Structure and mechanism of the mammalian fructose transporter GLUT5.

糖分を細胞内に輸送する膜たんぱく質の立体構造と動きを解明 -肥満やがんの抑制策に役立つ新たな知見-. 京都大学プレスリリース. 2015-10-01.The altered activity of the fructose transporter GLUT5, an isoform of the facilitated-diffusion glucose transporter family, has been linked to disorders such as type 2 diabetes and obesity. GLUT5 is also overexpressed in certain tumour cells, and inhibitors are potential drugs for these conditions. Here we describe the crystal structures of GLUT5 from Rattus norvegicus and Bos taurus in open outward- and open inward-facing conformations, respectively. GLUT5 has a major facilitator superfamily fold like other homologous monosaccharide transporters. On the basis of a comparison of the inward-facing structures of GLUT5 and human GLUT1, a ubiquitous glucose transporter, we show that a single point mutation is enough to switch the substrate-binding preference of GLUT5 from fructose to glucose. A comparison of the substrate-free structures of GLUT5 with occluded substrate-bound structures of Escherichia coli XylE suggests that, in addition to global rocker-switch-like re-orientation of the bundles, local asymmetric rearrangements of carboxy-terminal transmembrane bundle helices TM7 and TM10 underlie a 'gated-pore' transport mechanism in such monosaccharide transporters

Conformational Change of a Tryptophan Residue in BtuF Facilitates Binding and Transport of Cobinamide by the Vitamin B12 Transporter BtuCD-F

BtuCD-F is an ABC transporter that mediates cobalamin uptake into Escherichia coli. Early in vivo data suggested that BtuCD-F might also be involved in the uptake of cobinamide, a cobalamin precursor. However, neither was it demonstrated that BtuCD-F indeed transports cobinamide, nor was the structural basis of its recognition known. We synthesized radiolabeled cyano-cobinamide and demonstrated BtuCD-catalyzed in vitro transport, which was ATP- and BtuF-dependent. The crystal structure of cobinamide-bound BtuF revealed a conformational change of a tryptophan residue (W66) in the substrate binding cleft compared to the structure of cobalamin-bound BtuF. High-affinity binding of cobinamide was dependent on W66, because mutation to most other amino acids substantially reduced binding. The structures of three BtuF W66 mutants revealed that tight packing against bound cobinamide was only provided by tryptophan and phenylalanine, in line with the observed binding affinities. In vitro transport rates of cobinamide and cobalamin were not influenced by the substitutions of BtuF W66 under the experimental conditions, indicating that W66 has no critical role in the transport reaction. Our data present the molecular basis of the cobinamide versus cobalamin specificity of BtuCD-F and provide tools for in vitro cobinamide transport and binding assays

ABC transporters::A structure-function perspective

Most ABC importers employ a soluble substrate-binding protein (SBP) to capture the ligand and donate the molecule to the translocator. The SBP can be a soluble periplasmic protein or tethered to the membrane via a lipid moiety or protein anchor or fused to the translocator. In the hybrid ABC transporters, multiple substrate-binding domains (SBDs) can be fused in tandem and provide several extracytoplasmic substrate-binding sites. The substrate is transferred from the SBP to the membrane domain, which translocates the substrate via alternating access of a membrane-embedded substrate-binding pocket. A subset of ABC transporters, known as the energy-coupling factor (ECF) transporters, employs a membrane-embedded S-component to capture the substrate. The S-component guided by the ECF module transports the substrate over the membrane via a so-called toppling mechanism. An overview of the mechanisms of transport by the different types of ABC importers is presented, together with structural information about the proteins

Structural basis of nanobody-mediated blocking of BtuF, the cognate substrate-binding protein of the Escherichia coli vitamin B12 transporter BtuCD

Abstract Bacterial ABC importers catalyze the uptake of essential nutrients including transition metals and metal-containing co-factors. Recently, an IgG antibody targeting the external binding protein of the Staphylococcus aureus Mn(II) ABC importer was reported to inhibit transport activity and reduce bacterial cell growth. We here explored the possibility of using alpaca-derived nanobodies to inhibit the vitamin B12 transporter of Escherichia coli, BtuCD-F, as a model system by generating nanobodies against the periplasmic binding protein BtuF. We isolated six nanobodies that competed with B12 for binding to BtuF, with inhibition constants between 10−6 and 10−9 M. Kinetic characterization of the nanobody-BtuF interactions revealed dissociation half-lives between 1.6 and 6 minutes and fast association rates between 104 and 106 M−1s−1. For the tightest-binding nanobody, we observed a reduction of in vitro transport activity of BtuCD-F when an excess of nanobody over B12 was used. The structure of BtuF in complex with the most effective nanobody Nb9 revealed the molecular basis of its inhibitory function. The CDR3 loop of Nb9 reached into the substrate-binding pocket of BtuF, preventing both B12 binding and BtuCD-F complex formation. Our results suggest that nanobodies can mediate ABC importer inhibition, providing an opportunity for novel antibiotic strategies