Molecular dynamics simulation of tropomyosin bound to actins/myosin in the closed and open states

Tropomyosin (Tpm) is a dimeric coiled-coil protein that binds to filamentous actin, and regulates actin-myosin interaction by moving between three positions corresponding to the blocked, closed, and open states. To elucidate how Tpm undergoes transitions between these functional states, we have built structural models and conducted extensive molecular dynamics simulations of the Tpm-actins/myosin complex in the closed and open states (total simulation time >1.4 μs). Based on the simulation trajectories, we have analyzed the dynamics and energetics of a truncated Tpm interacting with actins/myosin under the physiological conditions. Our simulations have shown distinct dynamics of four Tpm periods (P3-P6), featuring pronounced biased fluctuations of P4 and P5 toward the open position in the closed state, which is consistent with a conformational selection mechanism for Tpm-regulated myosin binding. Additionally, we have identified key residues of Tpm specifically binding to actins/myosin in the closed and open state. Some of them were validated as functionally important in comparison with past functional/clinical studies, and the rest will make promising targets for future mutational experiments.     

Extracellular loops 1 and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function

The first and third extracellular loops (ECL) of G protein-coupled receptors (GPCRs) have been implicated in ligand binding and receptor function. This study describes the results of an alanine/leucine scan of ECLs 1 and 3 and loop-associated transmembrane (TM) domains of the secretin-like GPCR calcitonin receptor-like receptor which associates with receptor activity modifying protein 1 to form the CGRP receptor. Leu195Ala, Val198Ala and Ala199Leu at the top of TM2 all reduced αCGRP-mediated cAMP production and internalization; Leu195Ala and Ala199Leu also reduced αCGRP binding. These residues form a hydrophobic cluster within an area defined as the “minor groove” of rhodopsin-like GPCRs. Within ECL1, Ala203Leu and Ala206Leu influenced the ability of αCGRP to stimulate adenylate cyclase. In TM3, His219Ala, Leu220Ala and Leu222Ala have influences on αCGRP binding and cAMP production; they are likely to indirectly influence the binding site for αCGRP as well as having an involvement in signal transduction. On the exofacial surfaces of TMs 6 and 7, a number of residues were identified that reduced cell surface receptor expression, most noticeably Leu351Ala and Glu357Ala in TM6. The residues may contribute to the RAMP1 binding interface. Ile360Ala impaired αCGRP-mediated cAMP production. Ile360 is predicted to be located close to ECL2 and may facilitate receptor activation. Identification of several crucial functional loci gives further insight into the activation mechanism of this complex receptor system and may aid rational drug design.