2020同步年報

Life Science 045 The PiT contains a unique internal highly conserved domain named PD001131 present at the N- and C-termini. Within this domain are four highly conserved sequences: ΦNDΦ, GxxxxGxxVxxT, PΦSxT and IxxxWΦ (x: any amino acid; Φ: hydrophobic residue). Thermotoga maritima is a hyper- thermophilic bacterium that makes it an ideal model organ- ism for an integration of biochemical and structural exper- imental approaches. The authors show that Tm PiT belongs to the SLC20 family; its transmembrane domain exhibits protein characteristics and sequence homology similar to that of h PiT (similarity/identity 62%/38% for h PiT1 and 61%/39% for h PiT2). To investigate the structural information for a sodium- dependent phosphate transporter, a research team led by Yuh-Ju Sun (National Tsing Hua University) solved the crystal structure of Tm PiT, a homology of h PiT. They mea- sured the phosphate-binding affinity as 57.0 ± 1.1 µM and determined the uptake ability of Tm PiT being driven by sodium. The crystal structure of Tm PiT bound to sodium and phosphate ( Tm PiT-Na/Pi) is in an inward occluded state and exits as a dimer, with the two subunits showing distinct conformations. All diffraction data sets were collected at TPS 05A in NSRRC. 2 The Tm PiT-Pi/Na complex is shown in Figs. 1(a) and 1(b) ; the transport and scaffold domains are formed with 12 transmembrane helices. The transport domain of Tm PiT assumes a “5+5” fold and is arranged into two inverted repeats annotated as PD001131, N-PD001131 (TM1/TM2/ HP1a-HP1b/TM3) and C-PD001131 (TM6/TM7/HP2a- HP2b/TM8). HP1a-HP1b (HP1) and HP2a-HP2b (HP2) are re-entrant helical hairpins that have been reported in the transporters of elevator type, such as aspartate transporter Gltph and dicarboxylate transporter VcINDY. Tm PiT is a dimer with dimensions 83 Å × 61 Å × 55 Å; the N- and through two aspartates, D22 and D258. A phosphate was located 4.8 Å from each sodium ion. This phosphate was tightly bound via 12 interactions with eight conserved residues, including D22 (TM1), D258 (TM6) and six polar residues, S105/T106/T107 (HP1b) and S345/T346/T347 (HP2b) ( Fig. 1(e) ). Both Na1 and Na2 are bound within a penta-coordinated interaction with conserved residues Asp/Asn/Thr. All these Pi-2Na-binding residues are highly conserved in PiT families; D22 and D258 are involved in both Pi and Na binding. In addition to Pi-2Na binding, a third sodium, Na fore, was identified near TM1, TM6 and HP2a with penta-coordination through residues T29, Q243, S247 and D327 ( Fig. 1(f) ), which are highly or partially conserved in the PiT. In the Tm PiT-Pi/Na complex, there are significant structural differences between subunits A and B, which are reflected in a root-mean-square deviation 1.8 Å for the Cα atoms mainly in TM8 and intracellular loops L7 and LHP2. The ac- cessible volumes of this region, calculated using CASTp, are 68 Å 3 and 252 Å 3 for subunits A and B, respectively ( Figs. 2(a) and 2(b) ). Conformational changes in the Tm PiT-Pi/ Na complex in loops L7, LHP2 and TM8 between subunits A and B indicate that the subunits might have distinct functional states. They might control the inner gate during phosphate and sodium release on assuming closed and open states in subunits A and B, respectively. Interestingly, particular structural characteristics of Tm PiT reflect those reported in disease-associated mutations in h PiT. Several mutations in h PiT2 have been associated with neuropsychiatric disorders and primary familial brain calci- fication. 3 To understand how these mutations might affect h PiT function, the authors mapped these h PiT mutations onto the Tm PiT-Pi/Na complex structure ( Fig. 3 ); they found a significant correlation between their Tm PiT results and Fig. 2 : The structures of subunits A and B around inner gates are shown in (a) and (b), respec- tively. Two inverted, repeated domains are shown: HP1 from N-PD001131 (in magenta in both subunits) and HP2 and TM8 from C-PD001131 (in blue and cyan for subunits A and B, respectively). The structures shown include Pi and Na, and residues K314 and W378. The Pi and Na ions are shown in CPK and as purple spheres, respectively. Residues K314 and W378 are shown in CPK. The accessible volumes of the exit region were calculated with CASTp30 and are shown in brown. [Reproduced from Ref. 2] C-termini are located in the extracellu- lar region ( Fig. 1(c) ). The dimer inter- face between two subunits (A and B) is formed with TM2/7 from the transport domain and TM4/5 from the scaffold domain and has a buried area 1283.7 Å 2 . In the Tm PiT-Pi/Na complex struc- ture, three sodium ions were found ( Fig. 1(d) ): two sodium ions and one phosphate (hereafter Pi-2Na) were located at the core of Tm PiT; the third sodium (Na fore ) was situated near the inner membrane boundary. The Pi-2Na binding site is formed with TM1, loop HP1a-HP1b l(HP1 tip), TM6 and loop HP2a-HP2b (HP2 tip) ( Figs. 1(d) and 1(e) ). The phosphate is associated with two sodium ions as “Na1-Pi-Na2”