Biology subjects

Stix, R.

Publications and source records attributed to Stix, R..

6 recordsLinked to original sources

bioRxiv2025

The Role of Chloride Ions in Serotonin Transport

The human serotonin (5-HT+) transporter SERT facilitates 5-HT+ transport into cells by coupling it to Na+ symport and K+ antiport. Although extracellular Cl- is also essential for transport, Cl-co-transport has been disputed, raising questions about the role of Cl- ions and why they are required. We show that Cl- gradients do not impact 5-HT+ accumulation, indicating that Cl- does not provide a driving force for uptake and arguing against stoichiometric Cl- symport. The presence of Cl- had only a small effect on Na+-mediated cytoplasmic pathway closure but markedly reduced the accessibility of residues in the extracellular pathway, consistent with modulation of the outward-facing states. Simulations illustrate that Cl- interacts strongly with a bound Na+ ion and stabilizes helix packing on the extracellular side. We propose that Cl- acts as an essential architectural cofactor by enhancing Na+ affinity and interactions between helices, thereby facilitating transport-related conformational transitions. Significance statementThe serotonin transporter (SERT) clears serotonin from synapses and is the target of widely used antidepressants. Although chloride ions are known to be required for SERT function, whether chloride is transported together with serotonin has remained unresolved for decades. We show that chloride gradients do not drive serotonin uptake, arguing against chloride symport. Instead, chloride stabilizes specific conformations of the transporter by strengthening sodium binding and extracellular helix packing. These findings redefine chloride not as a transported substrate, but as an architectural cofactor that facilitates conformational transitions required for transport. This reinterpretation reshapes our understanding of ion dependence in SERT and suggests a broader principle: ion requirements in related transporters may reflect structural stabilization rather than obligatory symport.

biochemistry↗

bioRxiv2024

Distinct mechanisms of inhibition of Kv2 potassium channels by tetraethylammonium and RY785

Voltage-gated K+ channels play central roles in human physiology, both in health and disease. A repertoire of inhibitors that are both potent and specific would therefore be of great value, not only as pharmacological agents but also as research tools. The small molecule RY785 has been described as particularly promising in this regard, as it selectively inhibits channels in the Kv2 subfamily with high potency. Kv2 channels are expressed in multiple cell types in humans, and are of particular importance for neuronal function. The mechanism of action of RY785 has not yet been determined at the molecular level, but functional studies indicate it differs from that of less specific inhibitors, such as quaternary-ammonium compounds or aminopyridines; RY785 is distinct also in that it is electroneutral. To examine this mechanism at the single-molecule level, we have carried out a series of all-atom molecular dynamics simulations based on the experimental structure of the Kv2.1 channel in the activated, open state. First, we report a 25-microsecond trajectory calculated in the absence of any inhibitor, under an applied voltage of 100 mV, which demonstrates outward K+ flow under simulation conditions at rates comparable to experimental measurements. Additional simulations in which either RY785 or tetraethylammonium (TEA) is introduced in solution and allowed to interact with the pore domain show both inhibitors spontaneously enter the channel interior through the cytoplasmic gate, but with distinct effects. In agreement with prior structural studies, we observe that TEA binds to a site adjacent to the selectivity filter, on the pore axis, thereby blocking the flow of K+ ions. RY785, by contrast, binds to the channel walls, off-axis, and allows K+ flow while the cytoplasmic gate remains open. The observed mode of RY785 binding, however, indicates that its mechanism of action is to stabilize and occlude a semi-open state of the gate, by bridging hydrophobic protein-protein interactions therein; this hypothesis would explain the puzzling experimental observation that RY785 recognition influences the gating currents generated by the voltage sensors, 3 nm away.

biochemistry↗

bioRxiv2021

Structure of the Shaker Kv channel and mechanism of slow C-type inactivation

Voltage-activated potassium (Kv) channels open upon membrane depolarization and proceed to spontaneously inactivate. Inactivation controls neuronal firing rates and serves as a form of short-term memory, and is implicated in various human neurological disorders. Here, we use high-resolution cryo-electron microscopy and computer simulations to determine one of the molecular mechanisms underlying this physiologically crucial process. Structures of the activated Shaker Kv channel and of its W434F mutant in lipid bilayers demonstrate that C-type inactivation entails the dilation of the ion selectivity filter, and the repositioning of neighboring residues known to be functionally critical. Microsecond-scale molecular dynamics trajectories confirm these changes inhibit rapid ion permeation through the channel. This long-sought breakthrough establishes how eukaryotic K+ channels self-regulate their functional state through the plasticity of their selectivity filters. One-Sentence SummaryStructures of the Shaker Kv channel reveal the mechanism of slow C-type inactivation involves dilation of the selectivity filter.

biophysics↗

bioRxiv2021

Gaussian accelerated molecular dynamics reveals that a proline-rich signaling peptide frequently samples cis conformations when unbound

Disordered proline-rich motifs are common across the proteomes of many species and are often involved in protein-protein interactions. Proline is a unique amino acid due to the covalent bond between the backbone nitrogen and the proline side chain. The resulting five-membered ring allows proline to sample the cis state about its peptide bond, which other residues cannot do as readily. Because proline-rich disordered sequences exist as ensembles that likely include structures with the proline peptide bond in cis, a robust methodology to accurately account for these conformations in the overall ensemble is crucial. Observing the cis conformations of proline in a disordered sequence is challenging both experimentally and computationally. Nitrogen-hydrogen NMR spectroscopy cannot directly observe proline residues, which lack an amide bond, and computational methods struggle to overcome the large kinetic barrier between the cis and trans states, since isomerization usually occurs on the order of seconds. In the current work, Gaussian accelerated molecular dynamics was used to overcome this free energy barrier and simulate proline isomerization in a tetrapeptide (KPTP) and in the 12-residue proline-rich SH3 binding peptide, ArkA. We found that Gaussian accelerated molecular dynamics, when combined with a lowered peptide bond dihedral angle potential energy barrier (15 kcal/mol), allowed sufficient sampling of the proline cis and trans states on a microsecond timescale. All ArkA prolines spend a significant fraction of time in cis, leading to a more compact ensemble with less polyproline II helix structure than an ArkA ensemble with all peptide bonds in trans. The ensemble containing cis prolines also matches more closely to in vitro circular dichroism data than the all-trans ensemble. The ability of the ArkA prolines to isomerize likely affects the peptides ability to bind its partner SH3 domain, and should be studied further. This is the first molecular dynamics simulation study of proline isomerization in a biologically relevant proline-rich sequence that we know of, and a similar protocol could be applied to study multi-proline isomerization in other proline-containing proteins to improve conformational diversity and agreement with in vitro data.

biophysics↗

bioRxiv2020

A disordered encounter complex is central to the yeast Abp1p SH3 domain binding pathway

Protein-protein interactions are involved in a wide range of cellular processes. These interactions often involve intrinsically disordered proteins (IDPs) and protein binding domains. However, the details of IDP binding pathways are hard to characterize using experimental approaches, which can rarely capture intermediate states present at low populations. SH3 domains are common protein interaction domains that typically bind proline-rich disordered segments and are involved in cell signaling, regulation, and assembly. We hypothesized, given the flexibility of SH3 binding peptides, that their binding pathways include multiple steps important for function. Molecular dynamics simulations were used to characterize the steps of binding between the yeast Abp1p SH3 domain (AbpSH3) and a proline-rich IDP, ArkA. Before binding, the N-terminal segment 1 of ArkA is pre-structured and adopts a polyproline II helix, while segment 2 of ArkA (C-terminal) adopts a 310 helix, but is far less structured than segment 1. As segment 2 interacts with AbpSH3, it becomes more structured, but retains flexibility even in the fully engaged state. Binding simulations reveal that ArkA enters a flexible encounter complex before forming the fully engaged bound complex. In the encounter complex, transient nonspecific hydrophobic and long-range electrostatic contacts form between ArkA and the binding surface of SH3. The encounter complex ensemble includes conformations with segment 1 in both the forward and reverse orientation, suggesting that segment 2 may play a role in stabilizing the correct binding orientation. While the encounter complex forms quickly, the slow step of binding is the transition from the disordered encounter ensemble to the fully engaged state. In this transition, ArkA makes specific contacts with AbpSH3 and buries more hydrophobic surface. Simulating the binding between ApbSH3 and ArkA provides insight into the role of encounter complex intermediates and nonnative hydrophobic interactions for other SH3 domains and IDPs in general. Author SummaryComplex cellular processes are mediated by interactions between proteins, and to determine how these interactions affect cellular function and binding kinetics we often must understand the protein binding pathway. Many protein interaction domains, such as the SH3 domain, bind to intrinsically disordered proteins in a coupled folding and binding process. Using molecular dynamics simulations, we find that the binding of the disordered ArkA peptide to the yeast Abp1p SH3 domain proceeds through a flexible, disordered encounter complex before reaching a stable fully bound state. The encounter complex is stabilized by nonspecific long-range electrostatic interactions and nonspecific hydrophobic interactions between the peptide and domain. Our simulations highlight the important role of hydrophobic interactions in the entire SH3 binding process: both nonspecific hydrophobic contacts in the encounter complex and specific hydrophobic contacts in the fully bound complex. The encounter complex could be key to understanding the functional behavior of SH3 domain interactions because the encounter complex forms very quickly and the transition to the fully bound state is slower. In cells, an SH3 domain may form an encounter complex quickly and nonspecifically with many potential binding partners, allowing it to search for the correct recognition sequence before completing the binding process.

biophysics↗

bioRxiv2019

DHHC20 palmitoyl-transferase reshapes the membrane to foster catalysis

Cysteine palmitoylation, a form of S-acylation, is a key post-translational modification in cellular signaling. This type of reversible lipidation is catalyzed by a family of integral membrane proteins known as DHHC acyltransferases. The first step in the S-acylation process is the recognition of free acyl-CoA from the lipid bilayer. The DHHC enzyme then becomes auto-acylated, at a site defined by a conserved Asp-His-His-Cys motif. This reaction entails ionization of the catalytic Cys. Intriguingly, in known DHHC structures this catalytic Cys appears to be exposed to the hydrophobic interior of the lipid membrane, which would be highly unfavorable for a negatively charged nucleophile, thus hindering auto-acylation. Here, we use biochemical and computational methods to reconcile these seemingly contradicting facts. First, we experimentally demonstrate that human DHHC20 is active when reconstituted in POPC nanodiscs. Microsecond-long all-atom molecular dynamics simulations are then calculated for hDHHC20 and for different acyl-CoA forms, also in POPC. Strikingly, we observe that hDHHC20 induces a drastic deformation in the membrane, particularly on the cytoplasmic side where auto-acylation occurs. As a result, the catalytic Cys becomes hydrated and optimally positioned to encounter the cleavage site in acyl-CoA. In summary, we hypothesize that DHHC enzymes locally reshape the membrane to foster a morphology that is specifically adapted for acyl-CoA recognition and auto-acylation.\n\nSignificance StatementPalmitoylation, the most common form of S-acylation and the only reversible type of protein lipidation, is ubiquitous in eukaryotic cells. In humans, for example, it has been estimated that as much as [~]10% of the proteome becomes palmitoylated, i.e. thousands of proteins. Accordingly, protein palmitoylation touches every important aspect of human physiology, both in health and disease. Despite its biological and biomedical importance, little is known about the molecular mechanism of the enzymes that catalyze this post-translational modification, known as DHHC acyltransferases. Here, we leverage the recently-determined atomic-resolution structure of human DHHC20 to gain novel insights into the mechanism of this class of enzymes, using both experimental and computational approaches.

biophysics↗