Skp is a multivalent chaperone of outer-membrane proteins

Abstract

The trimeric chaperone Skp sequesters outer membrane proteins (OMPs) within a hydrophobic cage, preventing their aggregation during transport across the periplasm in Gram negative bacteria. Here, we study the interaction between Escherichia coli Skp and five OMPs of varying size. Investigations of the kinetics of OMP folding reveal that greater Skp:OMP ratios are required to prevent the folding of 16-stranded OMPs compared with their 8-stranded counterparts. Ion mobility spectrometry-mass spectrometry (IMS-MS) data, computer modelling and molecular dynamics simulations provide evidence that 10- to 16-stranded OMPs are encapsulated within an expanded Skp substrate cage. For OMPs which cannot be fully accommodated in the expanded cavity, sequestration is achieved by binding of an additional Skp trimer. The results suggest a new mechanism for Skp chaperone activity involving coordination of multiple copies of Skp to protect a single substrate from aggregation.

Mass spectrometry-enabled structural biology of membrane proteins

“…For example, since hydrophobic interactions are weakened in the gas phase, macromolecular complexes that rely on hydrophobic contacts for assembly may not survive intact during the transfer to the gas phase [82,83]. In addition, the loss of a solvation shell may result in structural reorganization, as evidenced for water soluble proteins, including intrinsically disordered proteins or complexes with 'open' topologies as found in extended polyproteins, such as IgGs or ring-like subunit assemblies [76,81,163]. In these cases it is advisable to perform molecular dynamics simulations to model the behavior of molecules in the gas phase.…”

“…5a). The assemblies of unfolded outer membrane proteins (OMPs) with the periplasmic chaperone Skp have also been studied by nano-ESI-IMS-MS, to understand how the chaperone accommodates its many client proteins of a range of sizes in its hydrophobic cavity [81]. Once chaperone bound, these complexes are water soluble, so no detergents are required for their characterization by IMS-MS.…”

“…Combining IMS with low resolution modelling [145], conventional, or steered molecular dynamics (MD) simulations [81,129] is also gaining traction as a method for obtaining structural insights into proteins/assemblies. IMS also enables structural perturbations, for example, as a result of ligand binding, to be studied (see below).…”

“…Indeed, there is evidence that structural compaction occurs in unstructured loop regions [77,78], in the case of intrinsically disordered proteins [79,80], and where large cavities may be structural features of proteins/assemblies [81]. Moreover, the lack of the hydrophobic effect in the gas-phase, which stabilizes protein structural states and protein complexes, means that some assemblies [89].…”

Magnifying ion mobility spectrometry–mass spectrometry measurements for biomolecular structure studies

“…Results from the gas-phase study suggest that lipid interactions stabilize the native structure. Other studies have used extensive MD simulations with CCS calculation using IMPACT to delineate the structural details of a trimeric chaperone and outer-membrane proteins [77]. …”