Publications by authors named "Nagarajan Vaidehi"

109 Publications

A universal allosteric mechanism for G protein activation.

Mol Cell 2021 Feb 20. Epub 2021 Feb 20.

Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address:

G proteins play a central role in signal transduction and pharmacology. Signaling is initiated by cell-surface receptors, which promote guanosine triphosphate (GTP) binding and dissociation of Gα from the Gβγ subunits. Structural studies have revealed the molecular basis of subunit association with receptors, RGS proteins, and downstream effectors. In contrast, the mechanism of subunit dissociation is poorly understood. We use cell signaling assays, molecular dynamics (MD) simulations, and biochemistry and structural analyses to identify a conserved network of amino acids that dictates subunit release. In the presence of the terminal phosphate of GTP, a glycine forms a polar network with an arginine and glutamate, putting torsional strain on the subunit binding interface. This "G-R-E motif" secures GTP and, through an allosteric link, discharges the Gβγ dimer. Replacement of network residues prevents subunit dissociation regardless of agonist or GTP binding. These findings reveal the molecular basis of the final committed step of G protein activation.
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http://dx.doi.org/10.1016/j.molcel.2021.02.002DOI Listing
February 2021

Kinase inhibitors allosterically disrupt a regulatory interaction to enhance PKCα membrane translocation.

J Biol Chem 2021 Jan 25:100339. Epub 2021 Jan 25.

Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota 55455, United States. Electronic address:

The eukaryotic kinase domain has multiple intrinsically disordered regions whose conformation dictates kinase activity. Small molecule kinase inhibitors (SMKIs) rely on disrupting the active conformations of these disordered regions to inactivate the kinase. While SMKIs are selected for their ability to cause this disruption, the allosteric effects of conformational changes in disordered regions is limited by a lack of dynamic information provided by traditional structural techniques. In this study, we integrated multi-scale molecular dynamics simulations with FRET sensors to characterize a novel allosteric mechanism that is selectively triggered by SMKI binding to the PKCα kinase domain. The indole maleimide inhibitors BimI and sotrastaurin were found to displace the Gly-rich loop (G-loop) that normally shields the ATP-binding site. Displacement of the Gly-rich loop interferes with a newly identified, structurally conserved binding pocket for the C1a domain on the N lobe of the kinase domain. This binding pocket, in conjunction with the N-terminal regulatory sequence, masks a diacylglycerol (DAG) binding site on the C1a domain. SMKI-mediated displacement of the Gly-rich loop released C1a and exposed the DAG binding site, enhancing PKCα translocation both to synthetic lipid bilayers and to live cell membranes in the presence of DAG. Inhibitor chemotype determined the extent of the observed allosteric effects on the kinase domain, and correlated with the extent of membrane recruitment. Our findings demonstrate the allosteric effects of SMKIs beyond the confines of kinase catalytic conformation, and provide an integrated computational-experimental paradigm to investigate parallel mechanisms in other kinases.
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http://dx.doi.org/10.1016/j.jbc.2021.100339DOI Listing
January 2021

Structure of the class D GPCR Ste2 dimer coupled to two G proteins.

Nature 2021 01 2;589(7840):148-153. Epub 2020 Dec 2.

MRC Laboratory of Molecular Biology, Cambridge, UK.

G-protein-coupled receptors (GPCRs) are divided phylogenetically into six classes, denoted A to F. More than 370 structures of vertebrate GPCRs (belonging to classes A, B, C and F) have been determined, leading to a substantial understanding of their function. By contrast, there are no structures of class D GPCRs, which are found exclusively in fungi where they regulate survival and reproduction. Here we determine the structure of a class D GPCR, the Saccharomyces cerevisiae pheromone receptor Ste2, in an active state coupled to the heterotrimeric G protein Gpa1-Ste4-Ste18. Ste2 was purified as a homodimer coupled to two G proteins. The dimer interface of Ste2 is formed by the N terminus, the transmembrane helices H1, H2 and H7, and the first extracellular loop ECL1. We establish a class D1 generic residue numbering system (CD1) to enable comparisons with orthologues and with other GPCR classes. The structure of Ste2 bears similarities in overall topology to class A GPCRs, but the transmembrane helix H4 is shifted by more than 20 Å and the G-protein-binding site is a shallow groove rather than a cleft. The structure provides a template for the design of novel drugs to target fungal GPCRs, which could be used to treat numerous intractable fungal diseases.
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http://dx.doi.org/10.1038/s41586-020-2994-1DOI Listing
January 2021

IFITM3 functions as a PIP3 scaffold to amplify PI3K signalling in B cells.

Nature 2020 12 4;588(7838):491-497. Epub 2020 Nov 4.

Center of Molecular and Cellular Oncology, Yale Cancer Center, Yale School of Medicine, New Haven, CT, USA.

Interferon-induced transmembrane protein 3 (IFITM3) has previously been identified as an endosomal protein that blocks viral infection. Here we studied clinical cohorts of patients with B cell leukaemia and lymphoma, and identified IFITM3 as a strong predictor of poor outcome. In normal resting B cells, IFITM3 was minimally expressed and mainly localized in endosomes. However, engagement of the B cell receptor (BCR) induced both expression of IFITM3 and phosphorylation of this protein at Tyr20, which resulted in the accumulation of IFITM3 at the cell surface. In B cell leukaemia, oncogenic kinases phosphorylate IFITM3 at Tyr20, which causes constitutive localization of this protein at the plasma membrane. In a mouse model, Ifitm3 naive B cells developed in normal numbers; however, the formation of germinal centres and the production of antigen-specific antibodies were compromised. Oncogenes that induce the development of leukaemia and lymphoma did not transform Ifitm3 B cells. Conversely, the phosphomimetic IFITM3(Y20E) mutant induced oncogenic PI3K signalling and initiated the transformation of premalignant B cells. Mechanistic experiments revealed that IFITM3 functions as a PIP3 scaffold and central amplifier of PI3K signalling. The amplification of PI3K signals depends on IFITM3 using two lysine residues (Lys83 and Lys104) in its conserved intracellular loop as a scaffold for the accumulation of PIP3. In Ifitm3 B cells, lipid rafts were depleted of PIP3, which resulted in the defective expression of over 60 lipid-raft-associated surface receptors, and impaired BCR signalling and cellular adhesion. We conclude that the phosphorylation of IFITM3 that occurs after B cells encounter antigen induces a dynamic switch from antiviral effector functions in endosomes to a PI3K amplification loop at the cell surface. IFITM3-dependent amplification of PI3K signalling, which in part acts downstream of the BCR, is critical for the rapid expansion of B cells with high affinity to antigen. In addition, multiple oncogenes depend on IFITM3 to assemble PIP3-dependent signalling complexes and amplify PI3K signalling for malignant transformation.
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http://dx.doi.org/10.1038/s41586-020-2884-6DOI Listing
December 2020

Activation Microswitches in Adenosine Receptor A Function as Rheostats in the Cell Membrane.

Biochemistry 2020 Oct 15;59(42):4059-4071. Epub 2020 Oct 15.

Department of Computational and Quantitative Medicine, Beckman Research Institute of the City of Hope, 1500 East Duarte Road, Duarte, California 91010, United States.

Although multiple components of the cell membrane modulate the stability and activation of G protein-coupled receptors (GPCRs), insights into the dynamics of GPCR structures come from biophysical studies conducted in detergents. This is because of the challenges of studying activation in a multicomponent lipid bilayer. To understand the role of cellular membrane lipids and cations in GPCR activation, we performed multiscale molecular dynamics simulations (56 μs) on three different conformational states of adenosine receptor AR, in both the cell membrane-like lipid bilayer and in detergent micelles. Molecular dynamics (MD) simulations show that the phosphatidylinositol bisphosphate (PIP2) interacts with the basic residues in the intracellular regions of AR, thereby reducing the flexibility of the receptor in the inactive state and limiting the transition to the active-intermediate state. In the G protein-coupled fully active state, PIP2 stabilizes the GPCR:G protein complex. Such stiffening effects are absent in non-ionic detergent micelles, and therefore, more transitions have been observed in detergents. The inter-residue distances that change significantly upon GPCR activation are known as activation microswitches. The activation microswitches show different levels of activation in the cell membrane, in the pure POPC bilayer, and in detergents. Thus, the temporal heat map of different activation microswitches calculated from the MD simulations suggests a rheostat model of GPCR activation microswitches rather than the binary switch model. These simulation results connect the chemistry of cell membrane lipids to receptor activity, which is useful for the design of detergents mimicking the cell membrane.
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http://dx.doi.org/10.1021/acs.biochem.0c00626DOI Listing
October 2020

Structural characterization of a dimeric complex between the short cytoplasmic domain of CEACAM1 and the pseudo tetramer of S100A10-Annexin A2 using NMR and molecular dynamics.

Biochim Biophys Acta Biomembr 2021 Jan 21;1863(1):183451. Epub 2020 Aug 21.

Department of Molecular Imaging and Therapy, , Beckman Research Institute of City of Hope, 1450 East Duarte Road, Duarte, CA 91010, United States of America. Electronic address:

AIIt, a heterotetramer of S100A10 (P11) and Annexin A2, plays a key role in calcium dependent, membrane associations with a variety of proteins. We previously showed that AIIt interacts with the short cytoplasmic domain (12 amino acids) of CEACAM1 (CEACAM1-SF). Since the cytoplasmic domains of CEACAM1 help regulate the formation of cis- or trans-dimers at the cell membrane, we investigated the possible role of their association with AIIt in this process. Using NMR and molecular dynamics, we show that AIIt and its pseudoheterodimer interacts with two molecules of short cytoplasmic domain isoform peptides, and that interaction depends on the binding motif 454-Phe-Gly-Lys-Thr-457 where Phe-454 binds in a hydrophobic pocket of AIIt, the null mutation Phe454Ala reduces binding by 2.5 fold, and the pseudophosphorylation mutant Thr457Glu reduces binding by three fold. Since these two residues in CEACAM1-SF were also found to play a role in the binding of calmodulin and G-actin at the membrane, we hypothesize a sequential set of three interactions are responsible for regulation of cis- to trans-dimerization of CEACAM1. The hydrophobic binding pocket in AIIt corresponds to a previously identified binding pocket for a peptide found in SMARCA3 and AHNAK, suggesting a conserved functional motif in AIIt allowing multiple proteins to reversibly interact with integral membrane proteins in a calcium dependent manner.
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http://dx.doi.org/10.1016/j.bbamem.2020.183451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7704867PMC
January 2021

How Do Branched Detergents Stabilize GPCRs in Micelles?

Biochemistry 2020 06 5;59(23):2125-2134. Epub 2020 Jun 5.

Department of Computational and Quantitative Medicine, Beckman Research Institute of the City of Hope, 1500 East Duarte Road, Duarte, California 91010, United States.

The structural and functional properties of G protein-coupled receptors (GPCRs) are often studied in a detergent micellar environment, but many GPCRs tend to denature or aggregate in short alkyl chain detergents. In our previous work [Lee, S., et al. (2016) , 15425-15433], we showed that GPCRs in alkyl glucosides were highly dynamic, resulting in the penetration of detergent molecules between transmembrane α-helices, which is the initial step in receptor denaturation. Although this was not observed for GPCRs in dodecyl maltoside (DDM, also known as lauryl maltoside), even this detergent is not mild enough to preserve the integrity of many GPCRs during purification. Lauryl maltose neopentylglycol (LMNG) detergents have been found to have significant advantages for purifying GPCRs in a native state as they impart more stability to the receptor than DDM. To gain insights into how they stabilize GPCRs, we used atomistic molecular dynamics simulations of wild type adenosine A receptor (WT-AR), thermostabilized AR (tAR), and wild type β-adrenoceptor (βAR) in a variety of detergents (LMNG, DMNG, OGNG, and DDM). Analysis of molecular dynamics simulations of tAR in LMNG, DMNG, and OGNG showed that this series of detergents exhibited behavior very similar to that of an analogous series of detergents DDM, DM, and OG in our previous study. However, there was a striking difference upon comparison of the behavior of LMNG to that of DDM. LMNG showed considerably less motion than DDM, which resulted in the enhanced density of the aliphatic chains around the hydrophobic regions of the receptor and considerably more hydrogen bond formation between the head groups. This contributed to enhanced interaction energies between both detergent molecules and between the receptor and detergent, explaining the enhanced stability of GPCRs purified in this detergent. Branched detergents occlude between transmembrane helices and reduce their flexibility. Our results provide a rational foundation to develop detergent variants for stabilizing membrane proteins.
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http://dx.doi.org/10.1021/acs.biochem.0c00183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7302508PMC
June 2020

Structural instability and divergence from conserved residues underlie intracellular retention of mammalian odorant receptors.

Proc Natl Acad Sci U S A 2020 02 23;117(6):2957-2967. Epub 2020 Jan 23.

Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710;

Mammalian odorant receptors are a diverse and rapidly evolving set of G protein-coupled receptors expressed in olfactory cilia membranes. Most odorant receptors show little to no cell surface expression in nonolfactory cells due to endoplasmic reticulum retention, which has slowed down biochemical studies. Here we provide evidence that structural instability and divergence from conserved residues of individual odorant receptors underlie intracellular retention using a combination of large-scale screening of odorant receptors cell surface expression in heterologous cells, point mutations, structural modeling, and machine learning techniques. We demonstrate the importance of conserved residues by synthesizing consensus odorant receptors that show high levels of cell surface expression similar to conventional G protein-coupled receptors. Furthermore, we associate in silico structural instability with poor cell surface expression using molecular dynamics simulations. We propose an enhanced evolutionary capacitance of olfactory sensory neurons that enable the functional expression of odorant receptors with cryptic mutations.
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http://dx.doi.org/10.1073/pnas.1915520117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7022149PMC
February 2020

Machine Learning for Prioritization of Thermostabilizing Mutations for G-Protein Coupled Receptors.

Biophys J 2019 12 24;117(11):2228-2239. Epub 2019 Oct 24.

Department of Computational and Quantitative Medicine, Beckman Research Institute of the City of Hope, Duarte, California. Electronic address:

Although the three-dimensional structures of G-protein coupled receptors (GPCRs), the largest superfamily of drug targets, have enabled structure-based drug design, there are no structures available for 87% of GPCRs. This is due to the stiff challenge in purifying the inherently flexible GPCRs. Identifying thermostabilized mutant GPCRs via systematic alanine scanning mutations has been a successful strategy in stabilizing GPCRs, but it remains a daunting task for each GPCR. We developed a computational method that combines sequence-, structure-, and dynamics-based molecular properties of GPCRs that recapitulate GPCR stability, with four different machine learning methods to predict thermostable mutations ahead of experiments. This method has been trained on thermostability data for 1231 mutants, the largest publicly available data set. A blind prediction for thermostable mutations of the complement factor C5a receptor 1 retrieved 36% of the thermostable mutants in the top 50 prioritized mutants compared to 3% in the first 50 attempts using systematic alanine scanning.
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http://dx.doi.org/10.1016/j.bpj.2019.10.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895739PMC
December 2019

Generation of dual specific bivalent BiTEs (dbBIspecific T-cell engaging antibodies) for cellular immunotherapy.

BMC Cancer 2019 Sep 5;19(1):882. Epub 2019 Sep 5.

Department of Molecular Imaging and Therapy, Beckman Research Institute City of Hope, Duarte, CA, 91010, USA.

Background: Bispecific T-cell engaging antibodies (BiTES), comprising dual anti-CD3 and anti-tumor antigen scFv fragments, are important therapeutic agents for the treatment of cancer. The dual scFv construct for BiTES requires proper protein folding while their small molecular size leads to rapid kidney clearance.

Methods: An intact (150 kDa) anti-tumor antigen antibody to CEA was joined in high yield (ca. 30%) to intact (150 kDa) anti-murine and anti-human CD3 antibodies using hinge region specific Click chemistry to form dual-specific, bivalent BiTES (dbBiTES, 300 kDa). dbBiTEs were tested in vitro by EM, flow cytometry and cell cytoxicity and in vivo by PET tumor imaging and redirected T-cell therapy.

Results: The interlocked hinge regions are compatible with a structural model that fits the electron micrographs of 300 kDa particles. Compared to intact anti-CEA antibody, dbBiTES exhibit high in vitro cytotoxicity, high in vivo tumor targeting as demonstrated by PET imaging, and redirected dbBiTE coated T-cells (1 microgram/10 million cells) that kill CEA target cells in vivo in CEA transgenic mice.

Conclusion: dbBiTE redirected T-cell therapy is a promising, efficient approach for targeting and killing cancer cells.
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http://dx.doi.org/10.1186/s12885-019-6056-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6727398PMC
September 2019

EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma.

Oncogenesis 2019 Sep 4;8(9):49. Epub 2019 Sep 4.

Department of Medical Oncology and Experimental Therapeutics, Comprehensive Cancer Center, City of Hope, Duarte, CA, USA.

Squamous cell carcinoma (SCC) and malignant pleural mesothelioma (MPM) are thoracic malignancies with very poor prognosis and limited treatment options. It is an established fact that most of the solid tumors have overexpression of EPHA2 receptor tyrosine kinase. EPHA2 is known to exhibit opposing roles towards cancer progression. It functions in inhibiting cancer survival and migration via a ligand and tyrosine kinase dependent signaling (Y772). Whereas it is known to promote tumor progression and cell migration through a ligand-independent signaling (S897). We analyzed the expression profile and mutational status of the ephrin receptor A2 (EPHA2) in SCC and MPM cell lines and primary patient specimens. The EPHA2 receptor was found to be either overexpressed, mutated or amplified in SCC and MPM. In particular, the EPHA2 mutants A859D and T647M were interesting to explore, A859D Y772 dead mutant exhibited lower levels of phosphorylation at Y772 compared to T647M mutant. Molecular Dynamics simulations studies suggested that differential changes in conformation might form the structural basis for differences in the level of EPHA2 activation. Consequently, A859D mutant cells exhibited increased proliferation as well as cell migration compared to controls and T647M mutant. Kinomics analysis demonstrated that the STAT3 and PDGF pathways were upregulated whereas signaling through CBL was suppressed. Considered together, the present work has uncovered the oncogenic characteristics of EPHA2 mutations in SSC and MPM reinstating the dynamics of different roles of EPHA2 in cancer. This study also suggests that a combination of doxazosin and other EPHA2 inhibitors directed to inhibit the pertinent signaling components may be a novel therapeutic strategy for MPM and Non-small cell lung cancer patients who have either EPHA2 or CBL alterations.
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http://dx.doi.org/10.1038/s41389-019-0159-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726628PMC
September 2019

Prediction of Conformation Specific Thermostabilizing Mutations for Class A G Protein-Coupled Receptors.

J Chem Inf Model 2019 09 27;59(9):3744-3754. Epub 2019 Aug 27.

Department of Computation and Quantitative Medicine , Beckman Research Institute of the City of Hope , 1500 E. Duarte Road , Duarte , California 91010 , United States.

G protein-coupled receptors (GPCRs) are highly flexible and prone to denaturation during protein extraction in detergents and purification. This poses a huge challenge to purify a conformationally homogeneous solution of GPCRs. Thermostabilizing mutations have been used widely to purify and obtain crystal structures of several GPCRs. However, identifying thermostabilizing mutations for GPCRs remains a tedious and expensive task as they are not transferable even among closely related GPCRs. Additionally, the mutations stabilizing one conformational state of a GPCR do not always stabilize other conformational state(s) of the same GPCR. Previously we developed a computational method, LiticonDesign, for rapid prediction of thermostabilizing mutations for a specific GPCR conformation. In this study, we have used LiticonDesign to predict thermostabilizing mutations for the agonist bound active-intermediate state of the human adenosine receptor (AR) using the structure of the inactive state of the same GPCR and vice versa. Our study shows that the thermostable mutation predictions using LiticonDesign, for an active-intermediate state of a GPCR (AR in our case), requires a homology model that is derived from an active/active-intermediate state GPCR structure as a template. Similarly, the homology models derived from inactive state GPCR conformations are better in predicting the thermostable mutations for the inactive state of AR. Overall, LiticonDesign method is not only efficient in predicting thermostabilizing mutations for a given GPCR sequence but also can recover conformation specific mutations for a state of interest, if a suitable starting structure of desired conformation is chosen.
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http://dx.doi.org/10.1021/acs.jcim.9b00175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6944194PMC
September 2019

Variable G protein determinants of GPCR coupling selectivity.

Proc Natl Acad Sci U S A 2019 06 29;116(24):12054-12059. Epub 2019 May 29.

Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, Augusta, GA 30912;

G protein-coupled receptors (GPCRs) activate four families of heterotrimeric G proteins, and individual receptors must select a subset of G proteins to produce appropriate cellular responses. Although the precise mechanisms of coupling selectivity are uncertain, the Gα subunit C terminus is widely believed to be the primary determinant recognized by cognate receptors. Here, we directly assess coupling between 14 representative GPCRs and 16 Gα subunits, including one wild-type Gα subunit from each of the four families and 12 chimeras with exchanged C termini. We use a sensitive bioluminescence resonance energy transfer (BRET) assay that provides control over both ligand and nucleotide binding, and allows direct comparison across G protein families. We find that the G- and G-coupled receptors we studied are relatively promiscuous and always couple to some extent to G heterotrimers. In contrast, G-coupled receptors are more selective. Our results with Gα subunit chimeras show that the Gα C terminus is important for coupling selectivity, but no more so than the Gα subunit core. The relative importance of the Gα subunit core and C terminus is highly variable and, for some receptors, the Gα core is more important for selective coupling than the C terminus. Our results suggest general rules for GPCR-G protein coupling and demonstrate that the critical G protein determinants of selectivity vary widely, even for different receptors that couple to the same G protein.
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http://dx.doi.org/10.1073/pnas.1905993116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6575158PMC
June 2019

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

Proc Natl Acad Sci U S A 2019 06 28;116(24):11956-11965. Epub 2019 May 28.

Irell & Manella Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, Duarte, CA 91010;

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 Gα, Gα, and Gα 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 β-adrenergic receptor stabilize binding of noncognate Gα protein in its latent cavity, allowing promiscuous signaling through both Gα and Gα 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.
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http://dx.doi.org/10.1073/pnas.1820944116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6575595PMC
June 2019

Leflunomide regulates c-Myc expression in myeloma cells through PIM targeting.

Blood Adv 2019 04;3(7):1027-1032

Department of Hematology and Hematopoietic Cell Transplantation, Judy and Bernard Briskin Center for Multiple Myeloma Research, Hematologic Malignancies and Stem Cell Transplantation Institute, and.

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http://dx.doi.org/10.1182/bloodadvances.2018027227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6457239PMC
April 2019

Efficient and Selective Electrochemically Driven Enzyme-Catalyzed Reduction of Carbon Dioxide to Formate using Formate Dehydrogenase and an Artificial Cofactor.

Acc Chem Res 2019 03 11;52(3):676-685. Epub 2019 Feb 11.

Loker Hydrocarbon Research Institute, Department of Chemistry , University of Southern California , Los Angeles , California 90089 , United States.

Increasing levels of carbon dioxide in the atmosphere and the growing need for energy necessitate a shift toward reliance on renewable energy sources and the utilization of carbon dioxide. Thus, producing carbonaceous fuel by the electrochemical reduction of carbon dioxide has been very appealing. We have focused on addressing the principal challenges of poor selectivity and poor energy efficiency in the electrochemical reduction of carbon dioxide. We have demonstrated here a viable pathway for the efficient and continuous electrochemical reduction of CO to formate using the metal-independent enzyme type of formate dehydrogenase (FDH) derived from C andida boidinii yeast. This type of FDH is attractive because it is commercially produced. In natural metabolic processes, this type of metal-independent FDH oxidizes formate to carbon dioxide using NAD as a cofactor. We show that FDH can catalyze the reverse process to generate formate when the natural cofactor NADH is replaced with an artificial cofactor, the methyl viologen radical cation. The methyl viologen radical cation is generated in situ, electrochemically. Our approach relies on the special properties of methyl viologen as a "unidirectional" redox cofactor for the conversion of CO to formate. Methyl viologen (in the oxidized form) does not catalyze formate oxidation, while the methyl viologen radical cation is an effective cofactor for the reduction of carbon dioxide. Thus, although the thermodynamic driving force is favorable for the oxidized form of methyl viologen to oxidize formate to carbon dioxide, the kinetic factors are not favorable. Only the reverse reaction of carbon dioxide reduction to formate is kinetically viable with the cofactor, methyl viologen radical cation. Binding free energy calculated from atomistic molecular dynamics (MD) simulations consolidate our understanding of these special binding properties of the methyl viologen radical cation and its ability to facilitate the two-electron reduction of carbon dioxide to formate in metal-independent FDH. By carrying out the reactions in a novel three-compartment cell, we have demonstrated the continuous production of formate at high energy efficiency and yield. This cell configuration uses judiciously selected ion-exchange membranes to separate the reaction compartments to preserve the yields of the methyl viologen radical cation and formate. By the electroregeneration of the methyl viologen radical cation at -0.44 V versus the normal hydrogen electrode, we could produce formate at 20 mV negative to the reversible electrode potential for carbon dioxide reduction to formate. Our results are in sharp contrast to the large overpotentials of -800 to -1000 mV required on metal catalysts, vindicating the selectivity and kinetic facility provided by FDH. Formate yields as high as 97% ± 1% could be realized by avoiding the adventitious reoxidation of the methyl viologen radical cation by molecular oxygen. We anticipate that the insights from the electrochemical studies and the MD simulations to be useful in redesigning the metal-independent FDH and alternate artificial cofactors to achieve even higher rates of conversion.
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http://dx.doi.org/10.1021/acs.accounts.8b00551DOI Listing
March 2019

Dynamic Role of the G Protein in Stabilizing the Active State of the Adenosine A Receptor.

Structure 2019 04 31;27(4):703-712.e3. Epub 2019 Jan 31.

Department of Computational and Quantitative Medicine, Beckman Research Institute of the City of Hope, 1500 E. Duarte Road, Duarte, CA 91010, USA. Electronic address:

Agonist binding in the extracellular region of the G protein-coupled adenosine A2A receptor increases its affinity to the G proteins in the intracellular region, and vice versa. The structural basis for this effect is not evident from the crystal structures of AR in various conformational states since it stems from the receptor dynamics. Using atomistic molecular dynamics simulations on four different conformational states of the adenosine A receptor, we observed that the agonists show decreased ligand mobility, lower entropy of the extracellular loops in the active-intermediate state compared with the inactive state. In contrast, the entropy of the intracellular region increases to prime the receptor for coupling the G protein. Coupling of the G protein to AR shrinks the agonist binding site, making tighter receptor agonist contacts with an increase in the strength of allosteric communication compared with the active-intermediate state. These insights provide a strong basis for structure-based ligand design studies.
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http://dx.doi.org/10.1016/j.str.2018.12.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6531377PMC
April 2019

NMR analysis of free and lipid nanodisc anchored CEACAM1 membrane proximal peptides with Ca/CaM.

Biochim Biophys Acta Biomembr 2019 04 10;1861(4):787-797. Epub 2019 Jan 10.

Department of Molecular Imaging and Therapy, Diabetes, Metabolism and Research Institute of City of Hope, 1450 East Duarte Road, Duarte, CA 91010, United States of America. Electronic address:

CEACAM1, a homotypic transmembrane receptor with 12 or 72 amino acid cytosolic domain isoforms, is converted from inactive cis-dimers to active trans-dimers by calcium-calmodulin (Ca/CaM). Previously, the weak binding of Ca/CaM to the human 12 AA cytosolic domain was studied using C-terminal anchored peptides. We now show the binding of N labeled Phe-454 cytosolic domain peptides in solution or membrane anchored using NMR demonstrates a significant role for the lipid bilayer. Although binding is increased by the mutation Phe454Ala, this mutation was previously shown to abrogate actin binding. On the other hand, Ca/CaM binding is abrogated by phosphorylation of nearby Thr-457, a post-translation modification required for actin binding and subsequent in vitro lumen formation. Binding of Ca/CaM to a membrane proximal peptide from the long 72 AA cytosolic domain anchored to lipid nanodiscs was very weak compared to lipid free conditions, suggesting membrane specific effects between the two isoforms. NMR analysis of N labeled Ca/CaM with unlabeled peptides showed the C-lobe of Ca/CaM is involved in peptide interactions, and hydrophobic residues such as Met-109, Val-142 and Met-144 play important roles in binding peptide. This information was incorporated into transmembrane models of CEACAM1 binding to Ca/CaM. The lack of Ca/CaM binding to phosphorylated Thr-457, a residue we have previously shown to be phosphorylated by CaMK2D, also dependent on Ca/CaM, suggests stepwise binding of the cytosolic domain first to Ca/CaM and then to actin.
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http://dx.doi.org/10.1016/j.bbamem.2019.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6524640PMC
April 2019

Structural insights into the subtype-selective antagonist binding to the M muscarinic receptor.

Nat Chem Biol 2018 12 12;14(12):1150-1158. Epub 2018 Nov 12.

Department of Cell Biology and Medical Chemistry, Graduate School of Medicine, Kyoto University, Konoe-cho, Yoshida, Sakyo-ku, Kyoto, Japan.

Human muscarinic receptor M is one of the five subtypes of muscarinic receptors belonging to the family of G-protein-coupled receptors. Muscarinic receptors are targets for multiple neurodegenerative diseases. The challenge has been designing subtype-selective ligands against one of the five muscarinic receptors. We report high-resolution structures of a thermostabilized mutant M receptor bound to a subtype-selective antagonist AF-DX 384 and a nonselective antagonist NMS. The thermostabilizing mutation S110R in M was predicted using a theoretical strategy previously developed in our group. Comparison of the crystal structures and pharmacological properties of the M receptor shows that the Arg in the S110R mutant mimics the stabilizing role of the sodium cation, which is known to allosterically stabilize inactive state(s) of class A GPCRs. Molecular dynamics simulations reveal that tightening of the ligand-residue contacts in M receptors compared to M receptors leads to subtype selectivity of AF-DX 384.
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http://dx.doi.org/10.1038/s41589-018-0152-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6462224PMC
December 2018

Engineering Salt Bridge Networks between Transmembrane Helices Confers Thermostability in G-Protein-Coupled Receptors.

J Chem Theory Comput 2018 Dec 6;14(12):6574-6585. Epub 2018 Nov 6.

Department of Molecular Imaging and Therapy , Beckman Research Institute of the City of Hope , 1500 East Duarte Road , Duarte , California 91010 , United States.

Introduction of specific point mutations has been an effective strategy in enhancing the thermostability of G-protein-coupled receptors (GPCRs). Our previous work showed that a specific residue position on transmembrane helix 6 (TM6) in class A GPCRs consistently yields thermostable mutants. The crystal structure of human chemokine receptor CCR5 also showed increased thermostability upon mutation of two positions, A233D and K303E. With the goal of testing the transferability of these two thermostabilizing mutations in other chemokine receptors, we tested the mutations A237D and R307E in human CCR3 for thermostability and aggregation properties in detergent solution. Interestingly, the double mutant exhibited a 6-10-fold decrease in the aggregation propensity of the wild-type protein. This is in stark contrast to the two single mutants whose aggregation properties resemble the wild type (WT). Moreover, unlike in CCR5, the two single mutants separately showed no increase in thermostability compared to the wild-type CCR3, while the double-mutant A237D/R307E confers an increase of 2.6 °C in the melting temperature compared to the WT. Extensive all-atom molecular dynamics (MD) simulations in detergent micelles show that a salt bridge network between transmembrane helices TM3, TM6, and TM7 that is absent in the two single mutants confers stability in the double mutant. The free energy surface of the double mutant shows conformational homogeneity compared to the single mutants. An annular n-dodecyl maltoside detergent layer packs tighter to the hydrophobic surface of the double-mutant CCR3 compared to the single mutants providing additional stability. The purification of other C-C chemokine receptors lacking such stabilizing residues may benefit from the incorporation of these two point mutations.
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http://dx.doi.org/10.1021/acs.jctc.8b00602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6549507PMC
December 2018

Bitopic Inhibition of ATP and Substrate Binding in Ser/Thr Kinases through a Conserved Allosteric Mechanism.

Biochemistry 2018 11 30;57(45):6387-6390. Epub 2018 Oct 30.

Department of Genetics, Cell Biology, and Development , University of Minnesota , Minneapolis , Minnesota 55455 , United States.

Protein kinases achieve substrate selective phosphorylation through their conformational flexibility and dynamic interaction with the substrate. Designing substrate selective or kinase selective small molecule inhibitors remains a challenge because of a lack of understanding of the dynamic mechanism by which substrates are selected by the kinase. Using a combination of all-atom molecular dynamics simulations and FRET sensors, we have delineated an allosteric mechanism that results in interaction among the DFG motif, G-loop, and activation loop and structurally links the nucleotide and substrate binding interfaces in protein kinase Cα and three other Ser/Thr kinases. ATP-competitive staurosporine analogues engage this allosteric switch region located just outside the ATP binding site to displace substrate binding to varying degrees. These inhibitors function as bitopic ligands by occupying the ATP binding site and interacting with the allosteric switch region. The conserved mechanism identified in this study can be exploited to select and design bitopic inhibitors for kinases.
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http://dx.doi.org/10.1021/acs.biochem.8b00729DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6432918PMC
November 2018

Allosteric Activation of Striatal-Enriched Protein Tyrosine Phosphatase (STEP, PTPN5) by a Fragment-like Molecule.

J Med Chem 2019 01 12;62(1):306-316. Epub 2018 Sep 12.

Department of Molecular Immunology , Beckman Research Institute of the City of Hope , 1500, E. Duarte Road , Duarte , California 91010 , United States.

Protein tyrosine phosphatase non-receptor type 5 (PTPN5, STEP) is a brain specific phosphatase that regulates synaptic function and plasticity by modulation of N-methyl-d-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) trafficking. Dysregulation of STEP has been linked to neurodegenerative and neuropsychiatric diseases, highlighting this enzyme as an attractive therapeutic target for drug discovery. Selective targeting of STEP with small molecules has been hampered by high conservation of the active site among protein tyrosine phosphatases. We report the discovery of the first small molecule allosteric activator for STEP that binds to the phosphatase domain. Allosteric binding is confirmed by both X-ray and N NMR experiments, and specificity has been demonstrated by an enzymatic test cascade. Molecular dynamics simulations indicate stimulation of enzymatic activity by a long-range allosteric mechanism. To allow the scientific community to make use of this tool, we offer to provide the compound in the course of an open innovation initiative.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00857DOI Listing
January 2019

C-NHEJ without indels is robust and requires synergistic function of distinct XLF domains.

Nat Commun 2018 06 27;9(1):2484. Epub 2018 Jun 27.

Department of Cancer Genetics and Epigenetics, Beckman Research Institute of the City of Hope, 1500 E Duarte Rd., Duarte, CA, 91010, USA.

To investigate the fidelity of canonical non-homologous end joining (C-NHEJ), we developed an assay to detect EJ between distal ends of two Cas9-induced chromosomal breaks that are joined without causing insertion/deletion mutations (indels). Here we find that such EJ requires several core C-NHEJ factors, including XLF. Using variants of this assay, we find that C-NHEJ is required for EJ events that use 1-2, but not ≥3, nucleotides of terminal microhomology. We also investigated XLF residues required for EJ without indels, finding that one of two binding domains is essential (L115 or C-terminal lysines that bind XRCC4 and KU/DNA, respectively), and that disruption of one of these domains sensitizes XLF to mutations that affect its dimer interface, which we examined with molecular dynamic simulations. Thus, C-NHEJ, including synergistic function of distinct XLF domains, is required for EJ of chromosomal breaks without indels.
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http://dx.doi.org/10.1038/s41467-018-04867-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6021437PMC
June 2018

Identifying Functional Hotspot Residues for Biased Ligand Design in G-Protein-Coupled Receptors.

Mol Pharmacol 2018 04 24;93(4):288-296. Epub 2018 Jan 24.

Department of Molecular Immunology, Beckman Research Institute of the City of Hope, Duarte, California (A.K.N., S.B., S.L., N.V.); Departments of Medicinal Chemistry (C.S.T.) and Immunology and Respiratory Diseases Research (I.K., T.K.), Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany; and Corporate Department of Business Development and Licensing, C.H. Boehringer Sohn, Ingelheim, Germany (P.C.)

G-protein-coupled receptors (GPCRs) mediate multiple signaling pathways in the cell, depending on the agonist that activates the receptor and multiple cellular factors. Agonists that show higher potency to specific signaling pathways over others are known as "biased agonists" and have been shown to have better therapeutic index. Although biased agonists are desirable, their design poses several challenges to date. The number of assays to identify biased agonists seems expensive and tedious. Therefore, computational methods that can reliably calculate the possible bias of various ligands ahead of experiments and provide guidance, will be both cost and time effective. In this work, using the mechanism of allosteric communication from the extracellular region to the intracellular transducer protein coupling region in GPCRs, we have developed a computational method to calculate ligand bias ahead of experiments. We have validated the method for several -arrestin-biased agonists in -adrenergic receptor (2AR), serotonin receptors 5-HT1B and 5-HT2B and for G-protein-biased agonists in the -opioid receptor. Using this computational method, we also performed a blind prediction followed by experimental testing and showed that the agonist carmoterol is -arrestin-biased in 2AR. Additionally, we have identified amino acid residues in the biased agonist binding site in both 2AR and -opioid receptors that are involved in potentiating the ligand bias. We call these residues functional hotspots, and they can be used to derive pharmacophores to design biased agonists in GPCRs.
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http://dx.doi.org/10.1124/mol.117.110395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5832328PMC
April 2018

How Does the Proliferating Cell Nuclear Antigen Modulate Binding Specificity to Multiple Partner Proteins?

J Chem Inf Model 2017 12 17;57(12):3011-3021. Epub 2017 Nov 17.

Department of Molecular Immunology and ‡Department of Molecular Medicine, Beckman Research Institute of the City of Hope , 1500 East Duarte Road, Duarte, California 91010, United States.

Proliferating cell nuclear antigen (PCNA) is a member of the family of sliding clamp proteins that serves as a clamp during DNA repair, DNA replication, cell cycle control, and multiple forms of chromatin modification. PCNA functions as a homotrimer and complexes with multiple proteins in order to carry out each of these varied functions. PCNA binds to different partner proteins in the same region of its structure, called the " interdomain connecting loop", but with different affinities. This interdomain connecting loop is an intrinsically disordered region that takes different conformations when binding to different partner proteins. In this work, we performed all-atom molecular dynamics simulations on PCNA trimer unbound to any partner protein, PCNA bound to peptides from different partner proteins, and PCNA bound to the full Fen 1 protein in two different conformations. Using this massive amount of simulation results, we analyzed whether PCNA in its free trimeric form samples conformations that are similar to those when it is bound to different partner proteins. We observed that PCNA samples many of these peptide-bound conformations even when not bound to the peptides and selects specific conformations when binding to partner proteins. We also identified PCNA-peptide interactions formed in the peptide bound simulation that play a crucial role in complex formation. The calculated binding energies correlate well with the measured binding affinities of various peptides to PCNA. Lastly, we studied the internal dynamics of PCNA and propose a mechanism through which PCNA recruits binding partners. This work highlights the functional role of intrinsically disordered regions in multifunctional proteins such as PCNA.
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http://dx.doi.org/10.1021/acs.jcim.7b00171DOI Listing
December 2017

Distinct structural mechanisms determine substrate affinity and kinase activity of protein kinase Cα.

J Biol Chem 2017 09 15;292(39):16300-16309. Epub 2017 Aug 15.

the Department of Genetics, Cell Biology, and Development, University of Minnesota, Twin Cities, Minneapolis, Minnesota 55455, and

Protein kinase Cα (PKCα) belongs to the family of AGC kinases that phosphorylate multiple peptide substrates. Although the consensus sequence motif has been identified and used to explain substrate specificity for PKCα, it does not inform the structural basis of substrate-binding and kinase activity for diverse substrates phosphorylated by this kinase. The transient, dynamic, and unstructured nature of this protein-protein interaction has limited structural mapping of kinase-substrate interfaces. Here, using multiscale MD simulation-based predictions and FRET sensor-based experiments, we investigated the conformational dynamics of the kinase-substrate interface. We found that the binding strength of the kinase-substrate interaction is primarily determined by long-range columbic interactions between basic (Arg/Lys) residues located N-terminally to the phosphorylated Ser/Thr residues in the substrate and by an acidic patch in the kinase catalytic domain. Kinase activity stemmed from conformational flexibility in the region C-terminal to the phosphorylated Ser/Thr residues. Flexibility of the substrate-kinase interaction enabled an Arg/Lys two to three amino acids C-terminal to the phosphorylated Ser/Thr to prime a catalytically active conformation, facilitating phosphoryl transfer to the substrate. The structural mechanisms determining substrate binding and catalytic activity formed the basis of diverse binding affinities and kinase activities of PKCα for 14 substrates with varying degrees of sequence conservation. Our findings provide insight into the dynamic properties of the kinase-substrate interaction that govern substrate binding and turnover. Moreover, this study establishes a modeling and experimental method to elucidate the structural dynamics underlying substrate selectivity among eukaryotic kinases.
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http://dx.doi.org/10.1074/jbc.M117.804781DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5625059PMC
September 2017

Cooperation of neurotrophin receptor TrkB and Her2 in breast cancer cells facilitates brain metastases.

Breast Cancer Res 2017 04 26;19(1):51. Epub 2017 Apr 26.

Division of Neurosurgery, Beckman Research Institute, City of Hope, 1500 E. Duarte Rd, Duarte, CA, 91010, USA.

Background: Patients with primary breast cancer that is positive for human epidermal growth factor receptor 2 (Her2+) have a high risk of developing metastases in the brain. Despite gains with systemic control of Her2+ disease using molecular therapies, brain metastases remain recalcitrant to therapeutic discovery. The clinical predilection of Her2+ breast cancer cells to colonize the brain likely relies on paracrine mechanisms. The neural niche poses unique selection pressures, and neoplastic cells that utilize the brain microenvironment may have a survival advantage.

Methods: Tropomyosin-related kinase B (TrkB), Her2, and downstream targets were analyzed in primary breast cancer, breast-to-brain metastasis (BBM) tissues, and tumor-derived cell lines using quantitative real-time PCR, western blot, and immunohistochemical assessment. TrkB function on BBM was confirmed with intracranial, intracardiac, or mammary fat pad xenografts in non-obese diabetic/severe combined immunodeficiency mice. The function of brain-derived neurotrophic factor (BDNF) on cell proliferation and TrkB/Her2 signaling and interactions were confirmed using selective shRNA knockdown and selective inhibitors. The physical interaction of Her2-TrkB was analyzed using electron microscopy, co-immunoprecipitation, and in silico analysis. Dual targeting of Her2 and TrkB was analyzed using clinically utilized treatments.

Results: We observed that patient tissues and cell lines derived from Her2+ human BBM displayed increased activation of TrkB, a neurotrophin receptor. BDNF, an extracellular neurotrophin, with roles in neuronal maturation and homeostasis, specifically binds to TrkB. TrkB knockdown in breast cancer cells led to decreased frequency and growth of brain metastasis in animal models, suggesting that circulating breast cancer cells entering the brain may take advantage of paracrine BDNF-TrkB signaling for colonization. In addition, we investigated a possible interaction between TrkB and Her2 receptors on brain metastatic breast cancer cells, and found that BDNF phosphorylated both its cognate TrkB receptor and the Her2 receptor in brain metastatic breast cancer cells.

Conclusion: Collectively, our findings suggest that heterodimerization of Her2 and TrkB receptors gives breast cancer cells a survival advantage in the brain and that dual inhibition of these receptors may hold therapeutic potential.
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http://dx.doi.org/10.1186/s13058-017-0844-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5406906PMC
April 2017

Structure and dynamics of a constitutively active neurotensin receptor.

Sci Rep 2016 12 7;6:38564. Epub 2016 Dec 7.

Membrane Protein Structure Function Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Department of Health and Human Services, Rockville, Maryland 20852, United States.

Many G protein-coupled receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in receptors have been implicated in diseases. To gain insight into mechanistic aspects of constitutive activity, we report here the 3.3 Å crystal structure of a constitutively active, agonist-bound neurotensin receptor (NTSR1) and molecular dynamics simulations of agonist-occupied and ligand-free receptor. Comparison with the structure of a NTSR1 variant that has little constitutive activity reveals uncoupling of the ligand-binding domain from conserved connector residues, that effect conformational changes during GPCR activation. Furthermore, molecular dynamics simulations show strong contacts between connector residue side chains and increased flexibility at the intracellular receptor face as features that coincide with robust signalling in cells. The loss of correlation between the binding pocket and conserved connector residues, combined with altered receptor dynamics, possibly explains the reduced neurotensin efficacy in the constitutively active NTSR1 and a facilitated initial engagement with G protein in the absence of agonist.
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http://dx.doi.org/10.1038/srep38564DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5141500PMC
December 2016

How Do Short Chain Nonionic Detergents Destabilize G-Protein-Coupled Receptors?

J Am Chem Soc 2016 11 15;138(47):15425-15433. Epub 2016 Nov 15.

Department of Molecular Immunology, Beckman Research Institute of the City of Hope , 1500 E. Duarte Road, Duarte, California 91010, United States.

Stability of detergent-solubilized G-protein-coupled receptors (GPCRs) is crucial for their purification in a biologically relevant state, and it is well-known that short chain detergents such as octylglucoside are more denaturing than long chain detergents such as dodecylmaltoside. However, the molecular basis for this phenomenon is poorly understood. To gain insights into the mechanism of detergent destabilization of GPCRs, we used atomistic molecular dynamics simulations of thermostabilized adenosine receptor (AR) mutants embedded in either a lipid bilayer or detergent micelles of alkylmaltosides and alkylglucosides. AR mutants in dodecylmaltoside or phospholipid showed low flexibility and good interhelical packing. In contrast, AR mutants in either octylglucoside or nonylglucoside showed decreased α-helicity in transmembrane regions, decreased α-helical packing, and the interpenetration of detergent molecules between transmembrane α-helices. This was not observed in octylglucoside containing phospholipid. Cholesteryl hemisuccinate in dodecylmaltoside increased the energetic stability of the receptor by wedging into crevices on the hydrophobic surface of AR, increasing packing interactions within the receptor and stiffening the detergent micelle. The data suggest a three-stage process for the initial events in the destabilization of GPCRs by octylglucoside: (i) highly mobile detergent molecules form small micelles around the receptor; (ii) loss of α-helicity and decreased interhelical packing interactions in transmembrane regions are promoted by increased receptor thermal motion; (iii) transient separation of transmembrane helices allowed penetration of detergent molecules into the core of the receptor. The relative hydration of the headgroup and alkyl chain correlates with detergent harshness and suggests new avenues to develop milder versions of octylglucoside for receptor crystallization.
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http://dx.doi.org/10.1021/jacs.6b08742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5148649PMC
November 2016

Allosteric communication pipelines in G-protein-coupled receptors.

Curr Opin Pharmacol 2016 10 4;30:76-83. Epub 2016 Aug 4.

Department of Molecular Immunology, Beckman Research Institute of the City of Hope, 1500 East Duarte Road, Duarte, CA 91010, USA.

The binding of ligands to G-protein-coupled receptors (GPCRs) in the extracellular region transmits the signal to the intracellular region to initiate coupling to effector proteins. The mechanism of this allosteric communication remains largely unexplored. Knowledge of the residues involved in the pipeline of the allosteric communication from the extracellular to the intracellular region will provide means to (a) design ligands with bias in potency towards one signaling pathway over others, and (b) design allosteric modulators that show subtype selectivity in GPCRs. In this review we describe the current state of the computational methods that provide insights into the allosteric communication in GPCRs and elucidate how this information can be used to design allosteric modulators.
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http://dx.doi.org/10.1016/j.coph.2016.07.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127785PMC
October 2016