LETTER doi:10.1038/nature09406 Structure of a fucose transporter in an outward-open conformation Shangyu Dang 1 , Linfeng Sun 1 *, Yongjian Huang 1 *, Feiran Lu 1 , Yufeng Liu 1 , Haipeng Gong 1 , Jiawei Wang 1 & Nieng Yan 1 The major facilitator superfamily (MFS) transporters are an ancient and widespread family of secondary active transporters 1 . In Escherichia coli, the uptake of L-fucose, a source of carbon for microorganisms, is mediated by an MFS proton symporter, FucP 2,3 . Despite intensive study of the MFS transporters, atomic structure information is only available on three proteins and the outward-open conformation has yet to be captured 4–6 . Here we report the crystal structure of FucP at 3.1 Å resolution, which shows that it contains an outward-open, amphipathic cavity. The similarly folded amino and carboxyl domains of FucP have con- trasting surface features along the transport path, with negative electrostatic potential on the N domain and hydrophobic surface on the C domain. FucP only contains two acidic residues along the transport path, Asp 46 and Glu 135, which can undergo cycles of protonation and deprotonation. Their essential role in active transport is supported by both in vivo and in vitro experiments. Structure-based biochemical analyses provide insights into energy coupling, substrate recognition and the transport mechanism of FucP. L-fucose is a major constituent of N-linked glycans on the cell sur- faces of microbes, plants and animals 7 . It can serve as the sole carbon source for some bacteria. In E. coli, the uptake of L-fucose is mediated by FucP, a fucose/H 1 symporter. FucP homologues include the glucose/ H 1 or mannose/H 1 symporter GlcP 8 , the 2-deoxy-D-ribose trans- porter DeoP 9 , the Na 1 -dependent sugar transporter HP1174 10 , and the Na 1 -dependent methyl a-glucoside transporter NaGLT1 11 (Sup- plementary Fig. 1). FucP and its homologues belong to the MFS family, the members of which exploit the electrochemical potential to shuttle substrates across cell membranes 1,12 . The MFS transporters are thought to use an alternating-access mechanism to upload and download sub- strate 13,14 . The best-characterized MFS transporter is LacY, the E. coli lactose/H 1 symporter 13,15,16 , for which structures of the substrate-free and substrate-bound states are known 4,17,18 . These LacY structures have a nearly identical, inward-open conformation. Similarly, the MFS transporter GlpT (a glycerol-3-phosphate/phosphate antiporter) also has an inward-open conformation 5 , whereas the multidrug resistance antiporter EmrD is in an intermediate state 6 . Although FucP and LacY are both MFS sugar/H 1 symporters, they share limited sequence homology (Supplementary Fig. 2a). It is therefore difficult to understand the functional mechanism of FucP using available structural and biochemical information on LacY. We sought to deter- mine the crystal structure of FucP. We overexpressed full-length FucP in E. coli and purified it to homogeneity. Consistent with the in vivo observations 2,3 , the recombinant FucP transported L-fucose in a pH- dependent manner (Supplementary Fig. 2b). Additional characteriza- tion confirmed that FucP selectively permeated L-fucose 2 (Fig. 1a). The FucP structure was determined by mercury-based, single-wavelength anomalous dispersion and refined at 3.14 Å resolution (Supplementary Table 1 and Supplementary Figs 3 and 4). Consistent with an earlier prediction 19 , FucP contains 12 transmem- brane segments, with both the N and the C termini located on the cytoplasmic side (Fig. 1b, left). Similar to known structures of MFS transporters 4–6 , the 12 transmembrane segments are arranged into two halves, the N and C domains, which can be superimposed with a root mean squared deviation (r.m.s.d.) of 2.97 Å over 138 Ca atoms and are related by a pseudo-two-fold symmetry axis that is perpendicular to the membrane bilayer (Supplementary Fig. 5a, b). The N and C domains each comprise a pair of internal structural repeats, which are related by an approximate 180u rotation around an axis parallel to the membrane bilayer (Supplementary Fig. 5c, d). Unlike any known MFS transporter structure 4–6 , that of FucP has an outward- open conformation. A central cavity, approximately 20Å in depth and 10Å in diameter at the periplasmic side, is surrounded by trans- membrane segments 1, 2, 4 and 5 of the N domain and transmembrane segments 7, 8, 10 and 11 of the C domain (Fig. 1b and Supplementary Fig. 5a). This cavity is probably an important part of the transport path for substrate molecules. Despite limited sequence similarity between FucP and LacY (Sup- plementary Fig. 2), the N domains of the two proteins can be super- imposed with an r.m.s.d. of 2.86 Å over 153 Ca atoms, whereas their C domains have an r.m.s.d. of 2.90Å over 173 Ca atoms (Fig. 2 and Supplementary Fig. 6). Thus, the overall conformations of the N and C domains seem to be ‘rigid’. To switch from outward-open to a LacY- like inward-open conformation, the N and C domains of FucP need to undergo a rotation of approximately 38u around an axis parallel to the membrane bilayer (Fig. 2). The long, flexible linker between trans- membrane segment 6 (TM6) and TM7 (Supplementary Fig. 2), which demarcates the N and C domains, allows the proposed rotation. Thus, we propose that the N and C domains of FucP undergo concentric, rigid-body rotations to achieve the two essential conformations required for alternating access: outward-open and inward-open. In the outward-open FucP, TM4 and TM10 interact with each other at the centre, where Glu135 of TM4 accepts a hydrogen bond from Tyr365 of TM10 (Supplementary Fig. 7a, b). To achieve the inward- open conformation, the interdomain contacts between the N and C halves must undergo considerable rearrangements. Notably, TM1 and TM7 do not directly contact each other in the outward-open con- formation of FucP, whereas they are at the centre of interdomain pack- ing in the inward-open structures of LacY 4 and GlpT 5 . Therefore, a rigid-body rotation of the N and C domains is likely to bring together TM1 and TM7 in the inward-open FucP (Supplementary Fig. 7c). The N and C domains of FucP have contrasting surface features, giving rise to an amphipathic cavity (Figs 1b and 3a). The N domain has a strip of negative electrostatic potential along the central cavity; whereas the C domain contains a hydrophobic patch in the cavity, capped by positively charged residues on the periplasmic side as well as the cytoplasmic side (Fig. 3a). The cavity-facing side of the N domain is enriched by Asn and Gln residues, with 14 Asn or Gln and four Asp or Glu residues (Fig. 3b, left). Only two of the four Asp or Glu residues are highly conserved among FucP homologues and located in the trans- port path. In contrast, residues of the C domain that line the central cavity are mostly hydrophobic, with only a few polar residues and two 1 State Key Laboratory of Bio-membrane and Membrane Biotechnology, Center for Structural Biology, School of Life Sciences and School of Medicine, Tsinghua University, Beijing 100084, China. *These authors contributed equally to this work. 734 | NATURE | VOL 467 | 7 OCTOBER 2010 Macmillan Publishers Limited. All rights reserved ©2010