TNNT2 mutations in the tropomyosin binding region of TNT1 disrupt its role in contractile inhibition and stimulate cardiac dysfunction.

Muscle contraction is regulated by the movement of end-to-end-linked troponin-tropomyosin complexes over the thin filament surface, which uncovers or blocks myosin binding sites along F-actin. The N-terminal half of troponin T (TnT), TNT1, independently promotes tropomyosin-based, steric inhibition of acto-myosin associations, in vitro. Recent structural models additionally suggest TNT1 may restrain the uniform, regulatory translocation of tropomyosin. Therefore, TnT potentially contributes to striated muscle relaxation; however, the in vivo functional relevance and molecular basis of this noncanonical role remain unclear. Impaired relaxation is a hallmark of hypertrophic and restrictive cardiomyopathies (HCM and RCM). Investigating the effects of cardiomyopathy-causing mutations could help clarify TNT1's enigmatic inhibitory property. We tested the hypothesis that coupling of TNT1 with tropomyosin's end-to-end overlap region helps anchor tropomyosin to an inhibitory position on F-actin, where it deters myosin binding at rest, and that, correspondingly, cross-bridge cycling is defectively suppressed under diastolic/low Ca2+ conditions in the presence of HCM/RCM lesions. The impact of TNT1 mutations on Drosophila cardiac performance, rat myofibrillar and cardiomyocyte properties, and human TNT1's propensity to inhibit myosin-driven, F-actin-tropomyosin motility were evaluated. Our data collectively demonstrate that removing conserved, charged residues in TNT1's tropomyosin-binding domain impairs TnT's contribution to inhibitory tropomyosin positioning and relaxation. Thus, TNT1 may modulate acto-myosin activity by optimizing F-actin-tropomyosin interfacial contacts and by binding to actin, which restrict tropomyosin's movement to activating configurations. HCM/RCM mutations, therefore, highlight TNT1's essential role in contractile regulation by diminishing its tropomyosin-anchoring effects, potentially serving as the initial trigger of pathology in our animal models and humans.

The Molecular Switching Mechanism at the Conserved D(E)RY Motif in Class-A GPCRs.

The disruption of ionic and H-bond interactions between the cytosolic ends of transmembrane helices TM3 and TM6 of class-A (rhodopsin-like) G protein-coupled receptors (GPCRs) is a hallmark for their activation by chemical or physical stimuli. In the bovine photoreceptor rhodopsin, this is accompanied by proton uptake at Glu(134) in the class-conserved D(E)RY motif. Studies on TM3 model peptides proposed a crucial role of the lipid bilayer in linking protonation to stabilization of an active state-like conformation. However, the molecular details of this linkage could not be resolved and have been addressed in this study by molecular dynamics (MD) simulations on TM3 model peptides in a bilayer of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). We show that protonation of the conserved glutamic acid alters the peptide insertion depth in the membrane, its side-chain rotamer preferences, and stabilizes the C-terminal helical structure. These factors contribute to the rise of the side-chain pKa (> 6) and to reduced polarity around the TM3 C terminus as confirmed by fluorescence spectroscopy. Helix stabilization requires the protonated carboxyl group; unexpectedly, this stabilization could not be evoked with an amide in MD simulations. Additionally, time-resolved Fourier transform infrared (FTIR) spectroscopy of TM3 model peptides revealed a different kinetics for lipid ester carbonyl hydration, suggesting that the carboxyl is linked to more extended H-bond clusters than an amide. Remarkably, this was seen as well in DOPC-reconstituted Glu(134)- and Gln(134)-containing bovine opsin mutants and demonstrates that the D(E)RY motif is a hydrated microdomain. The function of the D(E)RY motif as a proton switch is suggested to be based on the reorganization of the H-bond network at the membrane interface.

Lipid protein interactions couple protonation to conformation in a conserved cytosolic domain of G protein-coupled receptors.

The visual photoreceptor rhodopsin is a prototypical class I (rhodopsin-like) G protein-coupled receptor. Photoisomerization of the covalently bound ligand 11-cis-retinal leads to restructuring of the cytosolic face of rhodopsin. The ensuing protonation of Glu-134 in the class-conserved D(E)RY motif at the C-terminal end of transmembrane helix-3 promotes the formation of the G protein-activating state. Using transmembrane segments derived from helix-3 of bovine rhodopsin, we show that lipid protein interactions play a key role in this cytosolic "proton switch." Infrared and fluorescence spectroscopic pK(a) determinations reveal that the D(E)RY motif is an autonomous functional module coupling side chain neutralization to conformation and helix positioning as evidenced by side chain to lipid headgroup Foerster resonance energy transfer. The free enthalpies of helix stabilization and hydrophobic burial of the neutral carboxyl shift the side chain pK(a) into the range typical of Glu-134 in photoactivated rhodopsin. The lipid-mediated coupling mechanism is independent of interhelical contacts allowing its conservation without interference with the diversity of ligand-specific interactions in class I G protein-coupled receptors.

Secondary structure and compliance of a predicted flexible domain in kinesin-1 necessary for cooperation of motors.

Although the mechanism by which a kinesin-1 molecule moves individually along a microtubule is quite well-understood, the way that many kinesin-1 motor proteins bound to the same cargo move together along a microtubule is not. We identified a 60-amino-acid-long domain, termed Hinge 1, in kinesin-1 from Drosophila melanogaster that is located between the coiled coils of the neck and stalk domains. Its deletion reduces microtubule gliding speed in multiple-motor assays but not single-motor assays. Hinge 1 thus facilitates the cooperation of motors by preventing them from impeding each other. We addressed the structural basis for this phenomenon. Video-microscopy of single microtubule-bound full-length motors reveals the sporadic occurrence of high-compliance states alternating with longer-lived, low-compliance states. The deletion of Hinge 1 abolishes transitions to the high-compliance state. Based on Fourier transform infrared, circular dichroism, and fluorescence spectroscopy of Hinge 1 peptides, we propose that low-compliance states correspond to an unexpected structured organization of the central Hinge 1 region, whereas high-compliance states correspond to the loss of that structure. We hypothesize that strain accumulated during multiple-kinesin motility populates the high-compliance state by unfolding helical secondary structure in the central Hinge 1 domain flanked by unordered regions, thereby preventing the motors from interfering with each other in multiple-motor situations.

Flavonoids affect actin functions in cytoplasm and nucleus.

Based on the identification of actin as a target protein for the flavonol quercetin, the binding affinities of quercetin and structurally related flavonoids were determined by flavonoid-dependent quenching of tryptophan fluorescence from actin. Irrespective of differences in the hydroxyl pattern, similar Kd values in the 20 microM range were observed for six flavonoids encompassing members of the flavonol, isoflavone, flavanone, and flavane group. The potential biological relevance of the flavonoid/actin interaction in the cytoplasm and the nucleus was addressed using an actin polymerization and a transcription assay, respectively. In contrast to the similar binding affinities, the flavonoids exert distinct and partially opposing biological effects: although flavonols inhibit actin functions, the structurally related flavane epigallocatechin promotes actin activity in both test systems. Infrared spectroscopic evidence reveals flavonoid-specific conformational changes in actin which may mediate the different biological effects. Docking studies provide models of flavonoid binding to the known small molecule-binding sites in actin. Among these, the mostly hydrophobic tetramethylrhodamine-binding site is a prime candidate for flavonoid binding and rationalizes the high efficiency of quenching of the two closely located fluorescent tryptophans. The experimental and theoretical data consistently indicate the importance of hydrophobic, rather than H-bond-mediated, actin-flavonoid interactions. Depending on the rigidity of the flavonoid structures, different functionally relevant conformational changes are evoked through an induced fit.

Structure and pH sensitivity of the transmembrane segment 3 of rhodopsin.

Activation of G protein-coupled receptors (GPCRs) originates in ligand-induced protein conformational changes that are transmitted to the cytosolic receptor surface. In the photoreceptor rhodopsin, and possibly other rhodopsin-like GPCRs, protonation of a carboxylic acid in the conserved E(D)RY motif at the cytosolic end of transmembrane helix 3 (TM3) is coupled to receptor activation. Here, we have investigated the structure of synthetic peptides derived from rhodopsin TM3. Polarized FTIR spectroscopy reveals a helical structure of a 31-mer TM3 peptide reconstituted into PC vesicles with a large tilt of 40-50 degrees of the helical axis relative to the membrane normal. Helical structure is also observed for the TM3 peptide in detergent micelles and depends on pH, especially in the C-terminal sequence. In addition, the fluorescence emission of the single tyrosine of the D(E)RY motif in the TM3 peptide exhibits a pronounced pH sensitivity that is abolished when Glu is replaced by Gln, demonstrating that protonation of the conserved Glu side chain affects the structure in the environment of the D(E)RY motif of TM3. The pH regulation of the C-terminal TM3 structure may be an intrinsic feature of the E(D)RY motif in other class I receptors, allowing the coupling of protonation and conformation of membrane-exposed residues in full-length GPCRs.