The Trimeric Periplasmic Chaperone Skp of Escherichia coli Forms 1:1 Complexes with Outer Membrane Proteins via Hydrophobic and Electrostatic Interactions

Qu, Jian; Mayer, Christoph; Behrens, Susanne; Holst, Otto; Kleinschmidt, Jörg H.

doi:10.1016/j.jmb.2007.09.020

Cited by 107 publications

(174 citation statements)

References 48 publications

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“…In this case, a complex between Skp and OmpA is formed, which can be isolated by SEC (Fig. S1) (21). There was no indication of the complex falling apart during the chromatographic run, and the complex was stable for days as assayed by SEC (data not shown), which indicates that the complex formed between Skp and OmpA is stable and does not dissociate over time.…”

Section: Skp Prevents the Aggregation Of Ompa By Forming A Stable Commentioning

confidence: 92%

The cavity-chaperone Skp protects its substrate from aggregation but allows independent folding of substrate domains

Walton

Sandoval

Fowler

et al. 2009

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

113

116

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Outer membrane proteins (OMPs) of Gram-negative bacteria are synthesized in the cytosol and must cross the periplasm before insertion into the outer membrane. The 17-kDa protein (Skp) is a periplasmic chaperone that assists the folding and insertion of many OMPs, including OmpA, a model OMP with a membrane embedded ␤-barrel domain and a periplasmic ␣␤ domain. Structurally, Skp belongs to a family of cavity-containing chaperones that bind their substrates in the cavity, protecting them from aggregation. However, some substrates, such as OmpA, exceed the capacity of the chaperone cavity, posing a mechanistic challenge. Here, we provide direct NMR evidence that, while bound to Skp, the ␤-barrel domain of OmpA is maintained in an unfolded state, whereas the periplasmic domain is folded in its native conformation. Complementary cross-linking and NMR relaxation experiments show that the OmpA ␤-barrel is bound deep within the Skp cavity, whereas the folded periplasmic domain protrudes outside of the cavity where it tumbles independently from the rest of the complex. This domain-based chaperoning mechanism allows the transport of ␤-barrels across the periplasm in an unfolded state, which may be important for efficient insertion into the outer membrane.cavity-based ͉ outer membrane M any molecular chaperones have evolved cavity-like structures to bind their non-native substrates. Classic examples, such as the chaperonins, bind the substrate in the cavity formed by their open rings and protect them from aggregation. Cycles of ATP binding and hydrolysis, coupled with substrate binding and release, then help the substrate achieve its native conformation (1). Because the cavities of these chaperones have limited capacities, binding substrates larger than the cavity requires a portion of the substrate to be bound inside the cavity while the rest of the substrate remains outside. In these cases, however, the entire substrate remains unfolded while bound to the open ring of the chaperone (2-4).The 17-kDa protein (Skp) is a periplasmic chaperone present in many Gram-negative bacteria involved in the folding and insertion of proteins in the outer membrane (5-7). The crystal structure of Skp revealed a trimer with a ''jellyfish'' architecture where a central cavity is formed by long tentacle-like helical protrusions emanating from a body domain (Fig. 1A) (8, 9). The Skp structure was unexpectedly similar to that of prefoldin, a cytosolic molecular chaperone present in archea and eukarya (8-10). Although Skp is a trimer (8, 9, 11) and prefoldin is a hexamer (12, 13), both proteins share jellyfish architectures with a central cavity ( Fig. 1 A and B). In prefoldin, this cavity has been shown to bind substrate proteins (10, 13). Prefoldin thus belongs to a family of chaperones sometimes referred to as ''holdases.'' These chaperones are ATP independent and, in contrast to chaperonins, do not directly facilitate folding of their substrates. Instead they protect the substrates from aggregation by holding them in their cavities, and subs...

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Section: Skp Prevents the Aggregation Of Ompa By Forming A Stable Commentioning

confidence: 92%

The cavity-chaperone Skp protects its substrate from aggregation but allows independent folding of substrate domains

Walton

Sandoval

Fowler

et al. 2009

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

113

116

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“…The Skp or SurA binding data to several other unfolded OMPs (uOMPs) determined previously by two independent groups indicate that the energetics of chaperone/ uOMP interactions are similar (10,13). It is currently unknown whether Skp and SurA show client specificity, but they must be somewhat functionally redundant because the phenotypes for single deletions are relatively mild, whereas the Δskp/ΔsurA phenotype is synthetic lethal (23)(24)(25).…”

Section: Free Energy Of Folding May Be An Energy Sink For Sorting In Thementioning

confidence: 99%

“…To more fully understand the energetic potentials available for β-barrel maturation in bacterial cell surfaces, we therefore sought to determine the thermodynamic stabilities of several outer membrane proteins. Since the thermodynamics of folding leads polypeptide chains to adopt a set of conformations that are at their equilibrium free energy minimum, a comparison of folding stabilities to the energetics of binding interactions that OMPs have with chaperones as they transit the periplasm to the outer membranes should reveal unique insights into the forces involved in the biogenesis of bacterial outer membranes (10)(11)(12)(13).…”

mentioning

confidence: 99%

Membrane protein thermodynamic stability may serve as the energy sink for sorting in the periplasm

Moon

Zaccai

Fleming

et al. 2013

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

104

133

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Thermodynamic stabilities are pivotal for understanding structurefunction relationships of proteins, and yet such determinations are rare for membrane proteins. Moreover, the few measurements that are available have been conducted under very different experimental conditions, which compromises a straightforward extraction of physical principles underlying stability differences. Here, we have overcome this obstacle and provided structure-stability comparisons for multiple membrane proteins. This was enabled by measurements of the free energies of folding and the m values for the transmembrane proteins PhoP/PhoQ-activated gene product (PagP) and outer membrane protein W (OmpW) from Escherichia coli. Our data were collected in the same lipid bilayer and buffer system we previously used to determine those parameters for E. coli outer membrane phospholipase A (OmpLA). Biophysically, our results suggest that the stabilities of these proteins are strongly correlated to the water-to-bilayer transfer free energy of the lipid-facing residues in their transmembrane regions. We further discovered that the sensitivities of these membrane proteins to chemical denaturation, as judged by their m values, was consistent with that previously observed for water-soluble proteins having comparable differences in solvent exposure between their folded and unfolded states. From a biological perspective, our findings suggest that the folding free energies for these membrane proteins may be the thermodynamic sink that establishes an energy gradient across the periplasm, thus driving their sorting by chaperones to the outer membranes in living bacteria. Binding free energies of these outer membrane proteins with periplasmic chaperones support this energy sink hypothesis.protein folding | protein sorting | protein stability

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“…Although a Dskp surA::kan double null strain was synthetic lethal in rich media [28], the skp gene has not shown any genetic interactions with components of the BAM complex. Still, Skp appears to maintain uOMPs in a folding-competent, unfolded conformation because a uOMP captured by a Skp protein and subsequently presented with membranes will fold into those bilayers in vitro [11,29]. Despite the fact that these details are not well understood, it is clear that folding into outer membranes in vivo involves the BAM complex.…”

Section: Introduction: the Challenges Of Unfolded Outer Membrane Protmentioning

confidence: 99%

A combined kinetic push and thermodynamic pull as driving forces for outer membrane protein sorting and folding in bacteria

Fleming

2015

Phil. Trans. R. Soc. B

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One contribution of 12 to a theme issue 'The bacterial cell envelope'. In vitro folding studies of outer membrane beta-barrels have been invaluable in revealing the lipid effects on folding rates and efficiencies as well as folding free energies. Here, the biophysical results are summarized, and these kinetic and thermodynamic findings are considered in terms of the requirements for folding in the context of the cellular environment. Because the periplasm lacks an external energy source the only driving forces for sorting and folding available within this compartment are binding or folding free energies and their associated rates. These values define functions for periplasmic chaperones and suggest a biophysical mechanism for the BAM complex.

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The Trimeric Periplasmic Chaperone Skp of Escherichia coli Forms 1:1 Complexes with Outer Membrane Proteins via Hydrophobic and Electrostatic Interactions

Cited by 107 publications

References 48 publications

The cavity-chaperone Skp protects its substrate from aggregation but allows independent folding of substrate domains

The cavity-chaperone Skp protects its substrate from aggregation but allows independent folding of substrate domains

Membrane protein thermodynamic stability may serve as the energy sink for sorting in the periplasm

A combined kinetic push and thermodynamic pull as driving forces for outer membrane protein sorting and folding in bacteria

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