Structural Insights into Conformational Stability of Wild-Type and Mutant β1-Adrenergic Receptor

Abstract

Recent experiments to derive a thermally stable mutant of turkey beta-1-adrenergic receptor (beta1AR) have shown that a combination of six single point mutations resulted in a 20 degrees C increase in thermal stability in mutant beta1AR. Here we have used the all-atom force-field energy function to calculate a stability score to detect stabilizing point mutations in G-protein coupled receptors. The calculated stability score shows good correlation with the measured thermal stability for 76 single point mutations and 22 multiple mutants in beta1AR. We have demonstrated that conformational sampling of the receptor for various mutants improve the prediction of thermal stability by 50%. Point mutations Y227A5.58, V230A5.61, and F338M7.48 in the thermally stable mutant m23-beta1AR stabilizes key microdomains of the receptor in the inactive conformation. The Y227A5.58 and V230A5.61 mutations stabilize the ionic lock between R139(3.50) on transmembrane helix3 and E285(6.30) on transmembrane helix6. The mutation F338M7.48 on TM7 alters the interaction of the conserved motif NPxxY(x)5,6F with helix8 and hence modulates the interaction of TM2-TM7-helix8 microdomain. The D186-R317 salt bridge (in extracellular loops 2 and 3) is stabilized in the cyanopindolol-bound wild-type beta1AR, whereas the salt bridge between D184-R317 is preferred in the mutant m23. We propose that this could be the surrogate to a similar salt bridge found between the extracellular loop 2 and TM7 in beta2AR reported recently. We show that the binding energy difference between the inactive and active states is less in m23 compared to the wild-type, which explains the activation of m23 at higher norepinephrine concentration compared to the wild-type. Results from this work throw light into the mechanism behind stabilizing mutations. The computational scheme proposed in this work could be used to design stabilizing mutations for other G-protein coupled receptors.

Thermostabilization of the β1-Adrenergic Receptor Correlates with Increased Entropy of the Inactive State

“…In the dynamics of the wt-β 1 AR, we observed 27 two possible microstates for F338 7.48 -one in which it shows π stack interaction with Y343 7.53 which in turn π stacks with F349 7.59 ; and the other in which there is no such π stacking with Y343 7.53 . Y343 7.53 is part of the conserved NPxxY motif common to most class A GPCRs, and it is known to move upon activation in β 2 AR 39 .…”

“…S7) 39,45 . Therefore mutation of this residue leads to preservation and stabilization of the ionic lock and hence the inactive state and a substantial increase in thermostability 27 . We observe a reduction in coupling (37% less than wild type) between the sites of the receptor correlated to Y227 5.58 and this contributes to the increased entropy in m23-β 1 AR.…”

“…Thus reduced stress at P305 6.50 could lead to increased stability of the inactive state in m23-β 1 AR. The residue F353 H8 forms π stacking interaction with Y343 7.53 and could be responsible for the stabilization of TM7 and helix 8 in the inactive state 27 . Comparison of the inactive and active state crystal structures of β 2 AR (PDB: 2RH1 and 3SN6), shows that there is significant structural difference at the intracellular end of TM7 near the Y343 7.53 -F353 H8 interface (Fig.…”

“…MD simulations have been used in conjunction with NMR data to explore the mechanism of activation of rhodopsin 25 and they have also been used to sample the ensemble of conformations in a given potential energy minimum for GPCRs 26–27 . The role of thermostable mutations has been attributed to formation or breaking of inter-residue contacts at the site of mutation that stabilizes one selective receptor conformation over the others 8 .…”

How Can Mutations Thermostabilize G-Protein-Coupled Receptors?

“…These predicted mutants of β 1 AR were later tested using thermostability assays under saturating concentrations of the antagonist and, in the best cases, were found to improve thermostability of the receptor significantly [54]. In a different approach, we developed a systematic rapid screening computational method, LITiConDesign for predicting single point alanine mutations to thermostabilize GPCRs [55, 42]. It involves generating an ensemble of conformations using the conformational sampling method called LITiCon [55–57].…”