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 ...

Obesity is associated with metabolic inflammation and endoplasmic reticulum (ER) stress, both of which promote metabolic disease progression. Adipose tissue macrophages (ATMs) are key players orchestrating metabolic inflammation, and ER stress enhances macrophage activation. However, whether ER stress pathways underlie ATM regulation of energy homeostasis remains unclear. Here, we identified inositol-requiring enzyme 1α (IRE1α) as a critical switch governing M1-M2 macrophage polarization and energy balance. Myeloid-specific IRE1α abrogation in Ern1; Lyz2-Cre mice largely reversed high-fat diet (HFD)-induced M1-M2 imbalance in white adipose tissue (WAT) and blocked HFD-induced obesity, insulin resistance, hyperlipidemia and hepatic steatosis. Brown adipose tissue (BAT) activity, WAT browning and energy expenditure were significantly higher in Ern1; Lyz2-Cre mice. Furthermore, IRE1α ablation augmented M2 polarization of macrophages in a cell-autonomous manner. Thus, IRE1α senses protein unfolding and metabolic and immunological states, and consequently guides ATM polarization. The macrophage IRE1α pathway drives obesity and metabolic syndrome through impairing BAT activity and WAT browning.

Temperature profoundly affects aging in both poikilotherms and homeotherms. A general belief is that lower temperatures extend lifespan while higher temperatures shorten it. Though this “temperature law” is widely accepted, it has not been extensively tested. Here, we systematically evaluated the role of temperature in lifespan regulation in C. elegans. We found that while exposure to low temperatures at the adult stage prolongs lifespan, low temperature treatment at the larval stage surprisingly reduces lifespan. Interestingly, this differential effect of temperature on longevity in larvae and adults is mediated by the same thermosensitive TRP channel TRPA-1 that signals to the transcription factor DAF-16/FOXO. DAF-16/FOXO and TRPA-1 act in larva to shorten lifespan, but extend lifespan in adulthood. DAF-16/FOXO differentially regulates gene expression in larva and adult in a temperature-dependent manner. Our results uncover unexpected complexity underlying temperature modulation of longevity, demonstrating that temperature differentially regulates lifespan at different stages of life.

The G-protein-coupled receptors (GPCRs) are one of the largest super families of cell-surface receptors and play crucial roles in virtually every organ system. One particular family of GPCRs, the class C GPCRs, is distinguished by a characteristically large extracellular domain and constitutive dimerization. The structure and activation mechanism of this family result in potentially unique ligand recognition sites, thereby offering a variety of possibilities by which receptor activity might be modulated using novel compounds. In the present article, we aim to provide an overview of the exact sites and structural features involved in ligand recognition of the class C GPCRs. Furthermore, we demonstrate the precise steps that occur during the receptor activation process, which underlie the possibilities by which receptor function may be altered by different approaches. Finally, we use four typical family members to illustrate orthosteric and allosteric sites with representative ligands and their corresponding therapeutic potential.

Many cell surface receptors are multimeric proteins, composed of several structural domains, some involved in ligand recognition, whereas others are responsible for signal transduction. In most cases, the mechanism of how ligand interaction in the extracellular domains leads to the activation of effector domains remains largely unknown. Here we examined how the extracellular ligand binding to the venus flytrap (VFT) domains of the dimeric metabotropic glutamate receptors activate the seven transmembrane (7TM) domains responsible for G protein activation. These two domains are interconnected by a cysteine-rich domain (CRD). We show that any of the four disulfide bridges of the CRD are required for the allosteric coupling between the VFT and the 7TM domains. More importantly, we show that a specific association of the two CRDs corresponds to the active state of the receptor. Indeed, a specific crosslinking of the CRDs with intersubunit disulfide bridges leads to fully constitutively active receptors, no longer activated by agonists nor by allosteric modulators. These data demonstrate that intersubunit movement at the level of the CRDs represents a key step in metabotropic glutamate receptor activation.transmembrane signaling | G protein-coupled receptor | allosteric modulation M ost cell surface receptors are multimeric complexes of which each subunit is produced through the association of different domains throughout evolution (1-5). The activation of such receptor complexes is a result of coordinated conformational changes or movement of these different domains. Although an increasing amount of 3D crystal structures are becoming available, there is still limited information available on the structural basis of interdomain communication.Class C G protein-coupled receptors (GPCRs) represent key examples of such receptor complexes (6, 7). These receptors are obligatory dimers, either homo-or heterodimers, made by the association of two domains over evolutionary time; an extracellular bilobate venus flytrap (VFT) domain associated with a G protein activating 7 transmembrane (7TM) domain (Fig. 1A) (8). The VFTs are evolved from certain types of bacterial periplasmic binding proteins, especially those of the leucine-isoleucinevaline binding protein family involved in the transport of amino acids, sugars, or ions. Not surprisingly, class C GPCRs are activated by amino acids, i.e., the receptors for the two major neurotransmitters, the eight metabotropic glutamate receptors (mGluRs), and the GABA B receptor; sugar (the sweet taste receptors); or ions [the calcium-sensing receptor (CaSR)]. Accordingly, class C GPCRs represent exciting new targets for drug development for both the pharmaceutical and food industries, as illustrated by the number of drugs targeting these receptors already on the market (the GABA B receptor agonist baclofen, umami compounds, and various sweeteners such as aspartame, and cinacalcet, a positive allosteric modulator of the CaSR), and those in clinical trials (9-11).The precise mechanisms of how...