Membrane co-transport proteins that utilize a 5-helix inverted repeat motif have recently emerged as one of the largest structural class of secondary active transporters1,2. However, despite many structural advances there is no clear evidence as to how ion and substrate transport are coupled. Here, we report a comprehensive study of the Sodium-Galactose Transporter from Vibrio parahaemolyticus (vSGLT) consisting of molecular dynamics simulations, biochemical characterization, and a new crystal structure of the inward-open conformation at 2.7 Å resolution. Our data show that sodium exit causes a reorientation of transmembrane helix 1 (TM1) opening an inner gate required for substrate exit, while also triggering minor rigid body movements in two sets of transmembrane helical bundles. This cascade of events, initiated by sodium release, ensures proper timing of ion and substrate release. Once set in motion, these molecular changes weaken substrate binding to the transporter and allow galactose to readily enter the intracellular space. Additionally, we identify an allosteric pathway between the sodium binding sites, the unwound portion of TM1, and the substrate binding site that is essential in the coupling of co-transport.

Continuum electrostatic approaches have been extremely successful at describing the charged nature of soluble proteins and how they interact with binding partners. However, it is unclear whether continuum methods can be used to quantitatively understand the energetics of membrane protein insertion and stability. Recent translation experiments suggest that the energy required to insert charged peptides into membranes is much smaller than predicted by present continuum theories. Atomistic simulations have pointed to bilayer inhomogeneity and membrane deformation around buried charged groups as two critical features that are neglected in simpler models. Here, we develop a fully continuum method that circumvents both of these shortcomings by using elasticity theory to determine the shape of the deformed membrane and then subsequently uses this shape to carry out continuum electrostatics calculations. Our method does an excellent job of quantitatively matching results from detailed molecular dynamics simulations at a tiny fraction of the computational cost. We expect that this method will be ideal for studying large membrane protein complexes.on May 9, 2018 jgp.rupress.org Downloaded from

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The elasticity of alpha-helices is examined using equilibrium molecular-dynamics simulations. From the statistics of curvatures and twists, we compute the elastic moduli of several representative alpha-helices, both in the presence and absence of aqueous solvent. We discover that the bending modulus (persistence length) of the helices is independent of the amino-acid sequence, although helices in water are slightly softer than in vacuum. The response of the helices under the action of an external force is also computed and compared with continuum mechanics predictions. Within the time scale of our simulation, we show that the properties of alpha-helices are well reproduced by an elastic and isotropic rod. The persistence length (bending modulus) of most alpha-helices in water or vacuum is approximately 100 nm, roughly twice that of DNA.

It is well accepted that cotransporters facilitate water movement by two independent mechanisms: osmotic flow through a water channel in the protein and flow driven by ion/substrate cotransport. However, the molecular mechanism of transport-linked water flow is controversial. Some researchers believe that it occurs via cotransport, in which water is pumped along with the transported cargo, while others believe that flow is osmotic in response to an increase in intracellular osmolarity. In this letter, we report the results of a 200-ns molecular dynamics simulation of the sodium-dependent galactose cotransporter vSGLT. Our simulation shows that a significant number of water molecules cross the protein through the sugar-binding site in the presence as well as the absence of galactose, and 70-80 water molecules accompany galactose as it moves from the binding site into the intracellular space. During this event, the majority of water molecules in the pathway are unable to diffuse around the galactose, resulting in water in the inner half of the transporter being pushed into the intracellular space and replaced by extracellular water. Thus, our simulation supports the notion that cotransporters act as both passive water channels and active water pumps with the transported substrate acting as a piston to rectify the motion of water.

Secondary active transporters, such as those that adopt the leucinetransporter 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.T ransport of sugar molecules across membranes is virtually ubiquitous in biology, and it is of central importance in human health. The uptake of glucose is especially crucial due to its pivotal role in cellular metabolism and energy production. In mammals, glucose is absorbed in the small intestine and kidney via sodium-dependent glucose transporters (SGLTs), which localize to the apical membrane and concentrate glucose in the epithelia. SGLTs fall into the large leucine-transporter (LeuT) structural family of secondary active transporters that have evolved to concentrate a wide array of substrates across membranes using the energy stored in the Na + electrochemical potential gradient. For symporters in this family, transport occurs by an alternating access mechanism (1) in which the transporter first binds ligands in an outward-facing conformation, and then transitions to an inwardfacing conformation that releases the cargo to the cytoplasm. The order of ion and substrate binding and unbinding is likely tied to the function of the transporter, making it possible to convert the energy stored in the ion gradient into a substrate gradient, and vice versa when these proteins operate in reverse.Extensive biochemical uptake assays and electrophysiological studies of SGLTs have led to a view of Na + /glucose cotransport in which Na + binding precedes sugar binding on the external face of the transporter. Kinetic models adhering to this mechanism satisfactorily account fo...

Hydrophilic drugs are often poorly absorbed when administered orally. There has been considerable interest in the possibility of using absorption enhancers to promote absorption of polar molecules across membrane surfaces. The bile acids are one of the most widely investigated classes of absorption enhancers, but there is disagreement about what features of bile acid enhancers are responsible for their efficacy. We have designed a class of glycosylated bile acid derivatives to evaluate how increasing the hydrophilicity of the steroid nucleus affects the ability to transport polar molecules across membranes. Some of the glycosylated molecules are significantly more effective than taurocholate in promoting the intestinal absorption of a range of drugs, showing that hydrophobicity is not a critical parameter in transport efficacy, as previously suggested. Furthermore, the most effective glycosylated compound is also far less damaging to membranes than the best bile acid absorption promoters, presumably because it is more hydrophilic. The results reported here show that it is possible to decouple absorption-promoting activity from membrane damage, a finding that should spark interest in the design of new compounds to facilitate the delivery of polar drugs.

This article reports an all-atom molecular dynamics simulation to study a model pulmonary surfactant film interacting with a carbonaceous nanoparticle. The pulmonary surfactant is modeled as a dipalmitoylphosphatidylcholine monolayer with a peptide consisting of the first 25 residues from surfactant protein B. The nanoparticle model with a chemical formula C188H53 was generated using a computational code for combustion conditions. The nanoparticle has a carbon cage structure reminiscent of the buckyballs with open ends. A series of molecular-scale structural and dynamical properties of the surfactant film in the absence and presence of nanoparticle are analyzed, including radial distribution functions, mean-square displacements of lipids and nanoparticle, chain tilt angle, and the surfactant protein B peptide helix tilt angle. The results show that the nanoparticle affects the structure and packing of the lipids and peptide in the film, and it appears that the nanoparticle and peptide repel each other. The ability of the nanoparticle to translocate the surfactant film is one of the most important predictions of this study. The potential of mean force for dragging the particle through the film provides such information. The reported potential of mean force suggests that the nanoparticle can easily penetrate the monolayer but further translocation to the water phase is energetically prohibitive. The implication is that nanoparticles can interact with the lung surfactant, as supported by recent experimental data by Bakshi et al.