Crystal structure of Skp, a prefoldin-like chaperone that protects soluble and membrane proteins from aggregation

The Seventeen Kilodalton Protein (Skp) is a trimeric periplasmic chaperone that assists outer membrane proteins in their folding and insertion into membranes. Here we report the crystal structure of Skp from E. coli. The structure of the Skp trimer resembles a jellyfish with alpha-helical tentacles protruding from a beta barrel body defining a central cavity. The architecture of Skp is unexpectedly similar to that of Prefoldin/GimC, a cytosolic chaperone present in eukaria and archea, that binds unfolded substrates in its central cavity. The ability of Skp to prevent the aggregation of model substrates in vitro is independent of ATP. Skp can interact directly with membrane lipids and lipopolysaccharide (LPS). These interactions are needed for efficient Skp-assisted folding of membrane proteins. We have identified a putative LPS binding site on the outer surface of Skp and propose a model for unfolded substrate binding.     

Skp, a molecular chaperone of gram-negative bacteria, is required for the formation of soluble periplasmic intermediates of outer membrane proteins.

Using a cross-linking approach, we have analyzed the function of Skp, a presumed molecular chaperone of the periplasmic space of Escherichia coli, during the biogenesis of an outer membrane protein (OmpA). Following its transmembrane translocation, OmpA interacts with Skp in close vicinity to the plasma membrane. In vitro, Skp was also found to bind strongly and specifically to pOmpA nascent chains after their release from the ribosome suggesting the ability of Skp to recognize early folding intermediates of outer membrane proteins. Pulse labeling of OmpA in spheroplasts prepared from an skp null mutant revealed a specific requirement of Skp for the release of newly translocated outer membrane proteins from the plasma membrane. Deltaskp mutant cells are viable and show only slight changes in the physiology of their outer membranes. In contrast, double mutants deficient both in Skp and the periplasmic protease DegP (HtrA) do not grow at 37 degrees C in rich medium. We show that in the absence of an active DegP, a lack of Skp leads to the accumulation of protein aggregates in the periplasm. Collectively, our data demonstrate that Skp is a molecular chaperone involved in generating and maintaining the solubility of early folding intermediates of outer membrane proteins in the periplasmic space of Gram-negative bacteria.     

The Skp chaperone helps fold soluble proteins in vitro by inhibiting aggregation

The periplasmic seventeen kilodalton protein (Skp) chaperone has been characterized primarily for its role in outer membrane protein (OMP) biogenesis, during which the jellyfish-like trimeric protein encapsulates partially folded OMPs, protecting them from the aqueous environment until delivery to the BAM outer membrane protein insertion complex. However, Skp is increasingly recognized as a chaperone that also assists in folding soluble proteins in the bacterial periplasm. In this capacity, Skp coexpression increases the active yields of many recombinant proteins and bacterial virulence factors. Using a panel of single-chain antibodies and a single-chain T-cell receptor (collectively termed scFvs) possessing varying stabilities and biophysical characteristics, we performed in vivo expression and in vitro folding and aggregation assays in the presence or absence of Skp. For Skp-sensitive scFvs, the presence of Skp during in vitro refolding assays reduced aggregation but did not alter the observed folding rates, resulting in a higher overall yield of active protein. Of the proteins analyzed, Skp sensitivity in all assays correlated with the presence of folding intermediates, as observed with urea denaturation studies. These results are consistent with Skp acting as a holdase, sequestering partially folded intermediates and thereby preventing aggregation. Because not all soluble proteins are sensitive to Skp coexpression, we hypothesize that the presence of a long-lived protein folding intermediate renders a protein sensitive to Skp. Improved understanding of the bacterial periplasmic protein folding machinery may assist in high-level recombinant protein expression and may help identify novel approaches to block bacterial virulence.     

The periplasmic E. coli chaperone Skp is a trimer in solution: biophysical and preliminary crystallographic characterization

The 'seventeen kilodalton protein' Skp confers transient solubility on outer membrane proteins during biogenesis in Gram-negative bacteria. Here we report a first biophysical characterization of this chaperone itself, which also possesses biotechnological potential in the production of recombinant proteins. Using cross-linking and gel filtration methods, we found that Skp forms a stable homo-trimer in solution. Following thermal denaturation, monitored by CD spectroscopy, this chaperone refolds with high efficiency but exhibits a pronounced hysteresis between the un- and refolding transitions. Using the recombinant protein equipped with the Strep-tag II at its N-terminus, suitable crystallization conditions for Skp were found. A first data set was collected to 2.60 A resolution.     

The early interaction of the outer membrane protein phoe with the periplasmic chaperone Skp occurs at the cytoplasmic membrane

Spheroplasts were used to study the early interactions of newly synthesized outer membrane protein PhoE with periplasmic proteins employing a protein cross-linking approach. Newly translocated PhoE protein could be cross-linked to the periplasmic chaperone Skp at the periplasmic side of the inner membrane. To study the timing of this interaction, a PhoE-dihydrofolate reductase hybrid protein was constructed that formed translocation intermediates, which had the PhoE moiety present in the periplasm and the dihydrofolate reductase moiety tightly folded in the cytoplasm. The hybrid protein was found to cross-link to Skp, indicating that PhoE closely interacts with the chaperone when the protein is still in a transmembrane orientation in the translocase. Removal of N-terminal parts of PhoE protein affected Skp binding in a cumulative manner, consistent with the presence of two Skp-binding sites in that region. In contrast, deletion of C-terminal parts resulted in variable interactions with Skp, suggesting that interaction of Skp with the N-terminal region is influenced by parts of the C terminus of PhoE protein. Both the soluble as well as the membrane-associated Skp protein were found to interact with PhoE. The latter form is proposed to be involved in the initial interaction with the N-terminal regions of the outer membrane protein.     

Plasticity and transient binding are key ingredients of the periplasmic chaperone network

SurA, Skp, FkpA, and DegP constitute a chaperone network that ensures biogenesis of outer membrane proteins (OMPs) in Gram‐negative bacteria. Both Skp and FkpA are holdases that prevent the self‐aggregation of unfolded OMPs, whereas SurA accelerates folding and DegP is a protease. None of these chaperones is essential, and we address here how functional plasticity is manifested in nine known null strains. Using a comprehensive computational model of this network termed OMPBioM, our results suggest that a threshold level of steady state holdase occupancy by chaperones is required, but the cell is agnostic to the specific holdase molecule fulfilling this function. In addition to its foldase activity, SurA moonlights as a holdase when there is no expression of Skp and FkpA. We further interrogate the importance of chaperone–client complex lifetime by conducting simulations using lifetime values for Skp complexes that range in length by six orders of magnitude. This analysis suggests that transient occupancy of durations much shorter than the Escherichia coli doubling time is required. We suggest that fleeting chaperone occupancy facilitates rapid sampling of the periplasmic conditions, which ensures that the cell can be adept at responding to environmental changes. Finally, we calculated the network effects of adding multivalency by computing populations that include two Skp trimers per unfolded OMP. We observe only modest perturbations to the system. Overall, this quantitative framework of chaperone–protein interactions in the periplasm demonstrates robust plasticity due to its dynamic binding and unbinding behavior.     

Sgt1 associates with Hsp90: an initial step of assembly of the core kinetochore complex

The kinetochore, which consists of DNA sequence elements and structural proteins, is essential for high-fidelity chromosome transmission during cell division. In budding yeast, Sgt1, together with Skp1, is required for assembly of the core kinetochore complex (CBF3) via Ctf13 activation. Formation of the active Ctf13-Skp1 complex also requires Hsp90, a molecular chaperone. We have found that Sgt1 interacts with Hsp90 in yeast. We also have determined that Skp1 and Hsc82 (a yeast Hsp90 protein) bind to the N-terminal region of Sgt1 that contains tetratricopeptide repeat motifs. Results of sequence and phenotypic analyses of sgt1 mutants strongly suggest that the N-terminal region containing the Hsc82-binding and Skp1-binding domains of Sgt1 is important for the kinetochore function of Sgt1. We found that Hsp90's binding to Sgt1 stimulates the binding of Sgt1 to Skp1 and that Sgt1 and Hsp90 stimulate the binding of Skp1 to Ctf13, the F-box core kinetochore protein. Our results strongly suggest that Sgt1 and Hsp90 function in assembling CBF3 by activating Skp1 and Ctf13.     

Dissecting the Escherichia coli periplasmic chaperone network using differential proteomics

β-Barrel proteins, or outer membrane proteins (OMPs), perform many essential functions in Gram-negative bacteria, but questions remain about the mechanism by which they are assembled into the outer membrane (OM). In Escherichia coli, β-barrels are escorted across the periplasm by chaperones, most notably SurA and Skp. However, the contributions of these two chaperones to the assembly of the OM proteome remained unclear. We used differential proteomics to determine how the elimination of Skp and SurA affects the assembly of many OMPs. We have shown that removal of Skp has no impact on the levels of the 63 identified OM proteins. However, depletion of SurA in the skp strain has a marked impact on the OM proteome, diminishing the levels of almost all β-barrel proteins. Our results are consistent with a model in which SurA plays a primary chaperone role in E. coli. Furthermore, they suggest that while no OMPs prefer the Skp chaperone pathway in wild-type cells, most can use Skp efficiently when SurA is absent. Our data, which provide a unique glimpse into the protein content of the nonviable surA skp mutant, clarify the roles of the periplasmic chaperones in E. coli.     

The Agrobacterium tumefaciens DnaK: ATPase cycle, oligomeric state and chaperone properties

DnaK is a molecular chaperone that promotes cell survival during stress by preventing protein misfolding. The chaperone activity is regulated by nucleotide binding and hydrolysis events in the N-terminal ATPase domain, which in turn mediate substrate binding and release in the C-terminal substrate binding domain. In this study we determined that ATP hydrolysis was the rate limiting step in the ATPase cycle of Agrobacterium tumefaciens DnaK (Agt DnaK); however the data suggested that Agt DnaK had a significantly lower affinity for ATP than Escherichia coli DnaK. We show for the first time that Agt DnaK was very effective at preventing thermal aggregation of malate dehydrogenase (MDH) in a concentration dependent manner. This is in contrast to E. coli DnaK which was ineffective at preventing thermal aggregation of MDH. A mutant Agt DnaK-V431F, with a blocked hydrophobic pocket in the substrate binding domain, was unable to suppress the thermosensitivty of an E. coli dnaK103 deletion strain. However the mutation did not inhibit Agt DnaK-V431F from preventing the thermal aggregation of MDH. The oligomeric state of Agt DnaK was studied using size exclusion chromatography. We demonstrated that dilution of the Agt DnaK protein, the addition of ATP and the removal of the 10kDa C-terminal alpha-helical subdomain reduced higher order associations but did not abrogate dimerisation. Our research implies that the C-terminal alpha-helical subdomain is involved in higher order associations, while the substrate binding domain is possibly involved in dimerisation.     

Functional analysis of the enteropathogenic Escherichia coli type III secretion system chaperone CesT identifies domains that mediate substrate interactions

In many Gram-negative bacteria, a key indicator of pathogenic potential is the possession of a specialized type III secretion system, which is utilized to deliver virulence effector proteins directly into the host cell cytosol. Many of the proteins secreted from such systems require small cytosolic chaperones to maintain the secreted substrates in a secretion-competent state. One such protein, CesT, serves a chaperone function for the enteropathogenic Escherichia coli (EPEC) translocated intimin receptor (Tir) protein, which confers upon EPEC the ability to alter host cell morphology following intimate bacterial attachment. Using a combination of complementary biochemical approaches, functional domains of CesT that mediate intermolecular interactions, involved in both chaperone-chaperone and chaperone-substrate associations, were determined. The CesT N-terminal is implicated in chaperone dimerization, whereas the amphipathic alpha-helical region of the C-terminal, is intimately involved in substrate binding. By functional complementation of chaperone domains using the Salmonella SicA chaperone to generate chaperone chimeras, we show that CesT-Tir interaction proceeds by a mechanism potentially common to other type III secretion system chaperones.     

Escherichia coli skp chaperone coexpression improves solubility and phage display of single-chain antibody fragments

Expression of single-chain antibody fragments (scAb)in the periplasm of Escherichia coli often results in low soluble product yield and cell lysis. We have increased scAb solubility and prevented cell culture lysis by coexpressing the E. coli Skp chaperone gene. A mutant Skp cistron was linked to a bacteriophage T7 gene 10 translational initiation region and placed either downstream of a scAb gene within an isopropyl beta-d-thiogalactopyranoside-inducible expression cassette or on a separate colE1-compatible arabinose-inducible vector. Increases in scAb solubility reflected the amount of coexpressed Skp. A bacteriophage display vector that was also engineered to coexpress Skp permitted display of a virtually undisplayable scAb and should prove useful in expanding library sizes.     

Escherichia coliSkp Chaperone Coexpression Improves Solubility and Phage Display of Single-Chain Antibody Fragments

Expression of single-chain antibody fragments (scAb)in the periplasm ofEscherichia colioften results in low soluble product yield and cell lysis. We have increased scAb solubility and prevented cell culture lysis by coexpressing theE. coliSkp chaperone gene. A mutant Skp cistron was linked to a bacteriophage T7 gene 10 translational initiation region and placed either downstream of a scAb gene within an isopropyl β- -thiogalactopyranoside-inducible expression cassette or on a separate colE1-compatible arabinose-inducible vector. Increases in scAb solubility reflected the amount of coexpressed Skp. A bacteriophage display vector that was also engineered to coexpress Skp permitted display of a virtually undisplayable scAb and should prove useful in expanding library sizes.     

A negatively charged amino acid in Skp2 is required for Skp2-Cks1 interaction and ubiquitination of p27Kip1

Proteolysis of cyclin-dependent kinase inhibitor p27 occurs predominantly in the late G1 phase of the cell cycle through a ubiquitin-mediated protein degradation pathway. Ubiquitination of p27 requires the SCFSkp2 ubiquitin ligase and Skp2 F-box binding protein Cks1. The mechanisms by which Skp2 recognizes Cks1 to ubiquitylate p27 remain obscure. Here we show that Asp-331 in the carboxyl terminus of Skp2 is required for its association with Cks1 and ubiquitination of p27. Mutation of Asp-331 to Ala disrupts the interaction between Skp2 and Cks1. Although Asp-331 mutation negates the ability of the Skp1-Cullin-F-box protein (SCF) complex to ubiquitylate p27, such a mutation has no effect on Skp2 self-ubiquitination. A conservative change from Asp to Glu at position 331 of Skp2 does not affect Skp2-Cks1 interaction. Our results revealed a unique requirement for a negatively charged residue in the carboxyl-terminal region of Skp2 in recognition of Cks1 and ubiquitination of p27.     

Genetic Evidence for Parallel Pathways of Chaperone Activity in the Periplasm of Escherichia coli

The periplasm of Escherichia coli contains many proteins proposed to have redundant functions in protein folding. Using depletion analysis, we directly demonstrated that null mutations in skp and surA, as well as in degP and surA, result in synthetic phenotypes, suggesting that Skp, SurA, and DegP are functionally redundant. The Deltaskp surA::kan combination has a bacteriostatic effect and leads to filamentation, while the degP::Tn10 surA::kan combination is bactericidal. The steady-state levels of several envelope proteins are greatly reduced upon depletion of a wild-type copy of surA in both instances. We suggest that the functional redundancy of Skp, SurA, and DegP lies in the periplasmic chaperone activity. Taken together, our data support a model in which the periplasm of E. coli contains parallel pathways for chaperone activity. In particular, we propose that Skp and DegP are components of the same pathway and that SurA is a component of a separate pathway. The loss of either pathway has minimal effects on the cell, while the loss of both pathways results in the synthetic phenotypes observed.     

Chaperone Skp from <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> exhibits immunoglobulin G binding ability

A low-molecular-weight cationic protein that can bind human and rabbit immunoglobulins G has been isolated from Yersinia pseudotuberculosis cells. This immunoglobulin binding protein (IBP) interacts with IgG Fc-fragment, the association constant of the resulting complex being 3.1 μM^?1. MALDI-TOF mass spectrometry analysis of IBP revealed its molecular mass of 16.1 kDa, and capillary isoelectrofocusing analysis showed pI value of 9.2. N-Terminal sequence determination by Edman degradation revealed the sequence of the 15 terminal amino acid residues (ADKIAIVNVSSIFQ). Tryptic hydrolysate of IBP was subjected to MALDI-TOF mass spectrometry for proteolytic peptide profiling. Based on the peptide fingerprint, molecular mass, pI, and N-terminal sequence and using bioinformatic resources, IBP was identified as Y. pseudotuberculosis periplasmic chaperone Skp. Using the method of comparative modeling a spatial model of Skp has been built. This model was then used for modeling of Skp complexes with human IgG1 Fc-fragment by means of molecular docking.     

Structural plasticity of peptidyl-prolyl isomerase sFkpA is a key to its chaperone function as revealed by solution NMR

Intramolecular dynamics of periplasmic chaperone FkpA-deltaCT (sFkpA) and its complexes with partially structured substrates are studied by NMR in solution. The backbone amide 15N relaxation of sFkpA reveals flexibility in the relative orientation between the dimerization domain and two juxtaposed catalytic domains identified in the X-ray structure of sFkpA. This flexibility is attributed to the structural plasticity within the long alpha-helical arm (helix III) consisting of residues 84 and 91. Residual dipolar couplings (RDCs) indicate an absence of fixed orientation between the sFkpA domains. The substrate binding surface of sFkpA is defined on the X-ray structure by mapping of chemical shift perturbations introduced by complexation of sFkpA with its corresponding protein substrates: partially folded RNase A S-protein and reduced carboxymethylated bovine alpha-lactalbumin (RCM-la). A comparison of 15N relaxation of apo-sFkpA and its complex with RNase A S-protein indicates an increased rigidity within the long alpha-helix III and decreased interdomain mobility of the complex. We speculate that these dynamic properties may play a key role in the chaperone activity of sFkpA, since ability to bind different substrates potentially requires structural adaptations of the chaperone protein. We show that binding of sFkpA to RNase A S-protein greatly reduces the population of aggregated oligomeric species of RNase A S-protein. Finally, a molecular model, the so-called "mother's arms" model, is proposed to illustrate the mechanism of chaperone activity by FkpA.     

Aperiplasmic protein (Skp) of Escherichia coli selectively binds a class of outer membrane proteins

A search was performed for a periplasmic molecular chaperone which may assist outer membrane proteins of Escherichia coli on their way from the cytoplasmic to the outer membrane. Proteins of the periplasmic space were fractionated on an affinity column with sepharose-bound outer membrane porin OmpF. A 17kDa polypeptide was the predominant protein retained by this column. The corresponding gene was found in a gene bank; it encodes the periplasmic protein Skp. The protein was isolated and it could be demonstrated that it bound outer membrane proteins, following SDS-PAGE, with high selectivity. Among these were OmpA, OmpC, OmpF and the maltoporin LamB. The chromosomal skp gene was inactivated by a deletion causing removal of most of the signal peptide plus 107 residues of the 141-residue mature protein. The mutant was viable but possessed much-reduced concentrations of outer membrane proteins. This defect was fully restored by a plasmid-borne skp gene which may serve as a periplasmic chaperone.     

Protein phosphatase 2A stabilizes human securin, whose phosphorylated forms are degraded via the SCF ubiquitin ligase

Sister chromatid segregation is triggered at the metaphase-to-anaphase transition by the activation of the protease separase. For most of the cell cycle, separase activity is kept in check by its association with the inhibitory chaperone securin. Activation of separase occurs at anaphase onset, when securin is targeted for destruction by the anaphase-promoting complex or cyclosome E3 ubiquitin protein ligase. This results in the release of the cohesins from chromosomes, which in turn allows the segregation of sister chromatids to opposite spindle poles. Here we show that human securin (hSecurin) forms a complex with enzymatically active protein phosphatase 2A (PP2A) and that it is a substrate of the phosphatase, both in vitro and in vivo. Treatment of cells with okadaic acid, a potent inhibitor of PP2A, results in various hyperphosphorylated forms of hSecurin which are extremely unstable, due to the action of the Skp1/Cul1/F-box protein complex ubiquitin ligase. We propose that PP2A regulates hSecurin levels by counteracting its phosphorylation, which promotes its degradation. Misregulation of this process may lead to the formation of tumors, in which overproduction of hSecurin is often observed.     

Crystal structure and mutagenesis of the metallochaperone MeaB: insight into the causes of methylmalonic aciduria

MeaB is an auxiliary protein that plays a crucial role in the protection and assembly of the B(12)-dependent enzyme methylmalonyl-CoA mutase. Impairments in the human homologue of MeaB, MMAA, lead to methylmalonic aciduria, an inborn error of metabolism. To explore the role of this metallochaperone, its structure was solved in the nucleotide-free form, as well as in the presence of product, GDP. MeaB is a homodimer, with each subunit containing a central alpha/beta-core G domain that is typical of the GTPase family, as well as alpha-helical extensions at the N and C termini that are not found in other metalloenzyme chaperone GTPases. The C-terminal extension appears to be essential for nucleotide-independent dimerization, and the N-terminal region is implicated in protein-protein interaction with its partner protein, methylmalonyl-CoA mutase. The structure of MeaB confirms that it is a member of the G3E family of P-loop GTPases, which contains other putative metallochaperones HypB, CooC, and UreG. Interestingly, the so-called switch regions, responsible for signal transduction following GTP hydrolysis, are found at the dimer interface of MeaB instead of being positioned at the surface of the protein where its partner protein methylmalonyl-CoA mutase should bind. This observation suggests a large conformation change of MeaB must occur between the GDP- and GTP-bound forms of this protein. Because of their high sequence homology, the missense mutations in MMAA that result in methylmalonic aciduria have been mapped onto MeaB and, in conjunction with mutagenesis data, provide possible explanations for the pathology of this disease.     

Evolutionary Conservation in Biogenesis of β-Barrel Proteins Allows Mitochondria to Assemble a Functional Bacterial Trimeric Autotransporter Protein

Yersinia adhesin A (YadA) belongs to a class of bacterial adhesins that form trimeric structures. Their mature form contains a passenger domain and a C-terminal β-domain that anchors the protein in the outer membrane (OM). Little is known about how precursors of such proteins cross the periplasm and assemble into the OM. In the present study we took advantage of the evolutionary conservation in the biogenesis of β-barrel proteins between bacteria and mitochondria. We previously observed that upon expression in yeast cells, bacterial β-barrel proteins including the transmembrane domain of YadA assemble into the mitochondrial OM. In the current study we found that when expressed in yeast cells both the monomeric and trimeric forms of full-length YadA were detected in mitochondria but only the trimeric species was fully integrated into the OM. The oligomeric form was exposed on the surface of the organelle in its native conformation and maintained its capacity to adhere to host cells. The co-expression of YadA with a mitochondria-targeted form of the bacterial periplasmic chaperone Skp, but not with SurA or SecB, resulted in enhanced levels of both forms of YadA. Taken together, these results indicate that the proper assembly of trimeric autotransporter can occur also in a system lacking the lipoproteins of the BAM machinery and is specifically enhanced by the chaperone Skp.