J. Love

Structure of the agonist-bound neurotensin receptor

Neurotensin (NTS) is a 13-amino-acid peptide that functions as both a neurotransmitter and a hormone through the activation of the neurotensin receptor NTSR1, a G-protein-coupled receptor (GPCR). In the brain, NTS modulates the activity of dopaminergic systems, opioid-independent analgesia, and the inhibition of food intake; in the gut, NTS regulates a range of digestive processes. Here we present the structure at 2.8 Å resolution of Rattus norvegicus NTSR1 in an active-like state, bound to NTS8–13, the carboxy-terminal portion of NTS responsible for agonist-induced activation of the receptor. The peptide agonist binds to NTSR1 in an extended conformation nearly perpendicular to the membrane plane, with the C terminus oriented towards the receptor core. Our findings provide, to our knowledge, the first insight into the binding mode of a peptide agonist to a GPCR and may support the development of non-peptide ligands that could be useful in the treatment of neurological disorders, cancer and obesity.

Homologue structure of the SLAC1 anion channel for closing stomata in leaves

The plant SLAC1 anion channel controls turgor pressure in the aperture-defining guard cells of plant stomata, thereby regulating the exchange of water vapour and photosynthetic gases in response to environmental signals such as drought or high levels of carbon dioxide. Here we determine the crystal structure of a bacterial homologue (Haemophilus influenzae) of SLAC1 at 1.20 Å resolution, and use structure-inspired mutagenesis to analyse the conductance properties of SLAC1 channels. SLAC1 is a symmetrical trimer composed from quasi-symmetrical subunits, each having ten transmembrane helices arranged from helical hairpin pairs to form a central five-helix transmembrane pore that is gated by an extremely conserved phenylalanine residue. Conformational features indicate a mechanism for control of gating by kinase activation, and electrostatic features of the pore coupled with electrophysiological characteristics indicate that selectivity among different anions is largely a function of the energetic cost of ion dehydration.

Crystal structure of a potassium ion transporter, TrkH

The TrkH/TrkG/KtrB proteins mediate K+ uptake in bacteria and probably evolved from simple K+ channels by multiple gene duplications or fusions. Here we present the crystal structure of a TrkH from Vibrio parahaemolyticus. TrkH is a homodimer, and each protomer contains an ion permeation pathway. A selectivity filter, similar in architecture to those of K+ channels but significantly shorter, is lined by backbone and side-chain oxygen atoms. Functional studies showed that TrkH is selective for permeation of K+ and Rb+ over smaller ions such as Na+ or Li+. Immediately intracellular to the selectivity filter are an intramembrane loop and an arginine residue, both highly conserved, which constrict the permeation pathway. Substituting the arginine with an alanine significantly increases the rate of K+ flux. These results reveal the molecular basis of K+ selectivity and suggest a novel gating mechanism for this large and important family of membrane transport proteins.

Structure and mechanism of a pentameric formate channel

Formate transport across the inner membrane is a critical step in anaerobic bacterial respiration. Members of the formate/nitrite transport protein family function to shuttle substrate across the cytoplasmic membrane. In bacterial pathogens, the nitrite transport protein is involved in protecting bacteria from peroxynitrite released by host macrophages. We have determined the 2.13-Å structure of the formate channel FocA from Vibrio cholerae, which reveals a pentamer in which each monomer possesses its own substrate translocation pore. Unexpectedly, the fold of the FocA monomer resembles that found in water and glycerol channels. The selectivity filter in FocA consists of a cytoplasmic slit and a central constriction ring. A 2.5-Å high-formate structure shows two formate ions bound to the cytoplasmic slit via both hydrogen bonding and van der Waals interactions, providing a structural basis for the substrate selectivity of the channel.

The Structural Basis for the Specificity of Retinoid-X Receptor-selective Agonists: New Insights Into the Role of Helix H12

Ligands that specifically target retinoid-X receptors (RXRs) are emerging as potentially powerful therapies for cancer, diabetes, and the lowering of circulatory cholesterol. To date, RXR has only been crystallized in the absence of ligand or with the promiscuous ligand 9-cis retinoic acid, which also activates retinoic acid receptors. Here we present the structure of hRXRβ in complex with the RXR-specific agonist LG100268 (LG268). The structure clearly reveals why LG268 is specific for the RXR ligand binding pocket and will not activate retinoic acid receptors. Intriguingly, in the crystals, the C-terminal “activation” helix (AF-2/helix H12) is trapped in a novel position not seen in other nuclear receptor structures such that it does not cap the ligand binding cavity. Mammalian two-hybrid assays indicate that LG268 is unable to release co-repressors from RXR unless co-activators are also present. Together these findings suggest that RXR ligands may be inefficient at repositioning helix H12.

Structural insights into the interaction and activation of histone deacetylase 3 by nuclear receptor corepressors

SMRT (silencing mediator of retinoid acid and thyroid hormone receptor) and NCoR (nuclear receptor corepressor) are transcriptional corepressors that play an essential role in the regulation of development and metabolism. This role is achieved, in part, through the recruitment of a key histone deacetylase (HDAC3), which is itself indispensable for cell viability. The assembly of HDAC3 with the deacetylase activation domain (DAD) of SMRT and NCoR is required for activation of the otherwise inert deacetylase. The DAD comprises an N-terminal DAD-specific motif and a C-terminal SANT (SWI3/ADA2/NCoR/TFIIIB)-like domain. We report here the solution structure of the DAD from SMRT, which reveals a four-helical structure. The DAD differs from the SANT (and MYB) domains in that (i) it has an additional N-terminal helix and (ii) there is a notable hydrophobic groove on the surface of the domain. Structure-guided mutagenesis, combined with interaction assays, showed that residues in the vicinity of the hydrophobic groove are required for interaction with (and hence activation of) HDAC3. Importantly, one surface-exposed lysine is required for activation of HDAC3, but not for interaction. This lysine may play a uniquely important role in the mechanism of activating HDAC3.

Crystal structure of a phosphorylation-coupled saccharide transporter

Saccharides have a central role in the nutrition of all living organisms. Whereas several saccharide uptake systems are shared between the different phylogenetic kingdoms, the phosphoenolpyruvate-dependent phosphotransferase system exists almost exclusively in bacteria. This multi-component system includes an integral membrane protein EIIC that transports saccharides and assists in their phosphorylation. Here we present the crystal structure of an EIIC from Bacillus cereus that transports diacetylchitobiose. The EIIC is a homodimer, with an expansive interface formed between the amino-terminal halves of the two protomers. The carboxy-terminal half of each protomer has a large binding pocket that contains a diacetylchitobiose, which is occluded from both sides of the membrane with its site of phosphorylation near the conserved His 250 and Glu 334 residues. The structure shows the architecture of this important class of transporters, identifies the determinants of substrate binding and phosphorylation, and provides a framework for understanding the mechanism of sugar translocation.

Selective Electrodiffusion of Zinc Ions in a Zrt-, Irt-like Protein, ZIPB

All living cells need zinc ions to support cell growth. Zrt-, Irt-like proteins (ZIPs) represent a major route for entry of zinc ions into cells, but how ZIPs promote zinc uptake has been unclear. Here we report the molecular characterization of ZIPB from Bordetella bronchiseptica, the first ZIP homolog to be purified and functionally reconstituted into proteoliposomes. Zinc flux through ZIPB was found to be nonsaturable and electrogenic, yielding membrane potentials as predicted by the Nernst equation. Conversely, membrane potentials drove zinc fluxes with a linear voltage-flux relationship. Direct measurements of metal uptake by inductively coupled plasma mass spectroscopy demonstrated that ZIPB is selective for two group 12 transition metal ions, Zn2+ and Cd2+, whereas rejecting transition metal ions in groups 7 through 11. Our results provide the molecular basis for cellular zinc acquisition by a zinc-selective channel that exploits in vivo zinc concentration gradients to move zinc ions into the cytoplasm.

Panoramic view of a superfamily of phosphatases through substrate profiling

Significance Here, we examine the activity profile of the haloalkanoic acid dehalogenase (HAD) superfamily by screening a customized library against >200 enzymes from a broad sampling of the superfamily. From this dataset, we can infer the function of nearly 35% of the superfamily. Overall, the superfamily was found to show high substrate ambiguity, with 75% of the superfamily utilizing greater than five substrates. In addition, the HAD members with the least amount of structural accessorization of the Rossmann fold were found to be the most specific, suggesting that elaboration of the core domain may have led to increased substrate range of the superfamily. Large-scale activity profiling of enzyme superfamilies provides information about cellular functions as well as the intrinsic binding capabilities of conserved folds. Herein, the functional space of the ubiquitous haloalkanoate dehalogenase superfamily (HADSF) was revealed by screening a customized substrate library against >200 enzymes from representative prokaryotic species, enabling inferred annotation of ∼35% of the HADSF. An extremely high level of substrate ambiguity was revealed, with the majority of HADSF enzymes using more than five substrates. Substrate profiling allowed assignment of function to previously unannotated enzymes with known structure, uncovered potential new pathways, and identified iso-functional orthologs from evolutionarily distant taxonomic groups. Intriguingly, the HADSF subfamily having the least structural elaboration of the Rossmann fold catalytic domain was the most specific, consistent with the concept that domain insertions drive the evolution of new functions and that the broad specificity observed in HADSF may be a relic of this process.

The New York Consortium on Membrane Protein Structure (NYCOMPS): a high-throughput platform for structural genomics of integral membrane proteins

The New York Consortium on Membrane Protein Structure (NYCOMPS) was formed to accelerate the acquisition of structural information on membrane proteins by applying a structural genomics approach. NYCOMPS comprises a bioinformatics group, a centralized facility operating a high-throughput cloning and screening pipeline, a set of associated wet labs that perform high-level protein production and structure determination by x-ray crystallography and NMR, and a set of investigators focused on methods development. In the first three years of operation, the NYCOMPS pipeline has so far produced and screened 7,250 expression constructs for 8,045 target proteins. Approximately 600 of these verified targets were scaled up to levels required for structural studies, so far yielding 24 membrane protein crystals. Here we describe the overall structure of NYCOMPS and provide details on the high-throughput pipeline.