Protein-Protein Interaction between Sodium-Coupled Monocarboxylate Transporter 1 (SMCT1) and PDZ Domain-Containing Ring Finger Domain 3 (PDZRN3)

Sunday, February 12, 2017 cytosolic EIIB protein binds to EIIC and transfers a phosphate group to the incoming sugar that prevents the sugar from escaping the cell and at the same time primes the sugar for entering metabolic cycles. Little is known concerning how EIICs recognize and transport carbohydrates or how an EIIC coordinates with EIIB to achieve phosphate transfer. Crystal structures of a maltose transporter bcMalT[1] and a N-diacetylchitobiose transporter bcChbC[2] were solved recently, and the two structures appear to be in different states of a transport cycle: bcMalT in an outward facing state and bcChbC in an inward facing state. The bcChbC structure provides a template to build a model of bcMalT in an inward facing state, and vise versa. To examine the models, we designed pairs of cysteine residues that are distant in the crystal structures but are predicted to move close to each other in the alternate conformation. Several pairs of cysteines in in both bcMalT and bcChbC can be crosslinked by micromolar concentrations of mercury, indicating that these residues can move close to each other. We then solved the structure of the T280C/E54C bcMalT double cysteine ˚ resolution, and the structure is mutant in the crosslinked state to 3.6 A indeed in an inward-facing conformation. The new structure illustrates the large-scale movement of a structurally conserved domain in bcMalT, and shows that EIIC employs an elevator-like mechanism for substrate translocation. Further analyses suggest how the inward facing conformation could interact with an EIIB protein to achieve phosphate transfer. The structures also provide a solid starting point for investigating the dynamics of the EIIC protein using spectroscopic approaches. Reference [1]. Mccoy JG, Ren Z, Stanevich V, et al. The Structure of a Sugar Transporter of the Glucose EIIC Superfamily Provides Insight into the Elevator Mechanism of Membrane Transport. Structure. 2016;24(6):956-64. [2]. Cao Y, Jin X, Levin EJ, et al. Crystal structure of a phosphorylationcoupled saccharide transporter. Nature. 2011;473(7345):50-4. 637-Pos Board B402 Substrate-Induced Conformational Change in LeuT Yuan-Wei Zhang1, Lucy R. Forrest2, Gary Rudnick1. 1 Pharmacology, Yale University, New Haven, CT, USA, 2NINDS, NIH, Bethesda, MD, USA. LeuT is a prokaryotic amino acid transporter that has been used extensively as a model for neurotransmitter transport. We recently demonstrated that the conformational change induced by Naþ ions requires the Na2 site observed in LeuT crystal structures. We observed this conformational change using LeuT in E. coli membranes, in the absence of detergent, by a decrease in reactivity of a single cysteine (Y265C) in the cytoplasmic permeation pathway. We now show that this effect of Naþ is observed whether Kþ or NMDGþ is used as a control ion. In the presence of Naþ, addition of alanine, a substrate, induces the reverse conformational change, opening the cytoplasmic pathway and increasing Cys-265 reactivity. Three mutations in the substrate binding site each altered the affinity of LeuT for leucine and alanine, but did not interfere with the conformational change induced by Naþ. One of the mutations also blocked the substratedependent conformational change. The results suggest a mechanism by which substrate interactions with the central binding site of LeuT reverse the ability of Naþ to stabilize outward-facing conformations of this model transporter. 638-Pos Board B403 Membrane Remodeling by GltPh in the Inward- and Outward-Facing Conformations Explains Lack of Protomer Cooperativity Wenchang Zhou1, Claudio Anselmi1, Horacio Poblete1, Ali Karimi2, Lucy Forrest2, Jose Faraldo-Gomez1. 1 National Heart, Lung and Blood Institute, NIH, Bethesda, MD, USA, 2 National Institute of Neurological Disorders and Stroke, Bethesda, MD, USA. Membrane-embedded proteins can induce the remodeling of the adjacent lipid bilayers by promoting curvature, altering the membrane thickness and/or exposing hydrophobic groups. When these proteins undergo a function-related conformational transition, the energy cost associated with these membrane perturbations adds up to the free energy of each of the protein states and can therefore modulate their functional mechanisms. Here, we study the case of the Naþ-coupled aspartate transporter from Pyrococcus horikoshii (GltPh). GltPh has been crystallized as a trimer both in an inward- and outward-facing conformations. In this outward-toinward conformational exchange, the so-called transport domain ˚ across the membrane, relative to the seemingly rigid trimerimoves ~20 A zation/scaffold domains, so as to expose the substrate and Naþ-binding sites to either the cytoplasm or the extracellular space. Using large-scale coarse-

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grained and all-atom molecular dynamics (MD) simulations, we characterized the membrane deformation induced by GltPh trimers in all possible permutations of inward-facing and outward-facing protomer states. The simulations show that when a protomer is in the outward-facing state, the surrounding membrane is largely unperturbed. However, in the inward-facing state, the transport domain induces a strong deformation on the lipid bilayer, ˚ along the direction perpendicular to the bending its average plane by ~10 A ˚ , but remarkmembrane plane. This perturbation extends radially for ~50 A ably, it is largely localized around each of the transport domains in the vicinity of the protein, i.e. the membrane shape is restored near the protomer-protomer interfaces. The specific protein-lipid contacts that explain this local deformation are identified from all-atom MD simulations. In summary, MD simulations demonstrate that the lipid membrane favors the outward-facing state of GltPh over the inward-facing state, owing to the long-range curvature deformation induced by the latter. However, these simulations also show that this large deformation does not imply a membrane-mediated protomer cross-talk, explaining the mystifying absence of measurable cooperativity among protomers in this trimeric transporter. 639-Pos Board B404 Protein-Protein Interaction between Sodium-Coupled Monocarboxylate Transporter 1 (SMCT1) and PDZ Domain-Containing Ring Finger Domain 3 (PDZRN3) Yusuke Otsuka. Pharmacology, Chiba University Graduate School of Medicine, Chiba-shi, Japan. Sodium-coupled monocarboxylate transporters (SMCTs) are membrane proteins which transport lactate on the apical side of kidney. In addition to renal lactate reabsorption, it is known that both SMCTs and urate/anion transporter1 (URAT1) bind to PDZK1 and that SMCTs and URAT1 functionally cooperate to transport urate. This indicates that these transporters reabsorb urate by exchange for sodium. Our recent study revealed that SMCT1 (SLC5A8), a higher-affinity transporter than SMCT2 (SLC5A12), bound to PDZ domain-containing RING finger domain 3 (PDZRN3) in a yeast two hybrid screening. But there is less information available on the interaction between SMCT1 and PDZRN3. In this study, we elucidated this protein-protein interaction between them to resolve the regulation mechanism of serum urate level. We performed coimmunoprecipitation study to confirm the binding between SMCT1 and PDZRN3, and performed [3H] nicotinate uptake study to reveal whether there was a functional change by this binding, using HEK293 cells transiently transfected with SMCT1, its mutant lacking the PDZ motif and PDZRN3. Coimmunoprecipitation study revealed that the wild type SMCT1, but not its mutant lacking the SMCT1 C-terminal PDZ motif, directly bound to PDZRN3. In uptake study, there was no significant difference in amount of nicotinate uptake via SMCT1 and its mutant lacking the PDZ motif, in the presence of PDZRN3 or not. These results showed the protein-protein bind between SMCT1 and PDZRN3, but no functional interaction. This suggests that PDZRN3 regulates urate reabsorption via URAT1 by preventing SMCT1 from binding to PDZK1. 640-Pos Board B405 It Runs in the Family: Determining the Transport Mechanism of Sodium/ Dicarboxylate Symporter hNaDC3 Alissa J. Becerril1, Cristina Fenollar-Ferrer2, Lucy R. Forrest2, Joseph A. Mindell1. 1 Membrane Transport Biophysics Unit, NINDS, NIH, Bethesda, MD, USA, 2 Computational Structural Biology Unit, NINDS, NIH, Bethesda, MD, USA. Members of the divalent anion:Na(þ) symporter (DASS) family play important roles in mammalian physiology, transporting divalent anions, including Krebs cycle intermediates and sulfate, across the plasma membrane. These transporters may be key contributors to determining urinary citrate levels, which, in turn, may affect kidney stone formation; they also have been implicated in metabolic regulation in both drosophila and mammals. It is therefore important to understand the relationships in these proteins between their structure and functional mechanisms. Though no structures are yet available for mammalian DASS family members, a crystal structure of a bacterial homolog, vcINDY, has been determined. We recently demonstrated that vcINDY, a Naþ-coupled succinate transporter, utilizes a dramatic ‘‘elevator’’ mechanism to transport substrate, involving a large-scale vertical movement of a protein domain perpendicular to the plane of the lipid bilayer membrane. Here, we sought to determine whether a mammalian family member utilizes a similar transport