User:Aritra Paul/Sandbox
From Proteopedia
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Structural insights into the mechanism of phosphate recognition and transport by XPR1 G. Wenhui Zhang, Yanke Chen, Zeyuan Guan, Yong Wang, Meng Tang, Zhangmeng Du, Jie Zhang, Meng Cheng, Jiaqi Zuo, Yan Liu, Qiang Wang, Yanjun Liu, Delin Zhang, Ping Yin, LingMa & Zhu Liu [1]
BackgroundXPR1 (SLC53A1) is the only known eukaryotic plasma-membrane phosphate (Pi) exporter; loss-of-function variants cause cytosolic Pi accumulation and primary familial brain calcification (PFBC)
Structural overviewCryo-EM reconstructions (overall 2.9–3.3 Å) produced a Pi-unbound map plus three Pi-bound states, and an atomic model of the transmembrane domain (TMD). The cytosolic SPX domain showed poor density (mobile) while the TMD is well resolved. XPR1 is a dimer in detergent, but each protomer contains a 10-TM architecture split into an N domain (TM1–5) and an EXS domain (TM6–10) that together create an aqueous, channel-like pore. TM9b bends into the pore and seems important for gating.
Phosphate recognition & the “relay” mechanismThree sequential Pi recognition sites line the pore (named Pi1 → Pi2 → Pi3), observed as non-protein densities in three different maps. The pore interior is polar and overall positively charged, optimized for an anion Site 1 (Pi1): coordinated primarily by D398, K482, Y483, D529, D533, R570, R604 and the indole N of W607; peripheral residues R459 and N401 help orient the binding network. Site 2 (Pi2): as Pi shifts, it disengages D529/D533/W607 and establishes contacts with R603 and N401; E600 rotates to form a salt bridge to R603 and position it for coordination Site 3 (Pi3, near the exit): Pi is coordinated by N401, S404, Q452, W573; R570 and I577 support W573 through cation-π / CH-π interactions that stabilize site geometry. The residues forming each site are conserved among EXS-domain proteins. Functional validation (quantitative)Proteoliposome transport assays: reconstituted XPR1 transports Pi in an external-conc dependent and time-dependent manner; transport is roughly linear up to 1 mM Pi → low-affinity behavior (Km in the millimolar range). Measured rate in assay ≈ 33 pmol Pi · µg⁻¹·min⁻¹ (≈3 pmol Pi·pmol protein⁻¹·min⁻¹) at 500 µM external Pi. Binding (MST): detergent-solubilized XPR1 binds Pi with Kd ≈ 334 ± 79 µM; key site mutations reduce affinity. Mutagenesis: alanine / conservative substitutions of D398, N401, Q452, R603, Y483, W573, E600 etc. impair transport in liposomes and fail to complement Pi-transport-deficient yeast — consistent orthogonal evidence tying those residues to function.  Fig 1: a.Cartoon representation of the structure. The N domain and EXS domain are colored in magenta and blue, respectively. TM9b is colored in yellow. b. Schematic topology diagram of the structure. The gray background indicates the membrane bilayer. The N-terminal SPX domain of XPR1 is invisible in the determined cryo-EM structure. MD simulations & dynamicsTwo independent 1,000 ns (1 µs) all-atom MD runs were performed (CHARMM36m, POPC bilayer) starting from Pi-unbound and Pi-bound models. To observe transport within tractable time, an external electric field (~400 mV) was applied in Pi-bound runs. Under that field, an export event was observed: Pi left site-1 → site-2 → site-3 and exited to the extracellular side at ~350 ns (simulation timescale). The pore remained hydrated throughout; only minor backbone rearrangements were required — supporting channel-like conduction rather than large alternating-access movements. Residues Q576, R448, R270 were implicated in facilitating exit after site-3. As a result, CHU-128 can partially stabilise the deep polar core, which is sufficient for Gs coupling and cAMP production, but it cannot support the conformational features required for the other pathways described before. This produces a strongly biased signaling profile in which only the cAMP pathway is robustly activated, explaining its highly selective Gs-biased agonism. Regulation, comparisons & open questionsThe cytosolic SPX domain likely gates access in response to PP-InsP signals (paper included InsP6 during prep to stabilize SPX). Other groups (Yan et al., Lu et al.) reported complementary XPR1 structures (closed/PP-InsP bound), supporting an SPX-regulated entry mechanism; the TMD itself appears capable of conducting Pi once intracellular entry is allowed. The physiological membrane potential and PP-InsP levels are expected to be central regulators in vivo; reconstituted liposomes lack a membrane potential, which likely underestimates true in-cell transport rates Implications & therapeutic ideasMechanistic framework: provides residue-level explanation for PFBC mutations and offers rational targets (e.g., small molecules that stabilize the closed SPX/TMD interface or block the pore) to modulate Pi efflux in disease or cancer contexts. Experimental next steps: single-channel electrophysiology in defined bilayers (with physiological voltages), PP-InsP titrations, high-resolution SPX-bound structures, and small-molecule screens focused on the extracellular vestibule or SPX-TMD interface.
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