Skp, a Molecular Chaperone of Gram-negative Bacteria, Is Required for the Formation of Soluble Periplasmic Intermediates of Outer Membrane Proteins

Schäfer, Ute; Beck, Konstanze; Müller, Matthias

doi:10.1074/jbc.274.35.24567

Cited by 204 publications

(178 citation statements)

References 43 publications

(48 reference statements)

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“…Both proteins may act in either the same or, as genetic evidence suggests (22), in a parallel folding pathway. Skp has been shown to interact with early OMP folding intermediates at the periplasmic side of the inner membrane and to be required for the release and the maintenance of soluble periplasmic intermediates (23,24). Whether Skp also acts in late, outer membrane-associated steps of OMP maturation is unknown.…”

Section: Discussionmentioning

confidence: 99%

The Periplasmic Chaperone SurA Exploits Two Features Characteristic of Integral Outer Membrane Proteins for Selective Substrate Recognition

Hennecke

Nolte

Volkmer

et al. 2005

Journal of Biological Chemistry

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The Escherichia coli periplasmic chaperone and peptidyl-prolyl isomerase (PPIase) SurA facilitates the maturation of outer membrane porins. Although the PPIase activity exhibited by one of its two parvulin-like domains is dispensable for this function, the chaperone activity residing in the non-PPIase regions of SurA, a sizable N-terminal domain and a short C-terminal tail, is essential. Unlike most cytoplasmic chaperones SurA is selective for particular substrates and recognizes outer membrane porins synthesized in vitro much more efficiently than other proteins. Thus, SurA may be specialized for the maturation of outer membrane proteins. We have characterized the substrate specificity of SurA based on its natural, biologically relevant substrates by screening cellulose-bound peptide libraries representing outer membrane proteins. We show that two features are critical for peptide binding by SurA: specific patterns of aromatic residues and the orientation of their side chains, which are found more frequently in integral outer membrane proteins than in other proteins. For the first time this sufficiently explains the capability of SurA to discriminate between outer membrane protein and non-outer membrane protein folding intermediates. Furthermore, peptide binding by SurA requires neither an active PPIase domain nor the presence of proline, indicating that the observed substrate specificity relates to the chaperone function of SurA. Finally, we show that SurA is capable of associating with the outer membrane. Together, our data support a model in which SurA is specialized to interact with nonnative periplasmic outer membrane protein folding intermediates and to assist in their maturation from early to late outer membrane-associated steps.

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Section: Discussionmentioning

confidence: 99%

The Periplasmic Chaperone SurA Exploits Two Features Characteristic of Integral Outer Membrane Proteins for Selective Substrate Recognition

Hennecke

Nolte

Volkmer

et al. 2005

Journal of Biological Chemistry

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“…Skp also binds to and inserts into monolayers of negatively charged lipids [51]. Skp binds to the NH 2 -terminal part of OmpA and is required for the release of OmpA into the periplasm [52,53]. Skp does not bind to folded OmpA [46], suggesting that Skp recognizes nonnative structures of OMPs.…”

Section: Role Of Periplasmic Proteins (Chaperones Isomerases)mentioning

confidence: 99%

Membrane protein folding on the example of outer membrane protein A of Escherichia coli

Kleinschmidt¹

2003

Cellular and Molecular Life Sciences (CMLS)

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Abstract. The biophysical principles and mechanisms by which membrane proteins insert and fold into a biomembrane have mostly been studied with bacteriorhodopsin and outer membrane protein A (OmpA). This review describes the assembly process of the monomeric outer membrane proteins of Gram-negative bacteria, for which OmpA has served as an example. OmpA is a two-domain outer membrane protein composed of a 171-residue eight-stranded b-barrel transmembrane domain and a 154-residue periplasmic domain. OmpA is translocated in an unstructured form across the cytoplasmic membrane into the periplasm. In the periplasm, unfolded OmpA is kept in solution in complex with the molecular chaperone Skp. After binding of periplasmic lipopolysaccharide, OmpA insertion and folding occur spontaneously upon interaction of the complex with the phospholipid bilayer. Insertion and folding of the b-barrel transmembrane domain into the lipid bilayer are highly synchronized, i.e. the formation of large amounts of b-sheet secondary structure and b-barrel tertiary structure take place in parallel with the same rate constants, while OmpA inserts into the hydrophobic core of the membrane. In vitro, OmpA can successfully fold into a range of model membranes of very different phospholipid compositions, i.e. into bilayers of lipids of different headgroup structures and hydrophobic chain lengths. Three membrane-bound folding intermediates of OmpA were discovered in folding studies with dioleoylphosphatidylcholine bilayers. Their formation was monitored by time-resolved distance determinations by fluorescence quenching, and they were structurally distinguished by the relative positions of the five tryptophan residues of OmpA in projection to the membrane normal. Recent studies indicate a chaperone-assisted, highly synchronized mechanism of secondary and tertiary structure formation upon membrane insertion of b-barrel membrane proteins such as OmpA that involves at least three structurally distinct folding intermediates.

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“…One model is that periplasmic chaperones like Skp maintain the ␤-barrel domain of OMPs in an unfolded state to facilitate their insertion in the membrane. However, some studies have suggested that Skp recognizes a conformational motif in its substrates rather than extended, unfolded polypeptides (7,19), implying that substrate proteins must be at least partially folded when bound by Skp. To directly address this issue, heteronuclear NMR experiments were used to monitor the folding state of the ␤-barrel domain of OmpA (amino acids 1-177; OmpA 177 ) in complex with Skp.…”

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

confidence: 99%

“…In vivo, Skp has been shown to be important for the folding and insertion of several OMPs (5,7,19,22). These OMPs share the signature ␤-barrel structure as their integral membrane domain.…”

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

confidence: 99%

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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Skp, a Molecular Chaperone of Gram-negative Bacteria, Is Required for the Formation of Soluble Periplasmic Intermediates of Outer Membrane Proteins

Cited by 204 publications

References 43 publications

The Periplasmic Chaperone SurA Exploits Two Features Characteristic of Integral Outer Membrane Proteins for Selective Substrate Recognition

The Periplasmic Chaperone SurA Exploits Two Features Characteristic of Integral Outer Membrane Proteins for Selective Substrate Recognition

Membrane protein folding on the example of outer membrane protein A of Escherichia coli

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

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