Faculty Bibliography
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112
Proceedings of the National Academy of Sciences of the United States of America (PNAS). 2026:123(2).DOI: 10.1073/pnas.2500723123
Elevator mechanism dynamics in a sodium-coupled dicarboxylate transporter
VcINDY, the sodium-dependent dicarboxylate transporter from
PMID: 41490488
ISSN: 1091-6490
CID: 5980652
On the mechanism of K+ transport through the inter-subunit tunnel of KdpFABC
KdpFABC is an ATP-dependent membrane complex that enables prokaryotes to maintain potassium homeostasis under potassium-limited conditions. It features a unique hybrid mechanism combining a channel-like selectivity filter in KdpA with the ATP-driven transport functionality of KdpB. A key unresolved question is whether K+ ions translocate through the inter-subunit tunnel as a queue of ions or individually within a hydrated environment. Using molecular dynamics simulations, metadynamics, anomalous X-ray scattering, and biochemical assays, we demonstrate that the tunnel is predominantly occupied by water molecules rather than multiple K+ ions. Our results identify only one stable intermediate binding site for K+ within the tunnel, apart from the canonical sites in KdpA and KdpB. Free energy calculations reveal a substantial barrier (∼22 kcal/mol) at the KdpA-KdpB interface, making spontaneous K+ translocation unlikely. Furthermore, mutagenesis and functional assays confirm previous findings that Phe232 at this interface plays a key role in coupling ATP hydrolysis to K+ transport. These findings challenge previous models containing a continuous wire of K+ ions through the tunnel and suggest the existence of an as-yet unidentified intermediate state or mechanistic detail that facilitates K+ movement into KdpB.
PMID: 41384914
ISSN: 1540-7748
CID: 5978042
Conduction pathway for potassium through the Escherichia 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 pseudo fourfold symmetry 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 hypothesized that K+ moves parallel to the membrane plane through a 40 Å 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 Å structure of the E1~P·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.
PMID: 41259220
ISSN: 2050-084x
CID: 5969302
Structures and mechanism of the AUX/LAX transporters involved in auxin import
Auxins are plant hormones that direct the growth and development of organisms on the basis of environmental cues. Indole-3-acetic acid (IAA) is the most abundant auxin in most plants. A variety of membrane transport proteins work together to distribute auxins. These include the AUX/LAX protein family that mediate auxin import from the apoplast to the cytosol. Here we use structural and biophysical approaches combined with molecular dynamics to study transport by Arabidopsis thaliana LAX3, which is essential for plant root formation. Transport assays document high-affinity transport of IAA, as well as competitive behaviour of the synthetic phenoxyacetic acid auxin herbicide 2,4-dichlorophenoxyacetic acid and the auxin transport inhibitors 1-naphthoxyacetic acid and 2-naphthoxyacetic acid. Four cryo-EM structures were solved with resolutions of 2.9-3.4 Å: an inward open apo structure, two inward semi-occluded structures in complex with IAA and 2,4-dichlorophenoxyacetic acid, and a fully occluded structure in complex with 2-naphthoxyacetic acid. Structurally, LAX3 consists of a bundle and a scaffold domain. The ligand-binding site is sandwiched between these domains with two histidines occupying positions analogous to the sodium-binding sites in distantly related sodium:neurotransmitter transporters. This architecture suggests that these histidines couple transport to the proton motive force. Molecular dynamics simulations are used to explore substrate binding and release, including their dependence on specific protonation states. This study advances our understanding of auxin recognition and transport by AUX/LAX, providing insights into a fundamental aspect of plant physiology and development.
PMID: 40759769
ISSN: 2055-0278
CID: 5904872
Transport of phenoxyacetic acid herbicides by PIN-FORMED auxin transporters
Auxins are a group of phytohormones that control plant growth and development. Their crucial role in plant physiology has inspired development of potent synthetic auxins that can be used as herbicides. 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. Here we show that PIN-FORMED auxin transporters transport a range of phenoxyacetic acid herbicides across the membrane. We go on to characterize the molecular determinants of substrate specificity using a variety of different substrates as well as protein mutagenesis to probe the binding site. Finally, we present cryogenic electron microscopy structures of Arabidopsis thaliana PIN8 bound to either 2,4-dichlorophenoxyacetic acid or 4-chlorophenoxyacetic acid. These structures represent five key states from the transport cycle, allowing us to describe conformational changes associated with the transport cycle. 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 provide a foundation for future development of novel synthetic auxins and for precision breeding of herbicide-resistant crop plants.
PMID: 40263580
ISSN: 2055-0278
CID: 5830192
Numerical model for electrogenic transport by the ATP-dependent potassium pump KdpFABC
In vitro assays of ion transport are an essential tool for understanding molecular mechanisms associated with ATP-dependent pumps. Because ion transport is generally electrogenic, principles of electrophysiology are applicable, but conventional tools like patch-clamp are ineffective due to relatively low turnover rates of the pumps. Instead, assays have been developed to measure either voltage or current generated by transport activity of a population of molecules either in cell-derived membrane fragments or after reconstituting purified protein into proteoliposomes. In order to understand the nuances of these assays and to characterize effects of various operational parameters, we have developed a numerical model to simulate data produced by two relevant assays: fluorescence from voltage-sensitive dyes and current recorded by capacitive coupling on solid supported membranes. Parameters of the model, which has been implemented in Python, are described along with underlying principles of the computational algorithm. Experimental data from KdpFABC, a K+ pump associated with P-type ATPases, are presented, and model parameters have been adjusted to mimic these data. In addition, effects of key parameters such as nonselective leak conductance and turnover rate are demonstrated. Finally, simulated data are used to illustrate the effects of capacitive coupling on measured current and to compare alternative methods for quantification of raw data.
PMCID:11304011
PMID: 38950825
ISSN: 2667-0747
CID: 5689652
Proceedings of the National Academy of Sciences of the United States of America (PNAS). 2024:121(15).DOI: 10.1073/pnas.2315575121
Conformational changes in the Niemann-Pick type C1 protein NCR1 drive sterol translocation
The membrane protein Niemann-Pick type C1 (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 supporting the presence of a glycocalyx in isolated S. cerevisiae vacuoles and report four cryo-EM structures of NCR1 in two distinct conformations, named tense and relaxed. These two conformations illustrate the movement of sterols through a tunnel formed by the luminal domains, thus bypassing the barrier presented by the glycocalyx. 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 proton motive force drives conformational changes in the transmembrane domains that are allosterically coupled to luminal/extracellular domains to promote sterol transport.
PMCID:11009665
PMID: 38568972
ISSN: 1091-6490
CID: 5726232
Substrate recognition and transport mechanism of the PIN-FORMED auxin exporters
Auxins are pivotal plant hormones that regulate plant growth and transmembrane polar auxin transport (PAT) direct patterns of development. The PIN-FORMED (PIN) family of membrane transporters mediate auxin export from the plant cell and play crucial roles in PAT. Here we describe the recently solved structures of PIN transporters, PIN1, PIN3, and PIN8, and also their mechanisms of substrate recognition and transport of auxin. We compare structures of PINs in both inward- and outward-facing conformations, as well as PINs with different binding configurations for auxin. By this comparative analysis, a model emerges for an elevator transport mechanism. Central structural elements necessary for function are identified, and we show that these are shared with other distantly related protein families.
PMCID:10592131
PMID: 37574372
ISSN: 0968-0004
CID: 5619142
Energy coupling and stoichiometry of Zn2+/H+ antiport by the prokaryotic cation diffusion facilitator YiiP
YiiP from Shewanella oneidensis 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+.
PMID: 37906094
ISSN: 2050-084x
CID: 5634882
Structures and mechanism of the plant PIN-FORMED auxin transporter
Auxins are hormones that have central roles and control nearly all aspects of growth and development in plants1-3. The proteins in the PIN-FORMED (PIN) family (also known as the auxin efflux carrier family) are key participants in this process and control auxin export from the cytosol to the extracellular space4-9. Owing to a lack of structural and biochemical data, the molecular mechanism of PIN-mediated auxin transport is not understood. Here we present biophysical analysis together with three structures of Arabidopsis thaliana PIN8: two outward-facing conformations with and without auxin, and one inward-facing conformation bound to the herbicide naphthylphthalamic acid. The structure forms a homodimer, with each monomer divided into a transport and scaffold domain with a clearly defined auxin binding site. Next to the binding site, a proline-proline crossover is a pivot point for structural changes associated with transport, which we show to be independent of proton and ion gradients and probably driven by the negative charge of the auxin. The structures and biochemical data reveal an elevator-type transport mechanism reminiscent of bile acid/sodium symporters, bicarbonate/sodium symporters and sodium/proton antiporters. Our results provide a comprehensive molecular model for auxin recognition and transport by PINs, link and expand on a well-known conceptual framework for transport, and explain a central mechanism of polar auxin transport, a core feature of plant physiology, growth and development.
PMID: 35768502
ISSN: 1476-4687
CID: 5281202
