BCH3125

Protein Assignment

Crystal Structure of a Concentrative Nucleoside Transported from Vibrio cholera at 2.4

PDB accession code: 3TIJ

Pubmed ID code: 22407322

By Michael Dysart

6358390

1. Transport of nucleosides into the cell is crucial for signal termination and DNA/RNA synthesis. Concentrative nucleoside transporters (CNTs) accomplish this using an ion gradient to assist secondary active transport, and play a role in transport of nucleoside derived drugs. A CNT from Vibrio cholera (vcCNT) that has 39% sequence homology to a human CNT (hCNT3) and also uses a Na+ gradient for transport has been crystallized in complex with Na+ and the uridine.

vcCNT is a homotrimer transmembrane protein with three identical subunits positioned in a triangle. Each subunit can bind and transport nucleosides. Each subunit is an alpha-helical protein containing mostly transmembrane alpha helices and a few that span the surface of the membrane (figure 1). The alpha helices are arranged in roughly two subdomains: the scaffold domain (red in figure 1f)(helices TM1, TM2, IH1, EH, TM3, and TM6) that forms a ring around the central transport domain which is formed of two groups of helices (IH2, HP1a/b, TM4a/b and TM5 in one, IH3, HP2a/b, TM7a/b, and TM8 in the other). These two groups have structural symmetry. The structural domain is responsible for maintaining the overall structure of the protein and aids trimerization, while the transport domain contains Na+ and uridine binding sites.

The surface diagram of the trimer (figure 2) shows many water molecules bound on the extracellular and cytoplasmic sides though few in the region that makes contact with the membrane. There are many water molecules positioned within a given monomer close to the Na+ ion. The helices of the transport domain can be observed to extend approximately 14 below the membrane (cytoplasmic side) at the periphery of the trimer. This leaves a basin in the middle of the trimer that is close to the membrane and interacts with water. This basin allows access to the uridine binding pocket (figure 2e). TM6 blocks access to this binding pocket from the extracellular side. Trimerization is mediated by residues in the scaffold domain. On the extracellular side several aromatic residues from helix IH1 and TM3 of each subunit form tight hydrophobic interactions (figure 3a). On the cytoplasmic side leucine and alanine residues from TM6 also form tight packing involving hydrophobic interactions and ridges into grooves packing (figure 3b). In particular, L257 packs between A265 and L261 of an adjacent monomer. Binding to uridine in the binding pocket is mediated by many hydrogen bonds to residues in the pocket and water molecules (figure 4a). The hydroxyl groups of ribose form tight hydrogen bonds (distances between 2.7-3.1 ) to residues found on TM7b (S371, N368) and HP2b (E332). One oxygen and nitrogen atom of uracil form hydrogen bonds to residues found on the HP1a to HP1b linker loop (Q154, T155, and E156, the latter two mediated by a water molecule). Sodium ion forms a coordination complex in an octahedral array with several hydrogen bonds. These bonds involve the backbone carbonyl oxygen atoms of N149, V152, and I184, along with the side chain oxygen atoms of S183 and N149, and a final hydrogen bond to a water molecule. These residues are positioned on the loops connecting TM4a to TM4b and HP1a to HP1b. The Na+ and uridine do not interact directly (figure 4b), though both their binding sites involve residues from the loop that connects HP1a and HP1b helices, along with residues from the TM4b helix (figure 4c). It is hypothesised that Na+ binding is required to position the HP1 loop in the correct orientation to generate the uridine binding site.

It is hypothesised that uridine transport involves movement of the transport domain relative to the scaffold domain (termed the alternative access mechanism). This movement would shift the protein to a different conformation where the uridine binding pocket is on the opposite side of the TM6 helix and allow the uridine in the binding pocket to diffuse to the opposite side of the membrane. It is thought that the solved structure is the cytoplasmic directed conformation (see figure 2e); therefore the transport helix must translocate upwards above the TM6 helix to expose the binding pocket to the extracellular side to bind uridine and transport it into the cell.

Figure 1 : Structure of the vcCNT monomer. a. cartoon representation of the extracellular view of vcCNT. It is rainbow coloured from N-terminus (blue) to C-terminus (red). A sodium ion is shown as a purple sphere, while the bound uridine is shown as a stick structure. Labels are shown for most helices. Helices whose label is only a number are referred to as TM helices in the text. b. cytoplasmic view. c-e. transmembrane view. The approximate position of the membrane is shown as black lines. f . extracellular view showing the scaffold domain (red) and transport domain (green). Note: The space between helices 5 and 6 (dashed line: c and d) was a disordered region that could not be solved.

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Figure 2: vcCNT trimer. a. extracellular view of vcCNT trimer. One subunit is shown in cartoon while the other two are surface diagrams. Water molecules are shown as magenta spheres. Uridine is shown as yellow spheres while Na+ is the larger purple sphere. b. cytoplasmic view. Each monomer has a bulge towards the exterior of the trimer, which forms a basin towards the middle. c-d. transmembrane view at two angles. The approximate position of the membrane is shown by black lines. e. view of the interior of the trimer (one subunit hidden). This shows the cytoplasmic basin of the trimer. In the blue subunit the cytoplasmic access to the uridine binding pocket is visible. TM6 is identified in red for the blue subunit and blocks the upper face of the binding pocket.

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Figure 3: Hydrophobic interactions that link the monomers of the vcCNT trimer. a. packing occurring between helices TM3 and IH1 towards the extracellular surface. b. packing occurring between TM6 helices towards the cytoplasmic side. Ridges into grooves packing are observed. Only residues on the green helix are labelled.

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Figure 4: Uridine and Na+ binding pockets of vcCNT. a. Uridine binding pocket showing the residues and water molecules (magenta spheres) involved in binding. Oxygen atoms are magenta, nitrogen atoms are blue, and in uridine carbon atoms are yellow. Helix labels are shown. Hydrogen bonds are shown as dashed lines with distance labels (in ). b. Na+ (purple sphere) binding pocket showing the residues and water molecules involved in binding. c. combined Na+ and uridine binding pocket showing residues, helix labels, and hydrogen bonds.

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