Biology subjects

Stokes, D. L.

Publications and source records attributed to Stokes, D. L..

10 recordsLinked to original sources

bioRxiv2026

Lipids are essential for potassium transport by KdpFABC from E. coli

KdpFABC is a hetero-tetrameric potassium pump that uses ATP to import potassium and thereby maintain homeostasis in bacteria under stress conditions. KdpA is a channel-like subunit with a selectivity filter that binds potassium from the periplasm. K+ then moves through a [~]40[A]-long intramembrane tunnel to reach a canonical binding site in KdpB. KdpB is a P-type ATPase that orchestrates conformational changes associated with the Post-Albers reaction cycle, involving E1 and E2 conformations and formation of an aspartyl phosphate intermediate as a way of coupling ATP hydrolysis to K+ transport. To elucidate the associated structural changes in a lipid environment, we reconstituted wild-type KdpFABC into lipid nanodiscs and used cryo-EM to image the complex under active turnover. The resulting six high resolution (2.1-2.7 [A]) structures provide new insight into the sequence of allosteric changes that produce (1) occlusion of K+ at the canonical binding site and (2) expulsion of K+ from this site and into a low-affinity release site. The structures also reveal two types of lipids bound to the complex. Specifically, two structural lipids bind at subunit interfaces and [~]20 annular lipids are seen at the periphery of the complex. In addition, we tested functional effects of mutations to residues at the KdpA/KdpB interface. ATPase and transport assays were used to document functional defects that reflect delipidation of structurally compromised complexes. We conclude that lipids play an integral role in structure and function of the KdpFABC complex. SignificanceKdpFABC uses ATP to transport potassium across the plasma membrane of E. coli. To further our understanding of its mechanism, we put purified KdpFABC molecules into membrane bilayers and used cryo-EM to capture structures during active transport. We have thereby produced structures representing all major states of the transport cycle with a high degree of precision. Analysis of these structures reveals new details about two key steps in this cycle and shows lipid molecules bound to the protein. We then introduced mutations at the interface between the two main subunits, which controls passage of potassium across the membrane. Activity measurements reveal how the protein depends on lipid to stabilize the structure and facilitate transport.

biophysics↗

bioRxiv2025

Conduction pathway for potassium through the E. coli pump KdpFABC

Under osmotic stress, bacteria express a heterotetrameric protein complex, KdpFABC, which functions as an ATP-dependent K+ pump to maintain intracellular potassium levels. The subunit KdpA belongs to the Superfamily of K+ Transporters and adopts a pseudo-tetrameric architecture with a membrane embedded selectivity filter as seen in K+ channels. KdpB belongs to the superfamily of P-type ATPases with a conserved binding site for ions within the membrane domain and three cytoplasmic domains that orchestrate ATP hydrolysis via an aspartyl phosphate intermediate. Previous work has hypothesized that K+ moves parallel to the membrane plane through a 40-[A] long tunnel that connects the selectivity filter of KdpA with a canonical binding site in KdpB. In the current work, we have reconstituted KdpFABC into lipid nanodiscs and used cryo-EM to image the wild-type pump under turnover conditions. We present a 2.1 [A] structure of the E1[~]P{middle dot}ADP conformation, which reveals new features of the conduction pathway. This map shows strong densities within the selectivity filter and at the canonical binding site, consistent with K+ bound at each of these sites in this conformation. Many water molecules occupy a vestibule and the proximal end of the tunnel, which becomes markedly hydrophobic and dewetted at the subunit interface. We go on to use ATPase and ion transport assays to assess effects of numerous mutations along this proposed conduction pathway. The results confirm that K+ ions pass through the tunnel and support the existence of a low affinity site in KdpB for releasing these ions to the cytoplasm. Taken together, these data shed new light on the unique partnership between a transmembrane channel and an ATP-driven pump in maintaining the large electrochemical K+ gradient essential for bacterial survival.

biophysics↗

bioRxiv2024

Transport of herbicides by PIN-FORMED auxin transporters

Auxins are a group of phytohormones that control plant growth and development 1. Their crucial role in plant physiology has inspired development of potent synthetic auxins that can be used as herbicides 2. Phenoxyacetic acid derivatives are a widely used group of auxin herbicides in agriculture and research. Despite their prevalence, the identity of the transporters required for distribution of these herbicides in plants is both poorly understood and the subject of controversial debate 3,4. Here we show that PIN-FORMED auxin transporters transport a range of phenoxyacetic acid herbicides across the membrane and we characterize the molecular determinants of this process using a variety of different substrates as well as protein mutagenesis to control substrate specificity. Finally, we present Cryo-EM structures of Arabidopsis thaliana PIN8 with 2,4-dichlorophenoxyacetic acid (2,4-D) or 4-chlorophenoxyacetic acid (4-CPA) bound. These structures represent five key states from the transport cycle, allowing us to describe conformational changes associated with substrate binding and transport across the membrane. Overall, our results reveal that phenoxyacetic acid herbicides use the same export machinery as endogenous auxins and exemplify how transporter binding sites undergo transformations that dictate substrate specificity. These results enable development of novel synthetic auxins and for guiding precision breeding of herbicide resistant crop plants.

biochemistry↗

bioRxiv2023

Conformational changes in the Niemann-Pick Type C1 protein NCR1 drive sterol translocation

The membrane protein Niemann-Pick Type C1 protein (NPC1, named NCR1 in yeast) is central to sterol homeostasis in eukaryotes. Saccharomyces cerevisiae NCR1 is localized to the vacuolar membrane, where it is suggested to carry sterols across the protective glycocalyx and deposit them into the vacuolar membrane. However, documentation of a vacuolar glycocalyx in fungi is lacking and the mechanism for sterol translocation has remained unclear. Here we provide evidence that a glycocalyx is indeed present inside isolated Saccharomyces cerevisiae vacuoles, and report four cryo-EM structures of NCR1 in two distinct conformations that elucidate how it moves sterol through the glycocalyx. The two conformations, named "tense" and "relaxed", illustrate movement of sterol through a tunnel formed by the luminal domains. Based on these structures and on comparison with other members of the Resistance-Nodulation-Division (RND) superfamily we propose a transport model that links changes in the luminal domains with a cycle of protonation and deprotonation within the transmembrane region of the protein. Our model suggests that NPC proteins work by a generalized RND mechanism where the transmembrane domains form a motor-unit that sequentially adopts a tense and relaxed conformation to drive changes in luminal/extracellular domains. SIGNIFICANCE STATEMENTNiemann-Pick Type C1 (NPC1, named NCR1 in yeast) proteins play a critical role in sterol homeostasis by facilitating the integration of sterols into membranes of acidic organelles like lysosomes and vacuoles. The inner surface of these organelles membranes is shielded by the glycocalyx. Here, we provide evidence that a glycocalyx is present in vacuoles from Saccharomyces cerevisiae and demonstrate that NCR1 transports sterols across it by undergoing conformational changes. Our structures suggest a transport model where sterol transport is linked to proton-driven changes in the transmembrane region. This work sheds light on the mechanism of NPC1 protein function and has broad implications for understanding lysosomal storage disorders and for mechanisms employed by members of the Resistance-Nodulation-Division (RND) superfamily.

biochemistry↗

bioRxiv2023

Energy Coupling and Stoichiometry of Zn2+/H+ Antiport by the Cation Diffusion Facilitator YiiP

YiiP is a prokaryotic Zn2+/H+ antiporter that serves as a model for the Cation Diffusion Facilitator (CDF) superfamily, members of which are generally responsible for homeostasis of transition metal ions. Previous studies of YiiP as well as related CDF transporters have established a homodimeric architecture and the presence of three distinct Zn2+ binding sites named A, B, and C. In this study, we use cryo-EM, microscale thermophoresis and molecular dynamics simulations to address the structural and functional roles of individual sites as well as the interplay between Zn2+ binding and protonation. Structural studies indicate that site C in the cytoplasmic domain is primarily responsible for stabilizing the dimer and that site B at the cytoplasmic membrane surface controls the structural transition from an inward facing conformation to an occluded conformation. Binding data show that intramembrane site A, which is directly responsible for transport, has a dramatic pH dependence consistent with coupling to the proton motive force. A comprehensive thermodynamic model encompassing Zn2+ binding and protonation states of individual residues indicates a transport stoichiometry of 1 Zn2+ to 2-3 H+ depending on the external pH. This stoichiometry would be favorable in a physiological context, allowing the cell to use the proton gradient as well as the membrane potential to drive the export of Zn2+.

biophysics↗

bioRxiv2022

Elevator mechanism dynamics in a sodium-coupled dicarboxylate transporter

VcINDY, the sodium-dependent dicarboxylate transporter from Vibrio cholerae, is responsible for C4- and C5-carboxylate uptake into cells. The molecular mechanism of how VcINDY physically moves substrates across the membrane, and does so in an energetically efficient manner, is unclear. Here, we use single-molecule fluorescence resonance energy transfer experiments to directly observe the individual mechanistic steps that VcINDY takes to translocate substrates across a lipid bilayer, and then test key predictions of transport cycle mechanistic models. Our data provide the first direct evidence that VcINDY undergoes stochastic, elevator-type conformational motions that enable substrate translocation. Kinetic analysis suggests that the two protomers of the VcINDY homodimer undergo those motions in a non-cooperative manner, and thus catalyze two independent transport reactions. The relative substrate independence of those motions supports the notion that the VcINDY transport cycle maintains strict co-substrate coupling using a cooperative binding mechanism. Finally, thermodynamic modeling provides insight into how such a cooperative binding mechanism provides a generalized approach to optimizing transport for many secondary active transporters. Significance StatementTransporter proteins use energy to move molecular materials into and out of cells. To be efficient, the transporter motions responsible for moving the molecules must be tightly choreographed to avoid wasting energy without transporting anything. By measuring the motions and kinetics of a prototypical transporter (VcINDY) at the single-molecule level, this study finds the first evidence that transporters like VcINDY achieve efficient transport by coordinating constantly dynamic, "elevator-type" motions while sitting in the cellular membrane. The efficiency of these surprisingly dynamic transporters is then revealed by thermodynamic modeling, which explains the molecular basis behind how highly cooperative, substrate binding reactions may have evolved as the optimal strategy for maximizing transporter efficiency.

biophysics↗

bioRxiv2021

An intracellular pathway controlled by the N-terminus of the pump subunit inhibits the bacterial KdpFABC ion pump in high K+ conditions

The heterotetrameric bacterial KdpFABC transmembrane protein complex is an ion channel-pump hybrid that consumes ATP to import K+ against its transmembrane chemical potential gradient in low external K+ environments. The KdpB ion-pump subunit of KdpFABC is a P-type ATPase, and catalyses ATP hydrolysis. Under high external K+ conditions, K+ can diffuse into the cells through passive ion channels. KdpFABC must therefore be inhibited in high K+ conditions to conserve cellular ATP. Inhibition is thought to occur via unusual phosphorylation of residue Ser162 of the TGES motif of the cytoplasmic A domain. It is proposed that phosphorylation most likely traps KdpB in an inactive E1-P like conformation, but the molecular mechanism of phosphorylation-mediated inhibition and the allosteric links between phosphorylation on the A domain and the inactivation of the pump remain unknown. Here, we employ molecular dynamics (MD) simulations of the dephosphorylated and phosphorylated versions of KdpFABC to demonstrate that phosphorylated KdpB is trapped in a conformation where the ion-binding site is hydrated by an intracellular pathway between transmembrane helices M1 and M2 which opens in response to the rearrangement of cytoplasmic domains resulting from phosphorylation. Cytoplasmic access of water to the ion-binding site is accompanied by a remarkable loss of secondary structure of the KdpB N-terminus and disruption of a key salt bridge between Glu87 in the A domain and Arg212 in the P domain. Our results provide the molecular basis of a unique mechanism of regulation amongst P-type ATPases, and suggest that the N-terminus has a significant role to play in the conformational cycle and regulation of KdpFABC

biophysics↗

bioRxiv2021

Structural basis for Potassium transport by KdpFABC

KdpFABC is an oligomeric K+ transport complex in prokaryotes that maintains ionic homeostasis under stress conditions. The complex comprises a channel-like subunit (KdpA) from the Superfamily of K+ Transporters and a pump-like subunit (KdpB) from the superfamily of P-type ATPases. Recent structural work has defined the architecture and generated contradictory hypotheses for the transport mechanism. Here, we use substrate analogs to stabilize four key intermediates in the reaction cycle and determine the corresponding structures by cryo-EM. We find that KdpB undergoes conformational changes consistent with other representatives from the P-type superfamily, whereas KdpA, KdpC and KdpF remain static. We observe a series of spherical densities that we assign as K+ or water and which define a pathway for K+ transport. This pathway runs through an intramembrane tunnel in KdpA and delivers ions to sites in the membrane domain of KdpB. Our structures suggest a mechanism where ATP hydrolysis is coupled to K+ transfer between alternative sites in the membrane domain of KdpB, ultimately reaching a low-affinity site where a water-filled pathway allows release of K+ to the cytoplasm.

biophysics↗

bioRxiv2020

Zinc Dependent Conformational Changes in the Cation Diffusion Facilitator YiiP from S. oneidensis

YiiP is a secondary transporter that couples Zn2+ transport to the proton motive force. Structural studies of YiiP from prokaryotes as well as Znt8 from humans revealed three different Zn2+ sites and a conserved homodimeric architecture. These structures define the inward-facing and outward-facing states that characterize the archetypal alternating access mechanism of transport. To study effects of Zn2+ binding on the conformational transition, we have used a YiiP/Fab complex for single-particle cryo-EM together with Molecular Dynamics simulation to compare structures of YiiP from S. oneidensis in presence and absence of Zn2+. Without Zn2+, YiiP exhibits enhanced flexibility and adopts a novel conformation that appears to be an intermediate state. The transition closes a hydrophobic gate and is controlled by the Zn2+ site at the cytoplasmic membrane interface. This work enhances our understanding of individual Zn2+ binding sites and their role in the conformational dynamics that governs the transport cycle.

biophysics↗

bioRxiv2020

Serine Phosphorylation Regulates the P-type Potassium pump KdpFABC

KdpFABC is an ATP-dependent K+ pump that ensures bacterial survival in K+-deficient environments. Whereas transcriptional activation of kdpFABC expression is well studied, a mechanism for down regulation when K+ levels are restored has not been described. Here we show that KdpFABC is inhibited when cells return to a K+-rich environment. The mechanism of inhibition involves phosphorylation of Ser162 on KdpB, which can be reversed in vitro by treatment with serine phosphatase. Mutating Ser162 to Alanine produces constitutive activity, whereas the phosphomimetic Ser162Asp mutation inactivates the pump. Analyses of the transport cycle show that serine phosphorylation uncouples the pump and blocks the cycle after formation of the catalytic aspartyl phosphate intermediate (E1[~]P). Molecular dynamics simulations show that serine phosphorylation has allosteric effects that potentially explain the uncoupling. This regulatory mechanism, unique amongst P-type pumps, furthers our understanding of how bacteria control potassium homeostasis to maintain cell volume and osmotic potential.

biophysics↗