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114


Substrate recognition and transport mechanism of the PIN-FORMED auxin exporters

Ung, Kien Lam; Schulz, Lukas; Stokes, David L; Hammes, Ulrich Z; Pedersen, Bjørn Panyella
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

Hussein, Adel; Fan, Shujie; Lopez-Redondo, Maria; Kenney, Ian; Zhang, Xihui; Beckstein, Oliver; Stokes, David L
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

Ung, Kien Lam; Winkler, Mikael; Schulz, Lukas; Kolb, Martina; Janacek, Dorina P; Dedic, Emil; Stokes, David L; Hammes, Ulrich Z; Pedersen, Bjørn Panyella
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

Zinc binding alters the conformational dynamics and drives the transport cycle of the cation diffusion facilitator YiiP

Lopez-Redondo, Maria; Fan, Shujie; Koide, Akiko; Koide, Shohei; Beckstein, Oliver; Stokes, David L
YiiP is a secondary transporter that couples Zn2+ transport to the proton motive force. Structural studies of YiiP from prokaryotes and Znt8 from humans have 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 the effects of Zn2+ binding on the conformational transition, we use cryo-EM together with molecular dynamics simulation to compare structures of YiiP from Shewanella oneidensis in the presence and absence of Zn2+. To enable single-particle cryo-EM, we used a phage-display library to develop a Fab antibody fragment with high affinity for YiiP, thus producing a YiiP/Fab complex. To perform MD simulations, we developed a nonbonded dummy model for Zn2+ and validated its performance with known Zn2+-binding proteins. Using these tools, we find that, in the presence of Zn2+, YiiP adopts an inward-facing conformation consistent with that previously seen in tubular crystals. After removal of Zn2+ with high-affinity chelators, YiiP exhibits enhanced flexibility and adopts a novel conformation that appears to be intermediate between inward-facing and outward-facing states. This conformation involves closure of a hydrophobic gate that has been postulated to control access to the primary transport site. Comparison of several independent cryo-EM maps suggests that the transition from the inward-facing state is controlled by occupancy of a secondary 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 govern the transport cycle.
PMCID:8282283
PMID: 34254979
ISSN: 1540-7748
CID: 4950422

Structural basis for potassium transport in prokaryotes by KdpFABC

Sweet, Marie E; Larsen, Casper; Zhang, Xihui; Schlame, Michael; Pedersen, Bjørn P; Stokes, David L
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 cryogenic electron microscopy. 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 KdpB, ultimately reaching a low-affinity site where a water-filled pathway allows release of K+ to the cytoplasm.
PMCID:8307911
PMID: 34272288
ISSN: 1091-6490
CID: 4947632

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

Dubey, Vikas; Stokes, David; Pedersen, Bjørn Panyella; Khandelia, Himanshu
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 remains 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.
PMID: 33951450
ISSN: 1089-8638
CID: 4874062

Serine phosphorylation regulates the P-type potassium pump KdpFABC

Sweet, Marie E; Zhang, Xihui; Erdjument-Bromage, Hediye; Dubey, Vikas; Khandelia, Himanshu; Neubert, Thomas A; Pedersen, Bjørn P; Stokes, David L
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 abolishes the K+-dependence of ATP hydrolysis and blocks the catalytic cycle after formation of the aspartyl phosphate intermediate (E1~P). This regulatory mechanism is unique amongst P-type pumps and this study furthers our understanding of how bacteria control potassium homeostasis to maintain cell volume and osmotic potential.
PMCID:7535926
PMID: 32955430
ISSN: 2050-084x
CID: 4650292

The KdpFABC complex - K+ transport against all odds

Pedersen, Bjørn P; Stokes, David L; Apell, Hans-Jürgen
In bacteria, K+ is used to maintain cell volume and osmotic potential. Homeostasis normally involves a network of constitutively expressed transport systems, but in K+ deficient environments, the KdpFABC complex uses ATP to pump K+ into the cell. This complex appears to be a hybrid of two types of transporters, with KdpA descending from the superfamily of K+ transporters and KdpB belonging to the superfamily of P-type ATPases. Studies of enzymatic activity documented a catalytic cycle with hallmarks of classical P-type ATPases and studies of ion transport indicated that K+ import into the cytosol occurred in the second half of this cycle in conjunction with hydrolysis of an aspartyl phosphate intermediate. Atomic structures of the KdpFABC complex from X-ray crystallography and cryo-EM have recently revealed conformations before and after formation of this aspartyl phosphate that appear to contradict the functional studies. Specifically, structural comparisons with the archetypal P-type ATPase, SERCA, suggest that K+ transport occurs in the first half of the cycle, accompanying formation of the aspartyl phosphate. Further controversy has arisen regarding the path by which K+ crosses the membrane. The X-ray structure supports the conventional view that KdpA provides the conduit, whereas cryo-EM structures suggest that K+ moves from KdpA through a long, intramembrane tunnel to reach canonical ion binding sites in KdpB from which they are released to the cytosol. This review discusses evidence supporting these contradictory models and identifies key experiments needed to resolve discrepancies and produce a unified model for this fascinating mechanistic hybrid.
PMID: 31259644
ISSN: 1464-5203
CID: 3967832

The KdpFABC complex-K+ transport against all odds

Pedersen, B P; Stokes, D L; Apell, H -J
In bacteria, K+ is used to maintain cell volume and osmotic potential. Homeostasis normally involves a network of constitutively expressed transport systems, but in K+ deficient environments, the KdpFABC complex uses ATP to pump K+ into the cell. This complex appears to be a hybrid of two types of transporters, with KdpA descending from the superfamily of K+ transporters and KdpB belonging to the superfamily of P-type ATPases. Studies of enzymatic activity documented a catalytic cycle with hallmarks of classical P-type ATPases and studies of ion transport indicated that K+ import into the cytosol occurred in the second half of this cycle in conjunction with hydrolysis of an aspartyl phosphate intermediate. Atomic structures of the KdpFABC complex from X-ray crystallography and cryo-EM have recently revealed conformations before and after formation of this aspartyl phosphate that appear to contradict the functional studies. Specifically, structural comparisons with the archetypal P-type ATPase, SERCA, suggest that K+ transport occurs in the first half of the cycle, accompanying formation of the aspartyl phosphate. Further controversy has arisen regarding the path by which K+ crosses the membrane. The X-ray structure supports the conventional view that KdpA provides the conduit, whereas cryo-EM structures suggest that K+ moves from KdpA through a long, intramembrane tunnel to reach canonical ion binding sites in KdpB from which they are released to the cytosol. This review discusses evidence supporting these contradictory models and identifies key experiments needed to resolve discrepancies and produce a unified model for this fascinating mechanistic hybrid.
Copyright
EMBASE:2006969908
ISSN: 0968-7688
CID: 4638972

Integrative structure and functional anatomy of a nuclear pore complex

Kim, Seung Joong; Fernandez-Martinez, Javier; Nudelman, Ilona; Shi, Yi; Zhang, Wenzhu; Raveh, Barak; Herricks, Thurston; Slaughter, Brian D; Hogan, Joanna A; Upla, Paula; Chemmama, Ilan E; Pellarin, Riccardo; Echeverria, Ignacia; Shivaraju, Manjunatha; Chaudhury, Azraa S; Wang, Junjie; Williams, Rosemary; Unruh, Jay R; Greenberg, Charles H; Jacobs, Erica Y; Yu, Zhiheng; de la Cruz, M Jason; Mironska, Roxana; Stokes, David L; Aitchison, John D; Jarrold, Martin F; Gerton, Jennifer L; Ludtke, Steven J; Akey, Christopher W; Chait, Brian T; Sali, Andrej; Rout, Michael P
Nuclear pore complexes play central roles as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm. However, their large size and dynamic nature have impeded a full structural and functional elucidation. Here we determined the structure of the entire 552-protein nuclear pore complex of the yeast Saccharomyces cerevisiae at sub-nanometre precision by satisfying a wide range of data relating to the molecular arrangement of its constituents. The nuclear pore complex incorporates sturdy diagonal columns and connector cables attached to these columns, imbuing the structure with strength and flexibility. These cables also tie together all other elements of the nuclear pore complex, including membrane-interacting regions, outer rings and RNA-processing platforms. Inwardly directed anchors create a high density of transport factor-docking Phe-Gly repeats in the central channel, organized into distinct functional units. This integrative structure enables us to rationalize the architecture, transport mechanism and evolutionary origins of the nuclear pore complex.
PMCID:6022767
PMID: 29539637
ISSN: 1476-4687
CID: 2994232