Site-Directed Mutations and the Polymorphic Variant Ala160Thr in the Human Thromboxane Receptor Uncover a Structural Role for Transmembrane Helix 4
Open Access
- 17 January 2012
- journal article
- research article
- Published by Public Library of Science (PLoS) in PLOS ONE
- Vol. 7 (1), e29996
- https://doi.org/10.1371/journal.pone.0029996
Abstract
The human thromboxane A2 receptor (TP), belongs to the prostanoid subfamily of Class A GPCRs and mediates vasoconstriction and promotes thrombosis on binding to thromboxane (TXA2). In Class A GPCRs, transmembrane (TM) helix 4 appears to be a hot spot for non-synonymous single nucleotide polymorphic (nsSNP) variants. Interestingly, A160T is a novel nsSNP variant with unknown structure and function. Additionally, within this helix in TP, Ala1604.53 is highly conserved as is Gly1644.57. Here we target Ala1604.53 and Gly1644.57 in the TP for detailed structure-function analysis. Amino acid replacements with smaller residues, A160S and G164A mutants, were tolerated, while bulkier beta-branched replacements, A160T and A160V showed a significant decrease in receptor expression (Bmax). The nsSNP variant A160T displayed significant agonist-independent activity (constitutive activity). Guided by molecular modeling, a series of compensatory mutations were made on TM3, in order to accommodate the bulkier replacements on TM4. The A160V/F115A double mutant showed a moderate increase in expression level compared to either A160V or F115A single mutants. Thermal activity assays showed decrease in receptor stability in the order, wild type>A160S>A160V>A160T>G164A, with G164A being the least stable. Our study reveals that Ala1604.53 and Gly1644.57 in the TP play critical structural roles in packing of TM3 and TM4 helices. Naturally occurring mutations in conjunction with site-directed replacements can serve as powerful tools in assessing the importance of regional helix-helix interactions.Keywords
This publication has 48 references indexed in Scilit:
- Structural and functional roles of small group-conserved amino acids present on helix-H7 in the β2-adrenergic receptorBiochimica et Biophysica Acta (BBA) - Biomembranes, 2011
- Mapping the Ligand-Binding Site on a G Protein-Coupled Receptor (GPCR) Using Genetically Encoded PhotocrosslinkersBiochemistry, 2011
- Making Structural Sense of Dimerization Interfaces of Delta Opioid Receptor HomodimersBiochemistry, 2011
- Constant Constraint Matrix Approximation: A Robust, Parallelizable Constraint Method for Molecular SimulationsJournal of Chemical Theory and Computation, 2010
- The importance of valine 114 in ligand binding in β2-adrenergic receptorProtein Science, 2009
- AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreadingJournal of Computational Chemistry, 2009
- Improved prediction of protein side‐chain conformations with SCWRL4Proteins, 2009
- Helix movement is coupled to displacement of the second extracellular loop in rhodopsin activationNature Structural & Molecular Biology, 2009
- A Specific Cholesterol Binding Site Is Established by the 2.8 Å Structure of the Human β2-Adrenergic ReceptorStructure, 2008
- SWISS‐MODEL and the Swiss‐Pdb Viewer: An environment for comparative protein modelingElectrophoresis, 1997