Structural basis for bacterial energy extraction from atmospheric hydrogen.

Diverse aerobic bacteria use atmospheric H2 as an energy source for growth and survival1. This globally significant process regulates the composition of the atmosphere, enhances soil biodiversity and drives primary production in extreme environments2,3. Atmospheric H2 oxidation is attributed to uncharacterized members of the [NiFe] hydrogenase superfamily4,5. However, it remains unresolved how these enzymes overcome the extraordinary catalytic challenge of oxidizing picomolar levels of H2 amid ambient levels of the catalytic poison O2 and how the derived electrons are transferred to the respiratory chain1. Here we determined the cryo-electron microscopy structure of the Mycobacterium smegmatis hydrogenase Huc and investigated its mechanism. Huc is a highly efficient oxygen-insensitive enzyme that couples oxidation of atmospheric H2 to the hydrogenation of the respiratory electron carrier menaquinone. Huc uses narrow hydrophobic gas channels to selectively bind atmospheric H2 at the expense of O2, and 3 [3Fe-4S] clusters modulate the properties of the enzyme so that atmospheric H2 oxidation is energetically feasible. The Huc catalytic subunits form an octameric 833 kDa complex around a membrane-associated stalk, which transports and reduces menaquinone 94 Å from the membrane. These findings provide a mechanistic basis for the biogeochemically and ecologically important process of atmospheric H2 oxidation, uncover a mode of energy coupling dependent on long-range quinone transport, and pave the way for the development of catalysts that oxidize H2 in ambient air.

Toward a Structural Understanding of Class B GPCR Peptide Binding and Activation.

Class B G protein-coupled receptors (GPCRs) are important therapeutic targets for major diseases. Here, we present structures of peptide and Gs-bound pituitary adenylate cyclase-activating peptide, PAC1 receptor, and corticotropin-releasing factor (CRF), (CRF1) receptor. Together with recently solved structures, these provide coverage of the major class B GPCR subfamilies. Diverse orientations of the extracellular domain to the receptor core in different receptors are at least partially dependent on evolutionary conservation in the structure and nature of peptide interactions. Differences in peptide interactions to the receptor core also influence the interlinked TM2-TM1-TM6/ECL3/TM7 domain, and this is likely important in their diverse signaling. However, common conformational reorganization of ECL2, linked to reorganization of ICL2, modulates G protein contacts. Comparison between receptors reveals ICL2 as a key domain forming dynamic G protein interactions in a receptor- and ligand-specific manner. This work advances our understanding of class B GPCR activation and Gs coupling.

Differential GLP-1R Binding and Activation by Peptide and Non-peptide Agonists.

Peptide drugs targeting class B1 G-protein-coupled receptors (GPCRs) can treat multiple diseases; however, there remains substantial interest in the development of orally delivered non-peptide drugs. Here, we reveal unexpected overlap between signaling and regulation of the glucagon-like peptide-1 (GLP-1) receptor by the non-peptide agonist PF 06882961 and GLP-1 that was not observed for another compound, CHU-128. Compounds from these patent series, including PF 06882961, are currently in clinical trials for treatment of type 2 diabetes. High-resolution cryoelectron microscopy (cryo-EM) structures reveal that the binding sites for PF 06882961 and GLP-1 substantially overlap, whereas CHU-128 adopts a unique binding mode with a more open receptor conformation at the extracellular face. Structural differences involving extensive water-mediated hydrogen bond networks could be correlated to functional data to understand how PF 06882961, but not CHU-128, can closely mimic the pharmacological properties of GLP-1. These findings will facilitate rational structure-based discovery of non-peptide agonists targeting class B GPCRs.

Cholesterol-dependent dynamic changes in the conformation of the type 1 cholecystokinin receptor affect ligand binding and G protein coupling.

Development of optimal therapeutics for disease states that can be associated with increased membrane cholesterol requires better molecular understanding of lipid modulation of the drug target. Type 1 cholecystokinin receptor (CCK1R) agonist actions are affected by increased membrane cholesterol, enhancing ligand binding and reducing calcium signaling, while agonist actions of the closely related CCK2R are not. In this work, we identified a set of chimeric human CCK1R/CCK2R mutations that exchange the cholesterol sensitivity of these 2 receptors, providing powerful tools when expressed in CHO and HEK-293 model cell lines to explore mechanisms. Static, low energy, high-resolution structures of the mutant CCK1R constructs, stabilized in complex with G protein, were not substantially different, suggesting that alterations to receptor dynamics were key to altered function. We reveal that cholesterol-dependent dynamic changes in the conformation of the helical bundle of CCK receptors affects both ligand binding at the extracellular surface and G protein coupling at the cytosolic surface, as well as their interrelationships involved in stimulus-response coupling. This provides an ideal setting for potential allosteric modulators to correct the negative impact of membrane cholesterol on CCK1R.

Molecular basis for control of antibiotic production by a bacterial hormone.

Actinobacteria produce numerous antibiotics and other specialized metabolites that have important applications in medicine and agriculture1. Diffusible hormones frequently control the production of such metabolites by binding TetR family transcriptional repressors (TFTRs), but the molecular basis for this remains unclear2. The production of methylenomycin antibiotics in Streptomyces coelicolor A3(2) is initiated by the binding of 2-alkyl-4-hydroxymethylfuran-3-carboxylic acid (AHFCA) hormones to the TFTR MmfR3. Here we report the X-ray crystal structure of an MmfR-AHFCA complex, establishing the structural basis for hormone recognition. We also elucidate the mechanism for DNA release upon hormone binding through the single-particle cryo-electron microscopy structure of an MmfR-operator complex. DNA binding and release assays with MmfR mutants and synthetic AHFCA analogues define the role of individual amino acid residues and hormone functional groups in ligand recognition and DNA release. These findings will facilitate the exploitation of actinobacterial hormones and their associated TFTRs in synthetic biology and in the discovery of new antibiotics.

Structural insight into selectivity of amylin and calcitonin receptor agonists.

Amylin receptors (AMYRs), heterodimers of the calcitonin receptor (CTR) and one of three receptor activity-modifying proteins, are promising obesity targets. A hallmark of AMYR activation by Amy is the formation of a 'bypass' secondary structural motif (residues S19-P25). This study explored potential tuning of peptide selectivity through modification to residues 19-22, resulting in a selective AMYR agonist, San385, as well as nonselective dual amylin and calcitonin receptor agonists (DACRAs), with San45 being an exemplar. We determined the structure and dynamics of San385-bound AMY3R, and San45 bound to AMY3R or CTR. San45, via its conjugated lipid at position 21, was anchored at the edge of the receptor bundle, enabling a stable, alternative binding mode when bound to the CTR, in addition to the bypass mode of binding to AMY3R. Targeted lipid modification may provide a single intervention strategy for design of long-acting, nonselective, Amy-based DACRAs with potential anti-obesity effects.

Activation of the GLP-1 receptor by a non-peptidic agonist.

Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, including diabetes and obesity1. Structures of active receptors reveal peptide agonists engage deep within the receptor core, leading to an outward movement of extracellular loop 3 and the tops of transmembrane helices 6 and 7, an inward movement of transmembrane helix 1, reorganization of extracellular loop 2 and outward movement of the intracellular side of transmembrane helix 6, resulting in G-protein interaction and activation2-6. Here we solved the structure of a non-peptide agonist, TT-OAD2, bound to the glucagon-like peptide-1 (GLP-1) receptor. Our structure identified an unpredicted non-peptide agonist-binding pocket in which reorganization of extracellular loop 3 and transmembrane helices 6 and 7 manifests independently of direct ligand interaction within the deep transmembrane domain pocket. TT-OAD2 exhibits biased agonism, and kinetics of G-protein activation and signalling that are distinct from peptide agonists. Within the structure, TT-OAD2 protrudes beyond the receptor core to interact with the lipid or detergent, providing an explanation for the distinct activation kinetics that may contribute to the clinical efficacy of this compound series. This work alters our understanding of the events that drive the activation of class B receptors.

Cryo-EM Structure of the Human Amylin 1 Receptor in Complex with CGRP and Gs Protein.

Inhibition of calcitonin gene-related peptide (CGRP) or its cognate CGRP receptor (CGRPR) has arisen as a major breakthrough in the treatment of migraine. However, a second CGRP-responsive receptor exists, the amylin (Amy) 1 receptor (AMY1R), yet its involvement in the pathology of migraine is poorly understood. AMY1R and CGRPR are heterodimers consisting of receptor activity-modifying protein 1 (RAMP1) with the calcitonin receptor (CTR) and the calcitonin receptor-like receptor (CLR), respectively. Here, we present the structure of AMY1R in complex with CGRP and Gs protein and compare it with the reported structures of the AMY1R complex with rat amylin (rAmy) and the CGRPR in complex with CGRP. Despite similar protein backbones observed within the receptors and the N- and C-termini of the two peptides bound to the AMY1R complexes, they have distinct organization in the peptide midregions (the bypass motif) that is correlated with differences in the dynamics of the respective receptor extracellular domains. Moreover, divergent conformations of extracellular loop (ECL) 3, intracellular loop (ICL) 2, and ICL3 within the CTR and CLR protomers are evident when comparing the CGRP bound to the CGRPR and AMY1R, which influences the binding mode of CGRP. However, the conserved interactions made by the C-terminus of CGRP to the CGRPR and AMY1R are likely to account for cross-reactivity of nonpeptide CGRPR antagonists observed at AMY1R, which also extends to other clinically used CGRPR blockers, including antibodies.

Lipid-Dependent Activation of the Orphan G Protein-Coupled Receptor, GPR3.

The class A orphan G protein-coupled receptor (GPCR), GPR3, has been implicated in a variety of conditions, including Alzheimer's and premature ovarian failure. GPR3 constitutively couples with Gαs, resulting in the production of cAMP in cells. While tool compounds and several putative endogenous ligands have emerged for the receptor, its endogenous ligand, if it exists, remains a mystery. As novel potential drug targets, the structures of orphan GPCRs have been of increasing interest, revealing distinct modes of activation, including autoactivation, presence of constitutively activating mutations, or via cryptic ligands. Here, we present a cryo-electron microscopy (cryo-EM) structure of the orphan GPCR, GPR3 in complex with DNGαs and Gß1γ2. The structure revealed clear density for a lipid-like ligand that bound within an extended hydrophobic groove, suggesting that the observed "constitutive activity" was likely due to activation via a lipid that may be ubiquitously present. Analysis of conformational variance within the cryo-EM data set revealed twisting motions of the GPR3 transmembrane helices that appeared coordinated with changes in the lipid-like density. We propose a mechanism for the binding of a lipid to its putative orthosteric binding pocket linked to the GPR3 dynamics.

Structural and functional diversity among agonist-bound states of the GLP-1 receptor.

Recent advances in G-protein-coupled receptor (GPCR) structural elucidation have strengthened previous hypotheses that multidimensional signal propagation mediated by these receptors depends, in part, on their conformational mobility; however, the relationship between receptor function and static structures is inherently uncertain. Here, we examine the contribution of peptide agonist conformational plasticity to activation of the glucagon-like peptide 1 receptor (GLP-1R), an important clinical target. We use variants of the peptides GLP-1 and exendin-4 (Ex4) to explore the interplay between helical propensity near the agonist N terminus and the ability to bind to and activate the receptor. Cryo-EM analysis of a complex involving an Ex4 analog, the GLP-1R and Gs heterotrimer revealed two receptor conformers with distinct modes of peptide-receptor engagement. Our functional and structural data, along with molecular dynamics (MD) simulations, suggest that receptor conformational dynamics associated with flexibility of the peptide N-terminal activation domain may be a key determinant of agonist efficacy.

Structures of the human cholecystokinin 1 (CCK1) receptor bound to Gs and Gq mimetic proteins provide insight into mechanisms of G protein selectivity.

G protein-coupled receptors (GPCRs) are critical regulators of cellular function acting via heterotrimeric G proteins as their primary transducers with individual GPCRs capable of pleiotropic coupling to multiple G proteins. Structural features governing G protein selectivity and promiscuity are currently unclear. Here, we used cryo-electron microscopy (cryo-EM) to determine structures of the cholecystokinin (CCK) type 1 receptor (CCK1R) bound to the CCK peptide agonist, CCK-8 and 2 distinct transducer proteins, its primary transducer Gq, and the more weakly coupled Gs. As seen with other Gq/11-GPCR complexes, the Gq-α5 helix (αH5) bound to a relatively narrow pocket in the CCK1R core. Surprisingly, the backbone of the CCK1R and volume of the G protein binding pocket were essentially equivalent when Gs was bound, with the Gs αH5 displaying a conformation that arises from "unwinding" of the far carboxyl-terminal residues, compared to canonically Gs coupled receptors. Thus, integrated changes in the conformations of both the receptor and G protein are likely to play critical roles in the promiscuous coupling of individual GPCRs.

Active site metals mediate an oligomeric equilibrium in Plasmodium M17 aminopeptidases.

M17 leucyl aminopeptidases are metal-dependent exopeptidases that rely on oligomerization to diversify their functional roles. The M17 aminopeptidases from Plasmodium falciparum (PfA-M17) and Plasmodium vivax (Pv-M17) function as catalytically active hexamers to generate free amino acids from human hemoglobin and are drug targets for the design of novel antimalarial agents. However, the molecular basis for oligomeric assembly is not fully understood. In this study, we found that the active site metal ions essential for catalytic activity have a secondary structural role mediating the formation of active hexamers. We found that PfA-M17 and Pv-M17 exist in a metal-dependent dynamic equilibrium between active hexameric species and smaller inactive species that can be controlled by manipulating the identity and concentration of metals available. Mutation of residues involved in metal ion binding impaired catalytic activity and the formation of active hexamers. Structural resolution of Pv-M17 by cryoelectron microscopy and X-ray crystallography together with solution studies revealed that PfA-M17 and Pv-M17 bind metal ions and substrates in a conserved fashion, although Pv-M17 forms the active hexamer more readily and processes substrates faster than PfA-M17. On the basis of these studies, we propose a dynamic equilibrium between monomer ↔ dimer ↔ tetramer ↔ hexamer, which becomes directional toward the large oligomeric states with the addition of metal ions. This sophisticated metal-dependent dynamic equilibrium may apply to other M17 aminopeptidases and underpin the moonlighting capabilities of this enzyme family.

Structure of the PCBP2/stem-loop IV complex underlying translation initiation mediated by the poliovirus type I IRES.

The poliovirus type I IRES is able to recruit ribosomal machinery only in the presence of host factor PCBP2 that binds to stem-loop IV of the IRES. When PCBP2 is cleaved in its linker region by viral proteinase 3CD, translation initiation ceases allowing the next stage of replication to commence. Here, we investigate the interaction of PCBP2 with the apical region of stem-loop IV (SLIVm) of poliovirus RNA in its full-length and truncated form. CryoEM structure reconstruction of the full-length PCBP2 in complex with SLIVm solved to 6.1 Å resolution reveals a compact globular complex of PCBP2 interacting with the cruciform RNA via KH domains and featuring a prominent GNRA tetraloop. SEC-SAXS, SHAPE and hydroxyl-radical cleavage establish that PCBP2 stabilizes the SLIVm structure, but upon cleavage in the linker domain the complex becomes more flexible and base accessible. Limited proteolysis and REMSA demonstrate the accessibility of the linker region in the PCBP2/SLIVm complex and consequent loss of affinity of PCBP2 for the SLIVm upon cleavage. Together this study sheds light on the structural features of the PCBP2/SLIV complex vital for ribosomal docking, and the way in which this key functional interaction is regulated following translation of the poliovirus genome.

Structure of Pseudomonas aeruginosa ribosomes from an aminoglycoside-resistant clinical isolate.

Resistance to antibiotics has become a major threat to modern medicine. The ribosome plays a fundamental role in cell vitality by the translation of the genetic code into proteins; hence, it is a major target for clinically useful antibiotics. We report here the cryo-electron microscopy structures of the ribosome of a pathogenic aminoglycoside (AG)-resistant Pseudomonas aeruginosa strain, as well as of a nonresistance strain isolated from a cystic fibrosis patient. The structural studies disclosed defective ribosome complex formation due to a conformational change of rRNA helix H69, an essential intersubunit bridge, and a secondary binding site of the AGs. In addition, a stable conformation of nucleotides A1486 and A1487, pointing into helix h44, is created compared to a non-AG-bound ribosome. We suggest that altering the conformations of ribosomal protein uL6 and rRNA helix H69, which interact with initiation-factor IF2, interferes with proper protein synthesis initiation.

Cryo-electron microscopy structure of the glucagon receptor with a dual-agonist peptide.

Unimolecular dual agonists of the glucagon (GCG) receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R) are a new class of drugs that are potentially superior to GLP-1R-specific agonists for the management of metabolic disease. The dual-agonist, peptide 15 (P15), is a glutamic acid 16 analog of GCG with GLP-1 peptide substitutions between amino acids 17 and 24 that has potency equivalent to those of the cognate peptide agonists at the GCGR and GLP-1R. Here, we have used cryo-EM to solve the structure of an active P15-GCGR-Gs complex and compared this structure to our recently published structure of the GCGR-Gs complex bound to GCG. This comparison revealed that P15 has a reduced interaction with the first extracellular loop (ECL1) and the top of transmembrane segment 1 (TM1) such that there is increased mobility of the GCGR extracellular domain and at the C terminus of the peptide compared with the GCG-bound receptor. We also observed a distinct conformation of ECL3 and could infer increased mobility of the far N-terminal His-1 residue in the P15-bound structure. These regions of conformational variance in the two peptide-bound GCGR structures were also regions that were distinct between GCGR structures and previously published peptide-bound structures of the GLP-1R, suggesting that greater conformational dynamics may contribute to the increased efficacy of P15 in activation of the GLP-1R compared with GCG. The variable domains in this receptor have previously been implicated in biased agonism at the GLP-1R and could result in altered signaling of P15 at the GCGR compared with GCG.

Structure and Membrane Topography of the Vibrio-Type Secretin Complex from the Type 2 Secretion System of Enteropathogenic Escherichia coli.

The ß-barrel assembly machinery (BAM) complex is the core machinery for the assembly of ß-barrel membrane proteins, and inhibition of BAM complex activity is lethal to bacteria. Discovery of integral membrane proteins that are key to pathogenesis and yet do not require assistance from the BAM complex raises the question of how these proteins assemble into bacterial outer membranes. Here, we address this question through a structural analysis of the type 2 secretion system (T2SS) secretin from enteropathogenic Escherichia coli O127:H6 strain E2348/69. Long ß-strands assemble into a barrel extending 17 Å through and beyond the outer membrane, adding insight to how these extensive ß-strands are assembled into the E. coli outer membrane. The substrate docking chamber of this secretin is shown to be sufficient to accommodate the substrate mucinase SteC.IMPORTANCE In order to cause disease, bacterial pathogens inhibit immune responses and induce pathology that will favor their replication and dissemination. In Gram-negative bacteria, these key attributes of pathogenesis depend on structures assembled into or onto the outer membrane. One of these is the T2SS. The Vibrio-type T2SS mediates cholera toxin secretion in Vibrio cholerae, and in Escherichia coli O127:H6 strain E2348/69, the same machinery mediates secretion of the mucinases that enable the pathogen to penetrate intestinal mucus and thereby establish deadly infections.

Delivery of femtolitre droplets using surface acoustic wave based atomisation for cryo-EM grid preparation.

Cryo-Electron Microscopy (cryo-EM) has become an invaluable tool for structural biology. Over the past decade, the advent of direct electron detectors and automated data acquisition has established cryo-EM as a central method in structural biology. However, challenges remain in the reliable and efficient preparation of samples in a manner which is compatible with high time resolution. The delivery of sample onto the grid is recognized as a critical step in the workflow as it is a source of variability and loss of material due to the blotting which is usually required. Here, we present a method for sample delivery and plunge freezing based on the use of Surface Acoustic Waves to deploy 6-8 µm droplets to the EM grid. This method minimises the sample dead volume and ensures vitrification within 52.6 ms from the moment the sample leaves the microfluidics chip. We demonstrate a working protocol to minimize the atomised volume and apply it to plunge freeze three different samples and provide proof that no damage occurs due to the interaction between the sample and the acoustic waves.

Rapid near-atomic resolution single-particle 3D reconstruction with SIMPLE.

Cryogenic electron microscopy (cryo-EM) and single-particle analysis enables determination of near-atomic resolution structures of biological molecules. However, large computational requirements limit throughput and rapid testing of new image processing tools. We developed PRIME, an algorithm part of the SIMPLE software suite, for determination of the relative 3D orientations of single-particle projection images. PRIME has primarily found use for generation of an initial ab initio 3D reconstruction. Here we show that the strategy behind PRIME, iterative estimation of per-particle orientation distributions with stochastic hill climbing, provides a competitive approach to near-atomic resolution single-particle 3D reconstruction. A number of mathematical techniques for accelerating the convergence rate are introduced, leading to a speedup of nearly two orders of magnitude. We benchmarked our developments on numerous publicly available data sets and conclude that near-atomic resolution ab initio 3D reconstructions can be obtained with SIMPLE in a matter of hours, using standard over-the-counter CPU workstations.

Crystal structure of the synergistic antibiotic pair, lankamycin and lankacidin, in complex with the large ribosomal subunit.

The structures of the large ribosomal subunit of Deinococcus radiodurans (D50S) in complex with the antibiotic lankamycin (3.2 Å) and a double antibiotic complex of lankamycin and lankacidin C (3.45 Å) have been determined, in continuation of previous crystallographic studies on lankacidin-D50S complex. These two drugs have been previously reported to inhibit ribosomal function with mild synergistic effect. Lankamycin, a member of the macrolide family, binds in a similar manner to erythromycin. However, when in complex with lankacidin, lankamycin is located so that it can form interactions with lankacidin in the adjacent ribosomal binding site. When compared to the well-documented synergistic antibiotics, Streptogramins A and B, the pair of lankacidin and lankamycin bind in similar sites, the peptidyl transferase center and nascent peptide exit tunnel, respectively. Herein, we discuss the structural basis for antibiotic synergism and highlight the key factors involved in ribosomal inhibition.

The evolution of new lipoprotein subunits of the bacterial outer membrane BAM complex.

The ß-barrel assembly machine (BAM) complex is an essential feature of all bacteria with an outer membrane. The core subunit of the BAM complex is BamA and, in Escherichia coli, four lipoprotein subunits: BamB, BamC, BamD and BamE, also function in the BAM complex. Hidden Markov model analysis was used to comprehensively assess the distribution of subunits of the BAM lipoproteins across all subclasses of proteobacteria. A patchwork distribution was detected which is readily reconciled with the evolution of the α-, ß-, γ-, δ- and ε-proteobacteria. Our findings lead to a proposal that the ancestral BAM complex was composed of two subunits: BamA and BamD, and that BamB, BamC and BamE evolved later in a distinct sequence of events. Furthermore, in some lineages novel lipoproteins have evolved instead of the lipoproteins found in E. coli. As an example of this concept, we show that no known species of α-proteobacteria has a homologue of BamC. However, purification of the BAM complex from the model α-proteobacterium Caulobacter crescentus identified a novel subunit we refer to as BamF, which has a conserved sequence motif related to sequences found in BamC. BamF and BamD can be eluted from the BAM complex under similar conditions, mirroring the BamC:D module seen in the BAM complex of γ-proteobacteria such as E. coli.