The major facilitator superfamily (MFS) is the largest family of secondary active transporters and is present in all life kingdoms. Detailed structural basis of the substrate transport and energycoupling mechanisms of these proteins remain to be elucidated. YajR is a putative proton-driven MFS transporter found in many Gram-negative bacteria. Here we report the crystal structure of Escherichia coli YajR at 3.15 Å resolution in an outward-facing conformation. In addition to having the 12 canonical transmembrane helices, the YajR structure includes a unique 65-residue C-terminal domain which is independently stable. The structure is unique in illustrating the functional role of "sequence motif A." This highly conserved element is seen to stabilize the outward conformation of YajR and suggests a general mechanism for the conformational change between the inward and outward states of the MFS transporters.T ransporters are a type of membrane protein essential for all living cells that actively up-take nutrition and export metabolic substances and toxic materials across cellular membranes. Transporters are divided into two major types based on their energy sources. Although primary active transporters directly consume energy from ATP hydrolysis to drive substrate transport, secondary active transporters use energy derived from the electrochemical potential across the cell membrane. The major facilitator superfamily (MFS) is the largest class of secondary transporters and is present in all life kingdoms (1). For example, 25% of prokaryotic transporters belong to the MFS family (2), and the human genome contains over 110 MFS proteins (3). Currently, 3D crystal structures of nine bacterial MFS transporters (4-13) and one from fungi (9) have been reported at medium-high resolution. These studies have shown that MFS proteins contain a 12-transmembrane (TM) helix core composed of two six-helix rigid domains forming a central TM channel, which transports substrates using a rocker-switch mechanism (5). In such a mechanism, MFS proteins are believed to switch between two major conformations, inward and outward, which differ by an ∼40°rotation of one domain relative to the other. Both conformations have been captured in MFS crystal structures. However, many questions remain to be addressed, particularly those related to energy coupling and functional roles of conserved motifs.YajR, a 49-kDa transporter of the MFS family, has putatively been classified as a drug efflux protein solely on the basis of amino acid sequence analysis (14). In Escherichia coli, YajR consists of 454 amino acid residues. Besides containing 12 TM helices, YajR is predicted to possess an extra domain of about 65 residues at the C-terminal. Of the MFS proteins with reported 3D structures, the TM core of YajR shares highest sequence homology (21% identity) with EmrD (SI Appendix, Fig. S1A), which belongs to the 12-TM drug-resistance H + -driven antiporter (DHA12) subfamily (15). The YajR gene is found in a number of Gram-negative bacteria, and it shows high ...

APPL1 is an effector of the small GTPase Rab5. Together, they mediate a signal transduction pathway initiated by ligand binding to cell surface receptors. Interaction with Rab5 is confined to the amino (N)-terminal region of APPL1. We report the crystal structures of human APPL1 N-terminal BAR-PH domain motif. The BAR and PH domains, together with a novel linker helix, form an integrated, crescent-shaped, symmetrical dimer. This BAR–PH interaction is likely conserved in the class of BAR-PH containing proteins. Biochemical analyses indicate two independent Rab-binding sites located at the opposite ends of the dimer, where the PH domain directly interacts with Rab5 and Rab21. Besides structurally supporting the PH domain, the BAR domain also contributes to Rab binding through a small surface region in the vicinity of the PH domain. In stark contrast to the helix-dominated, Rab-binding domains previously reported, APPL1 PH domain employs β-strands to interact with Rab5. On the Rab5 side, both switch regions are involved in the interaction. Thus we identified a new binding mode between PH domains and small GTPases.

Pro-apoptotic Bax induces mitochondrial outer membrane permeabilization (MOMP) by forming oligomers through a largely undefined process. Using site-specific disulfide crosslinking, compartment-specific chemical labeling, and mutational analysis, we found that activated integral membrane Bax proteins form a BH3-in-groove dimer interface on the MOM surface similar to that observed in crystals. However, after the a5 helix was released into the MOM, the remaining interface with a2, a3, and a4 helices was rearranged. Another dimer interface was formed inside the MOM by two intersected or parallel a9 helices. Combinations of these interfaces generated oligomers in the MOM. Oligomerization was initiated by BH3-in-groove dimerization, without which neither the other dimerizations nor MOMP occurred. In contrast, a9 dimerization occurred downstream and was required for release of large but not small proteins from mitochondria. Moreover, the release of large proteins was facilitated by a9 insertion into the MOM and localization to the pore rim. Therefore, the BH3-in-groove dimerization on the MOM nucleates the assembly of an oligomeric Bax pore that is enlarged by a9 dimerization at the rim.