The structural basis of function in Cys-loop receptors

Cys-loop receptors are membrane-spanning neurotransmitter-gated ion channels that are responsible for fast excitatory and inhibitory transmission in the peripheral and central nervous systems. The best studied members of the Cys-loop family are nACh, 5-HT_3, GABA_A and glycine receptors. All these receptors share a common structure of five subunits, pseudo-symmetrically arranged to form a rosette with a central ion-conducting pore. Some are cation selective (e.g. nACh and 5-HT_3) and some are anion selective (e.g. GABA_A and glycine). Each receptor has an extracellular domain (ECD) that contains the ligand-binding sites, a transmembrane domain (TMD) that allows ions to pass across the membrane, and an intracellular domain (ICD) that plays a role in channel conductance and receptor modulation. Cys-loop receptors are the targets for many currently used clinically relevant drugs (e.g. benzodiazepines and anaesthetics). Understanding the molecular mechanisms of these receptors could therefore provide the catalyst for further development in this field, as well as promoting the development of experimental techniques for other areas of neuroscience. In this review, we present our current understanding of Cys-loop receptor structure and function. The ECD has been extensively studied. Research in this area has been stimulated in recent years by the publication of high-resolution structures of nACh receptors and related proteins, which have permitted the creation of many Cys loop receptor homology models of this region. Here, using the 5-HT_3 receptor as a typical member of the family, we describe how homology modelling and ligand docking can provide useful but not definitive information about ligand interactions. We briefly consider some of the many Cys-loop receptors modulators. We discuss the current understanding of the structure of the TMD, and how this links to the ECD to allow channel gating, and consider the roles of the ICD, whose structure is poorly understood. We also describe some of the current methods that are beginning to reveal the differences between different receptor states, and may ultimately show structural details of transitions between them

Computational Protein Design and Molecular Dynamics Simulations: A Study of Membrane Proteins, Small Peptides and Molecular Systems

Molecular design and modeling can provide stringent assessment of our understanding of the structure and function of proteins. Due to the subtleness of the interactions that largely stabilize proteins, computational methods have been particularly valuable in establishing practical, formal and physically grounded protocols to study the structure and function of these biomolecules. Especifically, computational protein design seeks to identify sequences that fold into a desired structure and have specific structural and functional properties using computational methodologies. Among current techniques, an entropy-based formalism that efficiently determines the number and composition of sequences satisfying a predefined set of constraints seems particularly promising and powerful. Complementary to this methodology are the well-established molecular dynamics simulation techniques that have been extensively used to study structure, function and dynamics of biologically relevant systems. Herein different studies of systems using computational techniques to address particular molecular problems are described. Efforts to redesign membrane proteins to generate water-soluble variants were applied to a widely studied pentameric ligand-gated ion channel, the nicotinic acetylchoilne receptor (nAChR). NMR structures and binding studies demostrated the robustness and applicability of the computational design approach. Toward the creation of water-soluble variants of a G protein–coupled receptor (GPCR), comparative modeling and docking calculations were used to investigate the structure of the human μ opioid receptor and presented in light of previous mutagenesis studies of structure and agonist-induced activation. Candidate peptides for possible therapeutic agents were computationally analyzed. Peptide design, loop modeling and MD simulations were applied to investigate the stromal cell-derived factor-1&a; (SDF-1&a;). SDF-1&a; displays promising therapeutic benefits to treat blood-supply related heart disease and elicit growth of microvasculature. Simplified analogs of SDF-1&a; exhibit enhanced therapeutic properties in cell-based assays. MD simulations provide insights about the molecular features of this enhancement. One simplified peptide offers a potentially clinically translatable neovasculogenic therapy. Lastly, MD simulations were utilized to analyze a molecule with hindered internal rotors, a tribenzylamine hemicryptophane. The molecule was characterized by different experimental and computational techniques. The structural and dynamic features of the hemicryptophane molecule make it an attractive starting point for controlling internal rotation of aromatic rings within molecular systems

Structural and functional studies of a chimeric GABA-A receptor

GABA-A receptors are ligand-gated ion channels principally responsible for inhibitory neurotransmission in the mammalian CNS. GABA binding initiates a series of conformational changes causing the receptor to transition from inactive (shut/closed) to active (open) ion channel states; and during prolonged agonist exposure, to a desensitized (closed) state. Critical to the fine-tuning of inhibitory responses in vivo is the allosteric modulation of GABA-A receptors by an array of compounds, many of which impart their effect through binding within the receptor’s transmembrane domain. Beyond the importance of GABA-mediated inhibition in maintaining nervous system function, GABA-A receptors are established therapeutic targets for psychiatric and neurodevelopmental disorders. Despite this, an understanding of the structure of these receptors at atomic resolution is crucially lacking; particularly with regards to the structural elements underpinning channel gating and allosteric modulation. Therefore, GABA-A receptor ion channels were subjected to atomic-resolution structural analyses using chimeric receptors, in addition to comparative studies with bacterial ion channel homologues. A functional receptor was formed from chimeras between the extracellular domain of the prokaryotic ion channel GLIC and the transmembrane domain of GABA-A receptor α1 subunits. These receptors exhibited GABA-A receptor-like properties with respect to their response to brain neurosteroids. The amenability of this receptor to high-level expression and purification was assessed. The baculovirus-insect cell expression system was identified as an appropriate system for generating receptor of sufficient quantity and purity to generate diffracting protein crystals. Additional studies of GABA-A receptor modulators at the bacterial homologs GLIC and ELIC identified previously unreported effects prompting further structural investigation using X-ray crystallography, cryo-electron microscopy and native mass spectrometry. In conclusion, these studies reveal a new system for atomic structural resolution investigation of GABA-A receptor subunits, likely to be applicable to other receptors. These receptors are potentially powerful tools for understanding the mechanism of GABA-A receptor allosteric modulation

Molecular mechanisms of allosteric modulation of nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChR) are part of the Cys-loop family of ligand-gated ion channels, and are implicated in a wide variety of neurological disorders such as nicotine addiction, schizophrenia, and cognitive dysfunction. Therefore, they represent a critical molecular target for drug development and targeted therapeutic intervention. Positive allosteric modulators (PAMs) of ligandgated ion channels have a unique therapeutic potential because they enhance synaptic transmission without disrupting the endogenous timing mechanisms. This research focused on the neuronal α7 nicotinic receptor because they are located both pre- and postsynaptically and can modulate glutamatergic and dopaminergic release in the brain regions involved in drug-seeking behaviors. Understanding the molecular mechanisms by which allosteric modulators enhance activation of neuronal nicotinic acetylcholine receptors is therefore critically important to the development of new drugs for research and therapeutics. Experiments with the Substituted Cysteine Accessibility Method indicate that two chemically different positive allosteric modulators, PNU-120596 and permeable divalent cations, cause structural transitions (or changes in local electrostatic potential) in the extracellular ligand binding domain of the α7 nicotinic receptor that are similar but not identical to those caused by the agonist, acetylcholine. These results suggest that positive allosteric modulators share a conserved mechanism to enhance receptor gating that is unrelated to the chemical structure of the molecule. As an additional approach to study gating of the nicotinic receptors, I developed homology models derived from the structures of bacterial Cys-loop receptors in the closed and open states. A comparison of electrophysiological MTSEA modification data against in silico calculations of solvent accessibility and electrostatic potential showed that electrostatic potential in the extracellular ligandbinding domain of the α7 nAChR is a better predictor of receptor gating from the closed to open states. Overall, this body of work has shown that positive allosteric modulators and agonists of the α7 nAChR induce similar conformational changes in the extracellularligand binding domain of the receptor by reducing the large electronegative potential energy along the ion-permeation pathway. A unifying model of receptor gating (electrostatic compensation) and future experiments designed to test this model are discussed

Computational Perspective on Intricacies of Interactions, Enzyme Dynamics and Solvent Effects in the Catalytic Action of Cyclophilin A

Cyclophilin A (CypA) is the well-studied member of a group of ubiquitous and evolutionarily conserved families of enzymes called peptidyl–prolyl isomerases (PPIases). These enzymes catalyze the cis-trans isomerization of peptidyl-prolyl bond in many proteins. The distinctive functional path triggered by each isomeric state of peptidyl-prolyl bond renders PPIase-catalyzed isomerization a molecular switching mechanism to be used on physiological demand. PPIase activity has been implicated in protein folding, signal transduction, and ion channel gating as well as pathological condition such as cancer, Alzheimer’s, and microbial infections. The more than five order of magnitude speed-up in the rate of peptidyl–prolyl cis–trans isomerization by CypA has been the target of intense research. Normal and accelerated molecular dynamic simulations were carried out to understand the catalytic mechanism of CypA in atomistic details. The results reaffirm transition state stabilization as the main factor in the astonishing enhancement in isomerization rate by enzyme. The ensuing intramolecular polarization, as a result of the loss of pseudo double bond character of the peptide bond at the transition state, was shown to contribute only about −1.0 kcal/mol to stabilizing the transition state. This relatively small contribution demonstrates that routinely used fixed charge classical force fields can reasonably describe these types of biological systems. The computational studies also revealed that the undemanding exchange of the free substrate between β- and α-helical regions is lost in the active site of the enzyme, where it is mainly in the β-region. The resultant relative change in conformational entropy favorably contributes to the free energy of stabilizing the transition state by CypA. The isomerization kinetics is strongly coupled to the enzyme motions while the chemical step and enzyme–substrate dynamics are in turn buckled to solvent fluctuations. The chemical step in the active site of the enzyme is therefore not separated from the fluctuations in the solvent. Of special interest is the nature of catalysis in a more realistic crowded environment, for example, the cell. Enzyme motions in such complicated medium are subjected to different viscosities and hydrodynamic properties, which could have implications for allosteric regulation and function

Inhibiting inhibition: interactions amongst interneurons of the hippocampus

Cortical networks comprise excitatory principal cells and interneurons (IN); the latter showing large neurochemical, morphological and physiological heterogeneity. GABA release from IN axon terminals activates fast ionotropic GABAA or slow metabotropic GABAB receptors (GABABR); ionotropic GABA mechanisms are well described in INs, whereas GABABR activity is less well understood. The primary aim of this thesis is to ascertain GABABR mediated inhibition in different IN types containing the neurochemicals parvalbumin (PV), cholecystokinin (CCK) or somatostatin (SSt). Using immunocytochemical techniques, at light and electron microscopic levels, we examined the cellular and subcellular expression of GABAB1 receptor subunits in these INs. Application of whole-cell patch clamp techniques in acute slices, allowed analysis of GABABR effects pre- and postsynaptically; in response to endogenous GABA release or pharmacological activation. Light microscopy showed GABAB1 expression in INs containing CCK or SSt, equivalent to CA1 pyramidal cells; with low expression in PV INs. Using electron microscopy, we detected GABAB1 receptor subunits in dendrites of CCK and PV INs, with densities equivalent or higher than CA1 pyramidal cell dendrites. Unexpectedly, SSt containing dendrites showed a lower density of GABAB1 receptor subunits. In axon terminals of CCK and PV containing INs, we found comparable densities of GABAB1 receptor subunits. Electrophysiological recordings confirmed the presence of functional postsynaptic GABABR in PV and CCK INs. GABABR-mediated slow inhibitory postsynaptic currents (IPSCs) had typically large amplitudes, but with high cell-to-cell variability in both IN types. Morphological separation of PV or CCK INs revealed slow IPSC amplitudes which were large in perisomatic inhibitory (PI)cells (30.8 ± 8.6 pA and 39.2 ± 5.5 pA, respectively) and small in dendritic inhibitory (DI) cells (4.0 ± 1.7 pA and 11.6 ± 2.4 pA, respectively). Consistently, SSt-immunoreactive DI INs exhibited very small IPSCs (1.5 ± 0.2 pA). Pharmacological activation of GABAB R by the selective agonist baclofen revealed variable amplitude whole-cell currents, confirming differences between IN subtypes. Examining presynaptic GABABR activity; we minimally stimulated str. pyramidale evoking monosynaptic IPSCs in CA1 pyramidal cells. IPSCs mediated by CCK or PV PI axons were pharmacologically isolated by CB1 or M2 receptor activation. Both monosynaptic responses were reduced by baclofen, albeit differentially so. To further investigate this effect we performed paired-recordings from PV or CCK INs coupled synaptically to CA1 pyramidal cells. Baclofen inhibited PV and CCK basket cell mediated IPSCs by 51% and 98%, respectively; with a smaller effect in DI INs. In summary, we have shown that functional GABABRs are expressed pre- and postsynaptically in hippocampal GABAergic INs; with distinct populations of INs under differential GABABR control. Postsynaptic inhibition was strong in PI INs, but weak or absent in DI INs, a relationship conserved presynaptically. The observed differential expression of GABABRs is likely to play a fundamental role in regulating the excitability and activity of GABAergic INs, regulating synaptic output and potentially contributing to network and oscillatory activity. Consequentially, during periods of high GABA release, GABABR activation could act as a switch, allowing DI INs to play a greater role in network inhibition, due to GABABR mediated inhibition of perisomatic-targeting INs

Engineering and functional characterisation of pentameric concatenated (alpha 4)2 (beta 2)3 and (alpha 4)3 (beta 2)2 nicotinic acetylcholine receptors

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that influence neurotransmitter release, hence constituting a key component of the physiological mechanisms of neuronal signalling. This thesis is concerned with the properties of the a4P2 nAChR, the most abundant nAChR in the brain, and the major contributor to the central effects of nicotine. The a4P2 nAChR is made up of five subunits, which in heterologous systems can assemble into at least two different stoichiometries: the high sensitivity (HS) (a4h(P2)3 stoichiometry and the low sensitivity (LS) (a4)3(p2)2 stoichiometry, which might both exist in native tissues. Despite the attractiveness of the a.4P2 nAChR as a target for therapeutic intervention, progress in the development of a4P2 nAChR-selective drugs has been slowed, partly because of the lack of stoichiometricspecific receptor models. This study presents a strategy to express homogenous populations of a4P2 nAChRs with fixed stoichiometry. By using standard molecular biological techniques, pentameric concatenated (a4)2(P2)3 and (a4)3(P2)2 nAChRs were engineered. These receptors were expressed in Xenopus laevis oocytes and functional studies showed that their functional properties resembled those of their non-linked counterparts. Subsequent site-directed mutagenesis in combination with functional analysis allowed the identification of the agonist-binding subunits in both concatamers. Concatenated receptors proved to be suitable for comparative studies of the effects of receptor mutation linked to autosomal dominant nocturnal frontal lobe epilepsy. Studies carried out on non-linked receptors, showed that the properties of the (a4)3(p2)2 stoichiometry were affected more markedly than those of the (a4)2(p2)3 stoichiometry. Insertion of the mutation in concatenated receptors revealed that the mutation not only affected the functional properties of a.4P2 nAChRs but also altered the subunit composition of the receptor. These studies show that pentameric concatenated constructs are a powerful tool to study the function and structure of receptors that assemble in multimeric types in expression systems

Development and Application of Computational Biology tools for Biomedicine

Biomolecular simulation can be considered as a virtual microscope for molecular biology, allowing to gain insights into the sub-cellular mechanisms of biological relevance at spatial and temporal scales that are difficult to observe experimentally. It provides a powerful tool to link the laws of physics with the complex behavior of biological systems. Dramatic recent advancements in achievable simulation speed and the underlying physical models will increasingly lead to molecular views of large systems. These improvements may largely affect biological sciences. In this thesis, I have applied computational molecular biology approaches to different biological systems using state of the art structural bioinformatics and computational biophysics tools (Chapter 3). My principal focus was on the computational design of molecular imprinted polymers (MIPs), which have recently attracted significant attention as cost effective substitutes for natural antibodies and receptors in chromatography, sensors and assays. I have used molecular modelling in the optimization of polymer compositions to make high affinity synthetic receptors based on Molecular Imprinting. In particular, I developed a new free of charge protocol that can be performed within just few hours that outputs a list of candidate monomers which are capable of strong binding interactions with the template. Furthermore, I have produced a new computational method for the calculation of the ideal monomer: template stoichiometric ratio to be used in the lab for the MIPs synthesis. These protocols have been implemented as a webserver that is available at http://mirate.di.univr.it/. In parallel, I have also investigated the modelling of much more complex MIPs systems by the introduction of some factors e.g. solvent and cross-linker molecules that are also essential in the polymerisation process. A novel algorithm, which mimics a radical polymerization mechanism, has been written for application in the rational design of MIPs (Chapter 4). Moreover, I have been involved in the field of computational molecular biomedicine. Indeed, in Chapters 5 and 6 I describe the work done in collaboration with two labs at the Department of Neuroscience, Biomedicine and Movement Sciences, University of Verona. In Chapter 5, starting from unpublished experimental data I have computationally characterized the interaction of ACOT8 with HIV-1 Nef accessory protein. I have performed a detailed structural and functional characterization of these two proteins in order to infer any possible functional details about their interactions. The bioinformatics predictions were then confirmed by wet-lab experiments. I have also carried out a detailed structural and functional characterization of two pathogenic mutations of AGT-Mi (Chapter 6). In particular, I have used classical molecular dynamics (MD) simulations to study the possible interference with the dimerization process of AGT-Mi exerted by I244T-Mi and F152I-Mi mutants. Those variants are associated with Primary Hyperoxaluria type 1 disease. In Chapter 7, I present the coarse-grained MD simulations of Membrane/Human ileal bile-acid-binding protein Interactions. This study was carried out in collaboration with the NMR group at the University of Verona and it is a part of an extensive research aimed at better understanding of the main biomolecular interactions in crowded cellular environments. MD simulations results were in agreement with experimental findings

Investigation of murine calcium-activated, chloride channel 6 as a potential modifier of Cystic fibrosis intestinal disease in mice

Certain mouse models of Cystic fibrosis (CF) exhibit a severe intestinal phenotype, resulting in death soon after birth or at the time o f weaning. Marked variations in intestinal disease severity among different CF mouse models have suggested a prominent role for secondary genetic modifiers ofthe disease. Calcium-activated, chloride channels (CLCA) have been postulated as genetic modifiers of CF. It has been previously shown that mCLCA3, a secreted protein involved in mucus production, shows markedly reduced expression in goblet cells o f the intestines o f a CF mouse model, and when expression is corrected through transgenic manipulation, can significantly ameliorate the disease. Current studies were undertaken using quantitative RT-PCR to show that expression of mClca6 is significantly reduced (p\u3c0.005) in the ileum of a juvenile CF mouse model. Further quantitative RT-PCR studies were performed on the mildly-affected adult CF mice, showing significant differential expression of mClca6 in the jejunum (p\u3c0.005), but not in the ileum. Moreover, a CF mouse model which exhibits relatively mild lung and intestinal disease compared to other mouse models shows no significant differential expression of mClca6 in the ileum. Although the function of mCLCA6 remains unclear, these results provide support for it as a potential modifier of intestinal disease severit

FGFR1-5HT1AR heteroreceptor complexes differently modulate GIRK currents in the hippocampus and the raphe nucleus of control rats and of a genetic rat model of depression

The midbrain raphe serotonin neurons provide the main ascending serotonergic projection to the forebrain, including the hippocampus, which has a recognized role in the pathophysiology of depressive disorder. The activation of G protein-coupled inwardly-rectifying potassium (GIRK) channels by serotonin 5HT1A receptors at the soma-dendritic level of serotonergic raphe neurons and glutamatergic hippocampal pyramidal neurons reduces neuronal activity. The presence of FGFR1-5HT1A heteroreceptor complexes in this raphe-hippocampal serotonin neuron system has been demonstrated, but functional receptor-receptor interactions in the heterocomplexes have only been studied in CA1 pyramidal neurons of control Sprague Dawley (SD) rats. In the present research, the short-term effects of FGFR1-5HT1A complex activation were studied in hippocampal pyramidal neurons, both in CA1 and CA2 areas, and midbrain dorsal raphe serotonergic neurons of SD rats and a genetic rat model of depression, the Flinders sensitive line (FSL) rats selected from SD strain, using an electrophysiological technique. The results obtained demonstrate that FGFR1-5HT1A heteroreceptor activation by specific agonists reduced the ability of the 5HT1AR protomer to open the GIRK channels via the allosteric inhibitory interplay produced by agonist activation of the FGFR1 protomer, resulting in increased neuronal firing in the raphe-hippocampal 5HT system of SD rats. In contrast, apart from CA2 neurons, the inhibitory allosteric effects of FGFR1 agonist on the 5HT1AR protomer were unable to have this influence on GIRK channels in FSL rats. According to these data, 5HT1AR activation impaired hippocampal plasticity in both SD and FSL rats, as determined by long-term potentiation induction capability in the CA1 field, but not in SD rats following simultaneous FGFR1-5HT1A heterocomplex activation. While, due to the impairment in heterocomplex activation, long-term potentiation was precluded in FSL rats. It is thus hypothesized that in the genetic FSL model of depression, there is a considerable decrease of the allosteric inhibition mediated by the FGFR1 protomer on the 5HT1AR protomer, resulting in a reduced opening of the GIRK channels in the raphe-hippocampal serotonin pathway. The consequent increase in inhibition in dorsal raphe 5HT nerve cells and glutamatergic hippocampal CA1 pyramidal nerve cell firing may contribute to the onset of major depression