Structural insights into the elevator-type transport mechanism of a bacterial ZIP metal transporter

Abstract

The Zrt-/Irt-like protein (ZIP) family consists of ubiquitously expressed divalent metal transporters critically involved in maintaining systemic and cellular homeostasis of zinc, iron, and manganese. Here, we present a study on a prokaryotic ZIP from Bordetella bronchiseptica (BbZIP) by combining structural biology, evolutionary covariance, computational modeling, and a variety of biochemical assays to tackle the issue of the transport mechanism which has not been established for the ZIP family. The apo state structure in an inward-facing conformation revealed a disassembled transport site, altered inter-helical interactions, and importantly, a rigid body movement of a 4-transmembrane helix (TM) bundle relative to the other TMs. The computationally generated and biochemically validated outward-facing conformation model revealed a slide of the 4-TM bundle, which carries the transport site(s), by approximately 8 Å toward the extracellular side against the static TMs which mediate dimerization. These findings allow us to conclude that BbZIP is an elevator-type transporter.

High-resolution structure of a mercury cross-linked ZIP metal transporter reveals delicate motions and metal relay for regulated zinc transport

“…Overall structure of the Hg 2+ cross-linked A95C/A214C variant The crystal structure was solved at 1.95 Å, representing the highest resolution for BbZIP (Figure 2A, Table S1). As revealed in the structures of BbZIP in the apo state (31,32), the transporter consists of nine TMs with the N-terminal TM0 only weakly associated with TM3 and TM6. The variable interface between TM0 and the rest of the transporter strongly indicates that TM0 is unlikely to play a crucial role in transporter function.…”

“…Different from our previous structure solved in the apo state (PDB: 8CZJ), the very N-terminal amphipathic helix α0a was not resolved in the new structure, highlighting the structural flexibility of this highly variable region. The conserved eight TMs is composed of two domains -TM1/4/5/6 form the transport domain and TM2/3/7/8 form the scaffold domain, according to the proposed elevator-type transport mode (31,32). As expected, a Hg 2+ was found to coordinate with the two introduced cysteine residues (C95 from TM2 and C214 from TM5) with an ideal geometry for a S-Hg-S linkage (Figure 3), fixing the relative orientation between the transport domain and the scaffold domain, which likely accounts for the high resolution of this crystal structure.…”

“…When compared with the structures of BbZIP in the metal bound state, the metal-free state structures consistently showed a downward rotation of the transport domain relative to the scaffold domain (31,32), whereas the new Hg 2+ cross-linked structure revealed an upward rigidbody rotation of the transport domain relative to the scaffold domain (Figure 7A). Notably, when the scaffold domains of the representative IFC structures are aligned, a continuous displacement of the transport domain can be visualized -it undergoes a hinge motion around a fixed axis that is located at the extracellular terminus of TM6 (referred to as the extracellular axis).…”

“…The structure of a prototypic ZIP from Bordetella bronchiseptica (BbZIP) revealed the structural framework of the transmembrane domain that is conserved in the entire ZIP family (30). An elevator-type transport mode for BbZIP was later proposed based on the two-domain architecture, evolutionary covariance analysis, and an outward-facing conformation (OFC) model validated by chemical crosslinking and cysteine accessibility assay (31,32). According to the proposed mechanism, the four transmembrane helix (TM) bundle composed of TM1/4/5/6 (the transport domain), which carries the metal substrate, moves vertically by 8 Å relative to the static and wall-like scaffold domain (TM2/3/7/8) to achieve alternating access.…”

“…Although several structures of the inward-facing conformation (IFC) have been solved experimentally and an OFC model has been proposed, the upward movement of the transport domain relative to the scaffold domain, which is an essential step for the proposed conformational transition from the IFC to the OFC, has not been observed. Rather, by comparing the transporter in the apo (metal-free) state with the metal-bound state, two independent studies have revealed a downward movement of the transport domain, which is associated with metal release from the transport site (31,32).…”