Excitation-contraction (EC) coupling in skeletal muscle depends on protein interactions between the transverse tubule dihydropyridine receptor (DHPR) voltage sensor and intracellular ryanodine receptor (RyR1) calcium release channel. We present novel data showing that the C-terminal 35 residues of the β(1a) subunit adopt a nascent α-helix in which 3 hydrophobic residues align to form a hydrophobic surface that binds to RyR1 isolated from rabbit skeletal muscle. Mutation of the hydrophobic residues (L496, L500, W503) in peptide β(1a)V490-M524, corresponding to the C-terminal 35 residues of β(1a), reduced peptide binding to RyR1 to 15.2 ± 7.1% and prevented the 2.9 ± 0.2-fold activation of RyR1 by 10 nM wild-type peptide. An upstream hydrophobic heptad repeat implicated in β(1a) binding to RyR1 does not contribute to RyR1 activation. Wild-type β(1a)A474-A508 peptide (10 nM), containing heptad repeat and hydrophobic surface residues, increased RyR1 activity by 2.3 ± 0.2- and 2.2 ± 0.3-fold after mutation of the heptad repeat residues. We conclude that specific hydrophobic surface residues in the 35 residue β(1a) C-terminus bind to RyR1 and increase channel activity in lipid bilayers and thus may support skeletal EC coupling.
Viral ion channels or viroporins are short membrane proteins that participate in wide-ranging functions including virus replication and entry, assembly, and virus release. One such viroporin is the 81 amino acid residue Vpu protein derived from HIV-1. This protein consists of one transmembrane (TM) and two cytoplasmic helical domains, the former of which oligomerises to form cation-selective ion channels. In this study, we investigate the binding properties of amiloride compounds to Vpu embedded into liposomes using surface plasmon resonance (SPR). We explore the Vpu ion channel inhibitor, hexamethylene amiloride (HMA), as a molecular tool to examine the potential interactive role of key TM residues, Trp23, Ser24, and Glu29, in terms of positioning of these residues on the channel pore and the orientation of its constituent helices. The study provides experimental support that a direct interaction between Ser24 and HMA occurs and that this residue is most likely located in the channel pore. Mutation of Trp23 does not impact HMA affinity suggesting no direct involvement in binding and that this residue is lipid facing. These findings indicate that small molecules such as amilorides are capable of specifically interacting with Vpu ion channels. Although a correlation between ion channel and functional activity cannot be dismissed, alternative mechanisms involving protein-protein interactions may play an important role in the efficacy of these compounds.
It has recently emerged that glutathione transferase enzymes (GSTs) and other structurally related molecules can be translocated from the external medium into many different cell types. In this study we aim to explore in detail, the structural features that govern cell translocation and by dissecting the human GST enzyme GSTM2-2 we quantatively demonstrate that the α-helical C-terminal domain (GST-C) is responsible for this property. Attempts to further examine the constituent helices within GST-C resulted in a reduction in cell translocation efficiency, indicating that the intrinsic GST-C domain structure is necessary for maximal cell translocation capacity. In particular, it was noted that the α-6 helix of GST-C plays a stabilising role in the fold of this domain. By destabilising the conformation of GST-C, an increase in cell translocation efficiency of up to ∼2-fold was observed. The structural stability profiles of these protein constructs have been investigated by circular dichroism and differential scanning fluorimetry measurements and found to impact upon their cell translocation efficiency. These experiments suggest that the globular, helical domain in the ‘GST-fold’ structural motif plays a role in influencing cellular uptake, and that changes that affect the conformational stability of GST-C can significantly influence cell translocation efficiency.
more rigorous physical model combined with effective sampling of molecular configurations is critical for binding affinity prediction to chemical accuracy, which is defined as within one order of magnitude of the true equilibrium dissociation constant. We have demonstrated that electrostatic interactions, especially electronic polarization, are critical for protein-ligand recognition due to the significant change in electrostatic environments between bulk water and protein pockets and have achieved encouraging success in treating charged species using the polarizable Atomic Multipole Optimized Energetics for Biomolecular Applications (AMOEBA) force field. To maintain accuracy while also achieving efficiency, AMOEBA has been combined with the Orthogonal Space Random Walk enhanced alchemical free energy algorithm. Here we present applications of this strategy for the computation of protein-ligand binding affinities and, for the first time, drug solubility from alchemical simulations using the Force Field X software. 2083-Plat MloK1 LigandBinding Simulations Indicate an Induced-Fit Mechanism Many ion channels such as the MloK1 channel are steered by ligand binding via conformational changes. Mainly two binding mechanisms have been proposed, induced fit and conformational selection. Using molecular dynamics simulations, we studied ligand binding of cyclic adeonise monophosphate at the cyclic nucleotide binding domain of the MloK1 ion channel of Mesorizobium loti. For this binding domain, both crystal and NMR structures have been determined for both the ligand free as well as for the ligand bound conformation. In the simulations, spontaneous binding was observed, which enabled us to determine reaction coordinates for the ligand binding as well as for the associated conformational change of the protein. We used a combination of force probe simulations, umbrella sampling, and unbiased simulations to determine potentials of mean force along these reaction coordinates, transition rates, as well as free energy differences and barriers between the most relevant substates. Our results are compared with measured affinities and kinetics, and suggest an induced fit-mechanism.
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