The human large intestine is populated by a high density of microorganisms, collectively termed the colonic microbiota, which has an important role in human health and nutrition. The survival of microbiota members from the dominant Gram-negative phylum Bacteroidetes depends on their ability to degrade dietary glycans that cannot be metabolized by the host. The genes encoding proteins involved in the degradation of specific glycans are organized into co-regulated polysaccharide utilization loci, with the archetypal locus sus (for starch utilisation system) encoding seven proteins, SusA-SusG. Glycan degradation mainly occurs intracellularly and depends on the import of oligosaccharides by an outer membrane protein complex composed of an extracellular SusD-like lipoprotein and an integral membrane SusC-like TonB-dependent transporter. The presence of the partner SusD-like lipoprotein is the major feature that distinguishes SusC-like proteins from previously characterized TonB-dependent transporters. Many sequenced gut Bacteroides spp. encode over 100 SusCD pairs, of which the majority have unknown functions and substrate specificities. The mechanism by which extracellular substrate binding by SusD proteins is coupled to outer membrane passage through their cognate SusC transporter is unknown. Here we present X-ray crystal structures of two functionally distinct SusCD complexes purified from Bacteroides thetaiotaomicron and derive a general model for substrate translocation. The SusC transporters form homodimers, with each β-barrel protomer tightly capped by SusD. Ligands are bound at the SusC-SusD interface in a large solvent-excluded cavity. Molecular dynamics simulations and single-channel electrophysiology reveal a 'pedal bin' mechanism, in which SusD moves away from SusC in a hinge-like fashion in the absence of ligand to expose the substrate-binding site to the extracellular milieu. These data provide mechanistic insights into outer membrane nutrient import by members of the microbiota, an area of major importance for understanding human-microbiota symbiosis.

Gap junctions establish direct pathways for cell-to-cell communication, through the assembly of twelve subunits (connexins) that form intercellular channels connecting neighboring cells. Co-assembly of different connexin isoforms produces channels with unique properties, and enables communication across cell-types. To gain access into the structural underpinnings of connexin co-assembly, we used single particle CryoEM to determine the structure of native lens gap junction channels, composed of connexin-46 and connexin-50 (Cx46/50). We provide the first comparative analysis to connexin-26 (Cx26), which together with computational studies elucidates key energetic features governing gap junction perm-selectivity. Cx46/50 adopts an open-state conformation that is unique from the Cx26 crystal structure, yet appears to be stabilized by a conserved set of hydrophobic anchoring residues. ‘Hot spots’ of genetic mutations linked to hereditary cataract formation map to the core structural-functional elements identified in Cx46/50, rationalizing many of the disease-causing effects.

Membrane proteins reside in lipid bilayers and are typically extracted from this environment for study, which often compromises their integrity. Here we eject intact assemblies from membranes, without chemical disruption, and use mass spectrometry to define their composition. From E. coli outer membranes, we identify a chaperone-porin association and lipid interactions in the beta-barrel assembly machinery. Bridging inner and outer membranes we observe efflux pumps, and from inner membranes a pentameric pore of TonB, and the protein-conducting channel Sec YEG, in association with F1FO ATP-synthase. Intact mitochondrial membranes from Bos taurus yield respiratory complexes and fatty acid-bound dimers of the ADP/ATP transporter (ANT-1). These results highlight the importance of native membrane environments for retaining small-molecule binding, subunit interactions and associated chaperones of the membrane proteome.

The Fanconi Anemia (FA) pathway repairs DNA damage caused by endogenous and chemotherapy-induced DNA crosslinks, and responds to replication stress 1,2. Genetic inactivation of this pathway impairs development, prevents blood production and promotes cancer 1,3. The key molecular step in the FA pathway is the monoubiquitination of a pseudosymmetric heterodimer of FANCI-FANCD2 4,5 by the FA core complex-a megadalton multiprotein E3 ubiquitin ligase 6,7. Monoubiquitinated FANCD2 then recruits enzymes to remove the DNA crosslink or to stabilize the stalled replication fork. A molecular structure of the FA core complex would explain how it acts to maintain genome stability. Here we reconstituted an active, recombinant FA core complex, and used electron cryo-microscopy (cryoEM) and mass spectrometry to determine its structure. The FA core complex is comprised of two central dimers of the FANCB and FAAP100 subunits, flanked by two copies of the RING finger protein, FANCL. These two heterotrimers act as a scaffold to assemble the remaining five subunits, resulting in an extended asymmetric structure. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Focal adhesions (FAs) are large multi-protein complexes that act as transmembrane links between the extracellular matrix and the actin cytoskeleton. Recently, FAs were extensively characterized, yet the molecular mechanisms underlying their mechanical and signalling functions remain unresolved. To address this question, we isolated protein complexes containing different FA components, from chicken smooth muscle, and characterized their properties. Here we identified 'hybrid complexes' consisting of the actin-nucleating subunits of Arp2/3 and either vinculin or vinculin and a-actinin. We further show that suppression of p41-ARC, a central component of native Arp2/3, which is absent from the hybrid complexes, increases the levels of the Arp2/3-nucleating core in FA sites and stimulates FA growth and dynamics. In contrast, overexpression of p41-ARC adversely affects FAs. These results support the view that Arp2/3 can form modular 'hybrid complexes' containing an actinnucleating 'functional core', and 'anchoring domains' (vinculin/p41-ARC) that regulate its subcellular localization.