scholarly journals The Extreme C-Terminal Region of Gαs Differentially Couples to the Luteinizing Hormone and β2-Adrenergic Receptors

2011 ◽  
Vol 25 (8) ◽  
pp. 1416-1430 ◽  
Author(s):  
Geneva DeMars ◽  
Francesca Fanelli ◽  
David Puett
Keyword(s):  
G Protein ◽  
Comparative Modeling ◽  
Cell System ◽  
Amino Acid Residues ◽  
Intracellular Loop ◽  
C Terminus ◽  
Model Cell ◽  
Rigid Body Docking ◽  
Molecular Landscape ◽  
Dynamics Simulations

The mechanisms of G protein coupling to G protein-coupled receptors (GPCR) share general characteristics but may exhibit specific interactions unique for each GPCR/G protein partnership. The extreme C terminus (CT) of G protein α-subunits has been shown to be important for association with GPCR. Hypothesizing that the extreme CT of Gαs is an essential component of the molecular landscape of the GPCR, human LH receptor (LHR), and β2-adrenergic receptor (β2-AR), a model cell system was created for the expression and manipulation of Gαs subunits in LHR+ s49 ck cells that lack endogenous Gαs. On the basis of studies involving truncations, mutations, and chain extensions of Gαs, the CT was found to be necessary for LHR and β2-AR signaling. Some general similarities were found for the responses of the two receptors, but significant differences were also noted. Computational modeling was performed with a combination of comparative modeling, molecular dynamics simulations, and rigid body docking. The resulting models, focused on the Gαs CT, are supported by the experimental observations and are characterized by the interaction of the four extreme CT amino acid residues of Gαs with residues in LHR and β2-AR helix 3, (including R of the DRY motif), helix 6, and intracellular loop 2. This portion of Gαs recognizes the same regions of the two GPCR, although with differences in the details of selected interactions. The predicted longer cytosolic extensions of helices 5 and 6 of β2-AR are expected to contribute significantly to differences in Gαs recognition by the two receptors.

Polarity of TRH receptors in transfected MDCK cells is independent of endocytosis signals and G protein coupling

1996 ◽  
Vol 270 (3) ◽  
pp. C753-C762 ◽  
Author(s):  
C. Yeaman ◽  
M. Heinflink ◽  
E. Falck-Pedersen ◽  
E. Rodriguez-Boulan ◽  
M. C. Gershengorn
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Mdck Cells ◽  
Cell System ◽  
Heterotrimeric G Proteins ◽  
Intracellular Loop ◽  
Surface Receptors ◽  
Model Cell ◽  
G Protein Coupled ◽  
Trh Receptors

Information concerning the molecular sorting of G protein-coupled receptors in polarized epithelial cells is limited. Therefore, we have expressed the receptor for thyrotropin-releasing hormone (TRH) in Madin-Darby canine kidney (MDCK) cells by adenovirus-mediated gene transfer to determine its distribution in a model cell system and to begin analyzing the molecular information responsible for its distribution. Equilibrium binding of [methyl-3H]TRH to apical and basolateral surfaces of polarized MDCK cells reveals that TRH receptors are expressed predominantly (>80%) on the basolateral cell surface. Receptors undergo rapid endocytosis following agonist binding; up to 80% are internalized in 15 min. A mutant receptor missing the last 59 residues, C335Stop, is poorly internalized (<10%) but is nevertheless basolaterally expressed (>85%). A second mutant TRH receptor, delta218-263, lacks essentially all of the third intracellular loop and is not coupled to G proteins on binding agonist. This receptor internalizes TRH approximately half as efficiently as wild-type TRH receptors but is nevertheless strongly polarized to the basolateral surface (>90%). These results indicate that molecular sequences responsible for basolateral accumulation of TRH receptors can be segregated from signals for ligand-induced receptor endocytosis and coupling to heterotrimeric G proteins.

scholarly journals A structural basis for how ligand binding site changes can allosterically regulate GPCR signaling and engender functional selectivity

2020 ◽  
Vol 13 (617) ◽  
pp. eaaw5885 ◽  
Author(s):  
Marta Sanchez-Soto ◽  
Ravi Kumar Verma ◽  
Blair K. A. Willette ◽  
Elizabeth C. Gonye ◽  
Annah M. Moore ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
G Protein ◽  
Structural Basis ◽  
Protein Signaling ◽  
Ligand Binding Site ◽  
Intracellular Loop ◽  
Gpcr Signaling ◽  
Biased Signaling ◽  
Dynamics Simulations

Signaling bias is the propensity for some agonists to preferentially stimulate G protein–coupled receptor (GPCR) signaling through one intracellular pathway versus another. We previously identified a G protein–biased agonist of the D2 dopamine receptor (D2R) that results in impaired β-arrestin recruitment. This signaling bias was predicted to arise from unique interactions of the ligand with a hydrophobic pocket at the interface of the second extracellular loop and fifth transmembrane segment of the D2R. Here, we showed that residue Phe189 within this pocket (position 5.38 using Ballesteros-Weinstein numbering) functions as a microswitch for regulating receptor interactions with β-arrestin. This residue is relatively conserved among class A GPCRs, and analogous mutations within other GPCRs similarly impaired β-arrestin recruitment while maintaining G protein signaling. To investigate the mechanism of this signaling bias, we used an active-state structure of the β2-adrenergic receptor (β2R) to build β2R-WT and β2R-Y1995.38A models in complex with the full β2R agonist BI-167107 for molecular dynamics simulations. These analyses identified conformational rearrangements in β2R-Y1995.38A that propagated from the extracellular ligand binding site to the intracellular surface, resulting in a modified orientation of the second intracellular loop in β2R-Y1995.38A, which is predicted to affect its interactions with β-arrestin. Our findings provide a structural basis for how ligand binding site alterations can allosterically affect GPCR-transducer interactions and result in biased signaling.

TMEM16A–TMEM16B chimaeras to investigate the structure–function relationship of calcium-activated chloride channels

2013 ◽  
Vol 452 (3) ◽  
pp. 443-455 ◽  
Author(s):  
Paolo Scudieri ◽  
Elvira Sondo ◽  
Emanuela Caci ◽  
Roberto Ravazzolo ◽  
Luis J. V. Galietta
Keyword(s):  
Chloride Channels ◽  
Transmembrane Domain ◽  
Amino Acid Residues ◽  
Intracellular Loop ◽  
Transmembrane Segments ◽  
C Terminus ◽  
Third Intracellular Loop ◽  
Cl Channels ◽  
Function Relationship ◽  
Relationship Of

TMEM16A and TMEM16B proteins are CaCCs (Ca2+-activated Cl− channels) with eight putative transmembrane segments. As shown previously, expression of TMEM16B generates CaCCs characterized by a 10-fold lower Ca2+ affinity and by faster activation and deactivation kinetics with respect to TMEM16A. To investigate the basis of the different properties, we generated chimaeric proteins in which different domains of the TMEM16A protein were replaced by the equivalent domains of TMEM16B. Replacement of the N-terminus, TMD (transmembrane domain) 1–2, the first intracellular loop and TMD3–4 did not change the channel's properties. Instead, replacement of intracellular loop 3 decreased the apparent Ca2+ affinity by nearly 8-fold with respect to wild-type TMEM16A. In contrast, the membrane currents derived from chimaeras containing TMD7–8 or the C-terminus of TMEM16B showed higher activation and deactivation rates without a change in Ca2+ sensitivity. Significantly accelerated kinetics were also found when the entire C-terminus of the TMEM16A protein (77 amino acid residues) was deleted. Our findings indicate that the third intracellular loop of TMEM16A and TMEM16B is the site involved in Ca2+-sensitivity, whereas the C-terminal part, including TMD7–8, affect the rate of transition between the open and the closed state.

scholarly journals 4096 Refined structure of human ferroportin using restraints from mass spectrometry

2020 ◽  
Vol 4 (s1) ◽  
pp. 101-102
Author(s):  
Christian S. Parry ◽  
Andrey Ivanov ◽  
Guelaguetza Vazquez-meves ◽  
Fatemah A. Alhakami ◽  
Jessika Agyepong ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Structural Model ◽  
Structural Information ◽  
Comparative Modeling ◽  
Mass Spectrometry Data ◽  
Peptide Transporter ◽  
Transmembrane Helices ◽  
Intracellular Loop ◽  
Water Solvent ◽  
Dynamics Simulations

OBJECTIVES/GOALS: Mammals require iron for hemoglobin, respiration, immunity and as cofactor in enzymes. But free iron is toxic from the production of reactive oxygen species. Ferroportin is the sole exporter of cellular iron and it crucially determines cellular and systemic iron levels. Labile iron must be tightly regulated. This requires structural understanding. METHODS/STUDY POPULATION: We built structure of human ferroportin (FPN1) using the ab ignition prediction approaches of Rosetta/Robetta and by comparative modeling with distance restraints in MODELLER. Templates selected were from solute carrier protein families of distantly related orthologs and homologs including a proton coupled peptide transporter (PDB ID: 4IKV) and the bacterial iron transporter in outward-open and inward-open states, (PDB ID: 5AYM, 5AYO). Each model was validated by experimental mass spectrometry data. The energy minimized structural model was inserted into a lipid bilayer, placed in a rectangular simulation box, covered with TIP3P water solvent balanced with counterions and conditioned. Finally, we carried out 350 nanoseconds molecular dynamics simulations. RESULTS/ANTICIPATED RESULTS: Our first model of FPN1 (571aa), using Rosetta/Robetta ab initio approach, resembles the structure of the proton-dependent transporter, POT and consists of 12 transmembrane helices. The membrane spanning helices veer away from the orientation in the structure of 4IKV. The alternate model using MODELLER and the method of satisfaction of constraints, returned one template, the structure of Bdellovibrio bacteriovorus iron (Fe2+) transporter homolog (5AYN, 440aa) with sequence identity of 19%. Aligning FPN1 on the template sequence incorporating structural information revealed better conservation (29%). This model also comprises 12 transmembrane helices in two bundles separated by a large intracellular loop. The iron binding site predicted in both models match the structures of distant bacterial homologs. DISCUSSION/SIGNIFICANCE OF IMPACT: We are using these experimentally verified structures and functional data to answer questions about the mechanism of ferroportin iron transport, structural dynamics and the significance of mutations in ferroportin seen in different populations, especially the Q248H mutation found in Africans and black Americans with moderate to high prevalence.

scholarly journals Amino acid residues in third intracellular loop of melanocortin 1 receptor are involved in G-protein coupling

IUBMB Life ◽  
1998 ◽  
Vol 46 (5) ◽  
pp. 913-922
Author(s):  
Per-Anders Frändberg ◽  
Marina Doufexis ◽  
Supriya Kapas ◽  
Vijay Chhajlani
Keyword(s):  
Amino Acid ◽  
G Protein ◽  
Amino Acid Residues ◽  
Intracellular Loop ◽  
G Protein Coupling ◽  
Melanocortin 1 Receptor ◽  
Protein Coupling ◽  
Third Intracellular Loop

A bioactive peptide from the transmembrane 5 - intracellular loop 3 region of the human 5HT1a receptor

1998 ◽  
Vol 76 (4) ◽  
pp. 657-660 ◽  
Author(s):  
Kenneth Hayataka ◽  
Mary-Frances O'Connor ◽  
Nancy Kinzler ◽  
John T Weber ◽  
Keith K Parker
Keyword(s):  
Amino Acid ◽  
G Protein ◽  
Carboxyl Terminus ◽  
Amino Acid Residues ◽  
Intracellular Loop ◽  
G Protein Coupling ◽  
5Ht1a Receptor ◽  
Peptide Receptor ◽  
Amino Acid Peptide ◽  
Camp Elevation

15 amino acid peptide from the transmembrane 5 - intracellular loop 3 region of the human 5HT1a receptor produced concentration-dependent decreases in agonist binding. This result is consistent with a competitive interaction between peptide, receptor, and G protein at the receptor - G protein interface. Bombesin and a 13 amino acid peptide from the carboxyl terminus region of the receptor were inactive. Additionally, the peptide decreased forskolin-mediated cAMP elevation. Overall, these results suggest that amino acid residues from this region of the receptor are involved in receptor - G protein coupling and that G protein is activated by the receptor.Key words: serotonin, 5HT1a receptor, G protein, cAMP, loop peptide.

scholarly journals Effects of the Q80K Polymorphism on the Physicochemical Properties of Hepatitis C Virus Subtype 1a NS3 Protease

Viruses ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 691
Author(s):  
Allan Peres-da-Silva ◽  
Deborah Antunes ◽  
André Luiz Quintanilha Torres ◽  
Ernesto Raul Caffarena ◽  
Elisabeth Lampe
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Physicochemical Properties ◽  
Comparative Modeling ◽  
Amino Acid Residues ◽  
Structural Protein ◽  
Virus Genotype ◽  
Genetic Barriers ◽  
Dynamics Simulations ◽  
Ns3 Protease

Hepatitis C virus genotype 1a (HCV-1a) comprises clades I and II. The Q80K polymorphism is found predominantly in clade I but rarely in clade II. Here, we investigated whether natural polymorphisms in HCV-1a clade II entailed structural protein changes when occurrence of the Q80K variant was simulated. Based on HCV-1a clade I and II protein sequences, the structure of the HCV-1a Q80K mutant NS3-4A was obtained by comparative modeling. Its physicochemical properties were studied by molecular dynamics simulations and network analysis. Results demonstrate that, in the presence of the K80 variant, clade II protease polymorphisms A91 and S/G174 led to variations in hydrogen bond occupancies. Structural analyses revealed differences in (i) flexibility of the H57 catalytic residue on the NS3 protease and (ii) correlations between amino acids on the NS3 protease and the NS4A cofactor. The latter indicated possible destabilization of interactions, resulting in increased separation of these proteins. The present findings describe how the relationships between different HCV-1a NS3 protease amino acid residues could affect the appearance of viral variants and the existence of distinct genetic barriers to HCV-1a isolates.

scholarly journals Conformational plasticity of the intracellular cavity of GPCR−G-protein complexes leads to G-protein promiscuity and selectivity

2019 ◽  
pp. 201820944 ◽  
Author(s):  
Manbir Sandhu ◽  
Anja M. Touma ◽  
Matthew Dysthe ◽  
Fredrik Sadler ◽  
Sivaraj Sivaramakrishnan ◽  
...  
Keyword(s):  
G Protein ◽  
Protein Complexes ◽  
Dependent Manner ◽  
Secondary Messenger ◽  
C Terminus ◽  
Conformational Plasticity ◽  
Small Ensemble ◽  
Dynamics Simulations ◽  
The Impact ◽  
Dose Dependent

While the dynamics of the intracellular surface in agonist-stimulated GPCRs is well studied, the impact of GPCR dynamics on G-protein selectivity remains unclear. Here, we combine molecular dynamics simulations with live-cell FRET and secondary messenger measurements, for 21 GPCR−G-protein combinations, to advance a dynamic model of the GPCR−G-protein interface. Our data show C terminus peptides of Gαs, Gαi, and Gαq proteins assume a small ensemble of unique orientations when coupled to their cognate GPCRs, similar to the variations observed in 3D structures of GPCR−G-protein complexes. The noncognate G proteins interface with latent intracellular GPCR cavities but dissociate due to weak and unstable interactions. Three predicted mutations in β2-adrenergic receptor stabilize binding of noncognate Gαq protein in its latent cavity, allowing promiscuous signaling through both Gαs and Gαq in a dose-dependent manner. This demonstrates that latent GPCR cavities can be evolved, by design or nature, to tune G-protein selectivity, giving insights to pluridimensional GPCR signaling.

Schistosomal Sulfotransferase Interaction with Oxamniquine Involves Hybrid Mechanism of Induced-fit and Conformational Selection

2020 ◽  
Vol 16 (4) ◽  
pp. 451-459 ◽  
Author(s):  
Fortunatus C. Ezebuo ◽  
Ikemefuna C. Uzochukwu
Keyword(s):  
Molecular Dynamics ◽  
Amino Acid ◽  
Binding Energy ◽  
Binding Site ◽  
Conformational Dynamics ◽  
Side Chain ◽  
Amino Acid Residues ◽  
Conformational Selection ◽  
Hybrid Mechanism ◽  
Dynamics Simulations

Background: Sulfotransferase family comprises key enzymes involved in drug metabolism. Oxamniquine is a pro-drug converted into its active form by schistosomal sulfotransferase. The conformational dynamics of side-chain amino acid residues at the binding site of schistosomal sulfotransferase towards activation of oxamniquine has not received attention. Objective: The study investigated the conformational dynamics of binding site residues in free and oxamniquine bound schistosomal sulfotransferase systems and their contribution to the mechanism of oxamniquine activation by schistosomal sulfotransferase using molecular dynamics simulations and binding energy calculations. Methods: Schistosomal sulfotransferase was obtained from Protein Data Bank and both the free and oxamniquine bound forms were subjected to molecular dynamics simulations using GROMACS-4.5.5 after modeling it’s missing amino acid residues with SWISS-MODEL. Amino acid residues at its binding site for oxamniquine was determined and used for Principal Component Analysis and calculations of side-chain dihedrals. In addition, binding energy of the oxamniquine bound system was calculated using g_MMPBSA. Results: The results showed that binding site amino acid residues in free and oxamniquine bound sulfotransferase sampled different conformational space involving several rotameric states. Importantly, Phe45, Ile145 and Leu241 generated newly induced conformations, whereas Phe41 exhibited shift in equilibrium of its conformational distribution. In addition, the result showed binding energy of -130.091 ± 8.800 KJ/mol and Phe45 contributed -9.8576 KJ/mol. Conclusion: The results showed that schistosomal sulfotransferase binds oxamniquine by relying on hybrid mechanism of induced fit and conformational selection models. The findings offer new insight into sulfotransferase engineering and design of new drugs that target sulfotransferase.

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