Biology subjects

Dutzler, R.

Publications and source records attributed to Dutzler, R..

11 recordsLinked to original sources

bioRxiv2025

Structural basis for lipid transport at membrane contact sites by the Ist2/Osh6 complex

Membrane contact sites (MCS) are hubs for inter-organellar lipid transport within eukaryotic cells. As one of the principal tethers bridging the ER and the plasma membrane in Saccharomyces cerevisiae, the protein Ist2 plays a major role during lipid transport between both compartments. Here, we present a comprehensive investigation elucidating structural and mechanistic properties of Ist2 and its interaction with the soluble lipid transport protein Osh6. The ER-embedded transmembrane domain of Ist2 is homologous to the TMEM16 family and acts as constitutively active lipid scramblase. The extended C-terminus binds to the plasma membrane and the PS/PI4P exchanger Osh6. Through cellular growth assays, biochemical and structural studies, we have elucidated the interaction between both proteins and provide evidence that Osh6 remains associated with Ist2 during lipid shuttling between membranes. These results highlight the role of the Ist2/Osh6 complex in lipid trafficking and offer new insights into the relevance of scramblases at MCSs.

biophysics↗

bioRxiv2024

Structural features of heteromeric channels composed of CALHM2 and CALHM4 paralogs

The CALHM proteins constitute a family of large pore channels that contains six closely related paralogs in human. Two family members, CALHM1 and 3, have been associated with the release of ATP during taste sensation. Both proteins form heteromeric channels that activate at positive potential and decreased extracellular Ca2+ concentration. Although the structures of several family members displayed large oligomeric organizations of different size, their function has in most cases remained elusive. Our previous study has identified the paralogs CALHM2, 4 and 6 to be highly expressed in the placenta and defined their structural properties as membrane proteins exhibiting features of large pore channels with unknown activation properties (Drozdzyk et al., 2020). Here we investigated whether these placental paralogs would form heteromers and characterized heteromeric complexes consisting of CALHM2 and CALHM4 subunits using specific binders as fiducial markers. Both proteins assemble with different stoichiometries with the largest population containing CALHM2 as predominant component. In these oligomers, the subunits segregate and reside in their preferred conformation found in homomeric channels. Our study has thus revealed the properties that govern the formation of CALHM heteromers in a process of potential relevance in a cellular context.

biophysics↗

bioRxiv2023

Structural and functional properties of the transporter SLC26A6 reveal mechanism of coupled anion exchange

Members of the SLC26 family constitute a conserved class of anion transport proteins, which encompasses uncoupled transporters with channel-like properties, coupled exchangers and motor proteins. Among the ten functional paralogs in humans, several participate in the secretion of bicarbonate in exchange with chloride and thus play an important role in maintaining pH homeostasis. Previously, we have elucidated the structure of murine SLC26A9 and defined its function as an uncoupled chloride transporter (Walter, Sawicka, & Dutzler, 2019). Here we have determined the structure of the closely related human transporter SLC26A6 and characterized it as a coupled exchanger of chloride with bicarbonate and presumably also oxalate. The structure defines an inward-facing conformation of the protein that generally resembles known structures of SLC26A9. The altered anion selectivity between both paralogs is a consequence of a remodeled ion binding site located in the center of a mobile unit of the membrane-inserted domain, which also accounts for differences in the coupling mechanism.

biophysics↗

bioRxiv2022

Structural and functional properties of a plant NRAMP related aluminum transporter

The transport of transition metal ions by members of the SLC11/NRAMP family constitutes a ubiquitous mechanism for the uptake of Fe2+ and Mn2+ across all kingdoms of life. Despite the strong conservation of the family, two of its branches have evolved a distinct substrate preference with one mediating Mg2+ uptake in prokaryotes and another the transport of Al3+ into plant cells. Our previous work on the SLC11 transporter from Eggerthella lenta revealed the basis for its Mg2+ selectivity (Ramanadane et al., 2022). Here we have addressed the structural and functional properties of a putative Al3+ transporter from Setaria italica. We show that the protein transports diverse divalent metal ions and binds the trivalent ions Al3+ and Ga3+, which are both presumable substrates. Its cryo-EM structure displays an occluded conformation that is closer to an inward-than an outward-facing state with a binding site that is remodeled to accommodate the increased charge density of its transported substrate.

biophysics↗

bioRxiv2022

Structures of ferroportin in complex with its specific inhibitor vamifeport

A central regulatory mechanism of iron homeostasis in humans involves ferroportin (FPN), the sole cellular iron exporter, and the peptide hormone hepcidin, which inhibits Fe2+ transport and induces internalization and degradation of FPN. Dysregulation of the FPN/hepcidin axis leads to diverse pathological conditions, and consequently, pharmacological compounds that inhibit FPN-mediated iron transport are of high clinical interest. Here, we describe the cryo-EM structures of human FPN in complex with synthetic nanobodies and vamifeport (VIT-2763), the first clinical-stage oral FPN inhibitor. Vamifeport competes with hepcidin for FPN binding and is currently in clinical development for {beta}-thalassemia and sickle cell disease. The structures display two distinct conformations of FPN, representing outward-facing and an occluded states of the transporter. The vamifeport site is located in the center of the protein, where the overlap with hepcidin interactions underlies the competitive relationship between the two molecules. The introduction of point mutations in the binding pocket of vamifeport reduces its affinity to FPN, emphasizing the relevance of the structural data. Together, our study reveals the conformational rearrangements of FPN on its transport cycle and it provides initial insight into the pharmacological targeting of this unique iron efflux transporter.

biochemistry↗

bioRxiv2022

Structure of a volume-regulated heteromeric LRRC8A/C channel

Volume-regulated anion channels participate in the cellular response to osmotic swelling. These heteromeric membrane proteins consist of LRRC8 family members, where the subunit composition determines permeation properties. Although structures of the obligatory LRRC8A subunit have previously defined the architecture of VRACs, the organization of heteromeric channels has remained elusive. Here we have closed this gap by the structural characterization of channels consisting of LRRC8A and LRRC8C. Like homomeric LRRC8A, these proteins assemble as hexamers. Despite twelve possible arrangements, we find a single predominant organization with an A:C ratio of two. In this assembly, four LRRCA subunits cluster in their preferred conformation observed in homomers as pairs of closely interacting proteins that stabilize a closed state of the channel. In contrast, the two interacting LRRC8C subunits show a larger flexibility, underlining their role in the destabilization of the tightly packed A subunits, thereby enhancing the activation properties of the protein.

biochemistry↗

bioRxiv2021

Structural and functional properties of a magnesium transporter of the SLC11/NRAMP family

Members of the ubiquitous SLC11/NRAMP family catalyze the uptake of divalent transition metal ions into cells. They have evolved to efficiently select these trace elements from a large pool of Ca2+ and Mg2+, which are both orders of magnitude more abundant, and to concentrate them in the cytoplasm aided by the cotransport of H+ serving as energy source. In the present study, we have characterized a member of a distant clade of the family found in prokaryotes, termed NRMTs, that were proposed to function as transporters of Mg2+. The protein transports Mg2+ and Mn2+ but not Ca2+ by a mechanism that is not coupled to H+. Structures determined by cryo-EM and X-ray crystallography revealed a generally similar protein architecture compared to classical NRAMPs, with a restructured ion binding site whose increased volume provides suitable interactions with ions that likely have retained much of their hydration shell.

biophysics↗

bioRxiv2021

Cryo-EM structures of the Caspase activated protein XKR9 involved in apoptotic lipid scrambling

The exposure of the negatively charged lipid phosphatidylserine on the cell-surface, catalyzed by lipid scramblases, is an important signal for the clearance of apoptotic cells by macrophages. The protein XKR9 is a member of a conserved family that has been associated with apoptotic lipid scrambling. Here, we describe structures of full-length and caspase-treated XKR9 in complex with a synthetic nanobody determined by cryo-electron microscopy. The 43 kDa monomeric membrane protein contains eight membrane-spanning helices, two segments that are partly inserted into the lipid bilayer and is organized as two structurally related repeats. In the full-length protein, the C-terminus interacts with a hydrophobic site located at the intracellular side acting as an inhibitor of protein function. Cleavage by caspase-3 at a specific site releases 16 residues of the C-terminus thus making the binding site accessible to the cytoplasm. Collectively, the work has revealed the unknown architecture of the XKR family and has provided initial insight into its activation by caspases.

biophysics↗

bioRxiv2020

Cryo-EM structures and functional properties of CALHM channels of the human placenta

The transport of substances across the placenta is essential for the development of the fetus. Here, we were interested in the role of channels of the calcium homeostasis modulator (CALHM) family in the human placenta. By transcript analysis, we found the paralogs CALHM2, 4, and 6 to be highly expressed in this organ and upregulated during trophoblast differentiation. Based on electrophysiology, we found that activation of these paralogs differs from the voltage- and calcium-gated channel CALHM1. Cryo-EM structures of CALHM4 display decameric and undecameric assemblies with large cylindrical pore, while in CALHM6 a conformational change has converted the pore shape into a conus that narrows at the intracellular side, thus describing distinct functional states of the channel. The pore geometry alters the distribution of lipids, which occupy the cylindrical pore of CALHM4 in a bilayer-like arrangement whereas they have redistributed in the conical pore of CALHM6 with potential functional consequences.

biophysics↗

bioRxiv2018

Cryo-EM structures and functional characterization of the lipid scramblase TMEM16F

The lipid scramblase TMEM16F initiates blood coagulation by catalyzing the exposure of phosphatidylserine in platelets. The protein is part of a family of membrane proteins, which encompasses calcium-activated channels for ions and lipids. Here, we reveal features of TMEM16F that underlie its function as lipid scramblase and ion channel. The cryo-EM structures of TMEM16F in Ca2+-bound and Ca2+-free states display a striking similarity to the scrambling-incompetent anion channel TMEM16A, yet with distinct differences in the catalytic site and in the conformational changes upon activation. In conjunction with functional data, we demonstrate the relationship between ion conduction and lipid scrambling. Although activated by a common mechanism, which likely resembles an equivalent process defined in the homologue nhTMEM16, both functions appear to be mediated by alternate protein conformations, which are at equilibrium in the ligand-bound state.

biochemistry↗

bioRxiv2018

Stepwise activation mechanism of the scramblase nhTMEM16 revealed by cryo-EM

Scramblases catalyze the movement of lipids between both leaflets of a bilayer. Whereas the X-ray structure of the protein nhTMEM16 has previously revealed the architecture of a Ca2+-dependent lipid scramblase, its regulation mechanism has remained elusive. Here, we have used cryo-electron microscopy and functional assays to address this question. Ca2+-bound and Ca2+-free conformations of nhTMEM16 in detergent and lipid nanodiscs illustrate the interactions with its environment and they reveal the conformational changes underlying its activation. In this process, Ca2+-binding induces a stepwise transition of the catalytic subunit cavity, converting a closed cavity that is shielded from the membrane in the absence of ligand, into a polar furrow that becomes accessible to lipid headgroups in the Ca2+-bound state. Additionally, our structures demonstrate how nhTMEM16 distorts the membrane at both entrances of the subunit cavity, thereby decreasing the energy barrier for lipid movement. Impact statementcryo-EM reveals the properties of distinct conformations occupied during activation of the lipid scramblase nhTMEM16 and provides new insights into its interactions with the lipid environment.

biochemistry↗