogenous calreticulin or by transfection with calreticulin. Thrombospondin forms complexes with the CRT-N-domain that are enhanced by physiologic levels of calcium and zinc. Consistent with thrombospondin/CRT-N-domain binding, only the CRT-N-domain blocks hep I- and thrombospondin-stimulated focal adhesion disassembly. A series of glutathione S-transferase-N-domain mutants were used to map the sequence within the N-domain that interacts with TSP/hep I. A construct containing aa 1-43 but not a construct of aa 1-31 supported thrombospondin binding and focal adhesion disassembly. A series of overlapping peptides were used to further map the thrombospondin-binding site. Peptides spanning aa 19-36 (RWIESKHKSDFGKFVLSS) blocked hep I-stimulated focal adhesion disassembly, indicating that the TSP/hep I-binding site is located to this sequence in calreticulin. A mutant fusion protein lacking aa 19-36 (glutathione S-transferase-CRTDeltahep I) failed to restore responsiveness to hep I in crt(-/-) cells, bind thrombospondin, or competitively block focal adhesion disassembly, providing evidence for the role of this calreticulin sequence in mediating thrombospondin signaling.     

The N-terminal domain of alphaB-crystallin is protected from proteolysis by bound substrate

Alpha-crystallin, a major structural protein of the lens can also function as a molecular chaperone by binding to unfolding substrate proteins. We have used a combination of limited proteolysis at low temperature, and mass spectrometry to identify the regions of alpha-crystallin directly involved in binding to the structurally compromised substrate, reduced alpha-lactalbumin. In the presence of trypsin, alpha-crystallin which had been pre-incubated with substrate showed markedly reduced proteolysis at the C-terminus compared with a control, indicating that the bound substrate restricted access of trypsin to R157, the main cleavage site. Chymotrypsin was able to cleave at residues in both the N- and C-terminal domains. In the presence of substrate, alpha-crystallin showed markedly reduced proteolysis at four sites in the N-terminal domain when compared with the control. Minor differences in cleavage were observed within the C-terminal domain suggesting that the N-terminal region of alpha-crystallin contains the major substrate interaction sites.     

The N-terminal domain of p53 is natively unfolded

p53 is one of the key molecules regulating cell proliferation, apoptosis and tumor suppression by integrating a wide variety of signals. The structural basis for this function is still poorly understood. p53 appears to exercise its function as a modular protein in which different functions are associated with distinct domains. Presumably, p53 contains both folded and partially structured parts. Here, we have investigated the structure of the isolated N-terminal part of p53 (amino acid residues 1-93) using biophysical techniques. We demonstrate that this domain is devoid of tertiary structure and largely missing secondary structure elements. It exhibits a large hydrodynamic radius, typical for unfolded proteins. These findings suggest strongly that the entire N-terminal part of p53 is natively unfolded under physiological conditions. Furthermore, the binding affinity to its functional antagonist Mdm2 was investigated. A comparison of the binding of human Mdm2 to the N-terminal part of p53 and full-length p53 suggests that unfolded and folded parts of p53 function synergistically.     

Crystal structure of the N-terminal domain of E. coli Lon protease

We report here the first crystal structure of the N-terminal domain of an A-type Lon protease. Lon proteases are ubiquitous, multidomain, ATP-dependent enzymes with both highly specific and non-specific protein binding, unfolding, and degrading activities. We expressed and purified a stable, monomeric 119-amino acid N-terminal subdomain of the Escherichia coli A-type Lon protease and determined its crystal structure at 2.03 A (Protein Data Bank [PDB] code 2ANE). The structure was solved in two crystal forms, yielding 14 independent views. The domain exhibits a unique fold consisting primarily of three twisted beta-sheets and a single long alpha-helix. Analysis of recent PDB depositions identified a similar fold in BPP1347 (PDB code 1ZBO), a 203-amino acid protein of unknown function from Bordetella parapertussis, crystallized as part of a structural genomics effort. BPP1347 shares sequence homology with Lon N-domains and with a family of other independently expressed proteins of unknown functions. We postulate that, as is the case in Lon proteases, this structural domain represents a general protein and polypeptide interaction domain.     

The N-terminal domain of mammalian Lysyl-tRNA synthetase is a functional tRNA-binding domain.

Lysyl-tRNA synthetase from higher eukaryotes possesses a lysine-rich N-terminal polypeptide extension appended to a classical prokaryotic-like LysRS domain. Band shift analysis showed that this extra domain provides LysRS with nonspecific tRNA binding properties. A N-terminally truncated derivative of LysRS, LysRS-DeltaN, displayed a 100-fold lower apparent affinity for tRNA(3)Lys and a 3-fold increase in K(m) for tRNA(3)Lys in the aminoacylation reaction, as compared with the native enzyme. The isolated N-domain of LysRS also displayed weak affinity for tRNA, suggesting that the catalytic and N-domains of LysRS act synergistically to provide a high affinity binding site for tRNA. A more detailed analysis revealed that LysRS binds and specifically aminoacylates an RNA minihelix mimicking the amino acid acceptor stem-loop structure of tRNA(3)Lys, whereas LysRS-DeltaN did not. As a consequence, merging an additional RNA-binding domain into a bacterial-like LysRS increases the catalytic efficiency of the enzyme, especially at the low concentration of deacylated tRNA prevailing in vivo. Our results provide new insights into tRNA(Lys) channeling in eukaryotic cells and shed new light on the possible requirement of native LysRS for triggering tRNA(3)Lys packaging into human immunodeficiency virus, type 1 viral particles.     

Cpx signal transduction is influenced by a conserved N-terminal domain in the novel inhibitor CpxP and the periplasmic protease DegP

In Escherichia coli, envelope stress can be overcome by three different envelope stress responses: the sigma(E) stress response and the Bae and Cpx two-component systems. The Cpx envelope stress response is controlled by the sensor kinase CpxA, the response regulator CpxR, and the novel periplasmic protein CpxP. CpxP mediates feedback inhibition of the Cpx pathway through a hypothetical interaction with the sensing domain of CpxA. No informative homologues of CpxP are known, and thus it is unclear how CpxP exerts this inhibition. Here, we identified six cpxP loss-of-function mutations using a CpxP-beta-lactamase (CpxP'-'Bla) translational fusion construct. These loss-of-function mutations identified a highly conserved, predicted alpha-helix in the N-terminal domain of CpxP that affects both the function and the stability of the protein. In the course of this study, we also found that CpxP'-'Bla stability is differentially controlled by the periplasmic protease DegP in response to inducing cues and that mutation of degP diminishes Cpx pathway activity. We propose that the N-terminal alpha-helix is an important functional domain for inhibition of the Cpx pathway and that CpxP is subject to DegP-dependent proteolysis.     

The N-terminal zinc binding domain of ClpX is a dimerization domain that modulates the chaperone function

Clp ATPases are unique chaperones that promote protein unfolding and subsequent degradation by proteases. The mechanism by which this occurs is poorly understood. Here we demonstrate that the N-terminal domain of ClpX is a C4-type zinc binding domain (ZBD) involved in substrate recognition. ZBD forms a very stable dimer that is essential for promoting the degradation of some typical ClpXP substrates such as lambdaO and MuA but not GFP-SsrA. Furthermore, experiments indicate that ZBD contains a primary binding site for the lambdaO substrate and for the cofactor SspB. Removal of ZBD from the ClpX sequence renders the ATPase activity of ClpX largely insensitive to the presence of ClpP, substrates, or the SspB cofactor. All these results indicate that ZBD plays an important role in the ClpX mechanism of function and that ATP binding and/or hydrolysis drives a conformational change in ClpX involving ZBD.     

Mutations in the N-terminal actin-binding domain of filamin C cause a distal myopathy

Linkage analysis of the dominant distal myopathy we previously identified in a large Australian family demonstrated one significant linkage region located on chromosome 7 and encompassing 18.6 Mbp and 151 genes. The strongest candidate gene was FLNC because filamin C, the encoded protein, is muscle-specific and associated with myofibrillar myopathy. Sequencing of FLNC cDNA identified a c.752T>C (p.Met251Thr) mutation in the N-terminal actin-binding domain (ABD); this mutation segregated with the disease and was absent in 200 controls. We identified an Italian family with the same phenotype and found a c.577G>A (p.Ala193Thr) filamin C ABD mutation that segregated with the disease. Filamin C ABD mutations have not been described, although filamin A and filamin B ABD mutations cause multiple musculoskeletal disorders. The distal myopathy phenotype and muscle pathology in the two families differ from myofibrillar myopathies caused by filamin C rod and dimerization domain mutations because of the distinct involvement of hand muscles and lack of pathological protein aggregation. Thus, like the position of FLNA and B mutations, the position of the FLNC mutation determines disease phenotype. The two filamin C ABD mutations increase actin-binding affinity in a manner similar to filamin A and filamin B ABD mutations. Cell-culture expression of the c.752T>C (p.Met251)Thr mutant filamin C ABD demonstrated reduced nuclear localization as did mutant filamin A and filamin B ABDs. Expression of both filamin C ABD mutants as full-length proteins induced increased aggregation of filamin. We conclude filamin C ABD mutations cause a recognizable distal myopathy, most likely through increased actin affinity, similar to the pathological mechanism of filamin A and filamin B ABD mutations.     

The pro region N-terminal domain provides specific interactions required for catalysis of alpha-lytic protease folding

The extracellular bacterial protease, alpha-lytic protease (alphaLP), is synthesized with a large, two-domain pro region (Pro) that catalyzes the folding of the protease to its native conformation. In the absence of its Pro folding catalyst, alphaLP encounters a very large folding barrier (DeltaG = 30 kcal mol(-1)) that effectively prevents the protease from folding (t(1/2) of folding = 1800 years). Although homology data, mutational studies, and structural analysis of the Pro.alphaLP complex suggested that the Pro C-terminal domain (Pro C-domain) serves as the minimum "foldase" unit responsible for folding catalysis, we find that the Pro N-terminal domain (Pro N-domain) is absolutely required for alphaLP folding. Detailed kinetic analysis of Pro N-domain point mutants and a complete N-domain deletion reveal that the Pro N-domain both provides direct interactions with alphaLP that stabilize the folding transition state and confers stability to the Pro C-domain. The Pro N- and C-domains make conflicting demands upon native alphaLP binding that are alleviated in the optimized interface of the folding transition state complex. From these studies, it appears that the extremely high alphaLP folding barrier necessitates the presence of both the Pro domains; however, alphaLP homologues with less demanding folding barriers may not require both domains, thus possibly explaining the wide variation in the pro region size of related pro-proteases.     

The N-terminal domain of the Flo11 protein from Saccharomyces cerevisiae is an adhesin without mannose-binding activity

The expression of the Flo11 flocculin in Saccharomyces cerevisiae offers the cell a wide range of phenotypes, depending on the strain and the environmental conditions. The most important are pseudohyphae development, invasive growth and flocculation. The mechanism of cellular adhesion mediated by Flo11p is not well understood. Therefore, the N-terminal domain of Flo11p was purified and studied. Although its amino acid sequence shows less similarity with the other flocculins, Flo11p belongs to the flocculin family. However, the N-terminal domain contains the 'Flo11-domain' (PF10181), but not the mannose-binding PA14 domain, which is present in the other flocculins (Flo1p, Flo5p, Flo9p and Flo10p). Structural and binding properties of the N-terminal domain of Flo11p were studied. It is shown that this domain is O-glycosylated and is structurally composed mainly of β-sheets, which is typical for the members of the flocculin family. Furthermore, fluorescence spectroscopy binding studies revealed that N-Flo11p does not bind mannose, which is in contrast to the other Flo proteins. However, surface plasmon resonance analysis showed that N-Flo11p self-interacts and explains the cell-cell interaction capacity of FLO11-expressing cells.     

The N-terminal domain of Janus kinase 2 is required for Golgi processing and cell surface expression of erythropoietin receptor.

We show that Janus kinase 2 (JAK2), and more specifically just its intact N-terminal domain, binds to the erythropoietin receptor (EpoR) in the endoplasmic reticulum and promotes its cell surface expression. This interaction is specific as JAK1 has no effect. Residues 32 to 58 of the JAK2 JH7 domain are required for EpoR surface expression. Alanine scanning mutagenesis of the EpoR membrane proximal region reveals two modes of EpoR-JAK2 interaction. A continuous block of EpoR residues is required for functional, ligand-independent binding to JAK2 and cell surface receptor expression, whereas four specific residues are essential in switching on prebound JAK2 after ligand binding. Thus, in addition to its kinase activity required for cytokine receptor signaling, JAK is also an essential subunit required for surface expression of cytokine receptors.     

The N-terminal domain of Homer/Vesl is a new class II EVH1 domain

Cellular activities controlled by signal transduction processes such as cell motility and cell growth depend on the tightly regulated assembly of multiprotein complexes. Adapter proteins that specifically interact with their target proteins are key components required for the formation of these assemblies. Ena/VASP-homology 1 (EVH1) domains are small constituents of large modular proteins involved in microfilament assembly that specifically recognize proline-rich regions. EVH1 domain-containing proteins are present in neuronal cells, like the Homer/Vesl protein family that is involved in memory-generating processes. Here, we describe the crystal structure of the murine EVH1 domain of Vesl 2 at 2.2 A resolution. The small globular protein consists of a seven-stranded antiparallel beta-barrel with a C-terminal alpha-helix packing alongside the barrel. A shallow groove running parallel with beta-strand VI forms an extended peptide-binding site. Using peptide library screenings, we present data that demonstrate the high affinity of the Vesl 2 EVH1 domain towards peptide sequences containing a proline-rich core sequence (PPSPF) that requires additional charged amino acid residues on either side for specific binding. Our functional data, substantiated by structural data, demonstrate that the ligand-binding of the Vesl EVH1 domain differs from the interaction characteristics of the previously examined EVH1 domains of the Evl/Mena proteins. Analogous to the Src homology 3 (SH3) domains that bind their cognate ligands in two distinct directions, we therefore propose the existence of two distinct classes of EVH1 domains.     

The N-terminal domain of the replication initiator protein RepE is a dimerization domain forming a stable dimer

The initiator protein RepE of the mini-F plasmid in Escherichia coli plays an essential role in DNA replication, which is regulated by the molecular chaperone-dependent oligomeric state (monomer or dimer). Crosslinking, ultracentrifugation, and gel filtration analyses showed that the solely expressed N-terminal domain (residues 1-144 or 1-152) exists in the dimeric state as in the wild-type RepE protein. This result indicates that the N-terminal domain functions as a dimerization domain of RepE and might be important for the interaction with the molecular chaperones. The N-terminal domain dimer has been crystallized in order to obtain structural insight into the regulation of the monomer/dimer conversion of RepE.     

The distal cytoplasmic tail of the influenza A M2 protein dynamically extends from the membrane

The influenza A M2 protein is a multifunctional membrane-associated homotetramer that orchestrates several essential events in the viral infection cycle. The monomeric subunits of the M2 homotetramer consist of an N-terminal ectodomain, a transmembrane domain, and a C-terminal cytoplasmic domain. The transmembrane domain forms a four-helix proton channel that promotes uncoating of virions upon host cell entry. The membrane-proximal region of the C-terminal domain forms a surface-associated amphipathic helix necessary for viral budding. The structure of the remaining ~34 residues of the distal cytoplasmic tail has yet to be fully characterized despite the functional significance of this region for influenza infectivity. Here, we extend structural and dynamic studies of the poorly characterized M2 cytoplasmic tail. We used SDSL-EPR to collect site-specific information on the mobility, solvent accessibility, and conformational properties of residues 61–70 of the full-length, cell-expressed M2 protein reconstituted into liposomes. Our analysis is consistent with the predominant population of the C-terminal tail dynamically extending away from the membranes surface into the aqueous medium. These findings provide insight into the hypothesis that the C-terminal domain serves as a sensor that regulates how M2 protein participates in critical events in the viral infection cycle.