Membrane mediated mechanical stimuli produces distinct active-like states in the AT1 receptor.

The Angiotensin II Type 1 (AT1) receptor is one of the most widely studied GPCRs within the context of biased signaling. While the AT1 receptor is activated by agonists such as the peptide AngII, it can also be activated by mechanical stimuli such as membrane stretch or shear in the absence of a ligand. Despite the importance of mechanical activation of the AT1 receptor in biological processes such as vasoconstriction, little is known about the structural changes induced by external physical stimuli mediated by the surrounding lipid membrane. Here, we present a systematic simulation study that characterizes the activation of the AT1 receptor under various membrane environments and mechanical stimuli. We show that stability of the active state is highly sensitive to membrane thickness and tension. Structural comparison of membrane-mediated vs. agonist-induced activation shows that the AT1 receptor has distinct active conformations. This is supported by multi-microsecond free energy calculations that show unique landscapes for the inactive and various active states. Our modeling results provide structural insights into the mechanical activation of the AT1 receptor and how it may produce different functional outcomes within the framework of biased agonism.

Mechanical Activation of MscL Revealed by a Locally Distributed Tension Molecular Dynamics Approach.

Membrane tension perceived by mechanosensitive (MS) proteins mediates cellular responses to mechanical stimuli and osmotic stresses, and it also guides multiple biological functions including cardiovascular control and development. In bacteria, MS channels function as tension-activated pores limiting excessive turgor pressure, with MS channel of large conductance (MscL) acting as an emergency release valve preventing cell lysis. Previous attempts to simulate gating transitions in MscL by either directly applying steering forces to the protein or by increasing the whole-system tension were not fully successful and often disrupted the integrity of the system. We present a novel, to our knowledge, locally distributed tension molecular dynamics (LDT-MD) simulation method that allows application of forces continuously distributed among lipids surrounding the channel using a specially constructed collective variable. We report reproducible and reversible transitions of MscL to the open state with measured parameters of lateral expansion and conductivity that exactly satisfy experimental values. The LDT-MD method enables exploration of the MscL-gating process with different pulling velocities and variable tension asymmetry between the inner and outer membrane leaflets. We use LDT-MD in combination with well-tempered metadynamics to reconstruct the tension-dependent free-energy landscape for the opening transition in MscL. The flexible definition of the LDT collective variable allows general application of our method to study mechanical activation of any membrane-embedded protein.

Direct Determination of Site-Specific Noncovalent Interaction Strengths of Proteins from NMR-Derived Fast Side Chain Motional Parameters.

A novel approach to accurately determine residue-specific noncovalent interaction strengths (ξ) of proteins from NMR-measured fast side chain motional parameters (Oaxis2) is presented. By probing the environmental sensitivity of side chain conformational energy surfaces of individual residues of a diverse set of proteins, the microscopic connections between ξ, Oaxis2, conformational entropy (Sconf), conformational barriers, and rotamer stabilities established here are found to be universal among proteins. The results reveal that side chain flexibility and conformational entropy of each residue decrease with increasing ξ and that for each residue type there exists a critical range of ξ, determined primarily by the mean side chain conformational barriers, within which flexibility of any residue can be reversibly tuned from highly flexible (with Oaxis2 ∼ 0) to highly restricted (with Oaxis2 ∼ 1) by increasing ξ by ∼3 kcal/mol. Beyond this critical range of ξ, both side chain flexibility and conformational entropy are insensitive to ξ. The interrelationships between conformational dynamics, conformational entropy, and noncovalent interactions of protein side chains established here open up new avenues to probe perturbation-induced (for example, ligand-binding, temperature, pressure) changes in fast side chain dynamics and thermodynamics of proteins by comparing their conformational energy surfaces in the native and perturbed states.