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.

In Bacteroidetes, one of the dominant phyla of the mammalian gut, active uptake of large nutrients across the outer membrane is mediated by SusCD protein complexes via a “pedal bin” transport mechanism. However, many features of SusCD function in glycan uptake remain unclear, including ligand binding, the role of the SusD lid and the size limit for substrate transport. Here we characterise the β2,6 fructo-oligosaccharide (FOS) importing SusCD from Bacteroides thetaiotaomicron (Bt1762-Bt1763) to shed light on SusCD function. Co-crystal structures reveal residues involved in glycan recognition and suggest that the large binding cavity can accommodate several substrate molecules, each up to ~2.5 kDa in size, a finding supported by native mass spectrometry and isothermal titration calorimetry. Mutational studies in vivo provide functional insights into the key structural features of the SusCD apparatus and cryo-EM of the intact dimeric SusCD complex reveals several distinct states of the transporter, directly visualising the dynamics of the pedal bin transport mechanism.

24In Bacteroidetes, one of the dominant phyla of the mammalian gut, active uptake of large 25 nutrients across the outer membrane is mediated by SusCD protein complexes via a "pedal 26 bin" transport mechanism. However, many features of SusCD function in glycan uptake remain 27 unclear, including ligand binding, the role of the SusD lid and the size limit for substrate 28 transport. Here we characterise the β2,6 fructo-oligosaccharide (FOS) importing SusCD from 29Bacteroides thetaiotaomicron (Bt1762-Bt1763) to shed light on SusCD function. Co-crystal 30 structures reveal residues involved in glycan recognition and suggest that the large binding 31 cavity can accommodate several substrate molecules, each up to ~2.5 kDa in size, a finding 32 supported by native mass spectrometry and isothermal titration calorimetry. Mutational studies 33 in vivo provide functional insights into the key structural features of the SusCD apparatus and 34 cryo-EM of the intact dimeric SusCD complex reveals several distinct states of the transporter, 35directly visualising the dynamics of the pedal bin transport mechanism. 36 37while SusD sits on top of the barrel, opening and closing like a lid to facilitate substrate binding. 59Previous structures of loaded SusCD complexes revealed a bound ligand which was 60 completely encapsulated by the closed pedal bin, indicating that SusCD transporters may 61 have a size limit for transport 15,17 . An investigation on the prototypical SusCD system revealed 62 that a mutant strain lacking the surface endo-amylase preferentially utilised malto-63 oligosaccharides with a degree of polymerisation (DP) of ~5-16 18 , suggesting this is the 64 preferred size range imported by the Bt3701-02 SusCD transporter. Direct evidence for this 65 notion is lacking, however, and this issue has not been explored for any other Sus systems 12 . 66Furthermore, many other key features of SusCD function remain unclear, including the role of 67 the SusD lid and other conserved structural elements, as well as the identity of the glycan 68 recognition elements of both SusC and SusD. 69 70One example of a typical PUL is the B. theta levan utilisation locus, spanning Bt1754-Bt1765 19 . 71Levan is a fructan polysaccharide comprised of β2,6-linked fructose units, with occasional 72 β2,1 fructose decorations and is produced mainly by bacteria as an exopolysaccharide, but 73 also by some cereal plants such as wheat 20,21 . For the levan utilisation system, the cell-surface 74 components are Bt1760 (a GH32 endo-levanase), Bt1761 (a surface glycan binding protein;; 75 SGBP), Bt1762 (SusD) and Bt1763 (SusC) 15,19,22 (Fig. 1a). The cell surface levanase Bt1760 76 and glycan binding proteins (Bt1761 and Bt1762) have been shown to be specific for levan, 77 with no activity against, or binding to, β2,1-linked inulin-type fructans that are common to many 78 plants 19,23 . 79 80In this study we characterise binding and uptake of β2,6 fructo-oligosaccharides (FOS) by the 81 Bt1762-63 SusCD transporter. Co-crystal structures of the cl...

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