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A Copper Relay System Involving Two Periplasmic Chaperones Drives cbb 3 -Type Cytochrome c Oxidase Biogenesis in Rhodobacter capsulatus
PccA and SenC are periplasmic copper chaperones required for the biogenesis of cbb 3 -type cytochrome c oxidase (cbb 3 -Cox) in Rhodobacter capsulatus at physiological Cu concentrations. However, both proteins are dispensable for cbb 3 -Cox assembly when the external Cu concentration is high. PccA and SenC bind Cu using Met and His residues and Cys and His residues as ligands, respectively, and both proteins form a complex during cbb 3 -Cox biogenesis. SenC also interacts directly with cbb 3 -Cox, as shown by chemical cross-linking. Here we determined the periplasmic concentrations of both proteins in vivo and analyzed their Cu binding stoichiometries and their Cu(I) and Cu(II) binding affinity constants (KD) in vitro. Our data show that both proteins bind a single Cu atom with high affinity. In vitro Cu transfer assays demonstrate Cu transfer both from PccA to SenC and from SenC to PccA at similar levels. We conclude that PccA and SenC constitute a Cu relay system that facilitates Cu delivery to cbb 3 -Cox.
Locking the Elbow: Improved Antibody Fab Fragments as Chaperones for Structure Determination
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Conformational equilibria in allosteric control of Hsp70 chaperones
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Membrane Chaperoning of a Thylakoid Protease Whose Structural Stability Is Modified by the Protonmotive Force
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The Escherichia coli P and Type 1 Pilus Assembly Chaperones PapD and FimC Are Monomeric in Solution
ABSTRACT The chaperone/usher pathway is used by Gram-negative bacteria to assemble adhesive surface structures known as pili or fimbriae. Uropathogenic strains of Escherichia coli use this pathway to assemble P and type 1 pili, which facilitate colonization of the kidney and bladder, respectively. Pilus assembly requires a periplasmic chaperone and outer membrane protein termed the usher. The chaperone allows folding of pilus subunits and escorts the subunits to the usher for polymerization into pili and secretion to the cell surface. Based on previous structures of mutant versions of the P pilus chaperone PapD, it was suggested that the chaperone dimerizes in the periplasm as a self-capping mechanism. Such dimerization is counterintuitive because the chaperone G1 strand, important for chaperone-subunit interaction, is buried at the dimer interface. Here, we show that the wild-type PapD chaperone also forms a dimer in the crystal lattice; however, the dimer interface is different from the previously solved structures. In contrast to the crystal structures, we found that both PapD and the type 1 pilus chaperone, FimC, are monomeric in solution. Our findings indicate that pilus chaperones do not sequester their G1 β-strand by forming a dimer. Instead, the chaperones may expose their G1 strand for facile interaction with pilus subunits. We also found that the type 1 pilus adhesin, FimH, is flexible in solution while in complex with its chaperone, whereas the P pilus adhesin, PapGII, is rigid. Our study clarifies a crucial step in pilus biogenesis and reveals pilus-specific differences that may relate to biological function. IMPORTANCE Pili are critical virulence factors for many bacterial pathogens. Uropathogenic E. coli relies on P and type 1 pili assembled by the chaperone/usher pathway to adhere to the urinary tract and establish infection. Studying pilus assembly is important for understanding mechanisms of protein secretion, as well as for identifying points for therapeutic intervention. Pilus biogenesis is a multistep process. This work investigates the oligomeric state of the pilus chaperone in the periplasm, which is important for understanding early assembly events. Our work unambiguously demonstrates that both PapD and FimC chaperones are monomeric in solution. We further demonstrate that the solution behavior of the FimH and PapGII adhesins differ, which may be related to functional differences between the two pilus systems.
Structural analysis of extracellular ATP-independent chaperones of streptococcal species and protein substrate interactions
ABSTRACT During infection, bacterial pathogens rely on secreted virulence factors to manipulate the host cell. However, in gram-positive bacteria, the molecular mechanisms underlying the folding and activity of these virulence factors after membrane translocation are not clear. Here, we solved the protein structures of two secreted parvulin and two secreted cyclophilin-like peptidyl-prolyl isomerase (PPIase) ATP-independent chaperones found in gram-positive streptococcal species. The extracellular parvulin-type PPIase, PrsA inStreptococcus pneumoniaeandStreptococcus mutansmaintain dimeric crystal structures reminiscent of folding catalysts that consist of two domains, a PPIase and foldase domain. Structural comparison of the two cyclophilin-like extracellular chaperones fromS. pneumoniaeandStreptococcus pyogeneswith other cyclophilins demonstrates that this group of cyclophilin-like chaperones has novel structural appendages formed by 9- and 24-residue insertions. Furthermore, we demonstrate that deletion ofprsAandslrAgenes impairs the secretion of the cholesterol-dependent pore-forming toxin, pneumolysin inS. pneumoniae. Using protein pull-down and biophysical assays, we demonstrate a direct interaction between PrsA and SlrA with Ply. Then, we developed chaperone-assisted folding assays that show that theS. pneumoniaePrsA and SlrA extracellular chaperones accelerate pneumolysin folding. In addition, we demonstrate that SlrA and, for the first time,S. pyogenes PpiA exhibit PPIase activity and can bind the immunosuppressive drug, cyclosporine A. Altogether, these findings suggest a mechanistic role for streptococcal PPIase chaperones in the activity and folding of secreted virulence factors such as pneumolysin. IMPORTANCE Streptococcal species are a leading cause of lower respiratory infections that annually affect millions of people worldwide. During infection, streptococcal species secrete a medley of virulence factors that allow the bacteria to colonize and translocate to deeper tissues. In many gram-positive bacteria, virulence factors are secreted from the cytosol across the bacterial membrane in an unfolded state. The bacterial membrane-cell wall interface is exposed to the potentially harsh extracellular environment, making it difficult for native virulence factors to fold before being released into the host. ATP-independent PPIase-type chaperones, PrsA and SlrA, are thought to facilitate folding and stabilization of several unfolded proteins to promote the colonization and spread of streptococci. Here, we present crystal structures of the molecular chaperones of PrsA and SlrA homologs from streptococcal species. We provide evidence that theStreptococcus pyogenesSlrA homolog, PpiA, has PPIase activity and binds to cyclosporine A. In addition, we show thatStreptococcus pneumoniaePrsA and SlrA directly interact and fold the cholesterol-dependent pore-forming toxin and critical virulence determinant, pneumolysin.
Calcium sensing receptor activates the NLRP3 inflammasome via a chaperone-assisted degradative pathway involving Hsp70 and LC3-II
Highlights: • CaSR activates the NLRP3-inflammasome via proteasome and lysosome. • CaSR signals via Gα{sub q} and Gβγ to activate the NLRP3 inflammasome. • CaSR stimulates Hsp70 expression leading to chaperone-assisted protein degradation. • CaSR promotes IL-1β processing via proteasome and lysosome-dependent pathways. • CaSR generates homeostasis-altering molecular processes. Calcium sensing receptor (CaSR) activates the NLRP3 inflammasome with consequences on homeostatic responses. However, little is known about how this process is orchestrated. Since proteolysis of critical regulators of NLRP3 inflammasome contribute to its activation, we aimed to understand how CaSR stimulates proteolytic pathways to activate the NLRP3 inflammasome. We found that proteasome and lysosome-dependent mechanisms are activated by CaSR to promote the degradation of important regulators of NLRP inflammasome. The pathway involves Gαq/PLC/PKC and Gβγ/PI3K signaling cascades and IRAK1 ubiquitination. In addition, CaSR stimulates Hsp70 expression activating a chaperone-assisted protein degradation that dictates the fate of ASC, NLRP3 (NOD-like receptor family protein 3), IRAK1 and TRAF6 proteins, turning on the NLRP3 inflammasome. In response to CaSR signaling, these proteins are degraded through the combination of CUPS (chaperone-assisted ubiquitin proteasome pathway) and CAEMI (chaperone-assisted endosomal microautophagy) systems being integrated by autophagosomes (chaperone-assisted macroautophagy, CAMA), as indicated by LC3-II, a classical marker for autophagy, that is induced in the process. Furthermore, CaSR triggers the proteolytic cleavage of pro-IL-1β (IL-1β, 31 kDa) into mature IL-1β (IL-1β, 17 kDa), via the proteasome. Taken together, our results indicate that CaSR promotes NLRP3 inflammasome activation and proteolytic maturation of IL-1β by inducing CUPS and CAEMI, chaperone-assisted degradation pathways. Overall, these results support the inclusion of CaSR as an activator of homeostasis-altering molecular processes.
Structural Variability of EspG Chaperones from Mycobacterial ESX-1, ESX-3, and ESX-5 Type VII Secretion Systems
Type VII secretion systems (ESX) are responsible for transport of multiple proteins in mycobacteria. How different ESX systems achieve specific secretion of cognate substrates remains elusive. In the ESX systems, the cytoplasmic chaperone EspG forms complexes with heterodimeric PE–PPE substrates that are secreted from the cells or remain associated with the cell surface. Here we report the crystal structure of the EspG 1 chaperone from the ESX-1 system determined using a fusion strategy with T4 lysozyme. EspG 1 adopts a quasi 2-fold symmetric structure that consists of a central β-sheet and two α-helical bundles. In addition, we describe the structures of EspG 3 chaperones from four different crystal forms. Alternate conformations of the putative PE–PPE binding site are revealed by comparison of the available EspG 3 structures. Analysis of EspG 1 , EspG 3 , and EspG 5 chaperones using small-angle X-ray scattering reveals that EspG 1 and EspG 3 chaperones form dimers in solution, which we observed in several of our crystal forms. Finally, we propose a model of the ESX-3 specific EspG 3 –PE5–PPE4 complex based on the small-angle X-ray scattering analysis.
Dynamic intramolecular regulation of the histone chaperone nucleoplasmin controls histone binding and release
Here, nucleoplasmin (Npm) is a highly conserved histone chaperone responsible for the maternal storage and zygotic release of histones H2A/H2B. Npm contains a pentameric N-terminal core domain and an intrinsically disordered C-terminal tail domain. Though intrinsically disordered regions are common among histone chaperones, their roles in histone binding and chaperoning remain unclear. Using an NMR-based approach, here we demonstrate that the Xenopus laevis Npm tail domain controls the binding of histones at its largest acidic stretch (A2) via direct competition with both the C-terminal basic stretch and basic nuclear localization signal. NMR and small-angle X-ray scattering (SAXS) structural analyses allowed us to construct models of both the tail domain and the pentameric complex. Functional analyses demonstrate that these competitive intramolecular interactions negatively regulate Npm histone chaperone activity in vitro. Together these data establish a potentially generalizable mechanism of histone chaperone regulation via dynamic and specific intramolecular shielding of histone interaction sites.
A widespread family of molecular chaperones promotes the intracellular stability of type VIIb secretion system– exported toxins
To survive in highly competitive environments, bacteria use specialized secretion systems to deliver antibacterial toxins into neighboring cells, thereby inhibiting their growth. In many Gram-positive bacteria, the export of such toxins requires a membrane-bound molecular apparatus known as the type VIIb secretion system (T7SSb). Recently, it was shown that toxin recruitment to the T7SSb requires a physical interaction between a toxin and two or more so-called targeting factors, which harbor key residues required for T7SS-dependent protein export. However, in addition to these targeting factors, some toxins additionally require a protein belonging to the DUF4176 protein family. Here, by examining two toxin–DUF4176 protein pairs, we demonstrate that DUF4176 constitutes a family of toxin-specific molecular chaperones. In addition to being required for toxin stability in producing cells, we find that DUF4176 proteins facilitate toxin export by specifically interacting with a previously uncharacterized intrinsically disordered region found in many T7SS toxins. Using X-ray crystallography, we determine structures of several DUF4176 chaperones in their unbound state, and of a DUF4176 chaperone in complex with the binding site of its cognate toxin. These structures reveal that this binding site consists of a disordered amphipathic α-helix that requires interaction with its cognate chaperone for proper folding. Overall, we have identified a family of secretion system associated molecular chaperones found throughout T7SSb-containing Gram-positive bacteria.
New insights into structural and functional relationships between LonA proteases and ClpB chaperones
LonA proteases and ClpB chaperones are key components of the protein quality control system in bacterial cells. LonA proteases form a unique family of ATPases associated with diverse cellular activities (AAA + ) proteins due to the presence of an unusual N-terminal region comprised of two domains: a β-structured N domain and an α-helical domain, including the coiled-coil fragment, which is referred to as HI(CC). The arrangement of helices in the HI(CC) domain is reminiscent of the structure of the H1 domain of the first AAA + module of ClpB chaperones. It has been hypothesized that LonA proteases with a single AAA + module may also contain a part of another AAA + module, the full version of which is present in ClpB. Here, we established and tested the structural basis of this hypothesis using the known crystal structures of various fragments of LonA proteases and ClpB chaperones, as well as the newly determined structure of the Escherichia coli LonA fragment (235–584). The similarities and differences in the corresponding domains of LonA proteases and ClpB chaperones were examined in structural terms. The results of our analysis, complemented by the finding of a singular match in the location of the most conserved axial pore-1 loop between the LonA NB domain and the NB2 domain of ClpB, support our hypothesis that there is a structural and functional relationship between two coiled–coil fragments and implies a similar mechanism of engagement of the pore-1 loops in the AAA + modules of LonAs and ClpBs.
On the nature of the optimal form of the holdase‐type chaperone stress response
The holdase paradigm of chaperone action involves preferential binding by the chaperone to the unfolded protein state, thereby preventing it from either, associating with other unstable proteins (to form large dysfunctional aggregates), or being degraded by the proteolytic machinery of the cell/organism. In this paper, we examine the necessary physical constraints imposed upon the holdase chaperone response in a cell‐like environment and use these limitations to comment on the likely nature of the optimal form of chaperone response in vivo .
Cooperative Subunit Refolding of a Light‐Harvesting Protein through a Self‐Chaperone Mechanism
Abstract The fold of a protein is encoded by its amino acid sequence, but how complex multimeric proteins fold and assemble into functional quaternary structures remains unclear. Here we show that two structurally different phycobiliproteins refold and reassemble in a cooperative manner from their unfolded polypeptide subunits, without biological chaperones. Refolding was confirmed by ultrafast broadband transient absorption and two‐dimensional electronic spectroscopy to probe internal chromophores as a marker of quaternary structure. Our results demonstrate a cooperative, self‐chaperone refolding mechanism, whereby the β‐subunits independently refold, thereby templating the folding of the α‐subunits, which then chaperone the assembly of the native complex, quantitatively returning all coherences. Our results indicate that subunit self‐chaperoning is a robust mechanism for heteromeric protein folding and assembly that could also be applied in self‐assembled synthetic hierarchical systems.
Cooperative Subunit Refolding of a Light-Harvesting Protein through a Self-Chaperone Mechanism
The fold of a protein is encoded by its amino acid sequence, but how complex multimeric proteins fold and assemble into functional quaternary structures remains unclear. Here we show that two structurally different phycobiliproteins refold and reassemble in a cooperative manner from their unfolded polypeptide subunits, without biological chaperones. Refolding was confirmed by ultrafast broadband transient absorption and two-dimensional electronic spectroscopy to probe internal chromophores as a marker of quaternary structure. Our results demonstrate a cooperative, self-chaperone refolding mechanism, whereby the β-subunits independently refold, thereby templating the folding of the α-subunits, which then chaperone the assembly of the native complex, quantitatively returning all coherences. Finally, our results indicate that subunit self-chaperoning is a robust mechanism for heteromeric protein folding and assembly that could also be applied in self-assembled synthetic hierarchical systems.
Patient mutations in human ATP:cob(I)alamin adenosyltransferase differentially affect its catalytic versus chaperone functions
Human ATP:cob(I)alamin adenosyltransferase (ATR) is a mitochondrial enzyme that catalyzes an adenosyl transfer to cob(I)alamin, synthesizing 5'-deoxyadenosylcobalamin (AdoCbl) or coenzyme B 12 . ATR is also a chaperone that escorts AdoCbl, transferring it to methylmalonyl-CoA mutase, which is important in propionate metabolism. Mutations in ATR lead to methylmalonic aciduria type B, an inborn error of B12 metabolism. Our previous studies have furnished insights into how ATR protein dynamics influence redox-linked cobalt coordination chemistry, controlling its catalytic versus chaperone functions. In this study, we have characterized three patient mutations at two conserved active site residues in human ATR, R190C/H, and E193K and obtained crystal structures of R190C and E193K variants, which display only subtle structural changes. All three mutations were found to weaken affinities for the cob(II)alamin substrate and the AdoCbl product and increase K M(ATP) . 31 P NMR studies show that binding of the triphosphate product, formed during the adenosylation reaction, is also weakened. However, although the k cat of this reaction is significantly diminished for the R190C/H mutants, it is comparable with the WT enzyme for the E193K variant, revealing the catalytic importance of Arg-190. Furthermore, although the E193K mutation selectively impairs the chaperone function by promoting product release into solution, its catalytic function might be unaffected at physiological ATP concentrations. In contrast, the R190C/H mutations affect both the catalytic and chaperoning activities of ATR. Because the E193K mutation spares the catalytic activity of ATR, our data suggest that the patients carrying this mutation are more likely to be responsive to cobalamin therapy.