Michael V. LeVine

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. Here, we introduce a novel analysis framework, called N-body Information Theory (NbIT) analysis, which is based on information theory and uses measures of configurational entropy in a biomolecular system to identify microdomains and individual residues that act as (i)-channels for long-distance information sharing between functional sites, and (ii)-coordinators that organize dynamics within functional sites. Application of the new method to molecular dynamics (MD) trajectories of the occluded state of the bacterial leucine transporter LeuT identifies a channel of allosteric coupling between the functionally important intracellular gate and the substrate binding sites known to modulate it. NbIT analysis is shown also to differentiate residues involved primarily in stabilizing the functional sites, from those that contribute to allosteric couplings between sites. NbIT analysis of MD data thus reveals rigorous mechanistic elements of allostery underlying the dynamics of biomolecular systems.

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. The mechanism involves PIP2-mediated electrostatic interactions between the N-term and the intracellular loops of the transporter molecule. Quantitative analyses of collective motions in the trajectories reveal that these interactions correlate with the inward-opening dynamics of hDAT and are allosterically coupled to the known functional sites of the transporter. The observed large-scale motions are enabled by specific reconfiguration of the network of ionic interactions at the intracellular end of the protein. The isomerization to the inward-facing state in hDAT is accompanied by concomitant movements in the extracellular vestibule and results in the release of an Na+ ion from the Na2 site and destabilization of the substrate dopamine in the primary substrate binding S1 site. The dynamic mechanism emerging from the findings highlights the involvement of the PIP2-regulated interactions between the N-term and the intracellular loop 4 in the functionally relevant conformational transitions that are also similar to those found to underlie state-to-state transitions in the leucine transporter (LeuT), a prototypical bacterial homologue of the NSS.

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. MD simulations offer a powerful complementary approach to elucidate the dynamic features associated with ligand-specific GPCR conformations. To shed light on the conformational elements and dynamics of the important aspect of GPCR functional selectivity, we carried out unbiased microsecond-length MD simulations of the human serotonin 2A receptor (5-HT2AR) in the absence of ligand and bound to four distinct serotonergic agonists. The 5-HT2AR is a suitable system to study the structural features involved in the ligand-dependent conformational heterogeneity of GPCRs because it is well-characterized experimentally and exhibits a strong agonist-specific phenotype in that some 5-HT2AR agonists induce LSD-like hallucinations, while others lack this psychoactive property entirely. Here we report evidence for structural and dynamic differences in 5-HT2AR interacting with such pharmacologically distinct ligands, hallucinogens, and nonhallucinogens obtained from all-atom MD simulations. Differential ligand binding contacts were identified for structurally similar hallucinogens and nonhallucinogens and found to correspond to different conformations in the intracellular loop 2 (ICL2). From the different ICL2 conformations, functional selective phenotypes are suggested through effects on dimerization and/or distinct direct interaction with effector proteins. The findings are presented in the context of currently proposed hallucinogenesis mechanisms, and ICL2 is proposed as a fine-tuning selective switch that can differentiates modes of 5-HT2AR activation.

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. Extensive atomistic molecular dynamics simulations of different protein/detergent/water number ratios revealed the formation of a proteomicelle characterized by a constant-sized shell of detergents surrounding LeuT protecting its transmembrane segments from unfavorable hydrophobic/hydrophilic exposure. Regardless of the DDM content in the simulated system, this shell consisted of a constant number of DDM molecules (∼120 measured at a 4 Å cutoff distance from LeuT). In contrast, the overall number of DDMs in the proteomicelle (aggregation number) was found to depend on the detergent concentration, reaching a saturation value of 226±17 DDMs in the highest concentration regime simulated. Remarkably, we found that at high detergent-to-protein ratios we observed two independent ways of DDM penetration into LeuT, both leading to a positioning of the DDM molecule in the second substrate (S2) binding site of LeuT. Consonant with several recent experimental studies demonstrating changes in functional properties of membrane proteins due to detergent, our findings highlight how the environment in which the membrane proteins are examined may affect the outcome and interpretation of their mechanistic features.

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. Here, we review some recent computational approaches that analyze allosteric coupling networks (ACNs) in transmembrane proteins, and in particular the recently developed Protein Interaction Analyzer (PIA) designed to study ACNs in the structural ensembles sampled by molecular dynamics simulations. The power of these computational approaches in interrogating the functional mechanisms of transmembrane proteins is illustrated with selected examples of recent experimental and computational studies pursued synergistically in the investigation of secondary active transporters and GPCRs. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov.

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. Because annular lipids can be retained during solubilization, we performed MD simulations of LeuT proteomicelles at various stages of the solubilization process. We find that at low DDM concentrations, lipids are retained around the protein and penetration of detergent into the S2 site does not occur, whereas at high concentrations, lipids are displaced and the probability of DDM binding in the S2 site is increased. This behavior is dependent on the type of detergent, however, as we find in the simulations that the detergent lauryl maltose-neopentyl glycol, which is approximately twice the size of DDM and structurally more closely resembles lipids, does not penetrate the protein even at very high concentrations. We present functional studies that confirm the computational findings, emphasizing the need for careful consideration of experimental conditions, and for cautious interpretation of data in gathering mechanistic information about membrane proteins.

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 (A2AR) that are deuterated apart from 1H/13C NMR probes at isoleucine δ1 methyl groups, which facilitated 1H/13C methyl TROSY NMR measurements with opposing ligands. Our data indicate that low [Na+] is required to allow large agonist-induced structural changes in A2AR, and that patterns of sidechain dynamics substantially differ between agonist (NECA) and inverse agonist (ZM241385) bound receptors, with the inverse agonist suppressing fast ps-ns timescale motions at the G protein binding site. Our approach to GPCR NMR creates a framework for exploring how different regions of a receptor respond to different ligands or signaling proteins through modulation of fast ps-ns sidechain dynamics.

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 allostery. Although allostery is ubiquitous in biological regulation and signal transduction, its treatment in theoretical models has mostly eschewed quantitative descriptions involving the systems underlying structural components and their interactions. Here, we show how Ising models can be used to formulate an approach to allostery in a structural context of interactions between the constitutive components by building simple allosteric constructs we termed Allosteric Ising Models (AIMs). We introduce the use of AIMs in analytical and numerical calculations that relate thermodynamic descriptions of allostery to the structural context, and then show that many fundamental properties of allostery, such as the multiplicative property of parallel allosteric channels, are revealed from the analysis of such models. The power of exploring mechanistic structural models of allosteric function in more complex systems by using AIMs is demonstrated by building a model of allosteric signaling for an experimentally well-characterized asymmetric homodimer of the dopamine D2 receptor.

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. Here, we report the use of single-molecule fluorescence resonance energy transfer (smFRET) techniques to image transitions between distinct conformational states on both the extracellular and intracellular sides of the prokaryotic NSS LeuT, including partially open intermediates associated with transport activity. The nature and functional context of these hitherto unidentified intermediate states shed new light on the allosteric mechanism that couples substrate and Na+ symport by the NSS family through conformational dynamics.Neurotransmitter:sodium symporters (NSS) modulate the duration and magnitude of signaling via the sodium-coupled reuptake of neurotransmitters. Here the authors describe quantitative single molecule imaging of ligand-induced, functional dynamics of both intracellular and extracellular surfaces of LeuT, further defining the mechanism for NSS transport.

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. In particular, we identify a prominent role in the allosteric coupling for the Na+-coordinating residue D421, where mutation has previously been associated with neurological disorders. Our results highlight the power of the TCF analysis to elucidate the molecular mechanism of complex allosteric processes in large biomolecular systems.