344 Citations
Generation of an electrochemical proton gradient is the first step of cell bioenergetics In prokaryotes the gradient is created by outward membrane protein proton pumps Inward plasma membrane native proton pumps are yet unknown We describe comprehensive functional studies of the representatives of the yet noncharacterized xenorhodopsins from Nanohaloarchaea family of microbial rhodopsins They are inward proton pumps as we demonstrate in model membrane systems Escherichia coli cells human embryonic kidney cells neuroblastoma cells and rat hippocampal neuronal cells We also solved the structure of a xenorhodopsin from the nanohalosarchaeon Nanosalina NsXeR and suggest a mechanism of inward proton pumping ... More
Generation of an electrochemical proton gradient is the first step of cell bioenergetics. In prokaryotes, the gradient is created by outward membrane protein proton pumps. Inward plasma membrane native proton pumps are yet unknown. We describe comprehensive functional studies of the representatives of the yet noncharacterized xenorhodopsins from Nanohaloarchaea family of microbial rhodopsins. They are inward proton pumps as we demonstrate in model membrane systems, Escherichia coli cells, human embryonic kidney cells, neuroblastoma cells, and rat hippocampal neuronal cells. We also solved the structure of a xenorhodopsin from the nanohalosarchaeon Nanosalina (NsXeR) and suggest a mechanism of inward proton pumping. We demonstrate that the NsXeR is a powerful pump, which is able to elicit action potentials in rat hippocampal neuronal cells up to their maximal intrinsic firing frequency. Hence, inwardly directed proton pumps are suitable for light-induced remote control of neurons, and they are an alternative to the well-known cation-selective channelrhodopsins. Less
NADH and NADPH are redox coenzymes broadly required for energy metabolism biosynthesis and detoxification Despite detailed knowledge of specific enzymes and pathways that utilize these coenzymes a holistic understanding of the regulation and compartmentalization of NADH and NADPH-dependent pathways is lacking in part because of a lack of tools with which to investigate them in living cells We previously reported the use of the naturally occurring Lactobacillus brevis H O-forming NADH oxidase LbNOX as a genetic tool for manipulation of the NAD NADH ratio in human cells Here we present TPNOX triphosphopyridine nucleotide oxidase a rationally designed and engineered mutant ... More
NADH and NADPH are redox coenzymes broadly required for energy metabolism, biosynthesis and detoxification. Despite detailed knowledge of specific enzymes and pathways that utilize these coenzymes, a holistic understanding of the regulation and compartmentalization of NADH and NADPH-dependent pathways is lacking, in part because of a lack of tools with which to investigate them in living cells. We previously reported the use of the naturally occurring Lactobacillus brevis H2O-forming NADH oxidase (LbNOX) as a genetic tool for manipulation of the NAD+/NADH ratio in human cells. Here we present TPNOX (triphosphopyridine nucleotide oxidase), a rationally designed and engineered mutant of LbNOX that is strictly specific towards NADPH. We characterize the effects of TPNOX expression on cellular metabolism and use it in combination with LbNOX to show how the redox states of mitochondrial NADPH and NADH pools are connected. Less
Monoclonal antibodies provide an attractive alternative to small-molecule therapies for a wide range of diseases Given the importance of G protein-coupled receptors GPCRs as pharmaceutical targets there has been an immense interest in developing therapeutic monoclonal antibodies that act on GPCRs Here we present the - resolution structure of a complex between the human -hydroxytryptamine B -HT B receptor and an antibody Fab fragment bound to the extracellular side of the receptor determined by serial femtosecond crystallography with an X-ray free-electron laser The antibody binds to a D epitope of the receptor that includes all three extracellular loops The -HT ... More
Monoclonal antibodies provide an attractive alternative to small-molecule therapies for a wide range of diseases. Given the importance of G protein-coupled receptors (GPCRs) as pharmaceutical targets, there has been an immense interest in developing therapeutic monoclonal antibodies that act on GPCRs. Here we present the 3.0-Å resolution structure of a complex between the human 5-hydroxytryptamine 2B (5-HT2B) receptor and an antibody Fab fragment bound to the extracellular side of the receptor, determined by serial femtosecond crystallography with an X-ray free-electron laser. The antibody binds to a 3D epitope of the receptor that includes all three extracellular loops. The 5-HT2B receptor is captured in a well-defined active-like state, most likely stabilized by the crystal lattice. The structure of the complex sheds light on the mechanism of selectivity in extracellular recognition of GPCRs by monoclonal antibodies. Less
The cannabinoid receptor CB is the principal target of the psychoactive constituent of marijuana the partial agonist -tetrahydrocannabinol -THC Here we report two agonist-bound crystal structures of human CB in complex with a tetrahydrocannabinol AM and a hexahydrocannabinol AM at and resolution respectively The two CB agonist complexes reveal important conformational changes in the overall structure relative to the antagonist-bound state including a reduction in the volume of the ligand-binding pocket and an increase in the surface area of the G-protein-binding region In addition a twin toggle switch of Phe and Trp superscripts denote Ballesteros Weinstein numbering is experimentally observed ... More
The cannabinoid receptor 1 (CB1) is the principal target of the psychoactive constituent of marijuana, the partial agonist Δ9-tetrahydrocannabinol (Δ9-THC)1. Here we report two agonist-bound crystal structures of human CB1 in complex with a tetrahydrocannabinol (AM11542) and a hexahydrocannabinol (AM841) at 2.80 Å and 2.95 Å resolution, respectively. The two CB1–agonist complexes reveal important conformational changes in the overall structure, relative to the antagonist-bound state2, including a 53% reduction in the volume of the ligand-binding pocket and an increase in the surface area of the G-protein-binding region. In addition, a ‘twin toggle switch’ of Phe2003.36 and Trp3566.48 (superscripts denote Ballesteros–Weinstein numbering3) is experimentally observed and appears to be essential for receptor activation. The structures reveal important insights into the activation mechanism of CB1 and provide a molecular basis for predicting the binding modes of Δ9-THC, and endogenous and synthetic cannabinoids. The plasticity of the binding pocket of CB1 seems to be a common feature among certain class A G-protein-coupled receptors. These findings should inspire the design of chemically diverse ligands with distinct pharmacological properties. Less
Clinical studies indicate that partial agonists of the G-protein-coupled free fatty acid receptor GPR enhance glucose-dependent insulin secretion and represent a potential mechanism for the treatment of type diabetes mellitus Full allosteric agonists AgoPAMs of GPR bind to a site distinct from partial agonists and can provide additional efficacy We report the - crystal structure of human GPR hGPR in complex with both the partial agonist MK- and an AgoPAM which exposes a novel lipid-facing AgoPAM-binding pocket outside the transmembrane helical bundle Comparison with an additional - structure of the hGPR MK- binary complex reveals an induced-fit conformational coupling between ... More
Clinical studies indicate that partial agonists of the G-protein-coupled, free fatty acid receptor 1 GPR40 enhance glucose-dependent insulin secretion and represent a potential mechanism for the treatment of type 2 diabetes mellitus. Full allosteric agonists (AgoPAMs) of GPR40 bind to a site distinct from partial agonists and can provide additional efficacy. We report the 3.2-Å crystal structure of human GPR40 (hGPR40) in complex with both the partial agonist MK-8666 and an AgoPAM, which exposes a novel lipid-facing AgoPAM-binding pocket outside the transmembrane helical bundle. Comparison with an additional 2.2-Å structure of the hGPR40–MK-8666 binary complex reveals an induced-fit conformational coupling between the partial agonist and AgoPAM binding sites, involving rearrangements of the transmembrane helices 4 and 5 (TM4 and TM5) and transition of the intracellular loop 2 (ICL2) into a short helix. These conformational changes likely prime GPR40 to a more active-like state and explain the binding cooperativity between these ligands. Less
PP C phosphatases control biological processes including stress responses development and cell division in all kingdoms of life Diverse regulatory domains adapt PP C phosphatases to specific functions but how these domains control phosphatase activity was unknown We present structures representing active and inactive states of the PP C phosphatase SpoIIE from Bacillus subtilis Based on structural analyses and genetic and biochemical experiments we identify an a-helical switch that shifts a carbonyl oxygen into the active site to coordinate a metal cofactor Our analysis indicates that this switch is widely conserved among PP C family members serving as a platform ... More
PP2C phosphatases control biological processes including stress responses, development, and cell division in all kingdoms of life. Diverse regulatory domains adapt PP2C phosphatases to specific functions, but how these domains control phosphatase activity was unknown. We present structures representing active and inactive states of the PP2C phosphatase SpoIIE from Bacillus subtilis. Based on structural analyses and genetic and biochemical experiments, we identify an a-helical switch that shifts a carbonyl oxygen into the active site to coordinate a metal cofactor. Our analysis indicates that this switch is widely conserved among PP2C family members, serving as a platform to control phosphatase activity in response to diverse inputs. Remarkably, the switch is shared with proteasomal proteases, which we identify as evolutionary and structural relatives of PP2C phosphatases. Although these proteases use an unrelated catalytic mechanism, rotation of equivalent helices controls protease activity by movement of the equivalent carbonyl oxygen into the active site. Less
The Smoothened receptor SMO belongs to the Class Frizzled of the G protein-coupled receptor GPCR superfamily constituting a key component of the Hedgehog signalling pathway Here we report the crystal structure of the multi-domain human SMO bound and stabilized by a designed tool ligand TC using an X-ray free-electron laser source at The structure reveals a precise arrangement of three distinct domains a seven-transmembrane helices domain TMD a hinge domain HD and an intact extracellular cysteine-rich domain CRD This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation By combining the structural data ... More
The Smoothened receptor (SMO) belongs to the Class Frizzled of the G protein-coupled receptor (GPCR) superfamily, constituting a key component of the Hedgehog signalling pathway. Here we report the crystal structure of the multi-domain human SMO, bound and stabilized by a designed tool ligand TC114, using an X-ray free-electron laser source at 2.9 Å. The structure reveals a precise arrangement of three distinct domains: a seven-transmembrane helices domain (TMD), a hinge domain (HD) and an intact extracellular cysteine-rich domain (CRD). This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation. By combining the structural data, molecular dynamics simulation, and hydrogen-deuterium-exchange analysis, we demonstrate that transmembrane helix VI, extracellular loop 3 and the HD play a central role in transmitting the signal employing a unique GPCR activation mechanism, distinct from other multi-domain GPCRs. Less
The glucagon-like peptide- receptor GLP- R and the glucagon receptor GCGR are members of the secretin-like class B family of G-protein-coupled receptors GPCRs and have opposing physiological roles in insulin release and glucose homeostasis The treatment of type diabetes requires positive modulation of GLP- R to inhibit glucagon secretion and stimulate insulin secretion in a glucose-dependent manner Here we report crystal structures of the human GLP- R transmembrane domain in complex with two different negative allosteric modulators PF- and NNC at and resolution respectively The structures reveal a common binding pocket for negative allosteric modulators present in both GLP- R ... More
The glucagon-like peptide-1 receptor (GLP-1R) and the glucagon receptor (GCGR) are members of the secretin-like class B family of G-protein-coupled receptors (GPCRs) and have opposing physiological roles in insulin release and glucose homeostasis1. The treatment of type 2 diabetes requires positive modulation of GLP-1R to inhibit glucagon secretion and stimulate insulin secretion in a glucose-dependent manner2. Here we report crystal structures of the human GLP-1R transmembrane domain in complex with two different negative allosteric modulators, PF-06372222 and NNC0640, at 2.7 and 3.0 Å resolution, respectively. The structures reveal a common binding pocket for negative allosteric modulators, present in both GLP-1R and GCGR3 and located outside helices V–VII near the intracellular half of the receptor. The receptor is in an inactive conformation with compounds that restrict movement of the intracellular tip of helix VI, a movement that is generally associated with activation mechanisms in class A GPCRs4,5,6. Molecular modelling and mutagenesis studies indicate that agonist positive allosteric modulators target the same general region, but in a distinct sub-pocket at the interface between helices V and VI, which may facilitate the formation of an intracellular binding site that enhances G-protein coupling. Less
Lipidic cubic phase LCP has been widely recognized as a promising membrane-mimicking matrix for biophysical studies of membrane proteins and their crystallization in a lipidic environment Application of this material to a wide variety of membrane proteins however is hindered due to a limited number of available host lipids mostly monoacylglycerols MAGs Here we designed synthesized and characterized a series of chemically stable lipids resistant to hydrolysis with properties complementary to the widely used MAGs In order to assess their potential to serve as host lipids for crystallization we characterized the phase properties and lattice parameters of mesophases made of ... More
Lipidic cubic phase (LCP) has been widely recognized as a promising membrane-mimicking matrix for biophysical studies of membrane proteins and their crystallization in a lipidic environment. Application of this material to a wide variety of membrane proteins, however, is hindered due to a limited number of available host lipids, mostly monoacylglycerols (MAGs). Here, we designed, synthesized and characterized a series of chemically stable lipids resistant to hydrolysis, with properties complementary to the widely used MAGs. In order to assess their potential to serve as host lipids for crystallization, we characterized the phase properties and lattice parameters of mesophases made of two most promising lipids at a variety of different conditions by polarized light microscopy and small-angle X-ray scattering. Both lipids showed remarkable chemical stability and an extended LCP region in the phase diagram covering a wide range of temperatures down to 4 °C. One of these lipids has been used for crystallization and structure determination of a prototypical membrane protein bacteriorhodopsin at 4 °C and 20 °C. Less
We describe a fast easy and potentially universal method for the de novo solution of the crystal structures of membrane proteins via iodide single-wavelength anomalous diffraction I-SAD The potential universality of the method is based on a common feature of membrane proteins the availability at the hydrophobic-hydrophilic interface of positively charged amino acid residues with which iodide strongly interacts We demonstrate the solution using I-SAD of four crystal structures representing different classes of membrane proteins including a human G protein coupled receptor GPCR and we show that I-SAD can be applied using data collection strategies based on either standard or ... More
We describe a fast, easy, and potentially universal method for the de novo solution of the crystal structures of membrane proteins via iodide–single-wavelength anomalous diffraction (I-SAD). The potential universality of the method is based on a common feature of membrane proteins—the availability at the hydrophobic-hydrophilic interface of positively charged amino acid residues with which iodide strongly interacts. We demonstrate the solution using I-SAD of four crystal structures representing different classes of membrane proteins, including a human G protein–coupled receptor (GPCR), and we show that I-SAD can be applied using data collection strategies based on either standard or serial x-ray crystallography techniques. Less
Angiotensin II receptors AT R and AT R serve as key components of the renin-angiotensin-aldosterone system While AT R plays a central role in the regulation of blood pressure the function of AT R is enigmatic with a variety of reported effects To elucidate the mechanisms for the functional diversity and ligand selectivity between these receptors we report crystal structures of the human AT R bound to an AT R-selective and an AT R AT R-dual ligand respectively capturing the receptor in an active-like conformation Unexpectedly helix VIII was found in a non-canonical position stabilizing the active-like state but at ... More
Angiotensin II receptors, AT1R and AT2R, serve as key components of the renin-angiotensin-aldosterone system. While AT1R plays a central role in the regulation of blood pressure, the function of AT2R is enigmatic with a variety of reported effects. To elucidate the mechanisms for the functional diversity and ligand selectivity between these receptors, we report crystal structures of the human AT2R bound to an AT2R-selective and an AT1R/AT2R-dual ligand, respectively, capturing the receptor in an active-like conformation. Unexpectedly, helix VIII was found in a non-canonical position, stabilizing the active-like state, but at the same time preventing the recruitment of G proteins/β-arrestins, in agreement with the lack of signaling responses in standard cellular assays. Structure-activity relationship, docking and mutagenesis studies revealed the interactions critical for ligand binding and selectivity. Our results thus provide insights into the structural basis for distinct functions of the angiotensin receptors, and may guide the design of novel selective ligands. Less
The Haemophilus surface fibril Hsf is an unusually large trimeric autotransporter adhesin TAA expressed by the most virulent strains of H influenzae Hsf is known to mediate adhesion between pathogen and host allowing the establishment of potentially deadly diseases such as epiglottitis meningitis and pneumonia While recent research has suggested that this TAA might adopt a novel hairpin-like architecture the characterization of Hsf has been limited to in silico modelling and electron micrographs with no high-resolution structural data available Here the crystal structure of Hsf putative domain PD is reported at resolution The structure corrects the previous domain annotation by ... More
The Haemophilus surface fibril (Hsf) is an unusually large trimeric autotransporter adhesin (TAA) expressed by the most virulent strains of H. influenzae. Hsf is known to mediate adhesion between pathogen and host, allowing the establishment of potentially deadly diseases such as epiglottitis, meningitis and pneumonia. While recent research has suggested that this TAA might adopt a novel ‘hairpin-like’ architecture, the characterization of Hsf has been limited to in silico modelling and electron micrographs, with no high-resolution structural data available. Here, the crystal structure of Hsf putative domain 1 (PD1) is reported at 3.3 Å resolution. The structure corrects the previous domain annotation by revealing the presence of an unexpected N-terminal TrpRing domain. PD1 represents the first Hsf domain to be solved, and thus paves the way for further research on the ‘hairpin-like’ hypothesis. Less
Membrane-like nanodiscs ND have become an important tool for the cell-free expression solubilization folding and in vitro structural and functional studies of membrane proteins MPs Direct crystallization of MPs embedded in NDs would be of high importance for structural biology However despite considerable efforts we have been as yet unable to obtain crystals suitable for X-ray crystallography In the present work we show that an ND-trapped MP can be transferred into the cubic phase and crystallized in meso Bacteriorhodopsin BR reconstituted into nanodiscs was mixed with a lipidic mesophase and crystallization was induced by adding a precipitant The resulting crystals ... More
Membrane-like nanodiscs (ND) have become an important tool for the cell-free expression, solubilization, folding, and in vitro structural and functional studies of membrane proteins (MPs). Direct crystallization of MPs embedded in NDs would be of high importance for structural biology. However, despite considerable efforts we have been as yet unable to obtain crystals suitable for X-ray crystallography. In the present work, we show that an ND-trapped MP can be transferred into the cubic phase and crystallized in meso. Bacteriorhodopsin (BR) reconstituted into nanodiscs was mixed with a lipidic mesophase and crystallization was induced by adding a precipitant. The resulting crystals diffract beyond 1.8 Å. The structure of BR was solved at 1.9 Å and found to be indistinguishable from previous structures obtained with the protein solubilized in detergent. We suggest the proposed protocol of in meso crystallization to be generally applicable to ND-trapped MPs. Less
Cannabinoid receptor CB is the principal target of -tetrahydrocannabinol THC a psychoactive chemical from Cannabis sativa with a wide range of therapeutic applications and a long history of recreational use CB is activated by endocannabinoids and is a promising therapeutic target for pain management inflammation obesity and substance abuse disorders Here we present the crystal structure of human CB in complex with AM a stabilizing antagonist synthesized and characterized for this structural study The structure of the CB -AM complex reveals key features of the receptor and critical interactions for antagonist binding In combination with functional studies and molecular modeling ... More
Cannabinoid receptor 1 (CB1) is the principal target of ?9-tetrahydrocannabinol (THC), a psychoactive chemical from Cannabis sativa with a wide range of therapeutic applications and a long history of recreational use. CB1 is activated by endocannabinoids and is a promising therapeutic target for pain management, inflammation, obesity, and substance abuse disorders. Here, we present the 2.8 � crystal structure of human CB1 in complex with AM6538, a stabilizing antagonist, synthesized and characterized for this structural study. The structure of the CB1-AM6538 complex reveals key features of the receptor and critical interactions for antagonist binding. In combination with functional studies and molecular modeling, the structure provides insight into the binding mode of naturally occurring CB1 ligands, such as THC, and synthetic cannabinoids. This enhances our understanding of the molecular basis for the physiological functions of CB1 and provides new opportunities for the design of next-generation CB1-targeting pharmaceuticals. Less
Human metapneumovirus hMPV is a paramyxovirus that is a common cause of bronchiolitis and pneumonia in children less than five years of age The hMPV fusion F glycoprotein is the primary target of neutralizing antibodies and is thus a critical vaccine antigen To facilitate structure-based vaccine design we stabilized the ectodomain of the hMPV F protein in the postfusion conformation and determined its structure to a resolution of by X-ray crystallography The structure resembles an elongated cone and is very similar to the postfusion F protein from the related human respiratory syncytial virus hRSV In contrast significant differences were apparent ... More
Human metapneumovirus (hMPV) is a paramyxovirus that is a common cause of bronchiolitis and pneumonia in children less than five years of age. The hMPV fusion (F) glycoprotein is the primary target of neutralizing antibodies and is thus a critical vaccine antigen. To facilitate structure-based vaccine design, we stabilized the ectodomain of the hMPV F protein in the postfusion conformation and determined its structure to a resolution of 3.3 Å by X-ray crystallography. The structure resembles an elongated cone and is very similar to the postfusion F protein from the related human respiratory syncytial virus (hRSV). In contrast, significant differences were apparent with the postfusion F proteins from other paramyxoviruses, such as human parainfluenza type 3 (hPIV3) and Newcastle disease virus (NDV). The high similarity of hMPV and hRSV postfusion F in two antigenic sites targeted by neutralizing antibodies prompted us to test for antibody cross-reactivity. The widely used monoclonal antibody 101F, which binds to antigenic site IV of hRSV F, was found to cross-react with hMPV postfusion F and neutralize both hRSV and hMPV. Despite the cross-reactivity of 101F and the reported cross-reactivity of two other antibodies, 54G10 and MPE8, we found no detectable cross-reactivity in the polyclonal antibody responses raised in mice against the postfusion forms of either hMPV or hRSV F. The postfusion-stabilized hMPV F protein did, however, elicit high titers of hMPV-neutralizing activity, suggesting that it could serve as an effective subunit vaccine. Structural insights from these studies should be useful for designing novel immunogens able to induce wider cross-reactive antibody responses. Less
Serial femtosecond X-ray crystallography SFX using an X-ray free electron laser XFEL is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins including G-protein coupled receptors GPCRs which often only produce microcrystals An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in Here we report the deposition of the XFEL data and provide further details on crystallization XFEL data collection and analysis structure determination and the validation of the structural model The rhodopsin-arrestin crystal structure solved with ... More
Serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in. Here we report the deposition of the XFEL data and provide further details on crystallization, XFEL data collection and analysis, structure determination, and the validation of the structural model. The rhodopsin-arrestin crystal structure solved with SFX represents the first near-atomic resolution structure of a GPCR-arrestin complex, provides structural insights into understanding of arrestin-mediated GPCR signaling, and demonstrates the great potential of this SFX-XFEL technology for accelerating crystal structure determination of challenging proteins and protein complexes. Less
Understanding the mechanism by which ligands impact receptor conformational equilibria is key in accelerating membrane protein structural biology In the case of G protein-coupled receptors GPCRs we currently pursue a brute force approach for identifying ligands that stabilize receptors and facilitate crystallogenesis The nociceptin orphanin FQ peptide receptor NOP is a member of the opioid receptor subfamily of GPCRs for which many structurally diverse ligands are available for screening We observed that antagonist potency is correlated with a ligand s ability to induce receptor stability Tm and crystallogenesis Using this screening strategy we solved two structures of NOP in complex ... More
Understanding the mechanism by which ligands impact receptor conformational equilibria is key in accelerating membrane protein structural biology. In the case of G protein-coupled receptors (GPCRs) we currently pursue a brute force approach for identifying ligands that stabilize receptors and facilitate crystallogenesis. The nociceptin/orphanin FQ peptide receptor (NOP) is a member of the opioid receptor subfamily of GPCRs for which many structurally diverse ligands are available for screening. We observed that antagonist potency is correlated with a ligand’s ability to induce receptor stability (Tm) and crystallogenesis. Using this screening strategy, we solved two structures of NOP in complex with top candidate ligands SB-612111 and C-35. Docking studies indicate that while potent, stabilizing antagonists strongly favor a single binding orientation, less potent ligands can adopt multiple binding modes, contributing to their low Tm values. These results suggest a mechanism for ligand-aided crystallogenesis whereby potent antagonists stabilize a single ligand-receptor conformational pair. Less
Angiotensin II type receptor AT R is the primary blood pressure regulator AT R blockers ARBs have been widely used in clinical settings as anti-hypertensive drugs and share a similar chemical scaffold although even minor variations can lead to distinct therapeutic efficacies toward cardiovascular etiologies The structural basis for AT R modulation by different peptide and non-peptide ligands has remained elusive Here we report the crystal structure of the human AT R in complex with an inverse agonist olmesartan BenicarTM a highly potent anti-hypertensive drug Olmesartan is anchored to the receptor primarily by the residues Tyr- Trp- and Arg- ECL ... More
Angiotensin II type 1 receptor (AT1R) is the primary blood pressure regulator. AT1R blockers (ARBs) have been widely used in clinical settings as anti-hypertensive drugs and share a similar chemical scaffold, although even minor variations can lead to distinct therapeutic efficacies toward cardiovascular etiologies. The structural basis for AT1R modulation by different peptide and non-peptide ligands has remained elusive. Here, we report the crystal structure of the human AT1R in complex with an inverse agonist olmesartan (BenicarTM), a highly potent anti-hypertensive drug. Olmesartan is anchored to the receptor primarily by the residues Tyr-351.39, Trp-842.60, and Arg-167ECL2, similar to the antagonist ZD7155, corroborating a common binding mode of different ARBs. Using docking simulations and site-directed mutagenesis, we identified specific interactions between AT1R and different ARBs, including olmesartan derivatives with inverse agonist, neutral antagonist, or agonist activities. We further observed that the mutation N1113.35A in the putative sodium-binding site affects binding of the endogenous peptide agonist angiotensin II but not the β-arrestin-biased peptide TRV120027. Less
A number of Gram-positive bacteria produce a class of bacteriocins called lantibiotics These lantibiotics are ribosomally synthesized peptides that possess high antimicrobial activity against Gram-positive bacteria including clinically challenging pathogens and are therefore potential alternatives to antibiotics All lantibiotic producer strains and some Gram-positive non producer strains express protein systems to circumvent a suicidal effect or to become resistant respectively Two-component systems consisting of a response regulator and a histidine kinase upregulate the expression of these proteins One of the best-characterized lantibiotics is nisin which is produced by Lactococcus lactis and possesses bactericidal activity against various Gram-positive bacteria including some ... More
A number of Gram-positive bacteria produce a class of bacteriocins called ‘lantibiotics’. These lantibiotics are ribosomally synthesized peptides that possess high antimicrobial activity against Gram-positive bacteria, including clinically challenging pathogens, and are therefore potential alternatives to antibiotics. All lantibiotic producer strains and some Gram-positive nonproducer strains express protein systems to circumvent a suicidal effect or to become resistant, respectively. Two-component systems consisting of a response regulator and a histidine kinase upregulate the expression of these proteins. One of the best-characterized lantibiotics is nisin, which is produced by Lactococcus lactis and possesses bactericidal activity against various Gram-positive bacteria, including some human pathogenic strains. Within many human pathogenic bacterial strains inherently resistant to nisin, a response regulator, NsrR, has been identified which regulates the expression of proteins involved in nisin resistance. In the present study, an expression and purification protocol was established for the NsrR protein from Streptococcus agalactiae COH1. The protein was successfully crystallized using the vapour-diffusion method, resulting in crystals that diffracted X-rays to 1.4 Å resolution. Less
G protein-coupled receptors GPCRs signal primarily through G proteins or arrestins Arrestin binding to GPCRs blocks G protein interaction and redirects signaling to numerous G protein-independent pathways Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin determined by serial femtosecond X-ray laser crystallography Together with extensive biochemical and mutagenesis data the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements including TM and Helix to recruit arrestin Correspondingly arrestin adopts the pre-activated conformation with a rotation between the ... More
G protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signaling to numerous G protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly, in which rhodopsin uses distinct structural elements, including TM7 and Helix 8 to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ~20° rotation between the N- and C- domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signaling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology. Less
Macromolecular X-ray crystallography has been the primary methodology for determining the three-dimensional structures of proteins nucleic acids and viruses Structural information has paved the way for structure-guided drug discovery and laid the foundations for structural bioinformatics However X-ray crystallography still has a few fundamental limitations some of which may be overcome and complemented using emerging methods and technologies in other areas of structural biology
Heterologous overexpression of functional membrane proteins is a major bottleneck of structural biology Bacteriorhodopsin from Halobium salinarum bR is a striking example of the difficulties in membrane protein overexpression We suggest a general approach with a finite number of steps which allows one to localize the underlying problem of poor expression of a membrane protein using bR as an example Our approach is based on constructing chimeric proteins comprising parts of a protein of interest and complementary parts of a homologous protein demonstrating advantageous expression This complementary protein approach allowed us to increase bR expression by two orders of magnitude ... More
Heterologous overexpression of functional membrane proteins is a major bottleneck of structural biology. Bacteriorhodopsin from Halobium salinarum (bR) is a striking example of the difficulties in membrane protein overexpression. We suggest a general approach with a finite number of steps which allows one to localize the underlying problem of poor expression of a membrane protein using bR as an example. Our approach is based on constructing chimeric proteins comprising parts of a protein of interest and complementary parts of a homologous protein demonstrating advantageous expression. This complementary protein approach allowed us to increase bR expression by two orders of magnitude through the introduction of two silent mutations into bR coding DNA. For the first time the high quality crystals of bR expressed in E. Coli were obtained using the produced protein. The crystals obtained with in meso nanovolume crystallization diffracted to 1.67 Å. Less
Nisin is a -amino-acid antimicrobial peptide produced by Lactococcus lactis belonging to the class of lantibiotics Nisin displays a high bactericidal activity against various Gram-positive bacteria including some human-pathogenic strains However there are some nisin-non-producing strains that are naturally resistant owing to the presence of the nsr gene within their genome The encoded protein NSR cleaves off the last six amino acids of nisin thereby reducing its bactericidal efficacy An expression and purification protocol has been established for the NSR protein from Streptococcus agalactiae COH The protein was successfully crystallized using the vapour-diffusion method in hanging and sitting drops resulting ... More
Nisin is a 34-amino-acid antimicrobial peptide produced by Lactococcus lactis belonging to the class of lantibiotics. Nisin displays a high bactericidal activity against various Gram-positive bacteria, including some human-pathogenic strains. However, there are some nisin-non-producing strains that are naturally resistant owing to the presence of the nsr gene within their genome. The encoded protein, NSR, cleaves off the last six amino acids of nisin, thereby reducing its bactericidal efficacy. An expression and purification protocol has been established for the NSR protein from Streptococcus agalactiae COH1. The protein was successfully crystallized using the vapour-diffusion method in hanging and sitting drops, resulting in crystals that diffracted X-rays to 2.8 and 2.2 Å, respectively. Less
Angiotensin II type receptor AT R is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance Although several anti-hypertensive drugs have been developed as AT R blockers ARBs the structural basis for AT R ligand-binding and regulation has remained elusive mostly due to the difficulties of growing high quality crystals for structure determination using synchrotron radiation By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser we successfully determined the room-temperature crystal structure of the human AT R in complex with its selective antagonist ZD at resolution The AT R-ZD ... More
Angiotensin II type 1 receptor (AT1R) is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance. Although several anti-hypertensive drugs have been developed as AT1R blockers (ARBs), the structural basis for AT1R ligand-binding and regulation has remained elusive, mostly due to the difficulties of growing high quality crystals for structure determination using synchrotron radiation. By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser, we successfully determined the room-temperature crystal structure of the human AT1R in complex with its selective antagonist ZD7155 at 2.9 � resolution. The AT1R-ZD7155 complex structure revealed key structural features of AT1R and critical interactions for ZD7155 binding. Docking simulations of the clinically used ARBs into the AT1R structure further elucidated both the common and distinct binding modes for these anti-hypertensive drugs. Our results thereby provide fundamental insights into AT1R structure-function relationship and structure-based drug design. Less
Recently the first known light-driven sodium pumps from the microbial rhodopsin family were discovered We have solved the structure of one of them Krokinobacter eikastus rhodopsin KR in the monomeric blue state and in two pentameric red states at resolutions of and and respectively The structures reveal the ion-translocation pathway and show that the sodium ion is bound outside the protein at the oligomerization interface that the ion-release cavity is capped by a unique N-terminal -helix and that the ion-uptake cavity is unexpectedly large and open to the surface Obstruction of the cavity with the mutation G F imparts KR ... More
Recently, the first known light-driven sodium pumps, from the microbial rhodopsin family, were discovered. We have solved the structure of one of them, Krokinobacter eikastus rhodopsin 2 (KR2), in the monomeric blue state and in two pentameric red states, at resolutions of 1.45 Å and 2.2 and 2.8 Å, respectively. The structures reveal the ion-translocation pathway and show that the sodium ion is bound outside the protein at the oligomerization interface, that the ion-release cavity is capped by a unique N-terminal α-helix and that the ion-uptake cavity is unexpectedly large and open to the surface. Obstruction of the cavity with the mutation G263F imparts KR2 with the ability to pump potassium. These results pave the way for the understanding and rational design of cation pumps with new specific properties valuable for optogenetics. Less
Crystallization of integral membrane proteins MPs is notoriously difficult given their poor stability outside native membrane environment and due to the interference of detergent micelles with crystallization process MP crystallization in a membrane mimetic matrix known as lipidic cubic phase LCP has recently started to gain popularity following successes in structure determination of G protein-coupled receptors GPCRs transporters and enzymes Unlike crystallization trials in aqueous solutions where protein molecules are free to move diffusion of MPs in LCP is restricted and thus a high level of protein mobility can serve as an early indication for subsequent crystallization success Prompted by ... More
Crystallization of integral membrane proteins (MPs) is notoriously difficult, given their poor stability outside native membrane environment and due to the interference of detergent micelles with crystallization process. MP crystallization in a membrane mimetic matrix, known as lipidic cubic phase (LCP), has recently started to gain popularity, following successes in structure determination of G protein-coupled receptors (GPCRs), transporters, and enzymes. Unlike crystallization trials in aqueous solutions where protein molecules are free to move, diffusion of MPs in LCP is restricted, and, thus, a high level of protein mobility can serve as an early indication for subsequent crystallization success. Prompted by our initial observations that precipitant conditions can dramatically affect diffusion of GPCRs in LCP, we have developed a simple precrystallization assay, based on measuring protein diffusion at a number of different conditions by fluorescence recovery after photobleaching (LCP-FRAP). Over the last few years, the LCP-FRAP assay was incorporated in our GPCR structure determination pipeline and proved as a powerful technique allowing for a faster identification of crystallization conditions for many different receptors. The assay is used to screen for the best protein constructs, ligands, LCP host lipids, precipitants, and additives, thereby focusing subsequent crystallization trials on the most promising parts of the multidimensional crystallization phase diagram, substantially increasing the likelihood of finding the right crystallization condition. Here, we describe our LCP-FRAP protocols for guiding GPCR crystallization, which can be adapted to any other MP, and discuss some of the critical considerations related to application of this assay. Less
Na -translocating NADH quinone oxidoreductase NQR is a redox-driven sodium pump operating in the respiratory chain of various bacteria including pathogenic species The enzyme has a unique set of redox active prosthetic groups which includes two covalently bound flavin mononucleotide FMN residues attached to threonine residues in subunits NqrB and NqrC The reason of FMN covalent bonding in the subunits has not been established yet In the current work binding of free FMN to the apo-form of NqrC from Vibrio harveyi was studied showing very low affinity of NqrC to FMN in the absence of its covalent bonding To study ... More
Na+-translocating NADH:quinone oxidoreductase (NQR) is a redox-driven sodium pump operating in the respiratory chain of various bacteria, including pathogenic species. The enzyme has a unique set of redox active prosthetic groups, which includes two covalently bound flavin mononucleotide (FMN) residues attached to threonine residues in subunits NqrB and NqrC. The reason of FMN covalent bonding in the subunits has not been established yet. In the current work, binding of free FMN to the apo-form of NqrC from Vibrio harveyi was studied showing very low affinity of NqrC to FMN in the absence of its covalent bonding. To study structural aspects of flavin binding in NqrC, its holo-form was crystallized and its 3D structure was solved at 1.56 Å resolution. It was found that the isoalloxazine moiety of the FMN residue is buried in a hydrophobic cavity and that its pyrimidine ring is squeezed between hydrophobic amino acid residues while its benzene ring is extended from the protein surroundings. This structure of the flavin-binding pocket appears to provide flexibility of the benzene ring, which can help the FMN residue to take the bended conformation and thus to stabilize the one-electron reduced form of the prosthetic group. These properties may also lead to relatively weak noncovalent binding of the flavin. This fact along with periplasmic location of the FMN-binding domains in the vast majority of NqrC-like proteins may explain the necessity of the covalent bonding of this prosthetic group to prevent its loss to the external medium. Less
Bi-functional - and - opioid receptor OR ligands are potential therapeutic alternatives to alkaloid opiate analgesics with diminished side effects We solved the structure of human -OR bound to the bi-functional -OR antagonist and -OR agonist tetrapeptide H-Dmt -Tic -Phe -Phe -NH DIPP-NH by serial femtosecond crystallography revealing a cis-peptide bond between H-Dmt and Tic The observed receptor-peptide interactions are critical to understand the pharmacological profiles of opioid peptides and to develop improved analgesics
Bacteriorhodopsins are a large family of seven-helical transmembrane proteins that function as light-driven proton pumps Here we present the crystal structure of a new member of the family Haloarcula marismortui bacteriorhodopsin I HmBRI D N mutant at the resolution of While the HmBRI retinal-binding pocket and proton donor site are similar to those of other archaeal proton pumps its proton release region is extended and contains additional water molecules The protein's fold is reinforced by three novel inter-helical hydrogen bonds two of which result from double substitutions relative to Halobacterium salinarum bacteriorhodopsin and other similar proteins Despite the expression in ... More
Bacteriorhodopsins are a large family of seven-helical transmembrane proteins that function as light-driven proton pumps. Here, we present the crystal structure of a new member of the family, Haloarcula marismortui bacteriorhodopsin I (HmBRI) D94N mutant, at the resolution of 2.5 Å. While the HmBRI retinal-binding pocket and proton donor site are similar to those of other archaeal proton pumps, its proton release region is extended and contains additional water molecules. The protein's fold is reinforced by three novel inter-helical hydrogen bonds, two of which result from double substitutions relative to Halobacterium salinarum bacteriorhodopsin and other similar proteins. Despite the expression in Escherichia coli and consequent absence of native lipids, the protein assembles as a trimer in crystals. The unique extended loop between the helices D and E of HmBRI makes contacts with the adjacent protomer and appears to stabilize the interface. Many lipidic hydrophobic tail groups are discernible in the membrane region, and their positions are similar to those of archaeal isoprenoid lipids in the crystals of other proton pumps, isolated from native or native-like sources. All these features might explain the HmBRI properties and establish the protein as a novel model for the microbial rhodopsin proton pumping studies. Less
Amphipols APols have become important tools for the stabilization folding and in vitro structural and functional studies of membrane proteins MPs Direct crystallization of MPs solubilized in APols would be of high importance for structural biology However despite considerable efforts it is still not clear whether MP APol complexes can form well-ordered crystals suitable for X-ray crystallography In the present work we show that an APol-trapped MP can be crystallized in meso Bacteriorhodopsin BR trapped by APol A - was mixed with a lipidic mesophase and crystallization was induced by adding a precipitant The crystals diffract beyond The structure of ... More
Amphipols (APols) have become important tools for the stabilization, folding, and in vitro structural and functional studies of membrane proteins (MPs). Direct crystallization of MPs solubilized in APols would be of high importance for structural biology. However, despite considerable efforts, it is still not clear whether MP/APol complexes can form well-ordered crystals suitable for X-ray crystallography. In the present work, we show that an APol-trapped MP can be crystallized in meso. Bacteriorhodopsin (BR) trapped by APol A8-35 was mixed with a lipidic mesophase, and crystallization was induced by adding a precipitant. The crystals diffract beyond 2 Å. The structure of BR was solved to 2 Å and found to be indistinguishable from previous structures obtained after transfer from detergent solutions. We suggest the proposed protocol of in meso crystallization to be generally applicable to APol-trapped MPs. Less
We have recently established a procedure for serial femtosecond crystallography in lipidic cubic phase LCP-SFX for protein structure determination at X-ray free electron lasers XFELs LCP-SFX uses the gel-like lipidic cubic phase LCP as a matrix for growth and delivery of membrane protein microcrystals for crystallographic data collection LCP is a liquid-crystalline mesophase composed of lipids and water It provides a membrane-mimicking environment that stabilizes membrane proteins and supports their crystallization Here we describe detailed procedures for the preparation and characterization of microcrystals for LCP-SFX applications The advantages of LCP-SFX over traditional crystallographic methods include the capability of collecting room ... More
We have recently established a procedure for serial femtosecond crystallography in lipidic cubic phase (LCP-SFX) for protein structure determination at X-ray free electron lasers (XFELs). LCP-SFX uses the gel-like lipidic cubic phase (LCP) as a matrix for growth and delivery of membrane protein microcrystals for crystallographic data collection. LCP is a liquid-crystalline mesophase, composed of lipids and water. It provides a membrane-mimicking environment that stabilizes membrane proteins and supports their crystallization. Here we describe detailed procedures for the preparation and characterization of microcrystals for LCP-SFX applications. The advantages of LCP-SFX over traditional crystallographic methods include the capability of collecting room temperature high-resolution data with minimal effects of radiation damage from sub-10 �m crystals of membrane and soluble proteins that are difficult to crystallize, while eliminating the need for crystal harvesting and cryo-cooling. Compared to SFX methods for microcrystals in solution using liquid injectors, LCP-SFX reduces protein consumption by 2�3 orders of magnitude for data collection at currently available XFELs. The whole procedure typically takes 3�5 days, including the time required for crystals to grow. Less
The Smoothened receptor SMO mediates signal transduction in the hedgehog pathway which is implicated in normal development and carcinogenesis SMO antagonists can suppress the growth of some tumors however mutations at SMO have been found to abolish their anti-tumor effects a phenomenon known as chemoresistance Here we report three crystal structures of human SMO bound to the antagonists SANT and Anta XV and the agonist SAG at resolution The long and narrow cavity in the transmembrane domain of SMO harbors multiple ligand binding sites where SANT binds at a deeper site as compared with other ligands Distinct interactions at D ... More
The Smoothened receptor (SMO) mediates signal transduction in the hedgehog pathway, which is implicated in normal development and carcinogenesis. SMO antagonists can suppress the growth of some tumors; however, mutations at SMO have been found to abolish their anti-tumor effects, a phenomenon known as chemoresistance. Here we report three crystal structures of human SMO bound to the antagonists SANT1 and Anta XV, and the agonist, SAG1.5, at 2.6–2.8Å resolution. The long and narrow cavity in the transmembrane domain of SMO harbors multiple ligand binding sites, where SANT1 binds at a deeper site as compared with other ligands. Distinct interactions at D4736.55 elucidated the structural basis for the differential effects of chemoresistance mutations on SMO antagonists. The agonist SAG1.5 induces a conformational rearrangement of the binding pocket residues, which could contribute to SMO activation. Collectively, these studies reveal the structural basis for the modulation of SMO by small molecules. Less
Lipidic cubic phase LCP is a gel-like liquid crystalline membrane-mimetic matrix It has been successfully used to stabilize and crystallize challenging membrane proteins such as G protein-coupled receptors the structure of which is often difficult to obtain by other methods Despite many advantages the LCP crystallization method has not been widely adopted because of difficulties associated with handling highly viscous LCP material Recent advances in the development of tools and instruments for LCP crystallization are aimed at facilitating the research in this area and to help structural biologists in integrating these technologies in their working routine
Phospholipids have major roles in the structure and function of all cell membranes Most integral membrane proteins from the large CDP-alcohol phosphatidyltransferase family are involved in phospholipid biosynthesis across the three domains of life They share a conserved sequence pattern and catalyse the displacement of CMP from a CDP-alcohol by a second alcohol Here we report the crystal structure of a bifunctional enzyme comprising a cytoplasmic nucleotidyltransferase domain IPCT fused with a membrane CDP-alcohol phosphotransferase domain DIPPS at resolution The bifunctional protein dimerizes through the DIPPS domains each comprising six transmembrane -helices The active site cavity is hydrophilic and widely ... More
Phospholipids have major roles in the structure and function of all cell membranes. Most integral membrane proteins from the large CDP-alcohol phosphatidyltransferase family are involved in phospholipid biosynthesis across the three domains of life. They share a conserved sequence pattern and catalyse the displacement of CMP from a CDP-alcohol by a second alcohol. Here we report the crystal structure of a bifunctional enzyme comprising a cytoplasmic nucleotidyltransferase domain (IPCT) fused with a membrane CDP-alcohol phosphotransferase domain (DIPPS) at 2.65 Å resolution. The bifunctional protein dimerizes through the DIPPS domains, each comprising six transmembrane α-helices. The active site cavity is hydrophilic and widely open to the cytoplasm with a magnesium ion surrounded by four highly conserved aspartate residues from helices TM2 and TM3. We show that magnesium is essential for the enzymatic activity and is involved in catalysis. Substrates docking is validated by mutagenesis studies, and a structure-based catalytic mechanism is proposed. Less
Lipidic cubic phase LCP crystallization has proven successful for high-resolution structure determination of challenging membrane proteins Here we present a technique for extruding gel-like LCP with embedded membrane protein microcrystals providing a continuously renewed source of material for serial femtosecond crystallography Data collected from sub- - m-sized crystals produced with less than mg of purified protein yield structural insights regarding cyclopamine binding to the Smoothened receptor
Opioids represent widely prescribed and abused medications although their signal transduction mechanisms are not well understood Here we present the high-resolution crystal structure of the human -opioid receptor -OR revealing the presence and fundamental role of a sodium ion mediating allosteric control of receptor functional selectivity and constitutive activity The distinctive -OR sodium ion site architecture is centrally located in a polar interaction network in the -transmembrane bundle core with the sodium ion stabilizing a reduced agonist affinity state and thereby modulating signal transduction Site-directed mutagenesis and functional studies reveal that changing the allosteric sodium site residue Asn to alanine ... More
Opioids represent widely prescribed and abused medications, although their signal transduction mechanisms are not well understood. Here we present the 1.8Å high-resolution crystal structure of the human δ-opioid receptor (δ-OR), revealing the presence and fundamental role of a sodium ion mediating allosteric control of receptor functional selectivity and constitutive activity. The distinctive δ-OR sodium ion site architecture is centrally located in a polar interaction network in the 7-transmembrane bundle core, with the sodium ion stabilizing a reduced agonist affinity state, and thereby modulating signal transduction. Site-directed mutagenesis and functional studies reveal that changing the allosteric sodium site residue Asn131 to alanine or valine augments constitutive arrestin-ergic signaling. Asp95Ala, Asn310Ala, and Asn314Ala mutations transform classical δ-opioid antagonists like naltrindole into potent β-arrestin-biased agonists. The data establish the molecular basis for allosteric sodium ion control in opioid signaling, revealing that sodium-coordinating residues act as “efficacy-switches” at a prototypic G protein-coupled receptor. Less
Nonlinear optical NLO instrumentation has been integrated with synchrotron X-ray diffraction XRD for combined single-platform analysis initially targeting applications for automated crystal centering Second-harmonic-generation microscopy and two-photon-excited ultraviolet fluorescence microscopy were evaluated for crystal detection and assessed by X-ray raster scanning Two optical designs were constructed and characterized one positioned downstream of the sample and one integrated into the upstream optical path of the diffractometer Both instruments enabled protein crystal identification with integration times between and s per pixel representing a -fold reduction in the per-pixel exposure time relative to X-ray raster scanning Quantitative centering and analysis of phenylalanine hydroxylase ... More
Nonlinear optical (NLO) instrumentation has been integrated with synchrotron X-ray diffraction (XRD) for combined single-platform analysis, initially targeting applications for automated crystal centering. Second-harmonic-generation microscopy and two-photon-excited ultraviolet fluorescence microscopy were evaluated for crystal detection and assessed by X-ray raster scanning. Two optical designs were constructed and characterized; one positioned downstream of the sample and one integrated into the upstream optical path of the diffractometer. Both instruments enabled protein crystal identification with integration times between 80 and 150 µs per pixel, representing a ∼103–104-fold reduction in the per-pixel exposure time relative to X-ray raster scanning. Quantitative centering and analysis of phenylalanine hydroxylase from Chromobacterium violaceum cPAH, Trichinella spiralis deubiquitinating enzyme TsUCH37, human κ-opioid receptor complex kOR-T4L produced in lipidic cubic phase (LCP), intimin prepared in LCP, and α-cellulose samples were performed by collecting multiple NLO images. The crystalline samples were characterized by single-crystal diffraction patterns, while α-cellulose was characterized by fiber diffraction. Good agreement was observed between the sample positions identified by NLO and XRD raster measurements for all samples studied. Less
Introduction X-ray crystallography is the main tool for macromolecular structure solution at atomic resolution It provides key information for the understanding of protein function opening opportunities for the modulation of enzymatic mechanisms and protein ligand interactions As a consequence macromolecular crystallography plays an essential role in drug design as well as in the a posteriori validation of drug mechanisms Areas covered The demand for method developments and also tools for macromolecular crystallography has significantly increased over the past years As a consequence access to the facilities required for these investigations such as synchrotron beamlines became more difficult and significant efforts ... More
Introduction: X-ray crystallography is the main tool for macromolecular structure solution at atomic resolution. It provides key information for the understanding of protein function, opening opportunities for the modulation of enzymatic mechanisms, and protein�ligand interactions. As a consequence, macromolecular crystallography plays an essential role in drug design, as well as in the a posteriori validation of drug mechanisms.
Areas covered: The demand for method developments and also tools for macromolecular crystallography has significantly increased over the past 10 years. As a consequence, access to the facilities required for these investigations, such as synchrotron beamlines, became more difficult and significant efforts were dedicated to the automation of the experimental setup in laboratories. In this article, the authors describe how this was accomplished and how robot-based systems contribute to the enhancement of the macromolecular structure solution pipeline.
Expert opinion: The evolution in robot technology, together with progress in X-ray beam performance and software developments, contributes to a new era in macromolecular X-ray crystallography. Highly integrated experimental environments open new possibilities for crystallography experiments. It is likely that it will also change the way this technique will be used in the future, opening the field to a larger community. Less
Areas covered: The demand for method developments and also tools for macromolecular crystallography has significantly increased over the past 10 years. As a consequence, access to the facilities required for these investigations, such as synchrotron beamlines, became more difficult and significant efforts were dedicated to the automation of the experimental setup in laboratories. In this article, the authors describe how this was accomplished and how robot-based systems contribute to the enhancement of the macromolecular structure solution pipeline.
Expert opinion: The evolution in robot technology, together with progress in X-ray beam performance and software developments, contributes to a new era in macromolecular X-ray crystallography. Highly integrated experimental environments open new possibilities for crystallography experiments. It is likely that it will also change the way this technique will be used in the future, opening the field to a larger community. Less
The smoothened SMO receptor a key signal transducer in the Hedgehog Hh signaling pathway is both responsible for the maintenance of normal embryonic development and implicated in carcinogenesis The SMO receptor is classified as a class Frizzled class F G protein-coupled receptor GPCR although the canonical Hh signaling pathway involves the transcription factor Gli and the sequence similarity with class A GPCRs is less than Here we report the crystal structure at resolution of the transmembrane domain of the human SMO receptor bound to the small molecule antagonist LY Although the SMO receptor shares the seven transmembrane helical TM fold ... More
The smoothened (SMO) receptor, a key signal transducer in the Hedgehog (Hh) signaling pathway is both responsible for the maintenance of normal embryonic development and implicated in carcinogenesis. The SMO receptor is classified as a class Frizzled (class F) G protein-coupled receptor (GPCR), although the canonical Hh signaling pathway involves the transcription factor Gli and the sequence similarity with class A GPCRs is less than 10%. Here we report the crystal structure at 2.5 Å resolution of the transmembrane domain of the human SMO receptor bound to the small molecule antagonist LY2940680. Although the SMO receptor shares the seven transmembrane helical (7TM) fold, most conserved motifs for class A GPCRs are absent, and the structure reveals an unusually complex arrangement of long extracellular loops stabilized by four disulfide bonds. The ligand binds at the extracellular end of the 7TM bundle and forms extensive contacts with the loops. Less
Structural studies of human G protein-coupled receptors GPCRs have recently been accelerated through the use of the T lysozyme fusion partner that was inserted into the third intracellular loop Using chimeras of the human -adrenergic and human A A adenosine receptors we present the methodology and data for the selection of five new fusion partners for crystallizing GPCRs In particular the use of the thermostabilized apocytochrome b RIL as a fusion partner displays certain advantages over the previously utilized T lysozyme resulting in a significant improvement in stability and structure in GPCR-fusion constructs
Members of the opioid receptor family of G-protein-coupled receptors GPCRs are found throughout the peripheral and central nervous system where they have key roles in nociception and analgesia Unlike the classical opioid receptors and -OR -OR and -OR which were delineated by pharmacological criteria in the s and s the nociceptin orphanin FQ N OFQ peptide receptor NOP also known as ORL- was discovered relatively recently by molecular cloning and characterization of an orphan GPCR Although it shares high sequence similarity with classical opioid GPCR subtypes NOP has a markedly distinct pharmacology featuring activation by the endogenous peptide N OFQ ... More
Members of the opioid receptor family of G-protein-coupled receptors (GPCRs) are found throughout the peripheral and central nervous system, where they have key roles in nociception and analgesia. Unlike the ‘classical’ opioid receptors, δ, κ and μ (δ-OR, κ-OR and μ-OR), which were delineated by pharmacological criteria in the 1970s and 1980s, the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP, also known as ORL-1) was discovered relatively recently by molecular cloning and characterization of an orphan GPCR1. Although it shares high sequence similarity with classical opioid GPCR subtypes (∼60%), NOP has a markedly distinct pharmacology, featuring activation by the endogenous peptide N/OFQ, and unique selectivity for exogenous ligands2,3. Here we report the crystal structure of human NOP, solved in complex with the peptide mimetic antagonist compound-24 (C-24) (ref. 4), revealing atomic details of ligand–receptor recognition and selectivity. Compound-24 mimics the first four amino-terminal residues of the NOP-selective peptide antagonist UFP-101, a close derivative of N/OFQ, and provides important clues to the binding of these peptides. The X-ray structure also shows substantial conformational differences in the pocket regions between NOP and the classical opioid receptors κ (ref. 5) and μ (ref. 6), and these are probably due to a small number of residues that vary between these receptors. The NOP–compound-24 structure explains the divergent selectivity profile of NOP and provides a new structural template for the design of NOP ligands. Less
Structure determination of biological macromolecules using x-ray crystallography has been greatly improved in recent years through the development of a number of key technologies and better integration with sample preparation steps This has enabled the crystallization of large numbers of proteins including integral membrane proteins This chapter discusses the crystallization process as well as the development of automation that has dramatically increased the likelihood of success Most noteworthy has been the introduction of crystallization protocols that use nanoliter protein solutions and the insight that production of high-quality crystals requires high protein sample quality The latter underscores the importance of the ... More
Structure determination of biological macromolecules using x-ray crystallography has been greatly improved in recent years through the development of a number of key technologies and better integration with sample preparation steps. This has enabled the crystallization of large numbers of proteins, including integral membrane proteins. This chapter discusses the crystallization process as well as the development of automation that has dramatically increased the likelihood of success. Most noteworthy has been the introduction of crystallization protocols that use nanoliter protein solutions and the insight that production of high-quality crystals requires high protein sample quality. The latter underscores the importance of the development of powerful biophysical characterization techniques. Less
Opioid receptors mediate the actions of endogenous and exogenous opioids on many physiological processes including the regulation of pain respiratory drive mood and in the case of -opioid receptor -OR dysphoria and psychotomimesis Here we report the crystal structure of the human -OR in complex with the selective antagonist JDTic arranged in parallel dimers at resolution The structure reveals important features of the ligand-binding pocket that contribute to the high affinity and subtype selectivity of JDTic for the human -OR Modelling of other important -OR-selective ligands including the morphinan-derived antagonists norbinaltorphimine and -guanidinonaltrindole and the diterpene agonist salvinorin A analogue ... More
Opioid receptors mediate the actions of endogenous and exogenous opioids on many physiological processes, including the regulation of pain, respiratory drive, mood, and—in the case of κ-opioid receptor (κ-OR)—dysphoria and psychotomimesis. Here we report the crystal structure of the human κ-OR in complex with the selective antagonist JDTic, arranged in parallel dimers, at 2.9 Å resolution. The structure reveals important features of the ligand-binding pocket that contribute to the high affinity and subtype selectivity of JDTic for the human κ-OR. Modelling of other important κ-OR-selective ligands, including the morphinan-derived antagonists norbinaltorphimine and 5′-guanidinonaltrindole, and the diterpene agonist salvinorin A analogue RB-64, reveals both common and distinct features for binding these diverse chemotypes. Analysis of site-directed mutagenesis and ligand structure–activity relationships confirms the interactions observed in the crystal structure, thereby providing a molecular explanation for κ-OR subtype selectivity, and essential insights for the design of compounds with new pharmacological properties targeting the human κ-OR. Less
Lipidic cubic phase LCP is a membrane-mimetic matrix suitable for stabilization and crystallization of membrane proteins in lipidic environment LCP technologies however have not been fully embraced by the membrane protein structural biology community primarily because of the difficulties associated with handling viscous materials Recent developments of pre-crystallization assays and improvements in crystal imaging successes in obtaining high resolution structures of G protein-coupled receptors GPCRs and commercial availability of LCP tools and instruments are beginning to attract structural biologists to integrate LCP technologies in their research This wider acceptance should translate to an increased number of otherwise difficult-to-crystallize membrane protein ... More
Lipidic cubic phase (LCP) is a membrane-mimetic matrix suitable for stabilization and crystallization of membrane proteins in lipidic environment. LCP technologies, however, have not been fully embraced by the membrane protein structural biology community, primarily because of the difficulties associated with handling viscous materials. Recent developments of pre-crystallization assays and improvements in crystal imaging, successes in obtaining high resolution structures of G protein-coupled receptors (GPCRs), and commercial availability of LCP tools and instruments are beginning to attract structural biologists to integrate LCP technologies in their research. This wider acceptance should translate to an increased number of otherwise difficult-to-crystallize membrane protein structures, shedding light on their functional mechanisms and on structural details of lipid-protein interactions. Less