Gianluigi Caltabiano

Tracking G-protein-coupled receptor activation using genetically encoded infrared probes

Rhodopsin is a prototypical heptahelical family A G-protein-coupled receptor (GPCR) responsible for dim-light vision. Light isomerizes rhodopsins retinal chromophore and triggers concerted movements of transmembrane helices, including an outward tilting of helix 6 (H6) and a smaller movement of H5, to create a site for G-protein binding and activation. However, the precise temporal sequence and mechanism underlying these helix rearrangements is unclear. We used site-directed non-natural amino acid mutagenesis to engineer rhodopsin with p-azido-l-phenylalanine residues incorporated at selected sites, and monitored the azido vibrational signatures using infrared spectroscopy as rhodopsin proceeded along its activation pathway. Here we report significant changes in electrostatic environments of the azido probes even in the inactive photoproduct Meta I, well before the active receptor state was formed. These early changes suggest a significant rotation of H6 and movement of the cytoplasmic part of H5 away from H3. Subsequently, a large outward tilt of H6 leads to opening of the cytoplasmic surface to form the active receptor photoproduct Meta II. Thus, our results reveal early conformational changes that precede larger rigid-body helix movements, and provide a basis to interpret recent GPCR crystal structures and to understand conformational sub-states observed during the activation of other GPCRs.

Membrane Protein Simulations Using AMBER Force Field and Berger Lipid Parameters.

AMBER force fields are among the most commonly used in molecular dynamics (MD) simulations of proteins. Unfortunately, they lack a specific set of lipid parameters, thus limiting its use in membrane protein simulations. In order to overcome this limitation we assessed whether the widely used united-atom lipid parameters described by Berger and co-workers could be used in conjunction with AMBER force fields in simulations of membrane proteins. Thus, free energies of solvation in water and in cyclohexane, and free energies of water to cyclohexane transfer, were computed by thermodynamic integration procedures for neutral amino acid side-chains employing AMBER99, AMBER03, and OPLS-AA amino acid force fields. In addition, MD simulations of three membrane proteins in a POPC lipid bilayer, the β2 adrenergic G protein-coupled receptor, Aquaporin-1, and the outer membrane protein Omp32, were performed with the aim of comparing the AMBER99SB/Berger combination of force fields with the OPLS-AA/Berger combination. We have shown that AMBER99SB and Berger force fields are compatible, they provide reliable free energy estimations relative to experimental values, and their combination properly describes both membrane and protein structural properties. We then suggest that the AMBER99SB/Berger combination is a reliable choice for the simulation of membrane proteins, which links the easiness of ligand parametrization and the ability to reproduce secondary structure of AMBER99SB force field with the largely validated Berger lipid parameters.

The specificity of binding of glycoprotein hormones to their receptors.

Abstract.The glycoprotein hormone receptor family is peculiar because, in contrast to other G protein-coupled receptors, a large N-terminal extracellular ectodomain is responsible for hormone recognition. Hormone-receptor pairs have evolved in such a manner that a limited number of positions both at the ‘seat-belt’ domain of the hormone and the leucine-rich repeats of the receptor, play attractive and repulsive interactions for binding and specificity, respectively. Surprisingly, the constitutive activity of the receptor, mostly modulated by highly conserved amino acids within the heptahelical domain of the receptor (i.e., outside the hormone binding region), also regulates effectiveness of hormone recognition by the extracellular part. In this review we analyze, at the molecular level, these important discriminating determinants for selective binding of glycoprotein hormones to their receptors, as well as natural mutations, observed in patients with gestational hyperthyroidism or ovarian hyperstimulation syndrome, that modify the selectivity of binding.

Impact of Helix Irregularities on Sequence Alignment and Homology Modeling of G Protein‐Coupled Receptors

Comparison of the crystal structures of G protein‐coupled receptors (GPCRs) revealed backbone irregularities in the majority of the transmembrane (TM) helices. Among these, wide (π bulge) and tight (310) helical turns on TM2 and TM5 deserve special attention because of their proximity to the ligand binding site. These irregularities are related to residue insertion or deletion (reflected by inclusion of gaps in sequence alignments) accumulated during the evolution of these two helices. These findings have direct implications for the sequence alignments, phylogeny reconstruction, and homology modeling of class A GPCRs.

New linezolid-like 1,2,4-oxadiazoles active against Gram-positive multiresistant pathogens.

The synthesis and the in vitro antibacterial activity of novel linezolid-like oxadiazoles are reported. Replacement of the linezolid morpholine C-ring with 1,2,4-oxadiazole results in an antibacterial activity against Staphylococcus aureus both methicillin-susceptible and methicillin-resistant comparable or even superior to that of linezolid. While acetamidomethyl or thioacetoamidomethyl moieties in the C(5) side-chain are required, fluorination of the phenyl B ring exhibits a slight effect on an antibacterial activity but its presence seems to reduce the compounds cytotoxicity. Molecular modeling performed using two different approaches - FLAP and Amber software - shows that in the binding pose of the newly synthesized compounds as compared with the crystallographic pose of linezolid, the 1,2,4-oxadiazole moiety seems to perfectly mimic the function of the morpholinic ring, since the H-bond interaction with U2585 is retained.

Analysis of Human Dopamine D3 Receptor Quaternary Structure.

Background: The dopamine D3 receptor can form dimers/oligomers, but the molecular basis for this is poorly defined. Results: Molecular modeling, mutagenesis, and analysis of inactive state receptor crystal structures allowed assessment of models of receptor organization. Conclusion: The dopamine D3 receptor can assume different dimeric and a rhombic tetrameric arrangements. Significance: These findings provide understanding of the molecular basis of D3 receptor quaternary structure. The dopamine D3 receptor is a class A, rhodopsin-like G protein-coupled receptor that can form dimers and/or higher order oligomers. However, the molecular basis for production of these complexes is not well defined. Using combinations of molecular modeling, site-directed mutagenesis, and homogenous time-resolved FRET, the interfaces that allow dopamine D3 receptor monomers to interact were defined and used to describe likely quaternary arrangements of the receptor. These were then compared with published crystal structures of dimeric β1-adrenoreceptor, μ-opioid, and CXCR4 receptors. The data indicate important contributions of residues from within each of transmembrane domains I, II, IV, V, VI, and VII as well as the intracellular helix VIII in the formation of D3-D3 receptor interfaces within homo-oligomers and are consistent with the D3 receptor adopting a β1-adrenoreceptor-like quaternary arrangement. Specifically, results suggest that D3 protomers can interact with each other via at least two distinct interfaces: the first one comprising residues from transmembrane domains I and II along with those from helix VIII and a second one involving transmembrane domains IV and V. Moreover, rather than existing only as distinct dimeric species, the results are consistent with the D3 receptor also assuming a quaternary structure in which two transmembrane domain I-II-helix VIII dimers interact to form a ”rhombic” tetramer via an interface involving residues from transmembrane domains VI and VII. In addition, the results also provide insights into the potential contribution of molecules of cholesterol to the overall organization and potential stability of the D3 receptor and possibly other GPCR quaternary structures.

Structural Determinants for the Interaction of Formyl Peptide Receptor 2 with Peptide Ligands

Background: Formyl peptide receptor 1 (FPR1) and FPR2 are highly homologous but bind fMet-Leu-Phe with very different affinities. Results: Asp-281 provides a negative charge that renders FPR2 more sensitive to the length and composition of formyl peptides than FPR1. Conclusion: Asp-281 is a major determinant for FPR2 binding. Significance: This work provides a structural basis for differential interaction between formyl peptides and their receptors. Unlike formyl peptide receptor 1 (FPR1), FPR2/ALX (FPR2) interacts with peptides of diverse sequences but has low affinity for the Escherichia coli-derived chemotactic peptide fMet-Leu-Phe (fMLF). Using computer modeling and site-directed mutagenesis, we investigated the structural requirements for FPR2 to interact with formyl peptides of different length and composition. In calcium flux assay, the N-formyl group of these peptides is necessary for activation of both FPR2 and FPR1, whereas the composition of the C-terminal amino acids appears more important for FPR2 than FPR1. FPR2 interacts better with pentapeptides (fMLFII, fMLFIK) than tetrapeptides (fMLFK, fMLFW) and tripeptide (fMLF) but only weakly with peptides carrying negative charges at the C terminus (e.g. fMLFE). In contrast, FPR1 is less sensitive to negative charges at the C terminus. A CXCR4-based homology model of FPR1 and FPR2 suggested that Asp-2817.32 is crucial for the interaction of FPR2 with certain formyl peptides as its negative charge may be repulsive with the terminal COO- group of fMLF and negatively charged Glu in fMLFE. Asp-2817.32 might also form a stable interaction with the positively charged Lys in fMLFK. Site-directed mutagenesis was performed to remove the negative charge at position 281 in FPR2. The D2817.32G mutant showed improved affinity for fMLFE and fMLF and reduced affinity for fMLFK compared with wild type FPR2. These results indicate that different structural determinants are used by FPR1 and FPR2 to interact with formyl peptides.

The Antiallergic Mast Cell Stabilizers Lodoxamide and Bufrolin as the First High and Equipotent Agonists of Human and Rat GPR35

Lack of high potency agonists has restricted analysis of the G protein–coupled receptor GPR35. Moreover, marked variation in potency and/or affinity of current ligands between human and rodent orthologs of GPR35 has limited their productive use in rodent models of physiology. Based on the reported modest potency of the antiasthma and antiallergic ligands cromolyn disodium and nedocromil sodium, we identified the related compounds lodoxamide and bufrolin as high potency agonists of human GPR35. Unlike previously identified high potency agonists that are highly selective for human GPR35, both lodoxamide and bufrolin displayed equivalent potency at rat GPR35. Further synthetic antiallergic ligands, either sharing features of the standard surrogate agonist zaprinast, or with lodoxamide and bufrolin, were also shown to display agonism at either human or rat GPR35. Because both lodoxamide and bufrolin are symmetric di-acids, their potential mode of binding was explored via mutagenesis based on swapping between the rat and human ortholog nonconserved arginine residues within proximity of a key conserved arginine at position 3.36. Computational modeling and ligand docking predicted the contributions of different arginine residues, other than at 3.36, in human GPR35 for these two ligands and were consistent with selective loss of potency of either bufrolin or lodoxamide at distinct arginine mutants. The computational models also suggested that bufrolin and lodoxamide would display reduced potency at a low-frequency human GPR35 single nucleotide polymorphism. This prediction was confirmed experimentally.

The Coexistence of a Novel Inactivating Mutant Thyrotropin Receptor Allele with Two Thyroid Peroxidase Mutations: A Genotype-Phenotype Correlation

CONTEXT TSH receptor (TSHR) and thyroid peroxidase (TPO) gene mutations occur independently. This is the first report of their coexistence in the same individuals. OBJECTIVES The objective of the study was to evaluate the genotype-phenotype correlations when mutations in both genes are present alone or together in the same individual. PATIENTS AND METHODS Thirty subjects from an extended Arab kindred underwent clinical investigation and molecular studies of the mutant TSHRs. RESULTS A novel mutant TSHR was identified, involving four nucleotides at three sites on the same allele, c.267G>T (L89L), c.269/270AG>CT (Q90P), and c.790C>T (P264S). In addition, two known TPO gene mutations, G493S and R540X, were identified. Thirteen heterozygotes for the mutant TSHR allele had mild hyperthyrotropinemia. In nine of theses, the coexistence of a TPO mutation in one allele did not magnify the hyperthyrotropinemia. Homozygotes for the mutant TSHR and a compound heterozygote for the TPO mutations presented frank hypothyroidism. In vitro studies showed increasing loss of function for Q90P less than P264S less than Q90P/P264S TSHR mutants, the latter being that expressed in the subjects under investigation. The two interchangeably used WT TSHR vectors, L87 and V87, although functionally identical, differed in structure and function in the presence of the Q90P mutation. CONCLUSIONS TSHR and TPO gene mutations were identified alone and together in individuals of a consanguineous kindred. Homozygotes for the TSHR and a compound heterozygote for the TPO mutations were hypothyroid. The mild hyperthyrotropinemia of heterozygotes for the mutant TSHR allele was not aggravated by the coexistence of a TPO defect in one allele.

The Parkinson's disease-associated GPR37 receptor-mediated cytotoxicity is controlled by its intracellular cysteine-rich domain

GPR37, also known as parkin‐associated endothelin‐like receptor (Pael‐R), is an orphan G protein‐coupled receptor (GPCR) that aggregates intracellularly in a juvenile form of Parkinsons disease. However, little is known about the structure or function of this receptor. Here, in order to better understand the functioning of this receptor, we focused on the GPR37 C‐terminal tail, in particular on a cystein‐enriched region. Thus, we aimed to reveal the role of these residues on receptor plasma membrane expression and function, and also whether the presence of this cysteine‐rich domain is linked to the previously described receptor‐mediated cytotoxicity. Interestingly, while the deletion of six cysteine residues within this region did not affect receptor internalization it promoted GPR37 plasma membrane expression and signaling. Furthermore, the removal of the C‐terminal cysteine‐rich domain protected against GPR37‐mediated apoptosis and cell death. Overall, we identified a GPR37 domain, namely the C‐terminal tail cysteine‐rich domain, which played a critical role in receptor cell surface expression, function and GPR37‐mediated cytotoxicity. These results might contribute to better comprehend the pathophysiology (i.e. in Parkinsons disease) of this rather unknown member of the GPCR family.