Brian Kloss

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.

Protein structure. Structure and activity of tryptophan-rich TSPO proteins.

Structural clues to protein function Translocator protein (TSPO) is a mitochondrial membrane protein thought to transport cholesterol and porphyrins. Its detailed function remains unclear, but interest in it is high because TSPO is involved in a variety of human diseases. Two papers now present crystal structures of bacterial TSPOs. Li et al. show that a mutant that mimics a human single polymorphism associated with psychiatric disorders has structural changes in a region implicated in cholesterol binding. Guo et al. suggest that TSPO may be more than a transporter. They show how it catalyzes the degradation of porphyrins, a function that could be important in protection against oxidative stress. Science, this issue p. 555, p. 551 Structures of bacterial homologs give insight into TSPO function in human diseases. Translocator proteins (TSPOs) bind steroids and porphyrins, and they are implicated in many human diseases, for which they serve as biomarkers and therapeutic targets. TSPOs have tryptophan-rich sequences that are highly conserved from bacteria to mammals. Here we report crystal structures for Bacillus cereus TSPO (BcTSPO) down to 1.7 Å resolution, including a complex with the benzodiazepine-like inhibitor PK11195. We also describe BcTSPO-mediated protoporphyrin IX (PpIX) reactions, including catalytic degradation to a previously undescribed heme derivative. We used structure-inspired mutations to investigate reaction mechanisms, and we showed that TSPOs from Xenopus and man have similar PpIX-directed activities. Although TSPOs have been regarded as transporters, the catalytic activity in PpIX degradation suggests physiological importance for TSPOs in protection against oxidative stress.

Structural basis of the alternating-access mechanism in a bile acid transporter

Bile acids are synthesized from cholesterol in hepatocytes and secreted through the biliary tract into the small intestine, where they aid in absorption of lipids and fat-soluble vitamins. Through a process known as enterohepatic recirculation, more than 90% of secreted bile acids are then retrieved from the intestine and returned to the liver for resecretion. In humans, there are two Na+-dependent bile acid transporters involved in enterohepatic recirculation, the Na+-taurocholate co-transporting polypeptide (NTCP; also known as SLC10A1) expressed in hepatocytes, and the apical sodium-dependent bile acid transporter (ASBT; also known as SLC10A2) expressed on enterocytes in the terminal ileum. In recent years, ASBT has attracted much interest as a potential drug target for treatment of hypercholesterolaemia, because inhibition of ASBT reduces reabsorption of bile acids, thus increasing bile acid synthesis and consequently cholesterol consumption. However, a lack of three-dimensional structures of bile acid transporters hampers our ability to understand the molecular mechanisms of substrate selectivity and transport, and to interpret the wealth of existing functional data. The crystal structure of an ASBT homologue from Neisseria meningitidis (ASBTNM) in detergent was reported recently, showing the protein in an inward-open conformation bound to two Na+ and a taurocholic acid. However, the structural changes that bring bile acid and Na+ across the membrane are difficult to infer from a single structure. To understand the structural changes associated with the coupled transport of Na+ and bile acids, here we solved two structures of an ASBT homologue from Yersinia frederiksenii (ASBTYf) in a lipid environment, which reveal that a large rigid-body rotation of a substrate-binding domain gives the conserved ‘crossover’ region, where two discontinuous helices cross each other, alternating accessibility from either side of the cell membrane. This result has implications for the location and orientation of the bile acid during transport, as well as for the translocation pathway for Na+.

Structure and selectivity in bestrophin ion channels.

Human bestrophin-1 (hBest1) is a calcium-activated chloride channel from the retinal pigment epithelium, where mutations are associated with vitelliform macular degeneration, or Best disease. We describe the structure of a bacterial homolog (KpBest) of hBest1 and functional characterizations of both channels. KpBest is a pentamer that forms a five-helix transmembrane pore, closed by three rings of conserved hydrophobic residues, and has a cytoplasmic cavern with a restricted exit. From electrophysiological analysis of structure-inspired mutations in KpBest and hBest1, we find a sensitive control of ion selectivity in the bestrophins, including reversal of anion/cation selectivity, and dramatic activation by mutations at the cytoplasmic exit. A homology model of hBest1 shows the locations of disease-causing mutations and suggests possible roles in regulation. A bacterial homolog structure gives insights into ion permeation, gating, and mutations that cause retinal degeneration. Insight into a retinal degeneration disease Human bestrophin 1 (hBest1) is a membrane protein that forms a chloride channel in the retinal pigment epithelium. Mutations in hBest1 can lead to a retinal degeneration disease known as Best disease. Yang et al. describe the structure of KpBest, a bacterial homolog of hBest1. KpBest forms a pentamer with an ion channel at its center. In contrast to hBest1, KpBest1 is a sodium channel. The structure suggests a mechanism for ion selectivity that was confirmed by mutagenesis of KpBest and hBest1. A model of the hBest1 channel structure based on the KpBest structure reveals how mutations cause disease. Science, this issue p. 355

Gating of the TrkH ion channel by its associated RCK protein TrkA.

TrkH belongs to a superfamily of K+ transport proteins required for growth of bacteria in low external K+ concentrations. The crystal structure of TrkH from Vibrio parahaemolyticus showed that TrkH resembles a K+ channel and may have a gating mechanism substantially different from K+ channels. TrkH assembles with TrkA, a cytosolic protein comprising two RCK (regulate the conductance of K+) domains, which are found in certain K+ channels and control their gating. However, fundamental questions on whether TrkH is an ion channel and how it is regulated by TrkA remain unresolved. Here we show single-channel activity of TrkH that is upregulated by ATP via TrkA. We report two structures of the tetrameric TrkA ring, one in complex with TrkH and one in isolation, in which the ring assumes two markedly different conformations. These results suggest a mechanism for how ATP increases TrkH activity by inducing conformational changes in TrkA.

Structural basis for a pH-sensitive calcium leak across membranes.

Allowing calcium to leak across a membrane Cells maintain a balance between calcium in the cytosol and calcium stored in organelles—too much stored calcium kills cells. Transmembrane Bax inhibitor motif (TMBIM) proteins form channels in organelle membranes that allow calcium to leak out. Chang et al. show that this calcium leak is pH-dependent. A bacterial homolog of TMBIM proteins converts between an open channel at low pH and a closed channel at high pH. Although the channel is open at low pH, calcium leakage is low because the inside of the channel remains at a neutral pH. Thus, at physiological pH, these channels will be in equilibrium between the open and closed states, so that excess calcium can leak through. Science, this issue p. 1131 pH sensing regulates calcium flow through a bacterial membrane channel that is a homolog of a human antiapoptotic protein. Calcium homeostasis balances passive calcium leak and active calcium uptake. Human Bax inhibitor–1 (hBI-1) is an antiapoptotic protein that mediates a calcium leak and is representative of a highly conserved and widely distributed family, the transmembrane Bax inhibitor motif (TMBIM) proteins. Here, we present crystal structures of a bacterial homolog and characterize its calcium leak activity. The structure has a seven-transmembrane-helix fold that features two triple-helix sandwiches wrapped around a central C-terminal helix. Structures obtained in closed and open conformations are reversibly interconvertible by change of pH. A hydrogen-bonded, pKa (where Ka is the acid dissociation constant)–perturbed pair of conserved aspartate residues explains the pH dependence of this transition, and biochemical studies show that pH regulates calcium influx in proteoliposomes. Homology models for hBI-1 provide insights into TMBIM-mediated calcium leak and cytoprotective activity.

Optimising the combination of thermostabilising mutations in the neurotensin receptor for structure determination.

Conformational thermostabilisation of G protein-coupled receptors is a successful approach for their structure determination. We have recently determined the structure of a thermostabilised neurotensin receptor NTS1 in complex with its peptide agonist and here we describe the strategy for the identification and combination of the 6 thermostabilising mutations essential for crystallisation. First, thermostability assays were performed on a panel of 340 detergent-solubilised Ala/Leu NTS1 mutants and the best 16 thermostabilising mutations were identified. These mutations were combined pair-wise in nearly all combinations (119 out of a possible 120 combinations) and each mutant was expressed and its thermostability was experimentally determined. A theoretical stability score was calculated from the sum of the stabilities measured for each double mutant and applied to develop 24 triple mutants, which in turn led to the construction of 14 quadruple mutants. Use of the thermostability data for the double mutants to predict further mutant combinations resulted in a greater percentage of the triple and quadruple mutants showing improved thermostability than if only the thermostability data for the single mutations was considered. The best quadruple mutant (NTS1-Nag36k) was further improved by including an additional 2 mutations (resulting in NTS1-GW5) that were identified from a complete Ala/Leu scan of Nag36k by testing the thermostability of the mutants in situ in whole bacteria. NTS1-GW5 had excellent stability in short chain detergents and could be readily purified as a homogenous sample that ultimately allowed crystallisation and structure determination.

Structure and activity of tryptophan-rich TSPO proteins

Structural clues to protein function Translocator protein (TSPO) is a mitochondrial membrane protein thought to transport cholesterol and porphyrins. Its detailed function remains unclear, but interest in it is high because TSPO is involved in a variety of human diseases. Two papers now present crystal structures of bacterial TSPOs. Li et al. show that a mutant that mimics a human single polymorphism associated with psychiatric disorders has structural changes in a region implicated in cholesterol binding. Guo et al. suggest that TSPO may be more than a transporter. They show how it catalyzes the degradation of porphyrins, a function that could be important in protection against oxidative stress. Science, this issue p. 555, p. 551 Structures of bacterial homologs give insight into TSPO function in human diseases. Translocator proteins (TSPOs) bind steroids and porphyrins, and they are implicated in many human diseases, for which they serve as biomarkers and therapeutic targets. TSPOs have tryptophan-rich sequences that are highly conserved from bacteria to mammals. Here we report crystal structures for Bacillus cereus TSPO (BcTSPO) down to 1.7 Å resolution, including a complex with the benzodiazepine-like inhibitor PK11195. We also describe BcTSPO-mediated protoporphyrin IX (PpIX) reactions, including catalytic degradation to a previously undescribed heme derivative. We used structure-inspired mutations to investigate reaction mechanisms, and we showed that TSPOs from Xenopus and man have similar PpIX-directed activities. Although TSPOs have been regarded as transporters, the catalytic activity in PpIX degradation suggests physiological importance for TSPOs in protection against oxidative stress.