Tag 1 | Beckstein Lab

Cryo-electron microscopy has become a powerful new structural technique to obtain atomic resolution structures of membrane proteins. By combining the experimental structures with molecular dynamics simulations of the protein in a realistic environment including a membrane and solvent (water and ions), new insights regarding the protein function can be obtained that are not available from a single technique alone.

NHE9 is a sodium/proton exchanger in mammals, part of a large family of secondary active transporters. Our collaborators (David Drew at the University of Stockholm) solved the structure of NHE9 (SLC9A9) from horse with cryo electron-microscopy to 3.2 Å resolution in an inward facing conformation, the first structure of a mammalian sodium/proton antiporter. Functional measurements, structural bioinformatics, and MD simulations all indicate that many components of the transport mechanism are similar between prokaryotic and mammalian sodium/proton antiporters.

Dr. Fiona Naughton won the Art of Science contest at the Meeting of the Biophysical Society Meeting in San Diego with her illustration of her current research project.

Transport of ions and small molecules across the cell membrane against electrochemical gradients is catalyzed by integral membrane proteins that use a source of free energy to drive the energetically uphill flux of the transported substrate. Here we review general principles that apply to most secondary active transporters, namely energy coupling through kinetic cycles and the thermodynamic driving forces, inverted repeat symmetry as a means to generate bi-stable systems that can function according to the alternating access model, and understanding transporters as gated pores.

Fiona was a postdoc in the lab. Her research interests include free energy calculations, enhanced sampling methods and software development for MD. Her current project aims to use computational methods to characterise the structural and energetic underpinnings of bile acid transporters.

Bor1p is a secondary transporter in yeast that is responsible for boron transport, likely powered by the proton gradient across the cell membrane. By combining cryo-electron microscopy and MD simulations we could generate models of Bor1p in inward facing and outward facing conformation, which suggest rigid body movements of the core domain relative to the gate domain, consistent with a rocking-bundle transport mechanism.

The APS 4 Corners Section Meeting is held annually and will be hosted at Colorado State University on October 20th and 21st. Oliver was invited to present the group’s research on Structure and function of electrogenic sodium/proton antiporter membrane proteins.

Undergraduate student Taylor Colburn was awarded the 2017 Tsong Prize for Undergraduate Research in the Department of Physics at Arizona State University for his work on Simulating the conformational transitions of the transmembrane symporter Mhp1.

We studied the process of sodium/proton antiport in the NapA transporter. Through a combination of X-ray crystallography, biochemistry and computer simulations we could show that the antiporter undergoes a large conformational transition that resembles a *elevator*-like movement whereby a single domain moves up- and down through the membrane and carries a sodium ion with it.

David Dotson was awarded a 2015 Education Committee Travel Award to present his research on Recent Structures and molecular dynamics simulations offer new perspective on Na⁺/H⁺ antiporters at the 59th Annual Meeting of the Biophysical Society in Baltimore, MD.

A new crystal structure of the Escherichia coli NhaA dimer reveals a previously unidentified salt bridge between two highly conserved residues at the putative binding site. The combination of structural data with molecular dynamics simulations yields new insights into the transport mechanism.

The hydantoin transporter Mhp1 is a sodium?coupled secondary active transport protein of the nucleobase?cation?symport family and shares the widespread 5?helix inverted repeat transporter architecture. Our previous work showed Mhp1 functions according to the alternating access mechanism. In our new paper in EMBO J , we elucidate detailed events of substrate binding, through a combination of crystallography, molecular dynamics, site?directed mutagenesis, biochemical/biophysical assays, and the design and synthesis of novel ligands.

We show that one of the best-studied secondary active transporters, the lactose permease LacY, goes through an occluded conformation during its transport cycle. We propose an atomically detailed model of the apo-occluded state. The simulations predict the formation of a transient salt bridge that has been hypothesized in the canonical model for transport of LacY. The simulations are validated by comparison to experimental EPR DEER data, using a new approach to simulate spin-label distance distributions through post-processing of molecular dynamics trajectories. We also define a set of order parameters that consistently classify all known MFS transporter structures as outward-open, occluded, or inward-open conformations.

We are reviewing the evidence for direct effects of lipids on the transport properties of ion channels and active transporters. For ion channels it has been convincingly shown that specific lipid-protein interactions can directly affect their function. For transporters, the evidence is more ambiguous. In all areas, however, the use of computer simulations extends the way in which we understand protein-membrane interactions.

In a combined X-ray crystallography/biochemistry/molecular simulation study published in Nature we present the structure of the sodium/proton antiporter NapA in its outward facing conformation. Together with the inward facing conformation of the related transporter NhaA we can now understand the conformational changes required for the sodium/proton antiport mechanism.

Structures of membrane proteins can be obtained by the experimental technique of X-ray crystallography. However, proteins are typically not crystallized in their native environment, the lipid membrane. Molecular dynamics simulations of the protein in the membrane provide a realistic model of the interactions between transporter and lipid bilayer.

Long Liang majored in Biology in his undergraduate study, and shifted to Physics for his PhD study. He hopes to combine his background in Biology and Physics to understand life in terms of the more fundamental Physical laws. In his rotation project he worked on constructing a validated model of a human neurotransmitter transporter. Long is now part of the Complex Materials Group and works on his PhD under Professor Yang Jiao.

David worked as a research software engineer in the lab. He left in June 2017 for industry.

Using molecular dynamics (MD) computer simulations we can study membrane proteins in atomic detail, down to the movements of individual water molecules. For example, the Mhp1 transporter protein shown here switches between three different functional states: A: outward facing; B: occluded; C: inward facing — as predicted byt the alternating access model.

Secondary transporters couple the free energy stored in an ionic gradient to the movement of solutes across the cell membrane. The coupling enables these transmembrane proteins to transport small molecules against their own concentration gradients. The transporters function by cycling between different conformational states in which access to the central binding site is switched from the extracellular solution to the intracellular compartment. Using experimental and computational approaches we could visualize for the first time how this process occurs for a secondary transporter.

Transporter proteins are used by cells to “pump” molecules or ions into or out of the cell. They are present in all cells (digestive system, nervous system, blood, …) with important roles in metabolism. Secondary active transporters move their substrates against a electrochemical potential gradient and therefore couple uptake/excretion to an energetically favourable flow of sodium ions or protons into the cell.

Many proteins in the living cell can be understood as molecular machines that use a source of energy to produce mechanical or chemical work. My lab’s primary interest is in those proteins located in the cell membrane that move nutrients, signalling molecules, or waste products into and out of the cell. We study their molecular mechanisms of action by detailed molecular dynamics simulations, which provide a “movie” of full atomic detail of a working protein.