Zinc transporters play critical roles in cellular zinc homeostatic control. The 2.9-Å resolution structure of the zinc transporter YiiP from Escherichia coli reveals a richly charged dimer-interface stabilized by zinc binding. Site-directed fluorescent resonance energy transfer (FRET) measurements and mutation-activity analysis suggest that zinc binding triggers hinge movements of two electrically repulsive cytoplasmic domains pivoting around four salt-bridges situated at the juncture of the cytoplasmic and transmembrane domains. These highly conserved salt-bridges interlock transmembrane helices at the dimer-interface, well positioned to transmit zinc-induced inter-domain movements to reorient transmembrane helices, thereby modulating coordination geometry of the active-site for zinc transport. The cytoplasmic domain of YiiP is a structural mimic of metal trafficking proteins and the metal-binding domains of metal-transporting P-type ATPases. The use of this common structural module to regulate metal coordination chemistry may enable a tunable transport activity in response to cytoplasmic metal fluctuations.

All living cells need zinc ions to support cell growth. Zrt-, Irt-like proteins (ZIPs) represent a major route for entry of zinc ions into cells, but how ZIPs promote zinc uptake has been unclear. Here we report the molecular characterization of ZIPB from Bordetella bronchiseptica, the first ZIP homolog to be purified and functionally reconstituted into proteoliposomes. Zinc flux through ZIPB was found to be nonsaturable and electrogenic, yielding membrane potentials as predicted by the Nernst equation. Conversely, membrane potentials drove zinc fluxes with a linear voltage-flux relationship. Direct measurements of metal uptake by inductively coupled plasma mass spectroscopy demonstrated that ZIPB is selective for two group 12 transition metal ions, Zn 2؉ and Cd 2؉ , whereas rejecting transition metal ions in groups 7 through 11. Our results provide the molecular basis for cellular zinc acquisition by a zinc-selective channel that exploits in vivo zinc concentration gradients to move zinc ions into the cytoplasm.Zinc is an essential element for all living organisms (1). Zinc chemistry is widely exploited to drive enzymatic catalysis, organize protein structures, and mediate macromolecular interactions (2). In known proteomes, zinc-containing metalloproteins account for ϳ10% of the total proteins (3). Zinc metabolism is also very high. In human, ϳ1% of the total body zinc content is replenished daily by the diet (4). The abundant zinc utilization and its rapid turnover necessitate highly efficient zinc uptake mechanisms by which cells accumulate zinc to a total concentration in the submillimolar range (5). The vast majority of cellular zinc is in complex with specific protein partners (6). Free zinc ions, on the other hand, must be strictly limited in the cytoplasm to prevent cytotoxic side effects (7). Zinc efflux transporters and intracellular buffering systems are evolved to maintain an extremely low level of cytoplasmic free zinc, probably in a femtomolar to picomolar range (8, 9). When a zinc supply is available in the extracellular medium, the free zinc concentrations in the cytoplasm are expected to be many orders of magnitude lower than the extracellular zinc concentrations (10, 11). The inward zinc concentration gradients would provide a powerful chemical driving force to draw extracellular zinc ions into the cytoplasm if a transmembrane zinc conduit would connect the external zinc availability and the high intracellular zinc binding capacity. Such a zinc-specific uptake channel has not heretofore been identified.A common assumption is that cellular zinc uptake is an active process mediated by metal transporters. In mammals, Zrt-, Irt-like protein (ZIP) 2 is the only zinc-specific uptake protein identified thus far (12). Although ZIPs supply zinc to meet cellular needs for growth, aberrant ZIP expressions have been linked to uncontrolled cell growth such as that occurring in cancer (13). Functionally, mammalian ZIPs promote zinc influx into the cytoplasm either from the extracellular medium or from zin...

The proton gradient is a principal energy source for respiration-dependent active transport, but the structural mechanisms of proton-coupled transport processes are poorly understood. YiiP is a proton-coupled zinc transporter found in the cytoplasmic membrane of E. coli, and the transport-site of YiiP receives protons from water molecules that gain access to its hydrophobic environment and transduces the energy of an inward proton gradient to drive Zn(II) efflux1,2. This membrane protein is a well characterized member3-7 of the protein family of cation diffusion facilitators (CDFs) that occurs at all phylogenetic levels8-10. X-ray mediated hydroxyl radical labeling of YiiP and mass spectrometric analysis showed that Zn(II) binding triggered a highly localized, all-or-none change of water accessibility to the transport-site and an adjacent hydrophobic gate. Millisecond time-resolved dynamics revealed a concerted and reciprocal pattern of accessibility changes along a transmembrane helix, suggesting a rigid-body helical reorientation linked to Zn(II) binding that triggers the closing of the hydrophobic gate. The gated water access to the transport-site enables a stationary proton gradient to facilitate the conversion of zinc binding energy to the kinetic power stroke of a vectorial zinc transport. The kinetic details provide energetic insights into a proton-coupled active transport reaction.

Zinc and cadmium are similar metal ions, but though Zn 2þ is an essential nutrient, Cd 2þ is a toxic and common pollutant linked to multiple disorders. Faster body turnover and ubiquitous distribution of Zn 2þ vs. Cd 2þ suggest that a mammalian metal transporter distinguishes between these metal ions. We show that the mammalian metal transporters, ZnTs, mediate cytosolic and vesicular Zn 2þ transport, but reject Cd 2þ , thus constituting the first mammalian metal transporter with a refined selectivity against Cd 2þ . Remarkably, the bacterial ZnT ortholog, YiiP, does not discriminate between Zn 2þ and Cd 2þ . A phylogenetic comparison between the tetrahedral metal transport motif of YiiP and ZnTs identifies a histidine at the mammalian site that is critical for metal selectivity. Residue swapping at this position abolished metal selectivity of ZnTs, and fully reconstituted selective Zn 2þ transport of YiiP. Finally, we show that metal selectivity evolves through a reduction in binding but not the translocation of Cd 2þ by the transporter. Thus, our results identify a unique class of mammalian transporters and the structural motif required to discriminate between Zn 2þ and Cd 2þ , and show that metal selectivity is tuned by a coordination-based mechanism that raises the thermodynamic barrier to Cd 2þ binding.Cd transport | metal binding site | zinc | Zn transporter | Cd toxicity Z n 2þ and Cd 2þ are both d 10 closed shell metals with similar outer electronic structures. However, while Zn 2þ is an essential micro nutrient (1), Cd 2þ is a common environmental pollutant associated with severe toxicity and linked to many disorders, including hypertension, cancer, infertility, thyroid, renal, and bone diseases (2-4). Due to their chemical similarity, Cd 2þ can exploit Zn 2þ uptake routes to enter cells through, for example, Zn 2þ influx transporters such as ZIPs (5-7). Similarly, Metallothioneins, the major cellular metal buffering proteins, bind both Cd 2þ and Zn 2þ , and the divalent metal transporter 1 (DMT1) catalyzes H þ cotransport of Cd 2þ and Zn 2þ as well as other heavy metals (8-10). In plants, most of the Heavy metal ATPases (HMA) classified P-type ATPase share the same nonselective metal ions transport (11). Although bacterial metal pumps HMA1 and plant OsHMA3 are selective for Zn 2þ over Cd 2þ and their selectivity is associated with transmembrane domains (12, 13), the structural basis for their metal selectivity is not fully understood.Mammalian cells do not possess such a wide repertoire of heavy metal pumps and only express the ATP7A and ATP7B P-type cupper selective pumps (14). Yet once inside mammalian tissues, Cd 2þ is trapped with a retention half-time of more than 30 y (15) in a tissue restricted manner whereas Zn 2þ undergoes rapid bodily dissemination through a sequence of secretion and reabsorption processes (16). This difference in the extrusion rates of Zn 2þ and Cd 2þ suggests that in addition to Cd 2þ buffering by metallothioneins (MTs), a mammalian class of metal transporters may selective...

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has created global health and economic emergencies. SARS-CoV-2 viruses promote their own spread and virulence by hijacking human proteins, which occurs through viral protein recognition of human targets. To understand the structural basis for SARS-CoV-2 viral-host protein recognition, here we use cryo-electron microscopy (cryo-EM) to determine a complex structure of the human cell junction protein PALS1 and SARS-CoV-2 viral envelope (E) protein. Our reported structure shows that the E protein C-terminal DLLV motif recognizes a pocket formed exclusively by hydrophobic residues from the PDZ and SH3 domains of PALS1. Our structural analysis provides an explanation for the observation that the viral E protein recruits PALS1 from lung epithelial cell junctions. In addition, our structure provides novel targets for peptide- and small-molecule inhibitors that could block the PALS1-E interactions to reduce E-mediated virulence.

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