Impact of holdase chaperones Skp and SurA on the folding of β-barrel outer-membrane proteins

Thoma, Johannes; Burmann, Björn M.; Hiller, Sebastian; Müller, Daniel J.

doi:10.1038/nsmb.3087

Cited by 113 publications

(141 citation statements)

References 62 publications

Supporting

Mentioning

126

Contrasting

Order By: Relevance

“…2). The importance of SurA in OMP biogenesis arises from its dual functions, both of which are incorporated in OMPBioM: (i) SurA can bind to uOMP, thereby preventing aggregation, and (ii) SurA can facilitate the folding of uOMP (13)(14)(15)(16)(17)22). Because SurA plays a prominent role in OMP folding, the loss of SurA in vivo results in the induction of the σ E stress response (11,13), a regulatory mechanism caused by the accumulation of unfolded protein in the periplasm.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Dynamic periplasmic chaperone reservoir facilitates biogenesis of outer membrane proteins

Costello

Plummer

Fleming

2016

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Outer membrane protein (OMP) biogenesis is critical to bacterial physiology because the cellular envelope is vital to bacterial pathogenesis and antibiotic resistance. The process of OMP biogenesis has been studied in vivo, and each of its components has been studied in isolation in vitro. This work integrates parameters and observations from both in vivo and in vitro experiments into a holistic computational model termed "Outer Membrane Protein Biogenesis Model" (OMPBioM). We use OMPBioM to assess OMP biogenesis mathematically in a global manner. Using deterministic and stochastic methods, we are able to simulate OMP biogenesis under varying genetic conditions, each of which successfully replicates experimental observations. We observe that OMPs have a prolonged lifetime in the periplasm where an unfolded OMP makes, on average, hundreds of short-lived interactions with chaperones before folding into its native state. We find that some periplasmic chaperones function primarily as quality-control factors; this function complements the folding catalysis function of other chaperones. Additionally, the effective rate for the β-barrel assembly machinery complex necessary for physiological folding was found to be higher than has currently been observed in vitro. Overall, we find a finely tuned balance between thermodynamic and kinetic parameters maximizes OMP folding flux and minimizes aggregation and unnecessary degradation. In sum, OMPBioM provides a global view of OMP biogenesis that yields unique insights into this essential pathway.T he cellular envelope of Gram-negative bacteria is comprised of two membranes separated by an aqueous compartment termed the "periplasm." The outer membrane of the cellular envelope contains integral β-barrel membrane proteins referred to as "outer membrane proteins" (OMPs) (1, 2). The outer membrane and OMPs provide the first barrier between bacteria and the environment and are essential to many important cellular processes including metabolic transport, bacterial virulence, and antibiotic resistance (3-5). Understanding the pathway by which OMPs traverse the periplasm and attain their native functional state is essential to an ability to manipulate this element of the bacterial cell.The OMP biogenesis process is distinct from the folding of cytosolic proteins because it involves a unique collection of obstacles. First, OMPs do not adopt their folded conformations while in an aqueous environment (6). Rather, unfolded OMPs (uOMPs) must be transported across the periplasm to reach their native membrane. Because of their marginal solubility in water, this process must be tightly controlled to avoid aggregation. Second, structures of folded OMPs (fOMPs) show that these proteins contain water-solvated residues in loops on the outer surfaces of bacteria. The desolvation and transport of these polar and ionizable side chains across the outer membrane represent significant kinetic barriers to OMP folding (7). Third, Gram-negative bacteria maintain a high density of fOMP in the expanding oute...

show abstract

Section: Resultsmentioning

confidence: 99%

“…Many in vitro and in vivo studies have investigated various components involved in this pathway (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). However, in vitro experiments typically characterize individual components in isolation, and in vivo studies are often incapable of deciphering how specific components are responsible for observed phenomena.…”

mentioning

confidence: 99%

Dynamic periplasmic chaperone reservoir facilitates biogenesis of outer membrane proteins

Costello

Plummer

Fleming

2016

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…proteins populate a dynamic ensemble of conformational states and transit between multiple short-lived interaction sites when bound to chaperones such as Skp, SurA, and Spy during trafficking through the bacterial periplasm (19)(20)(21). Nevertheless, the nature and dynamics of the targeting factors/chaperones in these complexes, and how these properties help guide substrate proteins to the target membrane, have not been addressed.…”

Section: Significancementioning

confidence: 99%

“…The TA-induced opening of Get3 further suggests that the TA substrate explores alternative sites and conformations for interacting with Get3 besides the previously observed docking of the TA-TMD at the well-defined hydrophobic groove in closed Get3, and that, collectively, these alternative Get3-TA interaction modes are energetically more favorable than the established mode. In analogy to membrane protein substrates bound to the Skp and SurA chaperones (19,20), it is plausible that TA substrates sample multiple transient interaction sites during Get3 opening, and TAs could be retained because interaction with alternative sites in Get3 is more favorable than with solvent. This model would explain, in part, the weak electron density for TATMDs in previous Get3•TA structures (13).…”

Section: -H)mentioning

confidence: 99%

A protean clamp guides membrane targeting of tail-anchored proteins

Chio

Chung

Weiss

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Proper localization of proteins to target membranes is a fundamental cellular process. How the nature and dynamics of the targeting complex help guide substrate proteins to the target membrane is not understood for most pathways. Here, we address this question for the conserved ATPase guided entry of tail-anchored protein 3 (Get3), which targets the essential class of tail-anchored proteins (TAs) to the endoplasmic reticulum (ER). Single-molecule fluorescence spectroscopy showed that, contrary to previous models of a static closed Get3•TA complex, Get3 samples open conformations on the submillisecond timescale upon TA binding, generating a fluctuating "protean clamp" that stably traps the substrate. Point mutations at the ATPase site bias Get3 toward closed conformations, uncouple TA binding from induced Get3•Get4/5 disassembly, and inhibit the ER targeting of the Get3•TA complex. These results demonstrate an essential role of substrate-induced Get3 dynamics in driving TA targeting to the membrane, and reveal a tightly coupled channel of communication between the TA-binding site, ATPase site, and effector interaction surfaces of Get3. Our results provide a precedent for large-scale dynamics in a substrate-bound chaperone, which provides an effective mechanism to retain substrate proteins with high affinity while also generating functional switches to drive vectorial cellular processes.protein targeting | chaperones | protein dynamics | single-molecule spectroscopy | ATPases O ver 35% of proteins need to be localized to the correct cellular destinations after their initial synthesis in the cytosol. These protein-targeting processes are essential for the establishment and maintenance of compartmentalization in all cells and pose complex mechanistic challenges to targeting machineries. To minimize improper exposure of substrate proteins in the cytosol, targeting factors must bind substrate proteins with high stability. This requirement is especially stringent during the targeting of integral membrane proteins, whose high aggregation propensity in the cytosol and other aqueous cellular environments demands that targeting factors also serve as effective chaperones to protect substrates from aggregation. Further, to minimize futile cycling of targeting factors, loading of substrates on the targeting factor must be tightly coupled to their delivery to membrane receptor sites. Finally, once at the target membrane, the targeting machinery must readily switch to a lowaffinity state to release substrate proteins to receptor complexes, translocases, or the phospholipid bilayer. With a few exceptions (1, 2), the nature and dynamics of protein targeting complexes and how their biophysical properties help meet these complex functional demands are not well understood, especially for posttranslational protein targeting pathways.

show abstract

“…The inner cavity of Skp may protect the denatured OmpA from DegP, but there is no apparent structure in SurA that may block DegP access (42,43). It was also reported that Skp binds to OMPs more tightly than SurA (44,45). Since SurA is more important for OMP biogenesis (35), the efficient degradation of OmpA by activated DegP may indicate that the lower levels of OMPs in the envelope stress is the result of not only the transcriptional downregulation of OMPs but also the direct DegP proteolysis of OMPs en route to the outer membrane.…”

Section: Discussionmentioning

confidence: 99%

A Small Periplasmic Protein with a Hydrophobic C-Terminal Residue Enhances DegP Proteolysis as a Suicide Activator

et al. 2018

View full text Add to dashboard Cite

DegP is a highly conserved protease that performs regulated proteolysis to selectively remove misfolded proteins in the periplasm of Escherichia coli. Binding of misfolded proteins is known to be the main mechanism of DegP activation, but it is unknown whether any native proteins can alter DegP activity. Here, we show that a small periplasmic protein, YjfN, which is highly upregulated by the Cpx envelope stress response, functions as a "suicide activator" for DegP and promotes efficient degradation of misfolded proteins. YjfN readily binds to and is degraded by DegP, for which a hydrophobic C-terminal residue and transient unfolding of YjfN are critical. YjfN also activates DegP in trans while it is being degraded and accelerates degradation of a denatured outer membrane protein, OmpA, that is not easily recognized by DegP. Although YjfN also prevents OmpA aggregation, the trans-activation effect is mainly responsible for efficient OmpA degradation. Overexpression of YjfN enhances the viability of cells in misfolded protein stress that is induced by the presence of a less-active variant of DegP at high temperature. Collectively, we suggest that YjfN can enhance DegP proteolysis for relieving envelope stresses that may generate toxic misfolded proteins.IMPORTANCE Proper degradation of toxic misfolded proteins is essential for bacterial survival. This function is mainly performed by a highly conserved protease, DegP, in the periplasm of Escherichia coli. It is known that binding of misfolded proteins is the main mechanism for activating the DegP protease. Here, we find that a small periplasmic protein, YjfN, can be a substrate and an activator of DegP. It is the first example of a native protein showing an ability to directly alter DegP activity. The YjfN-mediated trans activation of DegP promotes efficient degradation of misfolded proteins. Our results suggest that YjfN is a novel "suicide activator" for DegP that enhances DegP proteolysis under misfolded protein stress.

show abstract

Impact of holdase chaperones Skp and SurA on the folding of β-barrel outer-membrane proteins

Cited by 113 publications

References 62 publications

Dynamic periplasmic chaperone reservoir facilitates biogenesis of outer membrane proteins

Dynamic periplasmic chaperone reservoir facilitates biogenesis of outer membrane proteins

A protean clamp guides membrane targeting of tail-anchored proteins

A Small Periplasmic Protein with a Hydrophobic C-Terminal Residue Enhances DegP Proteolysis as a Suicide Activator

Contact Info

Product

Resources

About