How Can Mutations Thermostabilize G-Protein-Coupled Receptors?

Abstract

Structures of over 30 different G-protein-coupled receptors (GPCRs) have advanced our understanding of cell signaling and have provided a foundation for structure-guided drug design. This exciting progress has required the development of three complementary methods to facilitate GPCR crystallization, one of which is the thermostabilization of receptors by systematic mutagenesis. However, the reason why a particular mutation, or combination of mutations, stabilizes the receptor is not always evident from a static crystal structure. Molecular dynamics (MD) simulations have been used to identify and estimate the energetic factors that affect thermostability through comparing the dynamics of the thermostabilized receptors with structure-based models of the wild-type receptor. The data indicate that receptors are stabilized through a combination of factors, including an increase in receptor rigidity, a decrease in collective motion, reduced stress at specific residues, and the presence of ordered water molecules. Predicting thermostabilizing mutations computationally represents a major challenge for the field.

Keywords: GPCR; dynamics; structure; thermostability.

Figure 1

Schematic representation of the dynamics of the thermostable mutant GPCR compared to its wild-type receptor. This schematic is based on the analysis of the extensive MD simulation results on four thermostabilized GPCRs. TM5 and TM6 are more dynamic in the wild-type receptor compared to their thermostable mutant counterpart.

Figure 2

Role of water in thermostabilization of GPCRs. Clusters of tightly bound water molecules, shown as mesh in the figure, accumulate between the transmembrane helices of the thermostable mutant NTSR1-GW5 to stabilize the receptor, compared to the wild-type.

Figure 3

Experimentally measured thermostability scores ranging from blue to red (low to high) shown overlaid on the crystal structures of the thermostable mutants β₁AR-m23, A_2A-StaR2, A_2A-GL31 and NTSR1-GW5. Thermostability was measured using the following radioligands: the antagonist ³H-dihydroalprenolol for turkey β₁AR in the inactive state, the antagonist ³H-ZM241385 for A_2AR in the inactive state, the agonist ³H-NECA for the A_2AR in the active-like state, and the agonist ³H-neurotensin for NTSR1 in the active-like state. Each structure is shown in two mutually orthogonal views. The residues with high thermostability are marked in spheres and labeled in each receptor.