Yao SYM and Young JD (2018), Inward- and outward-facing homology modeling of...

XB-ART-55178

Channels (Austin) 2018 Jan 01;121:291-298. doi: 10.1080/19336950.2018.1506665.

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Inward- and outward-facing homology modeling of human concentrative nucleoside transporter 3 (hCNT3) predicts an elevator-type transport mechanism.

Yao SYM , Young JD .

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The human SLC28 family of concentrative (Na+-dependent) nucleoside transporters has three members, hCNT1, hCNT2 and hCNT3. Previously, we have used heterologous expression in Xenopus laevis oocytes in combination with an engineered cysteine-less hCNT3 protein hCNT3(C-) to undertake systematic substituted cysteine accessibility method (SCAM) analysis of the transporter using the membrane-impermeant thiol reactive reagent p-chloromercuribenzene sulfonate (PCMBS). A continuous sequence of more than 300 individual amino acid residue positions were investigated, including the entire transport domain of the protein, as well as important elements of the corresponding hCNT3 structural domain. We have now constructed 3D structural homology models of hCNT3 based upon inward-facing, intermediates and outward-facing crystal structures of the bacterial CNT Neisseria wadsworthii CNTNW to show that all previously identified PCMBS-sensitive residues in hCNT3 are located above (ie on the extracellular side of) the key diagonal barrier scaffold domain TM9 in the transporter's outward-facing conformation. In addition, both the Na+ and permeant binding sites of the mobile transport domain of hCNT3 are elevated from below the scaffold domain TM9 in the inward-facing conformation to above TM9 in the outward-facing conformation. The hCNT3 homology models generated in the present study validate our previously published PCMBS SCAM data, and confirm an elevator-type mechanism of membrane transport.

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Species referenced: Xenopus laevis
Genes referenced: tpm3

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	Figure 1. Topological models of hCNT3 and CNTNW. (a) Predicted membrane topology of human concentrative nucleoside transporter hCNT3 based upon that of its bacterial counterpart vcCNT from Vibrio cholera. PCMBS sensitive and uridine-protected residues Gln341, Thr342, Asn565 and Ile571 are shown as red circles. (b) Membrane topology of Neisseria wadsworthii CNT (CNTNW) [16]. The hCNT3 N-terminal transmembrane helices TM1, TM2 and TM3 and C-terminal extramembranous tail with glycosylation sites are not present in the bacterial protein.
	Figure 2. Homology models of hCNT3. Cartoon representations of the hCNT3 3D outward-facing conformation based upon the corresponding crystal structure of the bacterial nucleoside transporter CNTNW (Protein Data Bank entries 5L2A, protomer C) using the program SWISS-MODEL [17] and viewed parallel to the membrane. Molecular graphics and analyzes were performed using the UCSF Chimera package [18]. The extracellular boundaries of the hydrophobic core of the lipid bilayer were predicted using the PPM server [19], with residues outside of the hydrophobic boundaries of the bilayer shown in beige, and residues within the hydrophobic core of the bilayer shown in light blue. TM9 of the outer scaffold domain of hCNT3 is shown in purple. (A) Outward-facing homology model of hCNT3 with side-chains of PCMBS-sensitive residues shown in red. (B) Outward-facing homology model of hCNT3 with side-chains of PCMBS-sensitive and uridine-protected residues shown in red.
	Figure 3. Homology models of hCNT3 in different conformational states of the transport cycle. Cartoon representations of hCNT3 3D models are based upon the corresponding crystal structures of the bacterial nucleoside transporter CNTNW using the program SWISS-MODEL [17] and viewed parallel to the membrane. The extracellular boundaries of the hydrophobic core of the lipid bilayer were predicted using the PPM server [19], with residues outside of the hydrophobic boundaries of the bilayer shown in beige, and residues within the hydrophobic core of the bilayer shown in light blue. (a) Inward-facing, open state (Protein Data Bank entry 5L2A, protomer A) and outward-facing, open state (Protein Data Bank entry 5L2A, protomer C). (b) Intermediate-1 (Protein Data Bank entry 5L27, protomer C); Intermediate-2 (Protein Data Bank entry 5L24, protomer C); Intermediate-3 (Protein Data Bank entry 5U9W, protomer C). Side chains of PCMBS-sensitive, uridine-protected residues Gln341, Thr342, Asn565 and Ile571 are shown as red spheres. HP1 (residues 327 to 350 of hCNT3) are shown in green. TM9 of the outer scaffold domain (residues 432 to 475 of hCNT3) is shown in purple.
	Figure 4. Inward-facing and outward-facing homology models of hCNT3. Cartoon representations of hCNT3 3D inward-facing and outward-facing conformations are based upon the corresponding crystal structures of bacterial nucleoside transporter CNTNW (Protein Data Bank entries 5L2A, protomer A and protomer C, respectively) (a) Models of hCNT3 viewed parallel to the membrane. Side chains of residues involved in primary Na+-binding site are shown as blue spheres. (b) Models of hCNT3 viewed parallel to the membrane. Side chains of residues involved in primary Na+-binding site and substrate-binding site are shown as blue and red spheres, respectively. The extracellular boundaries of the hydrophobic core of the lipid bilayer were predicted using the PPM server [19], with residues outside of the hydrophobic boundaries of the bilayer shown in beige, and residues within the hydrophobic core of the bilayer shown in light blue. TM9 of the outer scaffold domain is shown in purple. (c) Models of hCNT3 viewed from the extracellular surface of the membrane. The outer scaffold domain of hCNT3 (TM6, TM9, IH1, IH2 and IH3) and inner transport domain of hCNT3 (TM4, TM5, HP1, TM7, TM8, HP2, TM10 and TM11) are shown in yellow and blue, respectively. Side chains of residues (Ile450 and Leu461) and (Leu480, Phe482 and Glu483) from the scaffold domain TM9 and linker region to IH3, respectively, are highlighted as red spheres.

References [+] :

Arimany-Nardi, Identification and Characterization of a Secondary Sodium-Binding Site and the Main Selectivity Determinants in the Human Concentrative Nucleoside Transporter 3. 2017, Pubmed