An In Vitro Assay for Outer Membrane Protein Assembly by the BAM Complex.

To elucidate the mechanism of a biochemical process it is often essential to reconstitute the reaction in vitro using the minimal set of factors required to drive the reaction to completion. Here, we describe a method to reconstitute the folding and membrane integration of bacterial outer membrane (OM) proteins that have a characteristic β-barrel structure. In this method the BAM complex, a heteroligomer that catalyzes the membrane integration of β-barrel proteins, is first purified and inserted into small lipid vesicles.

Remodeling of a delivery complex allows ClpS-mediated degradation of N-degron substrates.

The ClpS adaptor collaborates with the AAA+ ClpAP protease to recognize and degrade N-degron substrates. ClpS binds the substrate N-degron and assembles into a high-affinity ClpS-substrate-ClpA complex, but how the N-degron is transferred from ClpS to the axial pore of the AAA+ ClpA unfoldase to initiate degradation is not known. Here we demonstrate that the unstructured N-terminal extension (NTE) of ClpS enters the ClpA processing pore in the active ternary complex.

Reconstitution of bacterial autotransporter assembly using purified components.

Autotransporters are a superfamily of bacterial virulence factors consisting of an N-terminal extracellular ('passenger') domain and a C-terminal β barrel ('β') domain that resides in the outer membrane (OM). The mechanism by which the passenger domain is secreted is poorly understood. Here we show that a conserved OM protein insertase (the Bam complex) and a molecular chaperone (SurA) are both necessary and sufficient to promote the complete assembly of the Escherichia coli O157:H7 autotransporter EspP in vitro.

The ClpS adaptor mediates staged delivery of N-end rule substrates to the AAA+ ClpAP protease.

The ClpS adaptor delivers N-end rule substrates to ClpAP, an energy-dependent AAA+ protease, for degradation. How ClpS binds specific N-end residues is known in atomic detail and clarified here, but the delivery mechanism is poorly understood. We show that substrate binding is enhanced when ClpS binds hexameric ClpA.

Molecular basis of substrate selection by the N-end rule adaptor protein ClpS.

The N-end rule is a conserved degradation pathway that relates the stability of a protein to its N-terminal amino acid. Here, we present crystal structures of ClpS, the bacterial N-end rule adaptor, alone and engaged with peptides containing N-terminal phenylalanine, leucine, and tryptophan. These structures, together with a previous structure of ClpS bound to an N-terminal tyrosine, illustrate the molecular basis of recognition of the complete set of primary N-end rule amino acids.

The molecular basis of N-end rule recognition.

The N-end rule targets specific proteins for destruction in prokaryotes and eukaryotes. Here, we report a crystal structure of a bacterial N-end rule adaptor, ClpS, bound to a peptide mimic of an N-end rule substrate. This structure, which was solved at a resolution of 1.