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

Schlapschy, Martin; Dommel, Monica K.; Hadian, Kamyar; Fogarasi, Marton; Korndörfer, Ingo P.; Skerra, Arne

doi:10.1515/bc.2004.032

Cited by 34 publications

(27 citation statements)

References 29 publications

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“…10 Pulse labeling and biochemical reconstitution experiments suggested that LPS may be required for efficient assembly of OMPs such as trimeric PhoE, 16 and monomeric OmpA, 17 into outer membranes. Skp consists of 141 amino acid residues and forms a stable homotrimer, 18 consistent with the previously determined stoichiometry of Skp in complex with unfolded OmpA. 12 The trimer resembles a jellyfish and is formed of a tentacle domain with α-helical tentacles that protrude about 60 Å from a β-barrel body termed the association domain.…”

Section: Introductionsupporting

confidence: 83%

“…18 However, the binding stoichiometry may depend on the size of the OMP or its transmembrane domain. To obtain stoichiometries and to estimate the strength of Skp binding, we recorded fluorescence spectra of the aqueous forms of each OMP after dilution of the urea as a function of the molar Skp/OMP ratio until no further increase of the fluorescence intensity was observed ( Figure 2).…”

Section: Resultsmentioning

confidence: 99%

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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

Mayer

Behrens

et al. 2007

Journal of Molecular Biology

108

149

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The interactions of outer membrane proteins (OMPs) with the periplasmic chaperone Skp from Escherichia coli are not well understood. We have examined the binding of Skp to various OMPs of different origin, size, and function. These were OmpA, OmpG, and YaeT (Omp85) from Escherichia coli, the translocator domain of the autotransporter NalP from Neisseria meningitides, FomA from Fusobacterium nucleatum, and the voltagedependent anion-selective channel, human isoform 1 (hVDAC1) from mitochondria. Binding of Skp was observed for bacterial OMPs, but neither for hVDAC1 nor for soluble bovine serum albumin. The Skp trimer formed 1:1 complexes, OMP·Skp 3 , with bacterial OMPs, independent of their size or origin. The dissociation constants of these OMP·Skp 3 complexes were all in the nanomolar range, indicating that they are stable. Complexes of Skp 3 with YaeT displayed the smallest dissociation constants, complexes with NalP the largest. OMP binding to Skp 3 was pH-dependent and not observed when either Skp or OMPs were neutralized at very basic or very acidic pH. When the ionic strength was increased, the free energies of binding of Skp to OmpA or OmpG were reduced. Electrostatic interactions were therefore necessary for formation and stability of OMP·Skp 3 complexes. Light-scattering and circular dichroism experiments demonstrated that Skp 3 remained a stable trimer from pH 3 to pH 11. In the OmpA·Skp 3 complex, Skp efficiently shielded tryptophan residues of the transmembrane strands of OmpA against fluorescence quenching by aqueous acrylamide. Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, bound to OmpA·Skp 3 complexes at low stoichiometries. Acrylamide quenching of fluorescence indicated that in this ternary complex, the tryptophan residues of the transmembrane domain of OmpA were located closer to the surface than in binary OmpA·Skp 3 complexes. This may explain previous observations that folding of Skp-bound OmpA into lipid bilayers is facilitated in presence of LPS. Edited by I. B. HollandKeywords: Skp; outer membrane protein; membrane protein folding; molecular chaperone; protein-protein interactions *Corresponding author. E-mail address: joerg.helmut.kleinschmidt@uni-konstanz.de. Abbreviations used: BSA, bovine serum albumin; FomA, major outer membrane protein of Fusobacterium nucleatum; hVDAC1, voltage-dependent anion-selective channel human isoform-1; LPS, lipopolysaccharide; NalP, translocator domain of the autotransporter NalP from Neisseria meningitides; OM, outer membrane; OMP, outer membrane protein; OmpA, outer membrane protein A of E. coli; OmpG, outer membrane protein G of E. coli; PPIases, peptidyl-proly cis/trans isomerases; SurA, the survival factor A; TMD-OmpA, transmembrane domain of OmpA, i.e. amino acid residues 0 176; WT-OmpA, wild type OmpA; YaeT (Omp85), 89 kDa outer membrane protein of E. coli.Konstanzer Online-Publikations-System (KOPS)

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Section: Introductionsupporting

confidence: 83%

Section: Resultsmentioning

confidence: 99%

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

Mayer

Behrens

et al. 2007

Journal of Molecular Biology

108

149

View full text Add to dashboard Cite

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“…The Skp structure was unexpectedly similar to that of prefoldin, a cytosolic molecular chaperone present in archea and eukarya (8)(9)(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).…”

mentioning

confidence: 85%

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.

114

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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“…As previously mentioned, Skp is a homotrimeric periplasmic chaperone for newly synthesized OMPs. [15][16][17][18] Three hairpin-shaped α-helical extensions extend ~60 Å from the trimerization domain, which is composed of 3 intersubunit β-sheets that wind around a central axis. From observations of its secondary structure, the amino acid sequences of the Omp1-like protein from A. actinomycetemcomitans were not well matched with those of the Skp from E. coli, but both sets were composed mostly of α-helices.…”

Section: Resultsmentioning

confidence: 99%

Biological Characterization of the Omp1-like Protein from Actinobacillus actinomycetemcomitans

Ha¹,

Jeong²,

Jo³

et al. 2010

Bulletin of the Korean Chemical Society

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Actinobacillus actinomycetemcomitans is a gram-negative, nonmotile coccobacillus bacterium that is associated with several human diseases, including endocarditis, meningitis, osteomyelitis, subcutaneous abscesses and periodontal diseases. A full-length Omp1-like protein gene from A. actinomycetemcomitans was cloned into a pQE30 vector and overexpressed in Escherichia coli BL21(DE3) cells. The protein revealed sequence homologies to Seventeen kilodalton proteins (Skp) from Pasteurella multocida and E. coli that have been characterized as periplasmic chaperones. This soluble Omp1-like protein was successfully purified to homogeneity for further folding and functional studies. The purity, identity, and conformation of the protein were determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis, matrix-assisted laser desorption ionization mass spectrometry, circular dichroism, fluorescence spectroscopic, and differential scanning calorimetric studies. We showed that the protein formed an oligomer larger than a tetramer. We found, further, that it is comprised of mostly α-helices and boasts high thermal stability.

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

Cited by 34 publications

References 29 publications

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

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

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

Biological Characterization of the Omp1-like Protein from Actinobacillus actinomycetemcomitans

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