Predicting Transmembrane Dimerization and the Interfaces in Thyroid-Stimulating Hormone Receptor (TSHR) Using Brownian Dynamics Stimulation

Abstract

The TSHR receptor, a member of the GPCR protein family, has not been fully modeled. Only the crystal structure of the ecto-domain has been reported. Experimental data with truncated TSHRs indicate that the trans-membrane region has a major role in dimerization/ multimirization of this receptor. Biophysical (FRET) and biochemical (co-immunoprecipitation) studies from our laboratory have established that the TSH receptor, similar to other GPCRs, has the propensity to form dimers and multimers both in native and non-native cells. Recent experimental studies also from this laboratory have further reported that dimerization interfaces can reside in the extracellular domain. In addition, the TSHR, bereft of the ectodomain, shows similar characteristics, indicating that the transmembrane domains of TSHR are also involved in dimerization, receptor stability and trafficking.As server-based predictions for possible dimerization interfaces, have large uncertainties and wide variances precluding any definitive conclusions, we have resorted to the more robust and refined algorithm of Brownian dynamics in predicting transmembrane dimerization, a standard and established technique in such biological phenomena. A recently developed variant of Brownian dynamics has become an important computational tool to predict dimerization/multimerization of transmembrane proteins.The method is implemented in a program called Macrodox and involves complementing the Poisson-Boltzmann equation based calculation of the electrostatic interactions with the calculation of Van der Waals interactions. Furthermore, in this method the sampling efficiency is enhanced by restricting the protein movements within the membrane plane. Both translational (0.101x10−01 A 2/ps) and rotational diffusion constants (0.165x10−04 A 2/ps) and several other physical parameters (radius of gyration=18.17 A) have been computed for each TSHR monomer. We will present the results for contact residues from the trajectory of simulations and abrogation of dimerization following mutation of critical contact residues.