The allosteric mechanism leading to an open-groove lipid conductive state of the TMEM16F scramblase.

TMEM16F is a Ca-activated phospholipid scramblase in the TMEM16 family of membrane proteins. Unlike other TMEM16s exhibiting a membrane-exposed hydrophilic groove that serves as a translocation pathway for lipids, the experimentally determined structures of TMEM16F shows the groove in a closed conformation even under conditions of maximal scramblase activity. It is currently unknown if/how TMEM16F groove can open for lipid scrambling.

Development of Force Field Parameters for the Simulation of Single- and Double-Stranded DNA Molecules and DNA-Protein Complexes.

Although molecular dynamics (MD) simulations have been used extensively to study the structural dynamics of proteins, the role of MD simulation in studies of nucleic acid based systems has been more limited. One contributing factor to this disparity is the historically lower level of accuracy of the physical models used in such simulations to describe interactions involving nucleic acids. By modifying nonbonded and torsion parameters of a force field from the Amber family of models, we recently developed force field parameters for RNA that achieve a level of accuracy comparable to that of state-of-the-art protein force fields.

X-ray structure of LeuT in an inward-facing occluded conformation reveals mechanism of substrate release.

Neurotransmitter:sodium symporters (NSS) are conserved from bacteria to man and serve as targets for drugs, including antidepressants and psychostimulants. Here we report the X-ray structure of the prokaryotic NSS member, LeuT, in a Na/substrate-bound, inward-facing occluded conformation. To obtain this structure, we were guided by findings from single-molecule fluorescence spectroscopy and molecular dynamics simulations indicating that L-Phe binding and mutation of the conserved N-terminal Trp8 to Ala both promote an inward-facing state.

The allosteric mechanism of substrate-specific transport in SLC6 is mediated by a volumetric sensor.

Neurotransmitter:sodium symporters (NSSs) in the SLC6 family terminate neurotransmission by coupling the thermodynamically favorable transport of ions to the thermodynamically unfavorable transport of neurotransmitter back into presynaptic neurons. Results from many structural, functional, and computational studies on LeuT, a bacterial NSS homolog, have provided critical insight into the mechanism of sodium-coupled transport, but the mechanism underlying substrate-specific transport rates is still not understood. We present a combination of molecular dynamics simulations, single-molecule fluorescence resonance energy transfer (smFRET) imaging, and measurements of Na binding and substrate transport that reveals an allosteric substrate specificity mechanism.

A New Computational Method for Membrane Compressibility: Bilayer Mechanical Thickness Revisited.

Because lipid bilayers can bend and stretch in ways similar to thin elastic sheets, physical models of bilayer deformation have utilized mechanical constants such as the moduli for bending rigidity (κ) and area compressibility (K). However, the use of these models to quantify the energetics of membrane deformation associated with protein-membrane interactions, and the membrane response to stress is often hampered by the shortage of experimental data suitable for the estimation of the mechanical constants of various lipid mixtures. Although computational tools such as molecular dynamics simulations can provide alternative means to estimate K values, current approaches suffer significant technical limitations.

A partially-open inward-facing intermediate conformation of LeuT is associated with Na release and substrate transport.

Neurotransmitter:sodium symporters (NSS), targets of antidepressants and psychostimulants, clear neurotransmitters from the synaptic cleft through sodium (Na)-coupled transport. Substrate and Na are thought to be transported from the extracellular to intracellular space through an alternating access mechanism by coordinated conformational rearrangements in the symporter that alternately expose the binding sites to each side of the membrane. However, the mechanism by which the binding of ligands coordinates conformational changes occurring on opposite sides of the membrane is not well understood.

Thermodynamic Coupling Function Analysis of Allosteric Mechanisms in the Human Dopamine Transporter.

Allostery plays a crucial role in the mechanism of neurotransmitter-sodium symporters, such as the human dopamine transporter. To investigate the molecular mechanism that couples the transport-associated inward release of the Na ion from the Na2 site to intracellular gating, we applied a combination of the thermodynamic coupling function (TCF) formalism and Markov state model analysis to a 50-μs data set of molecular dynamics trajectories of the human dopamine transporter, in which multiple spontaneous Na release events were observed. Our TCF approach reveals a complex landscape of thermodynamic coupling between Na release and inward-opening, and identifies diverse, yet well-defined roles for different Na-coordinating residues.

Ligand modulation of sidechain dynamics in a wild-type human GPCR.

GPCRs regulate all aspects of human physiology, and biophysical studies have deepened our understanding of GPCR conformational regulation by different ligands. Yet there is no experimental evidence for how sidechain dynamics control allosteric transitions between GPCR conformations. To address this deficit, we generated samples of a wild-type GPCR (AR) that are deuterated apart from H/C NMR probes at isoleucine δ1 methyl groups, which facilitated H/C methyl TROSY NMR measurements with opposing ligands.

The Allostery Landscape: Quantifying Thermodynamic Couplings in Biomolecular Systems.

Allostery plays a fundamental role in most biological processes. However, little theory is available to describe it outside of two-state models. Here we use a statistical mechanical approach to show that the allosteric coupling between two collective variables is not a single number, but instead a two-dimensional thermodynamic coupling function that is directly related to the mutual information from information theory and the copula density function from probability theory.

Allosteric Mechanisms of Molecular Machines at the Membrane: Transport by Sodium-Coupled Symporters.

Solute transport across cell membranes is ubiquitous in biology as an essential physiological process. Secondary active transporters couple the unfavorable process of solute transport against its concentration gradient to the energetically favorable transport of one or several ions. The study of such transporters over several decades indicates that their function involves complex allosteric mechanisms that are progressively being revealed in atomistic detail.

Role of Annular Lipids in the Functional Properties of Leucine Transporter LeuT Proteomicelles.

Recent work has shown that the choice of the type and concentration of detergent used for the solubilization of membrane proteins can strongly influence the results of functional experiments. In particular, the amino acid transporter LeuT can bind two substrate molecules in low concentrations of n-dodecyl β-d-maltopyranoside (DDM), whereas high concentrations reduce the molar binding stoichiometry to 1:1. Subsequent molecular dynamics (MD) simulations of LeuT in DDM proteomicelles revealed that DDM can penetrate to the extracellular vestibule and make stable contacts in the functionally important secondary substrate binding site (S2), suggesting a potential competitive mechanism for the reduction in binding stoichiometry.

Computational approaches to detect allosteric pathways in transmembrane molecular machines.

Many of the functions of transmembrane proteins involved in signal processing and transduction across the cell membrane are determined by allosteric couplings that propagate the functional effects well beyond the original site of activation. Data gathered from breakthroughs in biochemistry, crystallography, and single molecule fluorescence have established a rich basis of information for the study of molecular mechanisms in the allosteric couplings of such transmembrane proteins. The mechanistic details of these couplings, many of which have therapeutic implications, however, have only become accessible in synergy with molecular modeling and simulations.

AIM for Allostery: Using the Ising Model to Understand Information Processing and Transmission in Allosteric Biomolecular Systems.

In performing their biological functions, molecular machines must process and transmit information with high fidelity. Information transmission requires dynamic coupling between the conformations of discrete structural components within the protein positioned far from one another on the molecular scale. This type of biomolecular "action at a distance" is termed .

Spontaneous inward opening of the dopamine transporter is triggered by PIP2-regulated dynamics of the N-terminus.

We present the dynamic mechanism of concerted motions in a full-length molecular model of the human dopamine transporter (hDAT), a member of the neurotransmitter/sodium symporter (NSS) family, involved in state-to-state transitions underlying function. The findings result from an analysis of unbiased atomistic molecular dynamics simulation trajectories (totaling >14 μs) of the hDAT molecule immersed in lipid membrane environments with or without phosphatidylinositol 4,5-biphosphate (PIP2) lipids. The N-terminal region of hDAT (N-term) is shown to have an essential mechanistic role in correlated rearrangements of specific structural motifs relevant to state-to-state transitions in the hDAT.

A functional selectivity mechanism at the serotonin-2A GPCR involves ligand-dependent conformations of intracellular loop 2.

With recent progress in determination of G protein-coupled receptor (GPCR) structure with crystallography, a variety of other experimental approaches (e.g., NMR spectroscopy, fluorescent-based assays, mass spectrometry techniques) are also being used to characterize state-specific and ligand-specific conformational states.

NbIT--a new information theory-based analysis of allosteric mechanisms reveals residues that underlie function in the leucine transporter LeuT.

Complex networks of interacting residues and microdomains in the structures of biomolecular systems underlie the reliable propagation of information from an input signal, such as the concentration of a ligand, to sites that generate the appropriate output signal, such as enzymatic activity. This information transduction often carries the signal across relatively large distances at the molecular scale in a form of allostery that is essential for the physiological functions performed by biomolecules. While allosteric behaviors have been documented from experiments and computation, the mechanism of this form of allostery proved difficult to identify at the molecular level.

The membrane protein LeuT in micellar systems: aggregation dynamics and detergent binding to the S2 site.

Structural and functional properties of integral membrane proteins are often studied in detergent micellar environments (proteomicelles), but how such proteomicelles form and organize is not well understood. This makes it difficult to evaluate the relationship between the properties of the proteins measured in such a detergent-solubilized form and under native conditions. To obtain mechanistic information about this relationship for the leucine transporter (LeuT), a prokaryotic homologue of the mammalian neurotransmitter/sodium symporters (NSSs), we studied the properties of proteomicelles formed by n-dodecyl-β,D-maltopyranoside (DDM) detergent.