BBC3034 Biomolecular Structure Continuous Assessment Exercise Questionnaire Student name: Artur Rego Costa Student No: 40104937 PROTEIN CHARACTERISTICS PDB ID : 3DH4; PROTEIN NAME : Sodium/galactose symporter; SIZE (mw and number of amino acid residues) : 543 residues with a total molecular weight of ~60 kDa; FUNCTION : Symport of ion sodium and carbohydrates; FAMILY : Solute Sodium Symporters; SECONDARY STRUCTURE OF TRANSMEMBRANE DOMAIN : -helix; NUMBER OF MEMBRANE CROSSINGS : 14; NUMBER OF POLYPEPTIDE CHAINS : One; QUATERNARY STRUCTURE IN VIVO : Monomer; SOURCE ORGANISM : Vibrio parahaemolyticus; EXPRESSION SYSTEM (if E. coli state strain; plasmid used) : E. coli XL1-Blue using VNH6A plasmid; AMINO ACID SEQUENCE (paste it here in FASTA format) : >3DH4:A|PDBID|CHAIN|SEQUENCE XXXXXXXXXXXXXXXXXAGKSLPWWAVGASLIAANISAEQFIGMSGSGYSIGLAIASYEWMSAITLIIVGKYFLPIFIEK GIYTIPEFVEKRFNKKLKTILAVFWISLYIFVNLTSVLYLGGLALETILGIPLMYSILGLALFALVYSIYGGLSAVVWTD VIQVFFLVLGGFMTTYMAVSFIGGTDGWFAGVSKMVDAAPGHFEMILDQSNPQYMNLPGIAVLIGGLWVANLYYWGFNQY IIQRTLAAKSVSEAQKGIVFAAFLKLIVPFLVVLPGIAAYVITSDPQLMASLGDIAATNLPSAANADKAYPWLTQFLPVG VKGVVFAALAAAIVSSLASMLNSTATIFTMDIYKEYISPDSGDHKLVNVGRTAAVVALIIACLIAPMLGGIGQAFQYIQE YTGLVSPGILAVFLLGLFWKKTTSKGAIIGVVASIPFALFLKFMPLSMPFMDQMLYTLLFTMVVIAFTSLSTSINDDDPK GISVTSSMFVTDRSFNIAAYGIMIVLAVLYTLFWVLYKSGGSPGHHHHHH ; STRUCTURE DETAILS PHASING METHOD : Multiple Isomorphous Replacement by incubation with methylmercuric acetate prior to crystallization; RESOLUTION () : 2.2 ; RESIDUES RESOLVED IN STRUCTURE : 47-178; 186-547 PROTEIN TOPOLOGY (location of N- and C-termini) : Both termini are exposed to the periplasm; CRYSTALLISATION / SOLUBILISATION / DATA COLLECTION CONDITIONS PROTEIN PURIFICATION METHODS : Ni2+ affinity chromatography and further size exclusion gel purification; CRYSTALLISATION METHOD : Hanging drop vapour diffusion; TEMPERATURE (OC) : 4.8OC; pH : 7,0;

DETERGENT USED IN SOUBILISATION (include the concentration) : 2% n-decyl--D-maltopyranoside (Anatrace); ADDITIVE USED IN CRYSTALLISATION : 1xcmc of Anzergent 3-12 PRECIPITANT USED IN CRYSTALLISATION : 20-25% PEG400 LIPID USED IF CRYSTALLISED IN CUBIC PHASE/BICELLES : - FACILITY USED FOR DATA COLLECTION : Advanced Light Source (Berkeley, California, USA) at the beamline 5.0.2; Advanced Photon Source (Argonne, Illinois, USA) at stations 23-ID and 24-ID ; and Swiss Light Source (Zurich, Switzerland) at the beamlines X10SA and X06SA; NON-PROTEIN COMPONENTS IN STRUCTURE ALL : D-Galactose, Na+ and Er3+ NATIVE LIGANDS : D-Galactose and Na+ CO-CRYSTALLANTS : Er3+ BIBLIOGRAPHIC INFORMATION CITATION TITLE : The Crystal Strucutre of a Sodium Galactose Transporter Reveals Mechanistic Insights into Na+/Sugar Symport; JOURNAL NAME : Science; VOLUME : 321; PAGES : 810-4; PUBLICATION YEAR : 2008; AUTHORS : Faham, Salem; Watanabe, Akira; Besserer, Gabriel Mercado; Cascio, Duilio; Specht, Alexandre; Hirayama, Bruce a; Wright, Ernest M; Abramson, Jeff; PDB DEPOSITION DATE : 16th June 2008.

A) A)

B) B)

C C N N

Figure 1. The structure of the V. paraehmolyticus Na+/sugar symporter (vSGLT PDB 3DH4) is colored as a rainbow from C-terminus (red) to N-terminus (blue). A) View of protein in the membrane plane. Brackets indicate boundaries of lipid bilayer. Extracellular and intracellular environments are above and under membrane region, respectively. B) Intracellular view of the protein depicting intracellular groove leading to substrate binding region. Ligands are shown as spheres. Galactose is represented as purple and red for C and O atoms, respectively. Na+ is yellow. Image of structure generated using PyMOL 1.3.

Function And Structure Of A Bacterial Na+/Sugar Symporter THE SYMPORTER

Since long it has been studied the widespread mechanism of sodium-dependency in the intake of certain solutes against an electrochemical gradient by cell membranes1. Crane was the first one to propose a hypothesis for the molecular basis of the process that included the coupling of the solute with sodium to enter through the cell membrane and the return of the ion to the extracellular environment2. The hypothesis was strongly supported and it is now established that the electrochemical gradient and the negative charge of the cell membrane drive the accumulation of certain solutes inside the cell. Back in the 90s, Sarker and colleagues described the first bacterial example of a sodium-dependent galactose/glucose transport system for Vibrio parahaemolyticus3,4. Much later than the its well-described orthologous, the mammalian Na+-dependent glucose transport system (SGLT1)2,5. The V. parahaemolyticus Na+/sugar symport unit (vSGLT) had then its activity characterized and its primary structure sequenced by the same group (although the nucleotide sequence would be later on corrected by Turk and colleagues)3,4,68. Although topological and structural properties of proteins can be reasonably assumed from primary structure, recent structural studies permit better understanding of the mechanism and function of the vSGLT. Moreover, the data sheds light on the general process of sodium-driven solute transport. PROTEIN ACTIVITY

With regard to the substrate, in vivo and in vitro inhibition assays demonstrated vSGLT has high affinity for both D-galactose and D-glucose, and to a lesser extent for D-fucose3,4,8. For sugar intake, coupled-ion can be Na+ or Li+, although efficiency for the former is much higher. Na+/substrate stoichiometry for vSGLT was demonstrated to be of 1:18. According to the kinetic models for sodium/solute symport, transportation through membrane is accomplished by means of the alternating-access mechanism9. First proposed in 1966, it consists of the protein having a binding site for the molecules to be transported and that it is alternatively exposed to the intracellular and extracellular environments by allosteric changes so that molecules can bind in one side and be released to the other10. PROTEIN STRUCTURE The structure of the vSGLT consists of 14 transmembrane (TM) helices with both termini exposed to periplasm11, as predicted by analysis of hydrophobicity, propensity for reverse-turns and freeze-fracture electron microscope7,8,12,13. Although the protein packed as a tight dimer when crystalized11, freeze-fracture electron microscopic studies indicate that the protein functions as a monomer 8,13. The vSGLT core structure is composed of 10 TM helices, in which TM2 to TM6 and TM7 to TM11 are structurally similar and related by a symmetry axis at the center of the cell membrane11,14. This same five-helices inverted motif has been lately described in other membrane proteins (BetP, LeuT, Mhp1) that do not share any identity at the

genetic or primary structure level15. These resemblances likely represent similar conformational changes undergone for solute transportation. The vSGLT core structure (as well as all those of other proteins that display similar core structure) possesses two discontinuous helices at the level of the membrane plane11,14,15. This feature has been shown to play important role in the mechanism of several transport proteins16. SUBSTRATE- AND ION-BINDING SITES In the vSGLT galactose is held tight in the center of the protein by coordination of its OH-groups through H-bonds with the core helices side-chains. In this protein conformation, substrate is occluded from the periplasm and cytoplasm by hydrophobic residues that form internal and external gates. Mutation assays proved these interactions to be essential for normal protein function11,14. The hydrophobic gate is formed by a pyranose ring (Try 263), and along with other flanking hydrophobic residues, prevents escape of substrate to a large hydrophilic groove that leads to the intracellular environment11. Displacement of this gate through conformational change allows water to interact with substrate, which releases disrupt H-bonds and prevents rebinding. The vSGLT has only one binding site for sodium, as predicted by its stoichiometry8. Structure comparison and mutation assays demonstrated Na+ is coordinated by interactions with side-chains within core region. Location is ~10 from the galactose-binding site, which indicates an allosteric pathway that could mediate substrate release11,14. TRANSPORTATION STEPS As discussed above, conformational changes are an essential part of the transportation mechanism. The association of the protein to the ion and the substrate enables it to change from an outward to an inward conformation that allows exposure of carried molecules to water and their consequential release to the intracellular environment. From kinetic data9 and modeling the vSGLT structure to the outward-facing LeuT structure16 it is possible to infer some characteristics of the process. The binding of Na+ causes the formation of the galactose-binding site, which when occupied will then induce inward conformation and formation of the hydrophobic gate11. Watanabe and colleagues were able to describe the ion and substrate release dynamics for the vSGLT symporter using a combination of data from structure, biochemical characterization and molecular dynamics simulations14. The Asn 64 residue is located in the unwound part of the TM2 helix11 and plays an important role in the stabilization of the galactose molecule and the hydrophobic gate (Tyr 263) by forming H-bonds with both of them14. Molecular dynamics simulations show that Na+ release causes conformational changes in the TM2 helix breaking Asn 64 and Tyr 263 H-bond leaving the latter to adopt an alternative rotamer conformation that opens pathways for the galactose molecule to the intracellular cavity. Release of both solute and ion causes protein to return to its outward-facing conformation ready for another cycle. It is very likely that this same mechanism applies to all similar membrane proteins14,15.

References

1. Schultz, S. G. & Curran, P. F. Coupled transport of sodium and organic solutes. Physiol. Rev. 50, 637718 (1970).

2. Crane, R. K., Miller, D. & Bihler, I. in Membr. Transp. Metab. (Kleinzeller, A. & Kotyk, A.) 43949 (Academic Press, 1960).

3. Sarker, R. I., Ogawa, W., Tsuda, M., Tanaka, S. & Tsuchiya, T. Characterization of a glucose transport system in Vibrio parahaemolyticus. J. Bacteriol. 176, 737882 (1994).

4. Sarker, R., Ogawa, W., Tsuda, M., Tanaka, S. & Tsuchiya, T. Properties of a Na+/galactose (glucose) symport system in Vibrio parahaemolyticus. Biochim. Biophys. Acta - Biomembr. 1279, 14956 (1996).

5. Wright, E. M., Loo, D. D. F. & Hirayama, B. A. Biology of human sodium glucose transporters. Physiol. Rev. 91, 73394 (2011).

6. Sarker, R., Okabe, Y., Tsuda, M. & Tsuchiya, T. Sequence of a Na+/glucose symporter gene and its flanking regions of Vibrio parahaemolyticus. Biochim. Biophys. Acta - Biomembr. 1281, 14 (1996).

7. Sarker, R., Ogawa, W., Shimamoto, T. & Tsuchiya, T. Primary Structure and Properties of the Na+/Glucose Symporter (SglS) of Vibrio parahaemolyticus. J. Bacteriol. 179, 18058 (1997).

8. Turk, E. et al. Molecular characterization of Vibrio parahaemolyticus vSGLT: a model for sodium-coupled sugar cotransporters. J. Biol. Chem. 275, 257116 (2000).

9. Loo, D. D. F., Hirayama, B. a, Karakossian, M. H., Meinild, A.-K. & Wright, E. M. Conformational dynamics of hSGLT1 during Na+/glucose cotransport. J. Gen. Physiol. 128, 70120 (2006).

10. Jardetzky, O. Simple allosteric model for membrane pumps. Nature 211, 96970 (1966).

11. Faham, S. et al. The crystal structure of a sodium galactose transporter reveals mechanistic insights into Na+/sugar symport. Science 321, 8104 (2008).

12. Turk, E. & Wright, E. M. Membrane Topology Motifs in the SGLT Cotransporter Family. J. Membr. Biol. 159, 120 (1997).

13. Eskandari, S., Wright, E. M., Kreman, M., Starace, D. M. & Zampighi, G. a. Structural analysis of cloned plasma membrane proteins by freeze-fracture electron microscopy. Proc. Natl. Acad. Sci. U. S. A. 95, 1123540 (1998).

14. Watanabe, A. et al. The mechanism of sodium and substrate release from the binding pocket of vSGLT. Nature 468, 98891 (2010).

15. Abramson, J. & Wright, E. M. Structure and function of Na(+)-symporters with inverted repeats. Curr. Opin. Struct. Biol. 19, 42532 (2009).

16. Yamashita, A., Singh, S. K., Kawate, T., Jin, Y. & Gouaux, E. Crystal structure of a bacterial homologue of Na+/Cl--dependent neurotransmitter transporters. Nature 437, 21523 (2005).