Transient receptor potential (TRP) melastatin 4 (TRPM4) is a widely expressed cation channel associated with a variety of cardiovascular disorders. TRPM4 is activated by increased intracellular calcium in a voltage dependent manner, but unlike many other TRP channels is permeable to monovalent cations only. Here we present two structures of full-length human TRPM4 embedded in lipid nanodiscs at ~3Å resolution as determined by single particle electron cryo-microscopy. These structures, with and without calcium bound, reveal a general architecture for this major subfamily of TRP channels and a well-defined calcium binding site within the intracellular side of the S1–S4 domain. The structures correspond to two distinct closed states. Calcium binding induces conformational changes that likely prime the channel for voltage-dependent opening.

Zinc is an essential micronutrient for all living organisms, required for signaling and proper function of a range of proteins involved in e.g. DNA-binding and enzymatic catalysis 1 . In prokaryotes and photosynthetic eukaryotes Zn 2+ -transporting P-type ATPases of class IB (ZntA) are crucial for cellular redistribution and detoxification of Zn 2+ and related elements 2,3 . Here we present crystal structures representing the phosphoenzyme ground state (E2P) and a dephosphorylation intermediate (E2.P i ) of ZntA from Shigella sonnei, determined at 3.2 and 2.7 Å resolution, respectively. The structures reveal a similar fold as the Cu + -ATPases with an amphipathic helix at the membrane interface. A conserved electronegative funnel connects this region to the intramembranous high-affinity ion-binding site and may promote specific uptake of cellular Zn 2+ ions. The E2P structure displays a wide extracellular release pathway reaching the invariant residues at the high-affinity site, including Cys392, Cys394 and Asp714. The pathway closes in the E2.P i state where Asp714 interacts with the conserved Lys693, which possibly stimulates Zn 2+ release as a built-in counter-ion, as also proposed for H + -ATPases. Indeed, transport studies in liposomes provide experimental support for ZntA activity without counter-

Monoamine transporters are responsible for termination of synaptic signaling and are involved in depression, control of appetite, and anxiety amongst other neurological processes. Despite extensive efforts, the structures of the monoamine transporters and the transport mechanism of ions and substrates are still largely unknown. Structural knowledge of the human serotonin transporter (hSERT) is much awaited for understanding the mechanistic details of substrate translocation and binding of antidepressants and drugs of abuse. The publication of the crystal structure of the homologous leucine transporter has resulted in homology models of the monoamine transporters. Here we present extended molecular dynamics simulations of an experimentally supported homology model of hSERT with and without the natural substrate yielding a total of more than 1.5 µs of simulation of the protein dimer. The simulations reveal a transition of hSERT from an outward-facing occluded conformation to an inward-facing conformation in a one-substrate-bound state. Simulations with a second substrate in the proposed symport effector site did not lead to conformational changes associated with translocation. The central substrate binding site becomes fully exposed to the cytoplasm leaving both the Na+-ion in the Na2-site and the substrate in direct contact with the cytoplasm through water interactions. The simulations reveal how sodium is released and show indications of early events of substrate transport. The notion that ion dissociation from the Na2-site drives translocation is supported by experimental studies of a Na2-site mutant. Transmembrane helices (TMs) 1 and 6 are identified as the helices involved in the largest movements during transport.

A highly alternating copolymer composed of acrylic acid and styrene (AASTY) is synthesized with controlled radical polymerization by exploiting the reactivity ratios of the monomers to control the monomer sequence. The AASTY copolymers are effective solubilizers of cellular membranes and their embedded proteins, which improves structural characterization by single-particle cryo-electron microscopy (cryo-EM). These copolymers are promising tools for exploring detergent-free membrane protein solubilization and direct formation of native nanodiscs, facilitating structural and functional analysis of the mammalian proteome.

The ability of styrene maleic acid copolymers to dissolve lipid membranes into nanosized lipid particles is a facile method of obtaining membrane proteins in solubilized lipid discs while conserving part of their native lipid environment. While the currently used copolymers can readily extract membrane proteins in native nanodiscs, their highly disperse composition is likely to influence the dispersity of the discs as well as the extraction efficiency. In this study, reversible addition-fragmentation chain transfer was used to control the polymer architecture and dispersity of molecular weights with a high-precision. Based on Monte Carlo simulations of the polymerizations, the monomer composition was predicted and allowed a structure-function analysis of the polymer architecture, in relation to their ability to assemble into lipid nanoparticles. We show that a higher degree of control of the polymer architecture generates more homogeneous samples. We hypothesize that low dispersity copolymers, with control of polymer architecture are an ideal framework for the rational design of polymers for customized isolation and characterization of integral membrane proteins in native lipid bilayer systems.

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The competitive inhibitor cocaine and the non-competitive inhibitor ibogaine induce different conformational states of the human serotonin transporter. It has been shown from accessibility experiments that cocaine mainly induces an outward-facing conformation, while the non-competitive inhibitor ibogaine, and its active metabolite noribogaine, have been proposed to induce an inward-facing conformation of the human serotonin transporter similar to what has been observed for the endogenous substrate, serotonin. The ligand induced conformational changes within the human serotonin transporter caused by these three different types of ligands, substrate, non-competitive and competitive inhibitors, are studied from multiple atomistic molecular dynamics simulations initiated from a homology model of the human serotonin transporter. The results reveal that diverse conformations of the human serotonin transporter are captured from the molecular dynamics simulations depending on the type of the ligand bound. The inward-facing conformation of the human serotonin transporter is reached with noribogaine bound, and this state resembles a previously identified inward-facing conformation of the human serotonin transporter obtained from molecular dynamics simulation with bound substrate, but also a recently published inward-facing conformation of a bacterial homolog, the leucine transporter from Aquifex Aoelicus. The differences observed in ligand induced behavior are found to originate from different interaction patterns between the ligands and the protein. Such atomic-level understanding of how an inhibitor can dictate the conformational response of a transporter by ligand binding may be of great importance for future drug design.

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