scholarly journals Ligand modulation of sidechain dynamics in a wild-type human GPCR

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Lindsay D Clark ◽  
Igor Dikiy ◽  
Karen Chapman ◽  
Karin EJ Rödström ◽  
James Aramini ◽  
...  
Keyword(s):  
Human Physiology ◽  
Structural Changes ◽  
Inverse Agonist ◽  
Methyl Groups ◽  
Signaling Proteins ◽  
Wild Type ◽  
Biophysical Studies ◽  
Methyl Trosy ◽  
Conformational Regulation ◽  
Methyl Trosy Nmr

GPCRs regulate all aspects of human physiology, and biophysical studies have deepened our understanding of GPCR conformational regulation by different ligands. Yet there is no experimental evidence for how sidechain dynamics control allosteric transitions between GPCR conformations. To address this deficit, we generated samples of a wild-type GPCR (A2AR) that are deuterated apart from 1H/13C NMR probes at isoleucine δ1 methyl groups, which facilitated 1H/13C methyl TROSY NMR measurements with opposing ligands. Our data indicate that low [Na+] is required to allow large agonist-induced structural changes in A2AR, and that patterns of sidechain dynamics substantially differ between agonist (NECA) and inverse agonist (ZM241385) bound receptors, with the inverse agonist suppressing fast ps-ns timescale motions at the G protein binding site. Our approach to GPCR NMR creates a framework for exploring how different regions of a receptor respond to different ligands or signaling proteins through modulation of fast ps-ns sidechain dynamics.

Detection of Chemical Exchange in Methyl Groups of Macromolecules

2019 ◽  
Author(s):  
Michelle Gill ◽  
Andrew Hsu ◽  
Arthur G. Palmer, III
Keyword(s):  
Relaxation Rate ◽  
Chemical Exchange ◽  
Double Quantum ◽  
Methyl Groups ◽  
Multiple Quantum ◽  
Relaxation Data ◽  
E Coli ◽  
Methyl Trosy ◽  
Dynamics Simulations ◽  
Exchange Broadening

<div> <div> <div> <p>The zero- and double-quantum methyl TROSY Hahn-echo and the methyl <sup>1</sup>H-<sup>1</sup>H dipole- dipole cross-correlation nuclear magnetic resonance experiments enable estimation of multiple quantum chemical exchange broadening in methyl groups in proteins. The two relaxation rate constants are established to be linearly dependent using molecular dynamics simulations and empirical analysis of experimental data. This relationship allows chemical exchange broadening to be recognized as an increase in the Hahn-echo relaxation rate constant. The approach is illustrated by analyzing relaxation data collected at three temperatures for <i>E. coli </i>ribonuclease HI and by analyzing relaxation data collected for different cofactor and substrate complexes of <i>E. coli </i>AlkB. </p> </div> </div> </div>

scholarly journals Genetic Deletion of Interleukin-15 Is Not Associated with Major Structural Changes Following Experimental Post-Traumatic Knee Osteoarthritis in Rats

2021 ◽  
Vol 11 (15) ◽  
pp. 7118
Author(s):  
Ermina Hadzic ◽  
Garth Blackler ◽  
Holly Dupuis ◽  
Stephen James Renaud ◽  
Christopher Thomas Appleton ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Subchondral Bone ◽  
Structural Changes ◽  
Cartilage Damage ◽  
Wild Type ◽  
Significant Difference ◽  
Post Traumatic ◽  
Interleukin 15 ◽  
Joint Trauma ◽  
Surgically Induced

Post-traumatic osteoarthritis (PTOA) is a degenerative joint disease, leading to articular cartilage breakdown, osteophyte formation, and synovitis, caused by an initial joint trauma. Pro-inflammatory cytokines increase catabolic activity and may perpetuate inflammation following joint trauma. Interleukin-15 (IL-15), a pro-inflammatory cytokine, is increased in OA patients, although its roles in PTOA pathophysiology are not well characterized. Here, we utilized Il15 deficient rats to examine the role of IL-15 in PTOA pathogenesis in an injury-induced model. OA was surgically induced in Il15 deficient Holtzman Sprague-Dawley rats and control wild-type rats to compare PTOA progression. Semi-quantitative scoring of the articular cartilage, subchondral bone, osteophyte size, and synovium was performed by two blinded observers. There was no significant difference between Il15 deficient rats and wild-type rats following PTOA-induction across articular cartilage damage, subchondral bone damage, and osteophyte scoring. Similarly, synovitis scoring across six parameters found no significant difference between genetic variants. Overall, IL-15 does not appear to play a key role in the development of structural changes in this surgically-induced rat model of PTOA.

scholarly journals Brown Spiders’ Phospholipases-D with Potential Therapeutic Applications: Functional Assessment of Mutant Isoforms

Biomedicines ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 320
Author(s):  
Thaís Pereira da Silva ◽  
Fernando Jacomini de Castro ◽  
Larissa Vuitika ◽  
Nayanne Louise Costacurta Polli ◽  
Bruno César Antunes ◽  
...  
Keyword(s):  
Structural Changes ◽  
Capillary Permeability ◽  
Structural Data ◽  
Histopathological Analysis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Antigenic Properties ◽  
Harmful Effects

Phospholipases-D (PLDs) found in Loxosceles spiders’ venoms are responsible for the dermonecrosis triggered by envenomation. PLDs can also induce other local and systemic effects, such as massive inflammatory response, edema, and hemolysis. Recombinant PLDs reproduce all of the deleterious effects induced by Loxosceles whole venoms. Herein, wild type and mutant PLDs of two species involved in accidents—L. gaucho and L. laeta—were recombinantly expressed and characterized. The mutations are related to amino acid residues relevant for catalysis (H12-H47), magnesium ion coordination (E32-D34) and binding to phospholipid substrates (Y228 and Y228-Y229-W230). Circular dichroism and structural data demonstrated that the mutant isoforms did not undergo significant structural changes. Immunoassays showed that mutant PLDs exhibit conserved epitopes and kept their antigenic properties despite the mutations. Both in vitro (sphingomyelinase activity and hemolysis) and in vivo (capillary permeability, dermonecrotic activity, and histopathological analysis) assays showed that the PLDs with mutations H12-H47, E32-D34, and Y228-Y229-W230 displayed only residual activities. Results indicate that these mutant toxins are suitable for use as antigens to obtain neutralizing antisera with enhanced properties since they will be based on the most deleterious toxins in the venom and without causing severe harmful effects to the animals in which these sera are produced.

scholarly journals Assembly of T-Antigen Double Hexamers on the Simian Virus 40 Core Origin Requires Only a Subset of the Available Binding Sites

1998 ◽  
Vol 18 (5) ◽  
pp. 2677-2687 ◽  
Author(s):  
Woo S. Joo ◽  
Henry Y. Kim ◽  
John D. Purviance ◽  
K. R. Sreekumar ◽  
Peter A. Bullock
Keyword(s):  
Structural Changes ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Wild Type ◽  
The Core ◽  
Structural Distortions ◽  
In The Wild ◽  
Sv40 Dna ◽  
Initial Assembly

ABSTRACT Initiation of simian virus 40 (SV40) DNA replication is dependent upon the assembly of two T-antigen (T-ag) hexamers on the SV40 core origin. To further define the oligomerization mechanism, the pentanucleotide requirements for T-ag assembly were investigated. Here, we demonstrate that individual pentanucleotides support hexamer formation, while particular pairs of pentanucleotides suffice for the assembly of T-ag double hexamers. Related studies demonstrate that T-ag double hexamers formed on “active pairs” of pentanucleotides catalyze a set of previously described structural distortions within the core origin. For the four-pentanucleotide-containing wild-type SV40 core origin, footprinting experiments indicate that T-ag double hexamers prefer to bind to pentanucleotides 1 and 3. Collectively, these experiments demonstrate that only two of the four pentanucleotides in the core origin are necessary for T-ag assembly and the induction of structural changes in the core origin. Since all four pentanucleotides in the wild-type origin are necessary for extensive DNA unwinding, we concluded that the second pair of pentanucleotides is required at a step subsequent to the initial assembly process.

scholarly journals Surface-Exposed Adeno-Associated Virus Vp1-NLS Capsid Fusion Protein Rescues Infectivity of Noninfectious Wild-Type Vp2/Vp3 and Vp3-Only Capsids but Not That of Fivefold Pore Mutant Virions

2007 ◽  
Vol 81 (15) ◽  
pp. 7833-7843 ◽  
Author(s):  
Joshua C. Grieger ◽  
Jarrod S. Johnson ◽  
Brittney Gurda-Whitaker ◽  
Mavis Agbandje-McKenna ◽  
R. Jude Samulski
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Wild Type ◽  
Dot Blot ◽  
Adeno Associated Virus ◽  
N Terminus ◽  
Localization Signal ◽  
Significant Effort

ABSTRACT Over the past 2 decades, significant effort has been dedicated to the development of adeno-associated virus (AAV) as a vector for human gene therapy. However, understanding of the virus with respect to the functional domains of the capsid remains incomplete. In this study, the goal was to further examine the role of the unique Vp1 N terminus, the N terminus plus the recently identified nuclear localization signal (NLS) (J. C. Grieger, S. Snowdy, and R. J. Samulski, J. Virol 80:5199-5210, 2006), and the virion pore at the fivefold axis in infection. We generated two Vp1 fusion proteins (Vp1 and Vp1NLS) linked to the 8-kDa chemokine domain of rat fractalkine (FKN) for the purpose of surface exposure upon assembly of the virion, as previously described (K. H. Warrington, Jr., O. S. Gorbatyuk, J. K. Harrison, S. R. Opie, S. Zolotukhin, and N. Muzyczka, J. Virol 78:6595-6609, 2004). The unique Vp1 N termini were found to be exposed on the surfaces of these capsids and maintained their phospholipase A2 (PLA2) activity, as determined by native dot blot Western and PLA2 assays, respectively. Incorporation of the fusions into AAV type 2 capsids lacking a wild-type Vp1, i.e., Vp2/Vp3 and Vp3 capsid only, increased infectivity by 3- to 5-fold (Vp1FKN) and 10- to 100-fold (Vp1NLSFKN), respectively. However, the surface-exposed fusions did not restore infectivity to AAV virions containing mutations at a conserved leucine (Leu336Ala, Leu336Cys, or Leu336Trp) located at the base of the fivefold pore. EM analyses suggest that Leu336 may play a role in global structural changes to the virion directly impacting downstream conformational changes essential for infectivity and not only have local effects within the pore, as previously suggested.

scholarly journals Genetic Analysis of Rare Human Variants of Regulators of G Protein Signaling Proteins and Their Role in Human Physiology and Disease

2018 ◽  
Vol 70 (3) ◽  
pp. 446-474 ◽  
Author(s):  
Katherine E. Squires ◽  
Carolina Montañez-Miranda ◽  
Rushika R. Pandya ◽  
Matthew P. Torres ◽  
John R. Hepler
Keyword(s):  
Human Physiology ◽  
Genetic Analysis ◽  
G Protein ◽  
G Protein Signaling ◽  
Signaling Proteins ◽  
Protein Signaling

Abstract 61: Ablation of BK Ca Channels Results in Cardiac Hypertrophy

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Harpreet Singh ◽  
Kajol Shah ◽  
Devsena Ponnalagu ◽  
Sanjay Chandrasekhar ◽  
Andrew R Kohut ◽  
...  
Keyword(s):  
Ejection Fraction ◽  
Cardiac Hypertrophy ◽  
Cardiac Function ◽  
Structural Changes ◽  
Cardiac Fibrosis ◽  
Ischemia Reperfusion Injury ◽  
Heart Diseases ◽  
Ischemia Reperfusion ◽  
Interventricular Septum ◽  
Wild Type

Expression and activation of the large conductance calcium and voltage-gated potassium (BK Ca ) channels encoded by Kcnma1 gene is shown to be vital in cardioprotection from ischemia-reperfusion injury. BK Ca channels present in SA node cells regulate the heart rate, and in blood vessels play an active role in vascular relaxation. However, the role of BK Ca in regulation of structure and function of the heart is not fully-established. Using Kcnma1 -/- mice, we have observed structural changes in cardiomyocytes and compromised cardiac function as compared to wild type mice. Absence of BK Ca resulted in significant increase in size of adult cardiomyocytes (from 7.95 + 0.1 um 2 to 9.68 + 0.1 um 2 , p < 0.01, n=480 cells each) and also increased cardiac fibrosis. Further to determine underlying signaling mechanisms in cardiac hypertrophy, we performed microarray analysis of RNAs isolated from wild type and Kcnma1 -/- mice (n=3) hearts. We found up regulation of a class of cardiac hypertrophy markers (myosin variants) and changes in the expression of several mitochondrial genes (such as ND4) directly associated with heart diseases in Kcnma1 -/- mice. To evaluate the functional consequence of absence of BK Ca , we performed high-resolution echocardiography on wild type and Kcnma1 -/- mice. Under anesthesia (1.5% isoflurane), left ventricle of Kcnma1 -/- mice showed significant reduction (p < 0.05) in ejection fraction (56 + 2 %, n=7) as compared to wild type (74 + 3 %, n=6) as well as fractional shortening (23 + 3 %, n=7, and 39 + 3 %, n=6, respectively). Similarly, right ventricle had a lower ejection fraction (35.7 + 4% vs 56.9 + 5 %, n > 5) in Kcnma1 -/- as compared to wild type mice. In agreement with our histopathology and microarray data, Kcnma1 -/- mice showed increased posterior wall thickness (0.75 + 0.3 mm vs 0.62 + 0.1 mm) and interventricular septum thickness (0.83 + 0.4 mm, n=7 vs 0.68 + 0.3 mm, n=6) . Together, these data imply that BK Ca plays a direct role in cardiac hypertrophy and cardiac function.

Abstract 15006: Serotonin Transporter (SERT) Knockout Leads to Increased Myocardial and Mitral Valvular Fibrosis in a Murine Model

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Alex D'Angelo ◽  
Estibaliz Castillero ◽  
Robert LEVY ◽  
Giovanni Ferrari
Keyword(s):  
Structural Changes ◽  
Smooth Muscle Actin ◽  
Heart Valve Disease ◽  
Ventricular Wall ◽  
Muscle Actin ◽  
Wild Type ◽  
Functional Differences ◽  
Ventricular Wall Thickness ◽  
Fractional Shortening ◽  
Ventricular Dimensions

Introduction: Heart valve disease attributed to serotonin (5HT) has been observed with 5HT-secreting carcinoid tumors and with the diet drug, Dexfenfluoramine, a 5HT transporter (SLC6A4, also known as SERT) inhibitor and 5HT receptor (HTR) 2B agonist. 5HT/SLC6A4 mechanisms had not been investigated in mitral valve (MV) regurgitation. SLC6A4 internalizes 5HT, limiting HTR signaling. Selective 5HT reuptake inhibitor (SSRI) antidepressants target SLC6A4 to increase 5HT. We showed that SLC6A4 reduction in human MV influences MV regurgitation through enhanced HTR signaling. We further investigate the cardiac impact of SLC6A4 deficiency in a SERT -/- mouse reported to have cardiac abnormalities. Hypothesis: Absence of SERT leads to increased myocardial and MV fibrosis. Methods: SERT -/- and wild type (WT) C57BL/6J mice underwent echocardiography at 8 and 16 weeks (w) of age (N=5 / group). Collagen staining was performed with Masson’s trichrome. Markers of myocardial fibrosis α-smooth muscle actin (αSMA) and collagen type1, alpha 1 (COL1A1) were measured by PCR. Results: 8w and 16w SERT -/- mice showed increased epicardial fibrosis and MV sub-valvular fibrosis, particularly at leaflet attachment points, and in 16w SERT -/- mice MV leaflet fibrosis was also present. At 8w, posterior ventricular wall thickness was greater in SERT -/- mice vs. WT, both in systole (1.30 ± 0.09mm vs. 1.61 ± 0.2mm, p=0.003) and diastole (0.78 ± 0.11mm vs. 1.22 ± 0.29mm, p=0.001). At 16 w, ventricular dimensions were larger in SERT -/- mice vs. WT at end-systole (2.29 ± 0.54mm vs. 3.0 ± 0.47mm, p=0.008) and end-diastole (3.48 ± 0.45mm vs. 4.07 ± 0.35, p=0.006). Fractional shortening was reduced in 16w SERT -/- mice vs. WT (35.20 ± 7.66 vs. 26.74 ± 6.14, p=0.017) and vs. 8w SERT -/- mice (38.85 ± 4.57 vs. 26.74 ± 6.14, p=0.002). PCR showed increased fibrosis markers αSMA and COL1A1 in SERT -/- mice (8w and 16w) vs. WT (all p<0.05). Conclusions: SERT -/- mice show pathologic structural changes and fibrosis in both myocardium and MV, corresponding to functional differences that worsen with age. As inhibition of SLC6A4, combined with active HTR2A/B signaling would hypothetically increase MV remodeling and regurgitation, these animals could be ideal to test 5HT pharmacology.

P219L substitution in human D-amino acid oxidase impacts the ligand binding and catalytic efficiency

2020 ◽  
Vol 168 (5) ◽  
pp. 557-567
Author(s):  
Wanitcha Rachadech ◽  
Yusuke Kato ◽  
Rabab M Abou El-Magd ◽  
Yuji Shishido ◽  
Soo Hyeon Kim ◽  
...  
Keyword(s):  
Amino Acid ◽  
Conformational Changes ◽  
Active Site ◽  
Structural Changes ◽  
Catalytic Efficiency ◽  
Acid Oxidase ◽  
Wild Type ◽  
Amino Acid Oxidase ◽  
Adenine Ring ◽  
The Impact

Abstract Human D-amino acid oxidase (DAO) is a flavoenzyme that is implicated in neurodegenerative diseases. We investigated the impact of replacement of proline with leucine at Position 219 (P219L) in the active site lid of human DAO on the structural and enzymatic properties, because porcine DAO contains leucine at the corresponding position. The turnover numbers (kcat) of P219L were unchanged, but its Km values decreased compared with wild-type, leading to an increase in the catalytic efficiency (kcat/Km). Moreover, benzoate inhibits P219L with lower Ki value (0.7–0.9 µM) compared with wild-type (1.2–2.0 µM). Crystal structure of P219L in complex with flavin adenine dinucleotide (FAD) and benzoate at 2.25 Å resolution displayed conformational changes of the active site and lid. The distances between the H-bond-forming atoms of arginine 283 and benzoate and the relative position between the aromatic rings of tyrosine 224 and benzoate were changed in the P219L complex. Taken together, the P219L substitution leads to an increase in the catalytic efficiency and binding affinity for substrates/inhibitors due to these structural changes. Furthermore, an acetic acid was located near the adenine ring of FAD in the P219L complex. This study provides new insights into the structure–function relationship of human DAO.

A Bcr-Abl Mutant Lacking Direct Binding Sites for the Grb2, Cbl and CrkL Adapter Proteins Fails to Induce Leukemia in Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 718-718
Author(s):  
Kara J. Johnson ◽  
Ian J. Griswold ◽  
Amie Corbin ◽  
Michael W.N. Deininger ◽  
Brian J. Druker
Keyword(s):  
Bone Marrow ◽  
Growth Factor ◽  
Cell Lines ◽  
Binding Sites ◽  
Signaling Proteins ◽  
Wild Type ◽  
Triple Mutant ◽  
Murine Bone Marrow ◽  
Direct Binding

Abstract The Bcr-Abl tyrosine kinase is detectable in greater than 95% of cases of chronic myelogenous leukemia (CML) and its kinase activity is required for induction of this disease. A number of signaling proteins are associated with and phosphorylated by Bcr-Abl. Proteins known to associate directly with Bcr-Abl include Grb2, c-Cbl, CrkL and p62Dok. Mutations of the direct binding sites for these proteins in Bcr-Abl abolish the direct interactions, but do not completely eliminate interactions, presumably due to the ability of many of these proteins to interact both directly and indirectly with Bcr-Abl. Individual mutations of the Grb2 and c-Cbl binding domains change the phenotype of disease induced in murine bone marrow transplantation assays from a myeloproliferative disorder to a B or T-lymphoid leukemia with prolonged latency. Thus, due to the complexity of Bcr-Abl signaling and a lack of a one to one correlation between direct binding sites, specific signaling proteins, and specific phenotypes, we created a triple mutant lacking the direct binding sites for Grb2, c-Cbl and CrkL. Stable myeloid cell lines were generated in the myeloid progenitor cell line, 32D, expressing the wild type and triple mutant forms of Bcr-Abl. Cell proliferation assays were performed in the presence and absence of WEHI (an IL-3 source) to assess growth factor requirements. Expression of the triple mutant in cell lines was able to confer growth factor dependence when expressed at levels comparable to wild type. Lysates from cell lines were analyzed by immunoprecipitation and immunoblotting and demonstrated that nearly all associations between Bcr-Abl and Grb2, c-Cbl, CrkL and p62Dok were eliminated in the triple mutant. Despite the lack of interaction, these proteins remained tyrosine phosphorylated at levels which correlate with Bcr-Abl expression. Phosphorylation was inhibited by treatment of cell lines with imatininb, indicating that the activity of Bcr-Abl is required for their phosphorylation, either directly or indirectly. Analysis of the activation of various signaling pathways (Akt, MAPK, MEK, Stat5), shows that only Stat5 remains phosphorylated in triple mutant cell lines. Despite inducing factor independent growth of 32D cells, the triple mutant was unable to induce the outgrowth of hematopoetic progenitors in B-cell lymphoid outgrowth assays. To test leukemogencity in vivo, murine bone marrow transplantation/transduction assays were also carried out using MSCV-MIGR1 vector constructs. The triple mutant failed to induce leukemia in the mice. In summary, a triple mutant of Bcr-Abl lacking the binding sites for Grb2, c-Cbl and CrkL is able to confer growth factor independence in cell lines. Although the associations of these proteins with Bcr-Abl are nearly eliminated, they are still tyrosine phosphorylated and this is dependent on the activity of Bcr-Abl. Despite its ability to transform cell lines, the triple mutant was not able to induce the outgrowth of hematopoetic progenitors in B-cell outgrowth assays, nor induce leukemia in mice. Although the phosphorylation of Stat5 correlates with factor independent growth, this is not sufficient to induce transformation in vivo suggesting that interactions with other signaling pathways disrupted in this triple mutant of Bcr-Abl are necessary. To our knowledge this is the only kinase active variant of Bcr-Abl that has failed to induce leukemia in vivo.

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