The CYTOLD and ERTOLD pathways for lipid droplet-protein targeting.

Lipid droplets (LDs) are the main organelles for lipid storage, and their surfaces contain unique proteins with diverse functions, including those that facilitate the deposition and mobilization of LD lipids. Among organelles, LDs have an unusual structure with an organic, hydrophobic oil phase covered by a phospholipid monolayer. The unique properties of LD monolayer surfaces require proteins to localize to LDs by distinct mechanisms. Here we review the two pathways known to mediate direct LD protein localization: the CYTOLD pathway mediates protein targeting from the cytosol toLDs, and the ERTOLD pathway functions in protein targeting from the endoplasmic reticulum toLDs. We describe the emerging principles for each targeting pathway in animal cells and highlight open questions in the field.

Lasso Peptides: Exploring the Folding Landscape of Nature's Smallest Interlocked Motifs.

Lasso peptides make up a class of natural products characterized by a threaded structure. Given their small size and stability, chemical synthesis would offer tremendous potential for the development of novel therapeutics. However, the accessibility of the pre-folded lasso architecture has limited this advance. To better understand the folding process de novo, simulations are used herein to characterize the folding propensity of microcin J25 (MccJ25), a lasso peptide known for its antimicrobial properties. New algorithms are developed to unambiguously distinguish threaded from nonthreaded precursors and determine handedness, a key feature in natural lasso peptides. We find that MccJ25 indeed forms right-handed pre-lassos, in contrast to past predictions but consistent with all natural lasso peptides. Additionally, the native pre-lasso structure is shown to be metastable prior to ring formation but to readily transition to entropically favored unfolded and nonthreaded structures, suggesting that de novo lasso folding is rare. However, by altering the ring forming residues and appending thiol and thioester functionalities, we are able to increase the stability of pre-lasso conformations. Furthermore, conditions leading to protonation of a histidine imidazole side chain further stabilize the modified pre-lasso ensemble. This work highlights the use of computational methods to characterize lasso folding and demonstrates that de novo access to lasso structures can be facilitated by optimizing sequence, unnatural modifications, and reaction conditions like pH.

Can membrane composition traffic toxins? Mycolactone and preferential membrane interactions.

Mycolactone is a cytotoxic and immunosuppressive macrolide produced by Mycobacterium ulcerans and the sole causative agent of the neglected tropical skin disease Buruli ulcer. The toxin acts by invading host cells and interacting with intracellular targets to disrupt multiple fundamental cellular processes. Mycolactone's amphiphilic nature enables strong interactions with lipophilic environments, including cellular membranes; however, the specificity of these interactions and the role of membranes in the toxin's pathogenicity remain unknown. It is likely that preferential interactions with lipophilic carriers play a key role in the toxin's distribution in the host, which, if understood, could provide insights to aid in the development of needed diagnostics for Buruli ulcer disease. In this work, molecular dynamics simulations were combined with enhanced free-energy sampling to characterize mycolactone's association with and permeation through models of the mammalian endoplasmic reticulum (ER) and plasma membranes (PMs). We find that increased order in the PMs not only leads to a different permeation mechanism compared with that in the ER membrane but also an energetic driving force for ER localization. Increased hydration, membrane deformation, and preferential interactions with unsaturated lipid tails stabilize the toxin in the ER membrane, while disruption of lipid packing is a destabilizing force in the PMs.

Improving the accuracy and convergence of drug permeation simulations via machine-learned collective variables.

Understanding the permeation of biomolecules through cellular membranes is critical for many biotechnological applications, including targeted drug delivery, pathogen detection, and the development of new antibiotics. To this end, computer simulations are routinely used to probe the underlying mechanisms of membrane permeation. Despite great progress and continued development, permeation simulations of realistic systems (e.g., more complex drug molecules or biologics through heterogeneous membranes) remain extremely challenging if not intractable. In this work, we combine molecular dynamics simulations with transition-tempered metadynamics and techniques from the variational approach to conformational dynamics to study the permeation mechanism of a drug molecule, trimethoprim, through a multicomponent membrane. We show that collective variables (CVs) obtained from an unsupervised machine learning algorithm called time-structure based Independent Component Analysis (tICA) improve performance and substantially accelerate convergence of permeation potential of mean force (PMF) calculations. The addition of cholesterol to the lipid bilayer is shown to increase both the width and height of the free energy barrier due to a condensing effect (lower area per lipid) and increase bilayer thickness. Additionally, the tICA CVs reveal a subtle effect of cholesterol increasing the resistance to permeation in the lipid head group region, which is not observed when canonical CVs are used. We conclude that the use of tICA CVs can enable more efficient PMF calculations with additional insight into the permeation mechanism.

The Surface and Hydration Properties of Lipid Droplets.

Lipid droplets (LDs) are energy storage organelles composed of neutral lipids, such as triacylglycerol (TG) and sterol esters, surrounded by a phospholipid (PL) monolayer. Their central role in metabolism, complex life cycle, and unique lipid monolayer surface have garnered great attention over the last decade. In this article, results from the largest and longest all-atom simulations to date suggest that 5-8% of the LD surface is occupied by TG molecules, a number that exceeds the maximal solubility reported for TGs in PL bilayers (2.8%). Two distinct classes of TG molecules that interact with the LD monolayer are found. Those at the monolayer surface (SURF-TG) are ordered like PLs with the glycerol moiety exposed to water, creating a significant amount of chemically unique packing defects, and the acyl chains extended toward the LD center. In contrast, the TGs that intercalate just into the PL tail region (CORE-TG) are disordered and increase the amount of PL packing defects and the PL tail order. The degree of interdigitation caused by CORE-TG is stable and determines the width of the TG-PL overlap, whereas that caused by SURF-TG fluctuates and is highly correlated with the area per PL or the expansion of the monolayer. Thus, when the supply of PLs is limited, SURF-TG may reduce surface tension by behaving as a secondary membrane component. The hydration properties of the simulated LD systems demonstrate ∼10 times more water in the LD core than previously reported. Collectively, the reported surface and hydration properties are expected to play a direct role in the mechanisms by which proteins target and interact with LDs.

Local conformational dynamics regulating transport properties of a Cl- /H+ antiporter.

ClC-ec1 is a Cl- /H+ antiporter that exchanges Cl- and H+ ions across the membrane. Experiments have demonstrated that several mutations, including I109F, decrease the Cl- and H+ transport rates by an order of magnitude. Using reactive molecular dynamics simulations of explicit proton transport across the central region in the I109F mutant, a two-dimensional free energy profile has been constructed that is consistent with the experimental transport rates. The importance of a phenylalanine gate formed by F109 and F357 and its influence on hydration connectivity through the central proton transport pathway is revealed. This work demonstrates how seemingly subtle changes in local conformational dynamics can dictate hydration changes and thus transport properties. © 2019 Wiley Periodicals, Inc.

Understanding the essential proton-pumping kinetic gates and decoupling mutations in cytochrome c oxidase.

Cytochrome c oxidase (CcO) catalyzes the reduction of oxygen to water and uses the released free energy to pump protons against the transmembrane proton gradient. To better understand the proton-pumping mechanism of the wild-type (WT) CcO, much attention has been given to the mutation of amino acid residues along the proton translocating D-channel that impair, and sometimes decouple, proton pumping from the chemical catalysis. Although their influence has been clearly demonstrated experimentally, the underlying molecular mechanisms of these mutants remain unknown. In this work, we report multiscale reactive molecular dynamics simulations that characterize the free-energy profiles of explicit proton transport through several important D-channel mutants. Our results elucidate the mechanisms by which proton pumping is impaired, thus revealing key kinetic gating features in CcO. In the N139T and N139C mutants, proton back leakage through the D-channel is kinetically favored over proton pumping due to the loss of a kinetic gate in the N139 region. In the N139L mutant, the bulky L139 side chain inhibits timely reprotonation of E286 through the D-channel, which impairs both proton pumping and the chemical reaction. In the S200V/S201V double mutant, the proton affinity of E286 is increased, which slows down both proton pumping and the chemical catalysis. This work thus not only provides insight into the decoupling mechanisms of CcO mutants, but also explains how kinetic gating in the D-channel is imperative to achieving high proton-pumping efficiency in the WT CcO.

Proton movement and coupling in the POT family of peptide transporters.

POT transporters represent an evolutionarily well-conserved family of proton-coupled transport systems in biology. An unusual feature of the family is their ability to couple the transport of chemically diverse ligands to an inwardly directed proton electrochemical gradient. For example, in mammals, fungi, and bacteria they are predominantly peptide transporters, whereas in plants the family has diverged to recognize nitrate, plant defense compounds, and hormones. Although recent structural and biochemical studies have identified conserved sites of proton binding, the mechanism through which transport is coupled to proton movement remains enigmatic. Here we show that different POT transporters operate through distinct proton-coupled mechanisms through changes in the extracellular gate. A high-resolution crystal structure reveals the presence of ordered water molecules within the peptide binding site. Multiscale molecular dynamics simulations confirm proton transport occurs through these waters via Grotthuss shuttling and reveal that proton binding to the extracellular side of the transporter facilitates a reorientation from an inward- to outward-facing state. Together these results demonstrate that within the POT family multiple mechanisms of proton coupling have likely evolved in conjunction with variation of the extracellular gate.

Mycolactone Toxin Membrane Permeation: Atomistic versus Coarse-Grained MARTINI Simulations.

Mycolactone, a cytotoxic and immunosuppressive macrolide produced by Mycobacterium ulcerans, is the central virulent factor in the skin disease Buruli ulcer. This multifunctional cytotoxin affects fundamental cellular processes such as cell adhesion, immune response, and cell death by targeting various cellular structures. Developing effective diagnostics that target mycolactone has been challenging, potentially because of suspected interactions with lipophilic architectures, including membranes. To better understand the pathogenesis of Buruli ulcer disease, aid in the development of diagnostics, and learn how amphiphiles in general use lipid trafficking to navigate the host environment, we seek to understand the nature of mycolactone-membrane interactions. Herein, we characterize how the two dominant isomers of mycolactone (A and B) interact with and permeate DPPC membranes with all-atom molecular dynamics simulations employing transition-tempered metadynamics and compare these results to those obtained by MARTINI coarse-grained simulations. Our all-atom simulations reveal that both isomers have a strong preference to associate with the membrane, although their mechanisms and energetics of membrane permeation differ slightly. Water molecules are found to play an important role in the permeation process. Although the MARTINI coarse-grained simulations give the correct free energy of membrane association, they fail to capture the mechanism of permeation and role of water during permeation as seen in all-atom simulations.

Dynamic Protonation Dramatically Affects the Membrane Permeability of Drug-like Molecules.

Permeability (Pm) across biological membranes is of fundamental importance and a key factor in drug absorption, distribution, and development. Although the majority of drugs will be charged at some point during oral delivery, our understanding of membrane permeation by charged species is limited. The canonical model assumes that only neutral molecules partition into and passively permeate across membranes, but there is mounting evidence that these processes are also facile for certain charged species. However, it is unknown whether such ionizable permeants dynamically neutralize at the membrane surface or permeate in their charged form. To probe protonation-coupled permeation in atomic detail, we herein apply continuous constant-pH molecular dynamics along with free energy sampling to study the permeation of a weak base propranolol (PPL), and evaluate the impact of including dynamic protonation on Pm. The simulations reveal that PPL dynamically neutralizes at the lipid-tail interface, which dramatically influences the permeation free energy landscape and explains why the conventional model overestimates the assigned intrinsic permeability. We demonstrate how fixed-charge-state simulations can account for this effect, and propose a revised model that better describes pH-coupled partitioning and permeation. Our results demonstrate how dynamic changes in protonation state may play a critical role in the permeation of ionizable molecules, including pharmaceuticals and drug-like molecules, thus requiring a revision of the standard picture.

Proton-Induced Conformational and Hydration Dynamics in the Influenza A M2 Channel.

The influenza A M2 protein is an acid-activated proton channel responsible for acidification of the inside of the virus, a critical step in the viral life cycle. This channel has four central histidine residues that form an acid-activated gate, binding protons from the outside until an activated state allows proton transport to the inside. While previous work has focused on proton transport through the channel, the structural and dynamic changes that accompany proton flux and enable activation have yet to be resolved. In this study, extensive Multiscale Reactive Molecular Dynamics simulations with explicit Grotthuss-shuttling hydrated excess protons are used to explore detailed molecular-level interactions that accompany proton transport in the +0, + 1, and +2 histidine charge states. The results demonstrate how the hydrated excess proton strongly influences both the protein and water hydrogen-bonding network throughout the channel, providing further insight into the channel's acid-activation mechanism and rectification behavior. We find that the excess proton dynamically, as a function of location, shifts the protein structure away from its equilibrium distributions uniquely for different pH conditions consistent with acid-activation. The proton distribution in the xy-plane is also shown to be asymmetric about the channel's main axis, which has potentially important implications for the mechanism of proton conduction and future drug design efforts.

Membrane perturbing properties of toxin mycolactone from Mycobacterium ulcerans.

Mycolactone is the exotoxin produced by Mycobacterium ulcerans and is the virulence factor behind the neglected tropical disease Buruli ulcer. The toxin has a broad spectrum of biological effects within the host organism, stemming from its interaction with at least two molecular targets and the inhibition of protein uptake into the endoplasmic reticulum. Although it has been shown that the toxin can passively permeate into host cells, it is clearly lipophilic. Association with lipid carriers would have substantial implications for the toxin's distribution within a host organism, delivery to cellular targets, diagnostic susceptibility, and mechanisms of pathogenicity. Yet the toxin's interactions with, and distribution in, lipids are unknown. Herein we have used coarse-grained molecular dynamics simulations, guided by all-atom simulations, to study the interaction of mycolactone with pure and mixed lipid membranes. Using established techniques, we calculated the toxin's preferential localization, membrane translocation, and impact on membrane physical and dynamical properties. The computed water-octanol partition coefficient indicates that mycolactone prefers to be in an organic phase rather than in an aqueous environment. Our results show that in a solvated membrane environment the exotoxin mainly localizes in the water-membrane interface, with a preference for the glycerol moiety of lipids, consistent with the reported studies that found it in lipid extracts of the cell. The calculated association constant to the model membrane is similar to the reported association constant for Wiskott-Aldrich syndrome protein. Mycolactone is shown to modify the physical properties of membranes, lowering the transition temperature, compressibility modulus, and critical line tension at which pores can be stabilized. It also shows a tendency to behave as a linactant, a molecule that localizes at the boundary between different fluid lipid domains in membranes and promotes inter-mixing of domains. This property has implications for the toxin's cellular access, T-cell immunosuppression, and therapeutic potential.

Multiscale simulations reveal key features of the proton-pumping mechanism in cytochrome c oxidase.

Cytochrome c oxidase (CcO) reduces oxygen to water and uses the released free energy to pump protons across the membrane. We have used multiscale reactive molecular dynamics simulations to explicitly characterize (with free-energy profiles and calculated rates) the internal proton transport events that enable proton pumping during first steps of oxidation of the fully reduced enzyme. Our results show that proton transport from amino acid residue E286 to both the pump loading site (PLS) and to the binuclear center (BNC) are thermodynamically driven by electron transfer from heme a to the BNC, but that the former (i.e., pumping) is kinetically favored whereas the latter (i.e., transfer of the chemical proton) is rate-limiting. The calculated rates agree with experimental measurements. The backflow of the pumped proton from the PLS to E286 and from E286 to the inside of the membrane is prevented by large free-energy barriers for the backflow reactions. Proton transport from E286 to the PLS through the hydrophobic cavity and from D132 to E286 through the D-channel are found to be strongly coupled to dynamical hydration changes in the corresponding pathways and, importantly, vice versa.

Acid activation mechanism of the influenza A M2 proton channel.

The homotetrameric influenza A M2 channel (AM2) is an acid-activated proton channel responsible for the acidification of the influenza virus interior, an important step in the viral lifecycle. Four histidine residues (His37) in the center of the channel act as a pH sensor and proton selectivity filter. Despite intense study, the pH-dependent activation mechanism of the AM2 channel has to date not been completely understood at a molecular level. Herein we have used multiscale computer simulations to characterize (with explicit proton transport free energy profiles and their associated calculated conductances) the activation mechanism of AM2. All proton transfer steps involved in proton diffusion through the channel, including the protonation/deprotonation of His37, are explicitly considered using classical, quantum, and reactive molecular dynamics methods. The asymmetry of the proton transport free energy profile under high-pH conditions qualitatively explains the rectification behavior of AM2 (i.e., why the inward proton flux is allowed when the pH is low in viral exterior and high in viral interior, but outward proton flux is prohibited when the pH gradient is reversed). Also, in agreement with electrophysiological results, our simulations indicate that the C-terminal amphipathic helix does not significantly change the proton conduction mechanism in the AM2 transmembrane domain; the four transmembrane helices flanking the channel lumen alone seem to determine the proton conduction mechanism.

Modulating the Chemical Transport Properties of a Transmembrane Antiporter via Alternative Anion Flux.

ClC-ec1 is a prototype of the ClC antiporters, proteins that stoichiometrically exchange Cl- and H+ ions in opposite directions across a membrane. It has been shown that other polyatomic anions, such as NO3- and SCN-, can also be transported by ClC-ec1, but with partially or completely uncoupled proton flux. Herein, with the help of multiscale computer simulations in which the Grotthuss mechanism of proton transport (PT) is treated explicitly, we demonstrate how the chemical nature of these anions alters the coupling mechanism and qualitatively explain the shifts in the ion stoichiometry. Multidimensional free energy profiles for PT and the coupled changes in hydration are presented for NO3- and SCN-. The calculated proton conductances agree with experiment, showing reduced or abolished proton flux. Surprisingly, the proton affinity of the anion is less influential on the PT, while its size and interactions with the protein significantly alter hydration and shift its influence on PT from facilitating to inhibiting. We find that the hydration of the cavity below the anion is relatively fast, but connecting the water network past the steric hindrance of these polyatomic anions is quite slow. Hence, the most relevant coordinate to the PT free energy barrier is the water connectivity along the PT pathway, but importantly only in the presence of the excess proton, and this coordinate is significantly affected by the nature of the bound anion. This work again demonstrates how PT is intrinsically coupled with protein cavity hydration changes as well as influenced by the protein environment. It additionally suggests ways in which ion exchange can be modulated and exchange stoichiometries altered.

Multiscale Kinetic Modeling Reveals an Ensemble of Cl-/H+ Exchange Pathways in ClC-ec1 Antiporter.

Despite several years of research, the ion exchange mechanisms in chloride/proton antiporters and many other coupled transporters are not yet understood at the molecular level. Here, we present a novel approach to kinetic modeling and apply it to ion exchange in ClC-ec1. Our multiscale kinetic model is developed by (1) calculating the state-to-state rate coefficients with reactive and polarizable molecular dynamics simulations, (2) optimizing these rates in a global kinetic network, and (3) predicting new electrophysiological results. The model shows that the robust Cl:H exchange ratio (2.2:1) can indeed arise from kinetic coupling without large protein conformational changes, indicating a possible facile evolutionary connection to chloride channels. The E148 amino acid residue is shown to couple chloride and proton transport through protonation-dependent blockage of the central anion binding site and an anion-dependent pKa value, which influences proton transport. The results demonstrate how an ensemble of different exchange pathways, as opposed to a single series of transitions, culminates in the macroscopic observables of the antiporter, such as transport rates, chloride/proton stoichiometry, and pH dependence.

Molecular transport through membranes: Accurate permeability coefficients from multidimensional potentials of mean force and local diffusion constants.

Estimating the permeability coefficient of small molecules through lipid bilayer membranes plays an important role in the development of effective drug candidates. In silico simulations can produce acceptable relative permeability coefficients for a series of small molecules; however, the absolute permeability coefficients from simulations are usually off by orders of magnitude. In addition to differences between the lipid bilayers used in vitro and in silico, the poor convergence of permeation free energy profiles and over-simplified diffusion models have contributed to these discrepancies. In this paper, we present a multidimensional inhomogeneous solubility-diffusion model to study the permeability of a small molecule drug (trimethoprim) passing through a POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) lipid bilayer. Our approach improves the permeation model in three ways: First, the free energy profile (potential of mean force, PMF) is two-dimensional in two key coordinates rather than simply one-dimensional along the direction normal to the bilayer. Second, the 2-D PMF calculation has improved convergence due to application of the recently developed transition-tempered metadynamics with randomly initialized replicas, while third, the local diffusivity coefficient was calculated along the direction of the minimum free energy path on the two-dimensional PMF. The permeability is then calculated as a line integral along the minimum free energy path of the PMF. With this approach, we report a considerably more accurate permeability coefficient (only 2-5 times larger than the experimental value). We also compare our approach with the common practice of computing permeability coefficients based only on the translation of the center of mass of the drug molecule. Our paper concludes with a discussion of approaches for minimizing the computational cost for the purpose of more rapidly screening a large number of drug candidate molecules.

Multiscale simulation reveals a multifaceted mechanism of proton permeation through the influenza A M2 proton channel.

The influenza A virus M2 channel (AM2) is crucial in the viral life cycle. Despite many previous experimental and computational studies, the mechanism of the activating process in which proton permeation acidifies the virion to release the viral RNA and core proteins is not well understood. Herein the AM2 proton permeation process has been systematically characterized using multiscale computer simulations, including quantum, classical, and reactive molecular dynamics methods. We report, to our knowledge, the first complete free-energy profiles for proton transport through the entire AM2 transmembrane domain at various pH values, including explicit treatment of excess proton charge delocalization and shuttling through the His37 tetrad. The free-energy profiles reveal that the excess proton must overcome a large free-energy barrier to diffuse to the His37 tetrad, where it is stabilized in a deep minimum reflecting the delocalization of the excess charge among the histidines and the cost of shuttling the proton past them. At lower pH values the His37 tetrad has a larger total charge that increases the channel width, hydration, and solvent dynamics, in agreement with recent 2D-IR spectroscopic studies. The proton transport barrier becomes smaller, despite the increased charge repulsion, due to backbone expansion and the more dynamic pore water molecules. The calculated conductances are in quantitative agreement with recent experimental measurements. In addition, the free-energy profiles and conductances for proton transport in several mutants provide insights for explaining our findings and those of previous experimental mutagenesis studies.

Multiscale Simulations Reveal Key Aspects of the Proton Transport Mechanism in the ClC-ec1 Antiporter.

Multiscale reactive molecular dynamics simulations are used to study proton transport through the central region of ClC-ec1, a widely studied ClC transporter that enables the stoichiometric exchange of 2 Cl(-) ions for 1 proton (H(+)). It has long been known that both Cl(-) and proton transport occur through partially congruent pathways, and that their exchange is strictly coupled. However, the nature of this coupling and the mechanism of antiporting remain topics of debate. Here multiscale simulations have been used to characterize proton transport between E203 (Glu(in)) and E148 (Glu(ex)), the internal and external intermediate proton binding sites, respectively. Free energy profiles are presented, explicitly accounting for the binding of Cl(-) along the central pathway, the dynamically coupled hydration changes of the central region, and conformational changes of Glu(in) and Glu(ex). We find that proton transport between Glu(in) and Glu(ex) is possible in both the presence and absence of Cl(-) in the central binding site, although it is facilitated by the anion presence. These results support the notion that the requisite coupling between Cl(-) and proton transport occurs elsewhere (e.g., during proton uptake or release). In addition, proton transport is explored in the E203K mutant, which maintains proton permeation despite the substitution of a basic residue for Glu(in). This collection of calculations provides for the first time, to our knowledge, a detailed picture of the proton transport mechanism in the central region of ClC-ec1 at a molecular level.

The Origin of Coupled Chloride and Proton Transport in a Cl-/H+ Antiporter.

The ClC family of transmembrane proteins functions throughout nature to control the transport of Cl- ions across biological membranes. ClC-ec1 from Escherichia coli is an antiporter, coupling the transport of Cl- and H+ ions in opposite directions and driven by the concentration gradients of the ions. Despite keen interest in this protein, the molecular mechanism of the Cl-/H+ coupling has not been fully elucidated. Here, we have used multiscale simulation to help identify the essential mechanism of the Cl-/H+ coupling. We find that the highest barrier for proton transport (PT) from the intra- to extracellular solution is attributable to a chemical reaction, the deprotonation of glutamic acid 148 (E148). This barrier is significantly reduced by the binding of Cl- in the "central" site (Cl-cen), which displaces E148 and thereby facilitates its deprotonation. Conversely, in the absence of Cl-cen E148 favors the "down" conformation, which results in a much higher cumulative rotation and deprotonation barrier that effectively blocks PT to the extracellular solution. Thus, the rotation of E148 plays a critical role in defining the Cl-/H+ coupling. As a control, we have also simulated PT in the ClC-ec1 E148A mutant to further understand the role of this residue. Replacement with a non-protonatable residue greatly increases the free energy barrier for PT from E203 to the extracellular solution, explaining the experimental result that PT in E148A is blocked whether or not Cl-cen is present. The results presented here suggest both how a chemical reaction can control the rate of PT and also how it can provide a mechanism for a coupling of the two ion transport processes.