Joshua L. Adelman

Constraint-based, Homology Model of the Extracellular Domain of the Epithelial Na Channel α Subunit Reveals a Mechanism of Channel Activation by Proteases

The epithelial Na+ channel (ENaC) mediates Na+ transport across high resistance epithelia. This channel is assembled from three homologous subunits with the majority of the proteins mass found in the extracellular domains. Acid-sensing ion channel 1 (ASIC1) is homologous to ENaC, but a key functional domain is highly divergent. Here we present molecular models of the extracellular region of α ENaC based on a large data set of mutations that attenuate inhibitory peptide binding in combination with comparative modeling based on the resolved structure of ASIC1. The models successfully rationalized the data from the peptide binding screen. We engineered new mutants that had not been tested based on the models and successfully predict sites where mutations affected peptide binding. Thus, we were able to confirm the overall general fold of our structural models. Further analysis suggested that the α subunit-derived inhibitory peptide affects channel gating by constraining motions within two major domains in the extracellular region, the thumb and finger domains.

Structure-guided simulations illuminate the mechanism of ATP transport through VDAC1.

The voltage-dependent anion channel (VDAC) mediates the flow of metabolites and ions across the outer mitochondrial membrane of all eukaryotic cells. The open channel passes millions of ATP molecules per second, whereas the closed state exhibits no detectable ATP flux. High-resolution structures of VDAC1 revealed a 19-stranded β-barrel with an α-helix partially occupying the central pore. To understand ATP permeation through VDAC, we solved the crystal structure of mouse VDAC1 (mVDAC1) in the presence of ATP, revealing a low-affinity binding site. Guided by these coordinates, we initiated hundreds of molecular dynamics simulations to construct a Markov state model of ATP permeation. These simulations indicate that ATP flows through VDAC through multiple pathways, in agreement with our structural data and experimentally determined physiological rates.

Simulations of the Alternating Access Mechanism of the Sodium Symporter Mhp1

Sodium coupled cotransporters of the five-helix inverted repeat (5HIR) superfamily use an alternating access mechanism to transport a myriad of small molecules across the cell membrane. One of the primary steps in this mechanism is the conformational transition from a state poised to bind extracellular substrates to a state that is competent to deliver substrate to the cytoplasm. Here, we construct a coarse-grained model of the 5HIR benzylhydantoin transporter Mhp1 that incorporates experimental structures of the outward- and inward-open states to investigate the mechanism of this conformational change. Using the weighted ensemble path-sampling method, we rigorously sample the outward- to inward-facing transition path ensemble. The transition path ensemble reveals a heterogeneous set of pathways connecting the two states and identifies two modes of transport: one consistent with a strict alternating access mechanism and another where decoupling of the inner and outer gates causes the transient formation of a continuous permeation pathway through the transporter. We also show that the conformational switch between the outward- and inward-open states results from rigid body motions of the hash motif relative to the substrate bundle, supporting the rocking bundle hypothesis. Finally, our methodology provides the groundwork for more chemically detailed investigations of the alternating mechanism.

WESTPA: An Interoperable, Highly Scalable Software Package for Weighted Ensemble Simulation and Analysis

The weighted ensemble (WE) path sampling approach orchestrates an ensemble of parallel calculations with intermittent communication to enhance the sampling of rare events, such as molecular associations or conformational changes in proteins or peptides. Trajectories are replicated and pruned in a way that focuses computational effort on underexplored regions of configuration space while maintaining rigorous kinetics. To enable the simulation of rare events at any scale (e.g., atomistic, cellular), we have developed an open-source, interoperable, and highly scalable software package for the execution and analysis of WE simulations: WESTPA (The Weighted Ensemble Simulation Toolkit with Parallelization and Analysis). WESTPA scales to thousands of CPU cores and includes a suite of analysis tools that have been implemented in a massively parallel fashion. The software has been designed to interface conveniently with any dynamics engine and has already been used with a variety of molecular dynamics (e.g., GROMACS, NAMD, OpenMM, AMBER) and cell-modeling packages (e.g., BioNetGen, MCell). WESTPA has been in production use for over a year, and its utility has been demonstrated for a broad set of problems, ranging from atomically detailed host–guest associations to nonspatial chemical kinetics of cellular signaling networks. The following describes the design and features of WESTPA, including the facilities it provides for running WE simulations and storing and analyzing WE simulation data, as well as examples of input and output.

Simulating rare events using a weighted ensemble-based string method

We introduce an extension to the weighted ensemble (WE) path sampling method to restrict sampling to a one-dimensional path through a high dimensional phase space. Our method, which is based on the finite-temperature string method, permits efficient sampling of both equilibrium and non-equilibrium systems. Sampling obtained from the WE method guides the adaptive refinement of a Voronoi tessellation of order parameter space, whose generating points, upon convergence, coincide with the principle reaction pathway. We demonstrate the application of this method to several simple, two-dimensional models of driven Brownian motion and to the conformational change of the nitrogen regulatory protein C receiver domain using an elastic network model. The simplicity of the two-dimensional models allows us to directly compare the efficiency of the WE method to conventional brute force simulations and other path sampling algorithms, while the example of protein conformational change demonstrates how the method can be used to efficiently study transitions in the space of many collective variables.

Structural Determinants of Water Permeation through the Sodium-Galactose Transporter vSGLT

Sodium-glucose transporters (SGLTs) facilitate the movement of water across the cell membrane, playing a central role in cellular homeostasis. Here, we present a detailed analysis of the mechanism of water permeation through the inward-facing state of vSGLT based on nearly 10 μs of molecular dynamics simulations. These simulations reveal the transient formation of a continuous water channel through the transporter that permits water to permeate the protein. Trajectories in which spontaneous release of galactose is observed, as well as those in which galactose remains in the binding site, show that the permeation rate, although modulated by substrate occupancy, is not tightly coupled to substrate release. Using a, to our knowledge, novel channel-detection algorithm, we identify the key residues that control water flow through the transporter and show that solvent gating is regulated by side-chain motions in a small number of residues on the extracellular face. A sequence alignment reveals the presence of two insertion sites in mammalian SGLTs that flank these outer-gate residues. We hypothesize that the absence of these sites in vSGLT may account for the high water permeability values for vSGLT determined via simulation compared to the lower experimental estimates for mammalian SGLT1.

The Mechanical Properties of PCNA: Implications for the Loading and Function of a DNA Sliding Clamp

Sliding clamps are toroidal proteins that encircle DNA and act as mobile platforms for DNA replication and repair machinery. To be loaded onto DNA, the eukaryotic sliding clamp Proliferating Cell Nuclear Antigen (PCNA) must be splayed open at one of the subunit-subunit interfaces by the ATP-dependent clamp loader, Replication Factor C, whose clamp-interacting sites form a right-handed spiral. Earlier molecular dynamics (MD) studies suggested that when PCNA opens, it preferentially adopts a right-handed spiral to match the spiral of the clamp loader. Here, analysis of considerably longer MD simulations shows that although the opened form of PCNA can achieve conformations matching the helical pitch of Replication Factor C, it is not biased toward a right-handed spiral structure. A coarse-grained elastic model was also built; its strong correspondence to the all-atom MD simulations of PCNA suggests that the behavior of the open clamp is primarily due to elastic deformation governed by the topology of the clamp domains. The elastic model was further used to construct the energy landscape of the opened PCNA clamp, including conformations that would allow binding to the clamp loader and loading onto double-stranded DNA. A picture of PCNA emerges of a rather flexible protein that, once opened, is mechanically compliant in the clamp opening process.

Accurate Estimation of Protein Folding and Unfolding Times: Beyond Markov State Models.

Because standard molecular dynamics (MD) simulations are unable to access time scales of interest in complex biomolecular systems, it is common to “stitch together” information from multiple shorter trajectories using approximate Markov state model (MSM) analysis. However, MSMs may require significant tuning and can yield biased results. Here, by analyzing some of the longest protein MD data sets available (>100 μs per protein), we show that estimators constructed based on exact non-Markovian (NM) principles can yield significantly improved mean first-passage times (MFPTs) for protein folding and unfolding. In some cases, MSM bias of more than an order of magnitude can be corrected when identical trajectory data are reanalyzed by non-Markovian approaches. The NM analysis includes “history” information, higher order time correlations compared to MSMs, that is available in every MD trajectory. The NM strategy is insensitive to fine details of the states used and works well when a fine time-discretization (i.e., small “lag time”) is used.

Stochastic steps in secondary active sugar transport

Significance The potential energy stored in ion gradients across cell membranes drives nutrients in and out of cells by cotransport proteins, e.g., uphill glucose accumulation in cells by sodium cotransporters. Insight into the mechanism of cotransport has been obtained from high-resolution atomic structures of the transporters, but further progress requires dynamic information about ion and substrate movements through the proteins. We have used multiple long molecular-dynamic simulations and electrophysiological assays to explore the dynamics of the transport cycle. Ligands bound to sodium-dependent glucose transporters are released to the cytoplasm stochastically, whereas release to the external solution is ordered with sugar first. The order of events is intimately tied to how the protein converts the energy stored in an ion gradient into a sugar gradient. Secondary active transporters, such as those that adopt the leucine-transporter fold, are found in all domains of life, and they have the unique capability of harnessing the energy stored in ion gradients to accumulate small molecules essential for life as well as expel toxic and harmful compounds. How these proteins couple ion binding and transport to the concomitant flow of substrates is a fundamental structural and biophysical question that is beginning to be answered at the atomistic level with the advent of high-resolution structures of transporters in different structural states. Nonetheless, the dynamic character of the transporters, such as ion/substrate binding order and how binding triggers conformational change, is not revealed from static structures, yet it is critical to understanding their function. Here, we report a series of molecular simulations carried out on the sugar transporter vSGLT that lend insight into how substrate and ions are released from the inward-facing state of the transporter. Our simulations reveal that the order of release is stochastic. Functional experiments were designed to test this prediction on the human homolog, hSGLT1, and we also found that cytoplasmic release is not ordered, but we confirmed that substrate and ion binding from the extracellular space is ordered. Our findings unify conflicting published results concerning cytoplasmic release of ions and substrate and hint at the possibility that other transporters in the superfamily may lack coordination between ions and substrate in the inward-facing state.

Expression of three topologically distinct membrane proteins elicits unique stress response pathways in the yeast Saccharomyces cerevisiae

Misfolded membrane proteins are retained in the endoplasmic reticulum (ER) and are subject to ER-associated degradation, which clears the secretory pathway of potentially toxic species. While the transcriptional response to environmental stressors has been extensively studied, limited data exist describing the cellular response to misfolded membrane proteins. To this end, we expressed and then compared the transcriptional profiles elicited by the synthesis of three ER retained, misfolded ion channels: The α-subunit of the epithelial sodium channel, ENaC, the cystic fibrosis transmembrane conductance regulator, CFTR, and an inwardly rectifying potassium channel, Kir2.1, which vary in their mass, membrane topologies, and quaternary structures. To examine transcriptional profiles in a null background, the proteins were expressed in yeast, which was previously used to examine the degradation requirements for each substrate. Surprisingly, the proteins failed to induce a canonical unfolded protein response or heat shock response, although messages encoding several cytosolic and ER lumenal protein folding factors rose when αENaC or CFTR was expressed. In contrast, the levels of these genes were unaltered by Kir2.1 expression; instead, the yeast iron regulon was activated. Nevertheless, a significant number of genes that respond to various environmental stressors were upregulated by all three substrates, and compared with previous microarray data we deduced the existence of a group of genes that reflect a novel misfolded membrane protein response. These data indicate that aberrant proteins in the ER elicit profound yet unique cellular responses.