The cavity-chaperone Skp protects its substrate from aggregation but allows independent folding of substrate domains.
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 beta-barrel domain and a periplasmic alphabeta 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 beta-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 beta-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 beta-barrels across the periplasm in an unfolded state, which may be important for efficient insertion into the outer membrane.
Walton
Troy A
TA
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309, USA.
Sandoval
Cristina M
CM
Fowler
C Andrew
CA
Pardi
Arthur
A
Sousa
Marcelo C
MC
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D006090:Gram-Negative Bacteria / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D021381:Protein Transport
D001425:Bacterial Outer Membrane Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; C072721:Skp protein, E coli; C078167:OMPA outer membrane proteins