Held at the University of Hertfordshire, U.K., 11 - 13 September 2014.
Periplasmic quality control in biogenesis of outer membrane proteins Zhi Xin Lyu* and Xin Sheng Zhao*1 *Beijing National Laboratory for Molecular Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Department of Chemical Biology, College of Chemistry and Molecular Engineering, and Biodynamic Optical Imaging Center (BIOPIC), Peking University, Beijing 100871, China
Abstract The β-barrel outer membrane proteins (OMPs) are integral membrane proteins that reside in the outer membrane of Gram-negative bacteria and perform a diverse range of biological functions. Synthesized in the cytoplasm, OMPs must be transported across the inner membrane and through the periplasmic space before they are assembled in the outer membrane. In Escherichia coli, Skp, SurA and DegP are the most prominent factors identified to guide OMPs across the periplasm and to play the role of quality control. Although extensive genetic and biochemical analyses have revealed many basic functions of these periplasmic proteins, the mechanism of their collaboration in assisting the folding and insertion of OMPs is much less understood. Recently, biophysical approaches have shed light on the identification of the intricate network. In the present review, we summarize recent advances in the characterization of these key factors, with a special emphasis on the multifunctional protein DegP. In addition, we present our proposed model on the periplasmic quality control in biogenesis of OMPs.
Introduction The cell envelope of Gram-negative bacteria consists of the inner membrane (IM), the outer membrane (OM) and the periplasm between them. As the first line of contact between bacterium and its external environment, the OM functions as a selective barrier that prevents the entry of many toxic molecules into the cell while simultaneously allows for the entry of nutrients required for cell survival. The β-barrel outer membrane proteins (OMPs) are integral membrane proteins that reside in the OM of bacteria with the β-barrel scaffolds. Despite the structural simplicity, OMPs have evolved to perform a plethora of biological functions including acting as adhesins, porins, transporters, receptors and enzymes [1]. OMPs are synthesized in the cytoplasm as precursors with an N-terminal signal sequence that directs OMPs Key words: outer membrane protein, periplasm, protease, chaperone, single molecule detection, FRET. Abbreviations: BAM, β-barrel assembly machinery; IM, inner membrane; OM, outer membrane; OMPs, outer membrane proteins; smFRET, single-molecule fluorescence resonance energy transfer. 1 To whom correspondence should be addressed (email [email protected]).
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through the Sec machinery in the IM. The molecular details of protein translocation across the IM are relatively well studied and have been reviewed [2]. After translocation and signal sequence cleavage, the newly exported proteins must be transported through the periplasmic space before reaching their final destination [3]. It is quite challenging that OMPs with hydrophobic nature go across the hydrophilic periplasm devoid of obvious energy source such as ATP. In Escherichia coli, Skp, SurA and DegP are the most prominent factors identified to guide OMPs across the periplasm and to play the role of quality control during such process [4]. High-resolution crystal structures are available for these periplasmic factors, providing a structural basis for understanding their functions. In addition, extensive genetic and biochemical analyses have revealed many underlying properties of these proteins. However, how they collaborate to safeguard OMPs across the periplasmic space is much less understood. Recently, biophysical approaches have shed light on the identification of the intricate network. The present review will demonstrate the progress made in the identification of interactive network through genetic, biochemical and biophysical approaches. C The
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Periplasmic quality control factors Given the great challenges for OMPs to pass through the periplasm, it is unsurprising that a range of periplasmic proteins function to facilitate the transportation and play the role of protein quality control. These proteins include proteases, molecular chaperones and folding catalysts [4], of which Skp, SurA and DegP are the most important ones in Escherichia coli.
Skp Skp is initially identified as a chaperone that selectively binds a class of OMPs in the bacterial periplasm [5]. Skp can prevent aggregation of its substrates by forming soluble periplasmic intermediate with dissociation constants in the nanomolar range [6]. The crystal structure of the trimeric Skp resembles a ‘jellyfish’ with the tentacles forming a cavity, which serves as a potential binding site for the unfolded substrate proteins [7]. NMR spectroscopy demonstrated that the β-barrel domain of OmpA is bound within the Skp cavity in an unfolded state, whereas the periplasmic domain folds independently outside the cavity [8]. Skp selectively binds the nascent OMPs at an early stage while they are still in contact with the Sec machinery [9]. Both the stopped-flow kinetics experiments and molecular dynamics simulations showed that the N-terminus of OMP enters the Skp cavity first [10], suggesting that Skp may interact with the N-terminal residues of the client proteins as they emerge from the translocon, and hence facilitates the translocation of the unfolded proteins across the IM. Conformational analysis of Skp-bound OmpX revealed that in the absence of a specific binding motif, the highaffinity interactions between Skp and OMPs arise from the formation of a range of simultaneous weak interactions that exist between the chaperone and its substrates [11]. In addition, the fast backbone dynamics of the Skp–OMP complex also provide a rationale for the OMP polypeptide transport towards downstream folding, which is in good agreement with current thermodynamic investigations [12]. The transient nature of the multitude of weak, local interactions has been hypothesized to facilitate handover of substrates from Skp to the β-barrel assembly machinery (BAM) complex [11]. However, there is no evidence that Skp has any direct interactions with BAM based on previous crosslinking assays [13].
SurA SurA is the primary periplasmic molecular chaperone that facilitates the folding and assembling of many OMPs in Gram-negative bacteria [14]. The SurA molecule consists of the N- and C-terminal parts connected by two peptidylprolyl isomerase (PPIase) domains, which are yet dispensable for its chaperone activity [15]. Crystal structure shows a core module formed by the N- and C-terminal segments and the first PPIase (P1) domain, with the other PPIase (P2) domain extending away from this core [16]. SurA tolerates a vast diversity of sequence compositions at its C The
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C-terminal segment, suggesting that the functional role of the C-terminal domain is more likely for structural support rather than substrate recognition and interactions [17]. Consistent with its proposed OMP-specific chaperone activity, SurA has been shown to bind to model peptides and unfolded OMPs with low micromolar affinity and prefer substrates rich in aromatic residues arranged in alternating sequences (Ar-X-Ar), a motif that is regularly present in OMPs [18]. However, such peptides are subsequently found to be recognized by the P1 domain of SurA, which is known to be dispensable for chaperone function, but not with the N-terminal segment suggested earlier [19]. This discrepancy can be explained if substrate selection and chaperone activity reside in different parts of SurA. Kinetic analysis of LamB assembly indicated that SurA binds with LamB prior to signal sequence cleavage, suggesting that SurA may interact with unfolded OMPs while they are translocating across the IM [20], as is the case for Skp. Additionally, SurA has been shown to be the only periplasmic folding factor that successfully cross-linked to BamA in vivo [13]. Together with genetic evidence, it has led to the hypothesis that SurA can escort OMP intermediates from one side of the periplasmic space to the other side [13], although there is still much to learn about the function of this protein at both cellular and molecular levels.
DegP DegP has received considerable attention as a quality control factor that protects cell from protein folding stress by its dual proteolytic and chaperone activities. Temperature changes were initially thought to cause the switch between the two activities of DegP, with the chaperone activity dominating at 28 ◦ C and the protease activity becoming dominant at 42 ◦ C [21]. Several in vivo substrates for DegP protease activity have been identified to be unfolded and mislocalized proteins that are improperly folded upon overproduction [22]. As a protease, DegP is regarded to degrade irreversibly misfolded proteins in the periplasm, thus reducing damage in the extracytoplasmic compartments under cellular stress [23]. Although the function of DegP as a protease is well documented [24], the physiological role of its chaperone activity is less clear. It has been shown that a proteasedeficient DegP protein can complement the surA-degP synthetic lethality at temperatures up to 37 ◦ C [25], which implies that the loss of the chaperone activity but not the protease activity of DegP is the cause of the synthetically lethal surA-degP phenotype. Each subunit of DegP contains a trypsin-like protease domain and two PDZ domains, which have been shown to be necessary for oligomer stabilization and substrate binding [26]. With a trimer as the fundamental structural unit, DegP exists in the form of various multimers, which are in dynamic equilibrium and perform specific functions to control the correct assembly of proteins in the cell. The crystal structure of DegP shows the protein to be arranged in an inactive hexamer composed of two staggered trimers [27]. During activation, the homohexameric resting state of Biochem. Soc. Trans. (2015) 43, 134–138; doi:10.1042/BST20140217
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Figure 1 Intramolecular smFRET of double-labelled uOmpC interacting with the quality control factors The smFRET efficiency distributions when double-labelled uOmpC was (A) in the PBS buffer, where uOmpC adopted a packed conformation; (B) in the presence of Skp, where uOmpC exhibited extended conformation; (C) in the presence of DegP(wt), where uOmpC was thoroughly degraded; (D) in the presence of Skp followed by addition of DegP(wt), where uOmpC was not degraded and kept the extended conformation. smFRET measurements were made as described previously [33]. Site-specific cysteine mutant of OmpC (OmpC-G8C/N336C) was double-labelled with AF555 and AF647. uOmpC was stocked in 8 M urea to keep it from aggregation, and was progressively diluted with buffer to less than 50 pM for smFRET measurements. The final concentrations of Skp and DegP(wt) were 0.2 and 5 μM, respectively. Estimated by using previously reported dissociation constants of uOmpC-Skp and uOmpC-DegP, more than 95 % of uOmpC should be transferred to DegP from Skp in Figure 2D. Notice the slight shift of the peak of the FRET efficiency from 0.23 (bound to Skp) in Figure 1B to 0.28 (bound to DegP) in Figure 1D. The peak around 0.1 is the background [33] (Lyu and Zhao, unpublished work).
DegP is converted into the large, cage-like 12-meric and 24-meric complexes. Multiple oligomers, including the 12mer, the 15-mer and the 18-mer, have been found to form bowl-shaped structures on the lipid interface as well [28]. Recent native mass spectrometry data indicated that the structural transition of DegP is initiated by the binding of the substrate to the PDZ1 domain, including a conformational change of the PDZ domains, followed by the dissociation from hexamer to trimer. The substrate-bound trimers then associate rapidly into large, cage-like structures through the transitory bowl-shaped oligomers such as the 9-mer, the 12mer, the 15-mer and the 18-mer [29]. DegP is a highly efficient, relatively nonspecific protease that binds and cleaves exposed hydrophobic residues. The likely reason for the many different oligomeric states of DegP is proposed to provide functional control of proteolytic activity. Recently, a cage-deficient mutant of DegP, which can only associate into trimers, was shown to be able to bind and degrade substrates without the need to form higher order oligomers [30]. Additionally, combining a mutation
that stabilizes active DegP with one that prevents cage formation converts the protein into a lethal rogue protease, which degrades certain critical periplasmic proteins with its excessive proteolytic ability [31]. These observations implies that the cage assembly is utilized to modulate proteolysis of active and inactive states via sequestering the active sites of DegP within the cage [31]. Alternatively, based on the finding that OMPs co-purify with cages of protease-deficient DegP [32], it is more reasonable to surmise that the cages of wild-type DegP function as chaperones to harbour the folded OMPs in their cavities. In summary, whether DegP possesses chaperone activity is still a critical issue yet to be resolved.
Model for periplasmic quality control in OMP biogenesis Little is known about how SurA, Skp and DegP in Escherichia coli work together within the networks of protein folding, repair and degradation. None of the three periplasmic C The
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Figure 2 Model for periplasmic quality control in biogenesis of OMPs in Escherichia coli OMPs are synthesized in the cytoplasm and transported across the IM through the Sec machinery. OMPs are then safeguarded by SurA/Skp or form aggregation in the periplasm without protection. OMPs can fall off from the OMP-chaperone complexes and become unprotected (dashed lines). DegP functions subsequently after SurA/Skp, either to safeguard the pretreated OMPs for further folding and assembly, or to degrade the misfolded OMPs, depending on whether the OMPs have been properly protected by SurA/Skp or not. DegP also captures the unprotected OMPs and degrades them. OMPs are assembled into the OM by BAM eventually. Meanwhile, SurA may safeguard OMPs to BAM independently.
factors is irreplaceable, since mutants in which these genes are singly inactive remain viable. However, the double deletion of surA-skp or surA-degP forms synthetic lethal phenotypes, in contrast to the skp-degP double deletion that can survive under normal temperatures [13]. Based on these genetic experiments, the existence of two parallel pathways of chaperone activity in the periplasm has been suggested: the SurA pathway transports most OMPs across the periplasm, whereas the Skp/DegP pathway rescues substrates that fall off the SurA pathway [13]. However, there are some doubts about the validity of this model. Firstly, DegP was shown to interact not only with Skp, but also with SurA [33]. Secondly, the fact that DegP is proved to be essential for survival of Escherichia coli at elevated-temperatures is inconsistent with the concept in above parallel pathways [34]. Alternative models have been proposed. For instance, it has been suggested that Skp and SurA cooperate sequentially in the same pathway: C The
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Skp interacts first with unfolded OMPs as they enter the periplasm, and SurA is involved in a later stage, assisting the folding of OMPs when they arrive at BAM [35]. Consistent with the latter model, in vivo site-specific photocrosslinking method was recently used to observe a sequential interaction of Skp and SurA/BamA with EspP (one OMP in Escherichia coli) during its assembly [36]. To answer fundamental questions about the role of these quality control factors, use of novel experimental approaches is desirable. Our group has recently characterized the kinetic and thermodynamic properties of interactions between unfolded OMPs and the three factors using the technique of single-molecule detection [33]. An unfolded OmpC (uOmpC) was shown to bind to Skp and SurA at a rate of 1000-fold faster than its binding to DegP. On the other hand, uOmpC–DegP complex is more stable than uOmpC–Skp and uOmpC–SurA. The kinetic preference of uOmpC binding to SurA and Skp and the thermodynamic
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advantage of uOmpC binding to DegP together make sure that uOmpC is captured by SurA or Skp first and then transferred to DegP. Moreover, results of three colour singlemolecule fluorescence resonance energy transfer (smFRET) experiment showed that DegP can form ternary complexes with uOmpC–Skp or uOmpC–SurA. The conformation of uOmpC directly bound to DegP differs from that of uOmpC pretreated by SurA or Skp, indicating that the induced conformational changes may allow uOmpC to avoid being acted upon by the protease activity of DegP. Our latest intramolecular smFRET experiments demonstrated that uOmpC adopts a packed conformation in a buffer but it becomes extended when bound to Skp or SurA, respectively. The packed uOmpC is degraded by DegP, whereas the uOmpC molecule pretreated by Skp or SurA remains intact after it binds to DegP (Figure 1), confirming the speculation that in such a case DegP has the chaperone-like activity [33]. Based on our study and previous results, we proposed an improved model for the periplasmic quality control in OMPs biogenesis (Figure 2). Previous biochemical assays suggested that Skp and SurA, rather than DegP, may interact with unfolded OMPs while they are translocating across the IM [9,20]. Our biophysical studies observed the kinetic preference of uOmpC binding to SurA and Skp and the thermodynamic advantage of uOmpC binding to DegP [33]. Combined with the existence of the SurA–OMP–DegP and Skp–OMP–DegP ternary complexes, we suppose that the three factors interact with the OMPs in a sequential fashion. SurA and Skp chaperones act as holding factors at an early stage of translocation, when the newly synthesized unfolded protein is secreted into the periplasm. Then, OMPs captured by SurA or Skp is transferred to DegP in a handover-hand manner, without being released into solution. DegP, as a bifunctional protease–chaperone, either safeguards the pretreated OMPs for further folding and assembly, or degrades the misfolded OMPs, depending on whether the OMPs have been properly protected by SurA/Skp or not. On the other hand, unprotected OMP molecules in the periplasm either coming from the Sec machinery or falling off from the OMP–chaperone complexes will be purged by DegP degradation. Meanwhile, as is proposed previously SurA forms an independent pathway in view of the double mutants [13]. Because the bacterial periplasm lacks obvious energy source such as ATP, it is interesting to understand the driving force for sorting OMPs across the periplasmic place to the OM. Recent thermodynamic investigations suggested that the large free energy release of OMPs folding provides an ‘energy sink’ that drives the release of these client proteins from chaperones and enables their subsequent transportation and assembly in the OM [12]. Although the thermodynamic stability of DegP–OMP complex is more favorable than that of SurA–OMP and Skp–OMP complexes, the OMP folding free energies are still much more favorable than any of these known chaperone–OMP interactions [12]. Our sequentialtransportation model for OMPs biogenesis is in excellent agreement with the ‘energy sink’ theory.
Conclusions and perspectives In the past few years, much progress has been made in the field of OMPs’ biogenesis with the identification of many new components involved in the process. Our review only touches a part of the networks. Apart from Skp, SurA and DegP, other periplasmic folding factors such as FkpA, PpiA, PpiD, DsbAand DsbC are involved in OMP assembly as well [37]. Several proteins combine various biochemical activities, leading to apparent functional redundancy in the periplasmic compartment. This fact reflects the importance and complexity of OMP biogenesis. Still, fundamental questions about the role of these periplasmic folding factors in OMP assembly remain elusive due to limited understanding of the networks. Addressing such conundrum requires the combination of diverse experimental and computational methods to overcome the inherent difficulties associated with membrane proteins. Currently, constructive mechanistic models proposed to comprehend chaperone-assisted OMP folding have benefited greatly from genetic and biochemical studies, and will increasingly get help from the biophysical approaches.
Funding We thank NKBRSF [grants numbers 2012CB917304 and 2010CB912302] and NSFC [grant number 21233002] for the financial support.
References 1 Fairman, J.W., Noinaj, N. and Buchanan, S.K. (2011) The structural biology of beta-barrel membrane proteins: a summary of recent reports. Curr. Opin. Struct. Biol. 21, 523–531 CrossRef PubMed 2 Driessen, A.J. and Nouwen, N. (2008) Protein translocation across the bacterial cytoplasmic membrane. Annu. Rev. Biochem. 77, 643–667 CrossRef PubMed 3 Ruiz, N., Kahne, D. and Silhavy, T.J. (2006) Advances in understanding bacterial outer-membrane biogenesis. Nat. Rev. Microbiol. 4, 57–66 CrossRef PubMed 4 Mogensen, J.E. and Otzen, D.E. (2005) Interactions between folding factors and bacterial outer membrane proteins. Mol. Microbiol. 57, 326–346 CrossRef PubMed 5 Chen, R. and Henning, U. (1996) A periplasmic protein (Skp) of Escherichia coli selectively binds a class of outer membrane proteins. Mol. Microbiol. 19, 1287–1294 CrossRef PubMed 6 Qu, J., Mayer, C., Behrens, S., Holst, O. and Kleinschmidt, J.H. (2007) The trimeric periplasmic chaperone Skp of Escherichia coli forms 1:1 complexes with outer membrane proteins via hydrophobic and electrostatic interactions. J. Mol. Biol. 374, 91–105 CrossRef PubMed 7 Korndorfer, I.P., Dommel, M.K. and Skerra, A. (2004) Structure of the periplasmic chaperone Skp suggests functional similarity with cytosolic chaperones despite differing architecture. Nat. Struct. Mol. Biol. 11, 1015–1020 CrossRef PubMed 8 Walton, T.A., Sandoval, C.M., Fowler, C.A., Pardi, A. and Sousa, M.C. (2009) The cavity-chaperone Skp protects its substrate from aggregation but allows independent folding of substrate domains. Proc. Nat. Acad. Sci. U.S.A. 106, 1772–1777 CrossRef 9 Harms, N., Koningstein, G., Dontje, W., Muller, M., Oudega, B., Luirink, J. and de Cock, H. (2001) The early interaction of the outer membrane protein PhoE with the periplasmic chaperone Skp occurs at the cytoplasmic membrane. J. Biol. Chem. 276, 18804–18811 CrossRef PubMed 10 Lyu, Z.X., Shao, Q., Gao, Y.Q. and Zhao, X.S. (2012) Direct observation of the uptake of outer membrane proteins by the periplasmic chaperone Skp. Plos ONE 7, e46068 CrossRef PubMed C The
C 2015 Biochemical Society Authors Journal compilation
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11 Burmann, B.M., Wang, C. and Hiller, S. (2013) Conformation and dynamics of the periplasmic membrane-protein-chaperone complexes OmpX-Skp and tOmpA-Skp. Nat. Struct. Mol. Biol. 20, 1265–1272 CrossRef PubMed 12 Moon, C.P., Zaccai, N.R., Fleming, P.J., Gessmann, D. and Fleming, K.G. (2013) Membrane protein thermodynamic stability may serve as the energy sink for sorting in the periplasm. Proc. Nat. Acad. Sci. U.S.A. 110, 4285–4290 CrossRef 13 Sklar, J.G., Wu, T., Kahne, D. and Silhavy, T.J. (2007) Defining the roles of the periplasmic chaperones SurA, Skp, and DegP in Escherichia coli. Gene Dev. 21, 2473–2484 CrossRef 14 Rouviere, P.E. and Gross, C.A. (1996) SurA, a periplasmic protein with peptidyl-prolyl isomerase activity, participates in the assembly of outer membrane porins. Gene Dev. 10, 3170–3182 CrossRef 15 Behrens, S., Maier, R., de Cock, H., Schmid, F.X. and Gross, C.A. (2001) The SurA periplasmic PPIase lacking its parvulin domains functions in vivo and has chaperone activity. EMBO J. 20, 285–294 CrossRef PubMed 16 Bitto, E. and McKay, D.B. (2002) Crystallographic structure of SurA, a molecular chaperone that facilitates folding of outer membrane porins. Structure 10, 1489–1498 CrossRef PubMed 17 Chai, Q., Ferrell, B., Zhong, M., Zhang, X., Ye, C. and Wei, Y. (2014) Diverse sequences are functional at the C-terminus of the E. coli periplasmic chaperone SurA. Protein Eng. Des. Sel. 27, 111–116 CrossRef PubMed 18 Bitto, E. and McKay, D.B. (2003) The periplasmic molecular chaperone protein SurA binds a peptide motif that is characteristic of integral outer membrane proteins. J. Biol. Chem. 278, 49316–49322 CrossRef PubMed 19 Xu, X., Wang, S., Hu, Y.X. and McKay, D.B. (2007) The periplasmic bacterial molecular chaperone SurA adapts its structure to bind peptides in different conformations to assert a sequence preference for aromatic residues. J. Mol. Biol. 373, 367–381 CrossRef PubMed 20 Ureta, A.R., Endres, R.G., Wingreen, N.S. and Silhavy, T.J. (2007) Kinetic analysis of the assembly of the outer membrane protein LamB in Escherichia coli mutants each lacking a secretion or targeting factor in a different cellular compartment. J. Bacteriol. 189, 446–454 CrossRef PubMed 21 Spiess, C., Beil, A. and Ehrmann, M. (1999) A temperature-dependent switch from chaperone to protease in a widely conserved heat shock protein. Cell 97, 339–347 CrossRef PubMed 22 Clausen, T., Southan, C. and Ehrmann, M. (2002) The HtrA family of proteases: implications for protein composition and cell fate. Mol. Cell 10, 443–455 CrossRef PubMed 23 Clausen, T., Kaiser, M., Huber, R. and Ehrmann, M. (2011) HTRA proteases: regulated proteolysis in protein quality control. Nat. Rev. Mol. Cell Biol. 12, 152–162 CrossRef PubMed 24 Ge, X., Wang, R., Ma, J., Liu, Y., Ezemaduka, A.N., Chen, P.R., Fu, X. and Chang, Z. (2014) DegP primarily functions as a protease for the biogenesis of beta-barrel outer membrane proteins in the Gram-negative bacterium Escherichia coli. FEBS J. 281, 1226–1240 CrossRef 25 Rizzitello, A.E., Harper, J.R. and Silhavy, T.J. (2001) Genetic evidence for parallel pathways of chaperone activity in the periplasm of Escherichia coli. J. Bacteriol. 183, 6794–6800 CrossRef PubMed
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C 2015 Biochemical Society Authors Journal compilation
26 Jiang, J., Zhang, X., Chen, Y., Wu, Y., Zhou, Z.H., Chang, Z. and Sui, S.F. (2008) Activation of DegP chaperone-protease via formation of large cage-like oligomers upon binding to substrate proteins. Proc. Nat. Acad. Sci. U.S.A. 105, 11939–11944 CrossRef 27 Krojer, T., Garrido-Franco, M., Huber, R., Ehrmann, M. and Clausen, T. (2002) Crystal structure of DegP (HtrA) reveals a new protease-chaperone machine. Nature 416, 455–459 CrossRef PubMed 28 Shen, Q.T., Bai, X.C., Chang, L.F., Wu, Y., Wang, H.W. and Sui, S.F. (2009) Bowl-shaped oligomeric structures on membranes as DegP’s new functional forms in protein quality control. Proc. Nat. Acad. Sci. U.S.A. 106, 4858–4863 CrossRef 29 Thompson, N.J., Merdanovic, M., Ehrmann, M., van Duijn, E. and Heck, A.J. (2014) Substrate occupancy at the onset of oligomeric transitions of DegP. Structure 22, 281–290 CrossRef PubMed 30 Kim, S. and Sauer, R.T. (2012) Cage assembly of DegP protease is not required for substrate-dependent regulation of proteolytic activity or high-temperature cell survival. Proc. Nat. Acad. Sci. U.S.A. 109, 7263–7268 CrossRef 31 Kim, S. and Sauer, R.T. (2014) Distinct regulatory mechanisms balance DegP proteolysis to maintain cellular fitness during heat stress. Gene Dev. 28, 902–911 CrossRef 32 Krojer, T., Sawa, J., Schafer, E., Saibil, H.R., Ehrmann, M. and Clausen, T. (2008) Structural basis for the regulated protease and chaperone function of DegP. Nature 453, 885–890 CrossRef PubMed 33 Wu, S., Ge, X., Lv, Z., Zhi, Z., Chang, Z. and Zhao, X.S. (2011) Interaction between bacterial outer membrane proteins and periplasmic quality control factors: a kinetic partitioning mechanism. Biochem. J. 438, 505–511 CrossRef PubMed 34 Strauch, K.L., Johnson, K. and Beckwith, J. (1989) Characterization of degP, a gene required for proteolysis in the cell envelope and essential for growth of Escherichia coli at high temperature. J. Bacteriol. 171, 2689–2696 PubMed 35 Walther, D.M., Rapaport, D. and Tommassen, J. (2009) Biogenesis of beta-barrel membrane proteins in bacteria and eukaryotes: evolutionary conservation and divergence. Cell. Mol. Life Sci. 66, 2789–2804 CrossRef PubMed 36 Pavlova, O., Peterson, J.H., Ieva, R. and Bernstein, H.D. (2013) Mechanistic link between beta barrel assembly and the initiation of autotransporter secretion. Proc. Nat. Acad. Sci. U.S.A. 110, 938–947 CrossRef 37 Merdanovic, M., Clausen, T., Kaiser, M., Huber, R. and Ehrmann, M. (2011) Protein quality control in the bacterial periplasm. Annu. Rev. Microbiol. 65, 149–168 CrossRef PubMed
Received 6 August 2014 doi:10.1042/BST20140217
Periplasmic quality control in biogenesis of outer membrane proteins Zhi Xin Lyu* and Xin Sheng Zhao*1 *Beijing National Laboratory for Molecular Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Department of Chemical Biology, College of Chemistry and Molecular Engineering, and Biodynamic Optical Imaging Center (BIOPIC), Peking University, Beijing 100871, China
Abstract The β-barrel outer membrane proteins (OMPs) are integral membrane proteins that reside in the outer membrane of Gram-negative bacteria and perform a diverse range of biological functions. Synthesized in the cytoplasm, OMPs must be transported across the inner membrane and through the periplasmic space before they are assembled in the outer membrane. In Escherichia coli, Skp, SurA and DegP are the most prominent factors identified to guide OMPs across the periplasm and to play the role of quality control. Although extensive genetic and biochemical analyses have revealed many basic functions of these periplasmic proteins, the mechanism of their collaboration in assisting the folding and insertion of OMPs is much less understood. Recently, biophysical approaches have shed light on the identification of the intricate network. In the present review, we summarize recent advances in the characterization of these key factors, with a special emphasis on the multifunctional protein DegP. In addition, we present our proposed model on the periplasmic quality control in biogenesis of OMPs.
Introduction The cell envelope of Gram-negative bacteria consists of the inner membrane (IM), the outer membrane (OM) and the periplasm between them. As the first line of contact between bacterium and its external environment, the OM functions as a selective barrier that prevents the entry of many toxic molecules into the cell while simultaneously allows for the entry of nutrients required for cell survival. The β-barrel outer membrane proteins (OMPs) are integral membrane proteins that reside in the OM of bacteria with the β-barrel scaffolds. Despite the structural simplicity, OMPs have evolved to perform a plethora of biological functions including acting as adhesins, porins, transporters, receptors and enzymes [1]. OMPs are synthesized in the cytoplasm as precursors with an N-terminal signal sequence that directs OMPs Key words: outer membrane protein, periplasm, protease, chaperone, single molecule detection, FRET. Abbreviations: BAM, β-barrel assembly machinery; IM, inner membrane; OM, outer membrane; OMPs, outer membrane proteins; smFRET, single-molecule fluorescence resonance energy transfer. 1 To whom correspondence should be addressed (email [email protected]).
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through the Sec machinery in the IM. The molecular details of protein translocation across the IM are relatively well studied and have been reviewed [2]. After translocation and signal sequence cleavage, the newly exported proteins must be transported through the periplasmic space before reaching their final destination [3]. It is quite challenging that OMPs with hydrophobic nature go across the hydrophilic periplasm devoid of obvious energy source such as ATP. In Escherichia coli, Skp, SurA and DegP are the most prominent factors identified to guide OMPs across the periplasm and to play the role of quality control during such process [4]. High-resolution crystal structures are available for these periplasmic factors, providing a structural basis for understanding their functions. In addition, extensive genetic and biochemical analyses have revealed many underlying properties of these proteins. However, how they collaborate to safeguard OMPs across the periplasmic space is much less understood. Recently, biophysical approaches have shed light on the identification of the intricate network. The present review will demonstrate the progress made in the identification of interactive network through genetic, biochemical and biophysical approaches. C The
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Periplasmic quality control factors Given the great challenges for OMPs to pass through the periplasm, it is unsurprising that a range of periplasmic proteins function to facilitate the transportation and play the role of protein quality control. These proteins include proteases, molecular chaperones and folding catalysts [4], of which Skp, SurA and DegP are the most important ones in Escherichia coli.
Skp Skp is initially identified as a chaperone that selectively binds a class of OMPs in the bacterial periplasm [5]. Skp can prevent aggregation of its substrates by forming soluble periplasmic intermediate with dissociation constants in the nanomolar range [6]. The crystal structure of the trimeric Skp resembles a ‘jellyfish’ with the tentacles forming a cavity, which serves as a potential binding site for the unfolded substrate proteins [7]. NMR spectroscopy demonstrated that the β-barrel domain of OmpA is bound within the Skp cavity in an unfolded state, whereas the periplasmic domain folds independently outside the cavity [8]. Skp selectively binds the nascent OMPs at an early stage while they are still in contact with the Sec machinery [9]. Both the stopped-flow kinetics experiments and molecular dynamics simulations showed that the N-terminus of OMP enters the Skp cavity first [10], suggesting that Skp may interact with the N-terminal residues of the client proteins as they emerge from the translocon, and hence facilitates the translocation of the unfolded proteins across the IM. Conformational analysis of Skp-bound OmpX revealed that in the absence of a specific binding motif, the highaffinity interactions between Skp and OMPs arise from the formation of a range of simultaneous weak interactions that exist between the chaperone and its substrates [11]. In addition, the fast backbone dynamics of the Skp–OMP complex also provide a rationale for the OMP polypeptide transport towards downstream folding, which is in good agreement with current thermodynamic investigations [12]. The transient nature of the multitude of weak, local interactions has been hypothesized to facilitate handover of substrates from Skp to the β-barrel assembly machinery (BAM) complex [11]. However, there is no evidence that Skp has any direct interactions with BAM based on previous crosslinking assays [13].
SurA SurA is the primary periplasmic molecular chaperone that facilitates the folding and assembling of many OMPs in Gram-negative bacteria [14]. The SurA molecule consists of the N- and C-terminal parts connected by two peptidylprolyl isomerase (PPIase) domains, which are yet dispensable for its chaperone activity [15]. Crystal structure shows a core module formed by the N- and C-terminal segments and the first PPIase (P1) domain, with the other PPIase (P2) domain extending away from this core [16]. SurA tolerates a vast diversity of sequence compositions at its C The
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C-terminal segment, suggesting that the functional role of the C-terminal domain is more likely for structural support rather than substrate recognition and interactions [17]. Consistent with its proposed OMP-specific chaperone activity, SurA has been shown to bind to model peptides and unfolded OMPs with low micromolar affinity and prefer substrates rich in aromatic residues arranged in alternating sequences (Ar-X-Ar), a motif that is regularly present in OMPs [18]. However, such peptides are subsequently found to be recognized by the P1 domain of SurA, which is known to be dispensable for chaperone function, but not with the N-terminal segment suggested earlier [19]. This discrepancy can be explained if substrate selection and chaperone activity reside in different parts of SurA. Kinetic analysis of LamB assembly indicated that SurA binds with LamB prior to signal sequence cleavage, suggesting that SurA may interact with unfolded OMPs while they are translocating across the IM [20], as is the case for Skp. Additionally, SurA has been shown to be the only periplasmic folding factor that successfully cross-linked to BamA in vivo [13]. Together with genetic evidence, it has led to the hypothesis that SurA can escort OMP intermediates from one side of the periplasmic space to the other side [13], although there is still much to learn about the function of this protein at both cellular and molecular levels.
DegP DegP has received considerable attention as a quality control factor that protects cell from protein folding stress by its dual proteolytic and chaperone activities. Temperature changes were initially thought to cause the switch between the two activities of DegP, with the chaperone activity dominating at 28 ◦ C and the protease activity becoming dominant at 42 ◦ C [21]. Several in vivo substrates for DegP protease activity have been identified to be unfolded and mislocalized proteins that are improperly folded upon overproduction [22]. As a protease, DegP is regarded to degrade irreversibly misfolded proteins in the periplasm, thus reducing damage in the extracytoplasmic compartments under cellular stress [23]. Although the function of DegP as a protease is well documented [24], the physiological role of its chaperone activity is less clear. It has been shown that a proteasedeficient DegP protein can complement the surA-degP synthetic lethality at temperatures up to 37 ◦ C [25], which implies that the loss of the chaperone activity but not the protease activity of DegP is the cause of the synthetically lethal surA-degP phenotype. Each subunit of DegP contains a trypsin-like protease domain and two PDZ domains, which have been shown to be necessary for oligomer stabilization and substrate binding [26]. With a trimer as the fundamental structural unit, DegP exists in the form of various multimers, which are in dynamic equilibrium and perform specific functions to control the correct assembly of proteins in the cell. The crystal structure of DegP shows the protein to be arranged in an inactive hexamer composed of two staggered trimers [27]. During activation, the homohexameric resting state of Biochem. Soc. Trans. (2015) 43, 134–138; doi:10.1042/BST20140217
Single Biomo lecules – in silico, in vitro and in vivo
Figure 1 Intramolecular smFRET of double-labelled uOmpC interacting with the quality control factors The smFRET efficiency distributions when double-labelled uOmpC was (A) in the PBS buffer, where uOmpC adopted a packed conformation; (B) in the presence of Skp, where uOmpC exhibited extended conformation; (C) in the presence of DegP(wt), where uOmpC was thoroughly degraded; (D) in the presence of Skp followed by addition of DegP(wt), where uOmpC was not degraded and kept the extended conformation. smFRET measurements were made as described previously [33]. Site-specific cysteine mutant of OmpC (OmpC-G8C/N336C) was double-labelled with AF555 and AF647. uOmpC was stocked in 8 M urea to keep it from aggregation, and was progressively diluted with buffer to less than 50 pM for smFRET measurements. The final concentrations of Skp and DegP(wt) were 0.2 and 5 μM, respectively. Estimated by using previously reported dissociation constants of uOmpC-Skp and uOmpC-DegP, more than 95 % of uOmpC should be transferred to DegP from Skp in Figure 2D. Notice the slight shift of the peak of the FRET efficiency from 0.23 (bound to Skp) in Figure 1B to 0.28 (bound to DegP) in Figure 1D. The peak around 0.1 is the background [33] (Lyu and Zhao, unpublished work).
DegP is converted into the large, cage-like 12-meric and 24-meric complexes. Multiple oligomers, including the 12mer, the 15-mer and the 18-mer, have been found to form bowl-shaped structures on the lipid interface as well [28]. Recent native mass spectrometry data indicated that the structural transition of DegP is initiated by the binding of the substrate to the PDZ1 domain, including a conformational change of the PDZ domains, followed by the dissociation from hexamer to trimer. The substrate-bound trimers then associate rapidly into large, cage-like structures through the transitory bowl-shaped oligomers such as the 9-mer, the 12mer, the 15-mer and the 18-mer [29]. DegP is a highly efficient, relatively nonspecific protease that binds and cleaves exposed hydrophobic residues. The likely reason for the many different oligomeric states of DegP is proposed to provide functional control of proteolytic activity. Recently, a cage-deficient mutant of DegP, which can only associate into trimers, was shown to be able to bind and degrade substrates without the need to form higher order oligomers [30]. Additionally, combining a mutation
that stabilizes active DegP with one that prevents cage formation converts the protein into a lethal rogue protease, which degrades certain critical periplasmic proteins with its excessive proteolytic ability [31]. These observations implies that the cage assembly is utilized to modulate proteolysis of active and inactive states via sequestering the active sites of DegP within the cage [31]. Alternatively, based on the finding that OMPs co-purify with cages of protease-deficient DegP [32], it is more reasonable to surmise that the cages of wild-type DegP function as chaperones to harbour the folded OMPs in their cavities. In summary, whether DegP possesses chaperone activity is still a critical issue yet to be resolved.
Model for periplasmic quality control in OMP biogenesis Little is known about how SurA, Skp and DegP in Escherichia coli work together within the networks of protein folding, repair and degradation. None of the three periplasmic C The
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Figure 2 Model for periplasmic quality control in biogenesis of OMPs in Escherichia coli OMPs are synthesized in the cytoplasm and transported across the IM through the Sec machinery. OMPs are then safeguarded by SurA/Skp or form aggregation in the periplasm without protection. OMPs can fall off from the OMP-chaperone complexes and become unprotected (dashed lines). DegP functions subsequently after SurA/Skp, either to safeguard the pretreated OMPs for further folding and assembly, or to degrade the misfolded OMPs, depending on whether the OMPs have been properly protected by SurA/Skp or not. DegP also captures the unprotected OMPs and degrades them. OMPs are assembled into the OM by BAM eventually. Meanwhile, SurA may safeguard OMPs to BAM independently.
factors is irreplaceable, since mutants in which these genes are singly inactive remain viable. However, the double deletion of surA-skp or surA-degP forms synthetic lethal phenotypes, in contrast to the skp-degP double deletion that can survive under normal temperatures [13]. Based on these genetic experiments, the existence of two parallel pathways of chaperone activity in the periplasm has been suggested: the SurA pathway transports most OMPs across the periplasm, whereas the Skp/DegP pathway rescues substrates that fall off the SurA pathway [13]. However, there are some doubts about the validity of this model. Firstly, DegP was shown to interact not only with Skp, but also with SurA [33]. Secondly, the fact that DegP is proved to be essential for survival of Escherichia coli at elevated-temperatures is inconsistent with the concept in above parallel pathways [34]. Alternative models have been proposed. For instance, it has been suggested that Skp and SurA cooperate sequentially in the same pathway: C The
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Skp interacts first with unfolded OMPs as they enter the periplasm, and SurA is involved in a later stage, assisting the folding of OMPs when they arrive at BAM [35]. Consistent with the latter model, in vivo site-specific photocrosslinking method was recently used to observe a sequential interaction of Skp and SurA/BamA with EspP (one OMP in Escherichia coli) during its assembly [36]. To answer fundamental questions about the role of these quality control factors, use of novel experimental approaches is desirable. Our group has recently characterized the kinetic and thermodynamic properties of interactions between unfolded OMPs and the three factors using the technique of single-molecule detection [33]. An unfolded OmpC (uOmpC) was shown to bind to Skp and SurA at a rate of 1000-fold faster than its binding to DegP. On the other hand, uOmpC–DegP complex is more stable than uOmpC–Skp and uOmpC–SurA. The kinetic preference of uOmpC binding to SurA and Skp and the thermodynamic
Single Biomo lecules – in silico, in vitro and in vivo
advantage of uOmpC binding to DegP together make sure that uOmpC is captured by SurA or Skp first and then transferred to DegP. Moreover, results of three colour singlemolecule fluorescence resonance energy transfer (smFRET) experiment showed that DegP can form ternary complexes with uOmpC–Skp or uOmpC–SurA. The conformation of uOmpC directly bound to DegP differs from that of uOmpC pretreated by SurA or Skp, indicating that the induced conformational changes may allow uOmpC to avoid being acted upon by the protease activity of DegP. Our latest intramolecular smFRET experiments demonstrated that uOmpC adopts a packed conformation in a buffer but it becomes extended when bound to Skp or SurA, respectively. The packed uOmpC is degraded by DegP, whereas the uOmpC molecule pretreated by Skp or SurA remains intact after it binds to DegP (Figure 1), confirming the speculation that in such a case DegP has the chaperone-like activity [33]. Based on our study and previous results, we proposed an improved model for the periplasmic quality control in OMPs biogenesis (Figure 2). Previous biochemical assays suggested that Skp and SurA, rather than DegP, may interact with unfolded OMPs while they are translocating across the IM [9,20]. Our biophysical studies observed the kinetic preference of uOmpC binding to SurA and Skp and the thermodynamic advantage of uOmpC binding to DegP [33]. Combined with the existence of the SurA–OMP–DegP and Skp–OMP–DegP ternary complexes, we suppose that the three factors interact with the OMPs in a sequential fashion. SurA and Skp chaperones act as holding factors at an early stage of translocation, when the newly synthesized unfolded protein is secreted into the periplasm. Then, OMPs captured by SurA or Skp is transferred to DegP in a handover-hand manner, without being released into solution. DegP, as a bifunctional protease–chaperone, either safeguards the pretreated OMPs for further folding and assembly, or degrades the misfolded OMPs, depending on whether the OMPs have been properly protected by SurA/Skp or not. On the other hand, unprotected OMP molecules in the periplasm either coming from the Sec machinery or falling off from the OMP–chaperone complexes will be purged by DegP degradation. Meanwhile, as is proposed previously SurA forms an independent pathway in view of the double mutants [13]. Because the bacterial periplasm lacks obvious energy source such as ATP, it is interesting to understand the driving force for sorting OMPs across the periplasmic place to the OM. Recent thermodynamic investigations suggested that the large free energy release of OMPs folding provides an ‘energy sink’ that drives the release of these client proteins from chaperones and enables their subsequent transportation and assembly in the OM [12]. Although the thermodynamic stability of DegP–OMP complex is more favorable than that of SurA–OMP and Skp–OMP complexes, the OMP folding free energies are still much more favorable than any of these known chaperone–OMP interactions [12]. Our sequentialtransportation model for OMPs biogenesis is in excellent agreement with the ‘energy sink’ theory.
Conclusions and perspectives In the past few years, much progress has been made in the field of OMPs’ biogenesis with the identification of many new components involved in the process. Our review only touches a part of the networks. Apart from Skp, SurA and DegP, other periplasmic folding factors such as FkpA, PpiA, PpiD, DsbAand DsbC are involved in OMP assembly as well [37]. Several proteins combine various biochemical activities, leading to apparent functional redundancy in the periplasmic compartment. This fact reflects the importance and complexity of OMP biogenesis. Still, fundamental questions about the role of these periplasmic folding factors in OMP assembly remain elusive due to limited understanding of the networks. Addressing such conundrum requires the combination of diverse experimental and computational methods to overcome the inherent difficulties associated with membrane proteins. Currently, constructive mechanistic models proposed to comprehend chaperone-assisted OMP folding have benefited greatly from genetic and biochemical studies, and will increasingly get help from the biophysical approaches.
Funding We thank NKBRSF [grants numbers 2012CB917304 and 2010CB912302] and NSFC [grant number 21233002] for the financial support.
References 1 Fairman, J.W., Noinaj, N. and Buchanan, S.K. (2011) The structural biology of beta-barrel membrane proteins: a summary of recent reports. Curr. Opin. Struct. Biol. 21, 523–531 CrossRef PubMed 2 Driessen, A.J. and Nouwen, N. (2008) Protein translocation across the bacterial cytoplasmic membrane. Annu. Rev. Biochem. 77, 643–667 CrossRef PubMed 3 Ruiz, N., Kahne, D. and Silhavy, T.J. (2006) Advances in understanding bacterial outer-membrane biogenesis. Nat. Rev. Microbiol. 4, 57–66 CrossRef PubMed 4 Mogensen, J.E. and Otzen, D.E. (2005) Interactions between folding factors and bacterial outer membrane proteins. Mol. Microbiol. 57, 326–346 CrossRef PubMed 5 Chen, R. and Henning, U. (1996) A periplasmic protein (Skp) of Escherichia coli selectively binds a class of outer membrane proteins. Mol. Microbiol. 19, 1287–1294 CrossRef PubMed 6 Qu, J., Mayer, C., Behrens, S., Holst, O. and Kleinschmidt, J.H. (2007) The trimeric periplasmic chaperone Skp of Escherichia coli forms 1:1 complexes with outer membrane proteins via hydrophobic and electrostatic interactions. J. Mol. Biol. 374, 91–105 CrossRef PubMed 7 Korndorfer, I.P., Dommel, M.K. and Skerra, A. (2004) Structure of the periplasmic chaperone Skp suggests functional similarity with cytosolic chaperones despite differing architecture. Nat. Struct. Mol. Biol. 11, 1015–1020 CrossRef PubMed 8 Walton, T.A., Sandoval, C.M., Fowler, C.A., Pardi, A. and Sousa, M.C. (2009) The cavity-chaperone Skp protects its substrate from aggregation but allows independent folding of substrate domains. Proc. Nat. Acad. Sci. U.S.A. 106, 1772–1777 CrossRef 9 Harms, N., Koningstein, G., Dontje, W., Muller, M., Oudega, B., Luirink, J. and de Cock, H. (2001) The early interaction of the outer membrane protein PhoE with the periplasmic chaperone Skp occurs at the cytoplasmic membrane. J. Biol. Chem. 276, 18804–18811 CrossRef PubMed 10 Lyu, Z.X., Shao, Q., Gao, Y.Q. and Zhao, X.S. (2012) Direct observation of the uptake of outer membrane proteins by the periplasmic chaperone Skp. Plos ONE 7, e46068 CrossRef PubMed C The
C 2015 Biochemical Society Authors Journal compilation
137
138
Biochemical Society Transactions (2015) Volume 43, part 2
11 Burmann, B.M., Wang, C. and Hiller, S. (2013) Conformation and dynamics of the periplasmic membrane-protein-chaperone complexes OmpX-Skp and tOmpA-Skp. Nat. Struct. Mol. Biol. 20, 1265–1272 CrossRef PubMed 12 Moon, C.P., Zaccai, N.R., Fleming, P.J., Gessmann, D. and Fleming, K.G. (2013) Membrane protein thermodynamic stability may serve as the energy sink for sorting in the periplasm. Proc. Nat. Acad. Sci. U.S.A. 110, 4285–4290 CrossRef 13 Sklar, J.G., Wu, T., Kahne, D. and Silhavy, T.J. (2007) Defining the roles of the periplasmic chaperones SurA, Skp, and DegP in Escherichia coli. Gene Dev. 21, 2473–2484 CrossRef 14 Rouviere, P.E. and Gross, C.A. (1996) SurA, a periplasmic protein with peptidyl-prolyl isomerase activity, participates in the assembly of outer membrane porins. Gene Dev. 10, 3170–3182 CrossRef 15 Behrens, S., Maier, R., de Cock, H., Schmid, F.X. and Gross, C.A. (2001) The SurA periplasmic PPIase lacking its parvulin domains functions in vivo and has chaperone activity. EMBO J. 20, 285–294 CrossRef PubMed 16 Bitto, E. and McKay, D.B. (2002) Crystallographic structure of SurA, a molecular chaperone that facilitates folding of outer membrane porins. Structure 10, 1489–1498 CrossRef PubMed 17 Chai, Q., Ferrell, B., Zhong, M., Zhang, X., Ye, C. and Wei, Y. (2014) Diverse sequences are functional at the C-terminus of the E. coli periplasmic chaperone SurA. Protein Eng. Des. Sel. 27, 111–116 CrossRef PubMed 18 Bitto, E. and McKay, D.B. (2003) The periplasmic molecular chaperone protein SurA binds a peptide motif that is characteristic of integral outer membrane proteins. J. Biol. Chem. 278, 49316–49322 CrossRef PubMed 19 Xu, X., Wang, S., Hu, Y.X. and McKay, D.B. (2007) The periplasmic bacterial molecular chaperone SurA adapts its structure to bind peptides in different conformations to assert a sequence preference for aromatic residues. J. Mol. Biol. 373, 367–381 CrossRef PubMed 20 Ureta, A.R., Endres, R.G., Wingreen, N.S. and Silhavy, T.J. (2007) Kinetic analysis of the assembly of the outer membrane protein LamB in Escherichia coli mutants each lacking a secretion or targeting factor in a different cellular compartment. J. Bacteriol. 189, 446–454 CrossRef PubMed 21 Spiess, C., Beil, A. and Ehrmann, M. (1999) A temperature-dependent switch from chaperone to protease in a widely conserved heat shock protein. Cell 97, 339–347 CrossRef PubMed 22 Clausen, T., Southan, C. and Ehrmann, M. (2002) The HtrA family of proteases: implications for protein composition and cell fate. Mol. Cell 10, 443–455 CrossRef PubMed 23 Clausen, T., Kaiser, M., Huber, R. and Ehrmann, M. (2011) HTRA proteases: regulated proteolysis in protein quality control. Nat. Rev. Mol. Cell Biol. 12, 152–162 CrossRef PubMed 24 Ge, X., Wang, R., Ma, J., Liu, Y., Ezemaduka, A.N., Chen, P.R., Fu, X. and Chang, Z. (2014) DegP primarily functions as a protease for the biogenesis of beta-barrel outer membrane proteins in the Gram-negative bacterium Escherichia coli. FEBS J. 281, 1226–1240 CrossRef 25 Rizzitello, A.E., Harper, J.R. and Silhavy, T.J. (2001) Genetic evidence for parallel pathways of chaperone activity in the periplasm of Escherichia coli. J. Bacteriol. 183, 6794–6800 CrossRef PubMed
C The
C 2015 Biochemical Society Authors Journal compilation
26 Jiang, J., Zhang, X., Chen, Y., Wu, Y., Zhou, Z.H., Chang, Z. and Sui, S.F. (2008) Activation of DegP chaperone-protease via formation of large cage-like oligomers upon binding to substrate proteins. Proc. Nat. Acad. Sci. U.S.A. 105, 11939–11944 CrossRef 27 Krojer, T., Garrido-Franco, M., Huber, R., Ehrmann, M. and Clausen, T. (2002) Crystal structure of DegP (HtrA) reveals a new protease-chaperone machine. Nature 416, 455–459 CrossRef PubMed 28 Shen, Q.T., Bai, X.C., Chang, L.F., Wu, Y., Wang, H.W. and Sui, S.F. (2009) Bowl-shaped oligomeric structures on membranes as DegP’s new functional forms in protein quality control. Proc. Nat. Acad. Sci. U.S.A. 106, 4858–4863 CrossRef 29 Thompson, N.J., Merdanovic, M., Ehrmann, M., van Duijn, E. and Heck, A.J. (2014) Substrate occupancy at the onset of oligomeric transitions of DegP. Structure 22, 281–290 CrossRef PubMed 30 Kim, S. and Sauer, R.T. (2012) Cage assembly of DegP protease is not required for substrate-dependent regulation of proteolytic activity or high-temperature cell survival. Proc. Nat. Acad. Sci. U.S.A. 109, 7263–7268 CrossRef 31 Kim, S. and Sauer, R.T. (2014) Distinct regulatory mechanisms balance DegP proteolysis to maintain cellular fitness during heat stress. Gene Dev. 28, 902–911 CrossRef 32 Krojer, T., Sawa, J., Schafer, E., Saibil, H.R., Ehrmann, M. and Clausen, T. (2008) Structural basis for the regulated protease and chaperone function of DegP. Nature 453, 885–890 CrossRef PubMed 33 Wu, S., Ge, X., Lv, Z., Zhi, Z., Chang, Z. and Zhao, X.S. (2011) Interaction between bacterial outer membrane proteins and periplasmic quality control factors: a kinetic partitioning mechanism. Biochem. J. 438, 505–511 CrossRef PubMed 34 Strauch, K.L., Johnson, K. and Beckwith, J. (1989) Characterization of degP, a gene required for proteolysis in the cell envelope and essential for growth of Escherichia coli at high temperature. J. Bacteriol. 171, 2689–2696 PubMed 35 Walther, D.M., Rapaport, D. and Tommassen, J. (2009) Biogenesis of beta-barrel membrane proteins in bacteria and eukaryotes: evolutionary conservation and divergence. Cell. Mol. Life Sci. 66, 2789–2804 CrossRef PubMed 36 Pavlova, O., Peterson, J.H., Ieva, R. and Bernstein, H.D. (2013) Mechanistic link between beta barrel assembly and the initiation of autotransporter secretion. Proc. Nat. Acad. Sci. U.S.A. 110, 938–947 CrossRef 37 Merdanovic, M., Clausen, T., Kaiser, M., Huber, R. and Ehrmann, M. (2011) Protein quality control in the bacterial periplasm. Annu. Rev. Microbiol. 65, 149–168 CrossRef PubMed
Received 6 August 2014 doi:10.1042/BST20140217
