Cells are enclosed by lipidic membranes that isolate them from each other and from their environment. Membranes exert the cellular sequestration due to their impermeability to most water-soluble molecules and ions. The ingress and egress of numerous molecules and ions to cells are governed by channels and transport proteins that are embedded in the membrane. As such, transporters and channels have a tremendous importance in the normal physiology of all living organisms. Moreover, 60% of all drugs are thought to act on membrane proteins and certain drugs have been shown to enter or leave cells through specific transporters. Our objectives are to gain a structural-based understanding of the dynamics of key human transporters, relate these to functional aspects of the transporters and harness these insights into the development of novel therapeutic approaches.
A Deeper Understanding
In order to shuttle their cargo across membranes, transporters shift between multiple conformations in a thermodynamic reversible manner. In simplistic terms, in order for a transporter to shuttle its cargo from the extracellular milieu into the cell, it samples a conformation in which the ligand binding site is situated at a cavity that is open to the extracellular space. Ligand binding causes motions in the transporter that occlude the ligand and finally, the ligand releases into the cell by motions that expose the ligand binding site to the cytoplasm. After a successful transport event, the transporter would isomerize once more to an extracellular-open state and the transport cycle could repeat over and over. A number of factors such as substrate and driving ion concentrations, and the membrane potential have been shown to shift the equilibrium between the different conformers a transporter adopts. We strive to characterize the most sampled conformations in the transport process, generate structural snapshots of these, and identify the principal pathways by which transporters isomerize by utilizing spectroscopic, structural and functional experimental systems.
Transporter mediated drug targeting
A number of transporters exhibit tissue specific distributions (e.g. Organic Anion-Transporting Polypeptide 1B1 and 1B3 in the liver), and in some tumors certain transporter genes are upregulated. Structural studies of these transporters in complex with known ligands could provide atomic-resolution understanding of the interactions between transporters and ligands, leading to a better definition of motifs that target the ligands to these transporters. In parallel, transport assays would provide affinity measurements and aid in refining the identified motifs. In turn, these data could lead to the design of novel tissue specific and anti-cancer drugs that enter or bind the desired target through these transporters.
Substrate-bound outward-open structure of a Na+-coupled sialic acid symporter reveals a new Na+ site. Wahlgren WY, Dunevall E, North RA, Paz A, Scalise M, Bisignano P, Bengtsson-Palme J, Goyal P, Claesson E, Caing-Carlsson R, Andersson R, Beis K, Nilsson UJ, Farewell A, Pochini L, Indiveri C, Grabe M, Dobson RCJ, Abramson J, Ramaswamy S, Friemann R. Nat Commun. 2018, PMID:29717135.
Conformational transitions of the sodium-dependent sugar transporter, vSGLT. Paz A, Claxton DP, Kumar JP, Kazmier K, Bisignano P, Sharma S, Nolte SA, Liwag TM, Nayak V, Wright EM, Grabe M, Mchaourab HS, Abramson J. Proc Natl Acad Sci USA. 2018, PMID: 29507231.
Active site voltage clamp fluorometry of the sodium glucose cotransporter hSGLT1. Gorraitz E, Hirayama BA, Paz A, Wright EM, Loo DDF. Proc Natl Acad Sci USA. 2017, PMID: 29087341.
A large Rab GTPase encoded by CRACR2A is a component of subsynaptic vesicles that transmit T cell activation signals. Srikanth S, Kim KD, Gao Y, Woo JS, Ghosh S, Calmettes G, Paz A, Abramson J, Jiang M, Gwack Y. Sci Signal. 2016, PMID: 27016526.
Identification of a second substrate-binding site in solute-sodium symporters. Li Z, Lee AS, Bracher S, Jung H, Paz A, Kumar JP, Abramson J, Quick M, Shi L. J Biol Chem. 2015, PMID: 25398883.
Structure-guided simulations illuminate the mechanism of ATP transport through VDAC1. Choudhary OP, Paz A, Adelman JL, Colletier JP, Abramson J, Grabe M. Nat Struct Mol Biol. 2014, PMID: 24908397.
High resolution structure and double electron-electron resonance of the zebrafish voltage-dependent anion channel 2 reveal an oligomeric population. Schredelseker J, Paz A, López CJ, Altenbach C, Leung CS, Drexler MK, Chen JN, Hubbell WL, Abramson J. J Biol Chem. 2014, PMID: 24627492.
The specific interaction of the photosensitizer methylene blue with acetylcholinesterase provides a model system for studying the molecular consequences of photodynamic therapy. Silman I, Roth E, Paz A, Triquigneaux MM, Ehrenshaft M, Xu Y, Shnyrov VL, Sussman JL, Deterding LJ, Ashani Y, Mason RP, Weiner L. Chem Biol Interact. 2013, PMID: 23159732.
Structure of flagellar motor proteins in complex allows for insights into motor structure and switching. Vartanian AS, Paz A, Fortgang EA, Abramson J, Dahlquist FW. J Biol Chem. 2012, PMID: 22896702.
Structural and functional characterization of the interaction of the photosensitizing probe methylene blue with Torpedo californica acetylcholinesterase. Paz A, Roth E, Ashani Y, Xu Y, Shnyrov VL, Sussman JL, Silman I, Weiner L. Protein Sci. 2012, PMID: 22674800.
Flexibility of the flap in the active site of BACE1 as revealed by crystal structures and molecular dynamics simulations. Xu Y, Li MJ, Greenblatt H, Chen W, Paz A, Dym O, Peleg Y, Chen T, Shen X, He J, Jiang H, Silman I, Sussman JL. Acta Crystallogr D Biol Crystallogr. 2012, PMID: 22194329.
Backbone and side chain NMR assignments for the intrinsically disordered cytoplasmic domain of human neuroligin-3. Wood K, Paz A, Dijkstra K, Scheek RM, Otten R, Silman I, Sussman JL, Mulder FA. Biomol NMR Assign. 2012, PMID: 21647611.
The quaternary structure of amalgam, a Drosophila neuronal adhesion protein, explains its dual adhesion properties. Zeev-Ben-Mordehai T, Mylonas E, Paz A, Peleg Y, Toker L, Silman I, Svergun DI, Sussman JL. Biophys J. 2009, PMID: 19843464.
Assessment of CASP8 structure predictions for template free targets. Ben-David M, Noivirt-Brik O, Paz A, Prilusky J, Sussman JL, Levy Y. Proteins. 2009, PMID: 19774550.
The crystal structure of a complex of acetylcholinesterase with a bis-(-)-nor-meptazinol derivative reveals disruption of the catalytic triad. Paz A, Xie Q, Greenblatt HM, Fu W, Tang Y, Silman I, Qiu Z, Sussman JL. J Med Chem. 2009, PMID: 19326912.
Amalgam, an axon guidance Drosophila adhesion protein belonging to the immunoglobulin superfamily: over-expression, purification and biophysical characterization. Zeev-Ben-Mordehai T, Paz A, Peleg Y, Toker L, Wolf SG, Rydberg EH, Sussman JL, Silman I. Protein Expr Purif. 2009, PMID: 18938249.
Biophysical characterization of the unstructured cytoplasmic domain of the human neuronal adhesion protein neuroligin 3. Paz A, Zeev-Ben-Mordehai T, Lundqvist M, Sherman E, Mylonas E, Weiner L, Haran G, Svergun DI, Mulder FA, Sussman JL, Silman I. Biophys J. 2008 PMID: 18456828
Operational definition of intrinsically unstructured protein sequences based on susceptibility to the 20S proteasome. Tsvetkov P, Asher G, Paz A, Reuven N, Sussman JL, Silman I, Shaul Y. Proteins. 2008, PMID: 17879262.
Structural biology. It’s all in the symmetry. Abramson J, Paz A, Philipson KD. Science. 2012, PMID: 22323810.
Structures of the prokaryotic galactose transporter vSGLT and their implications on alternating access mechanism in human SGLT1. Abramson J, Paz A, Vartanian AS. In Membrane Transport Mechanism 3D Structure and Beyond, 2014.
Purification of Intrinsically Disordered Proteins. Paz A, Zeev-Ben-Mordehai T, Sussman J, Silman I. In Instrumental Analysis of Intrinsically Disordered Proteins: Assessing Structure and Conformation, 2010.