Structural and functional studies of human porphyrin transporter ABCB6 and Aeribacillus pallidus γ-carbonic anhydrase

Abstract

Part I: Structural and functional analysis of human porphyrin transporter ABCB6 using cryo electron microscopy ATP-binding cassette transporter B6 (ABCB6), a protein essential for heme biosynthesis in mitochondria, also functions as a heavy metal efflux pump. Here, I present cryo-electron microscopy structures of human ABCB6 bound to a cadmium Cd(II) ion in the presence of antioxidant thiol peptides glutathione (GSH) and phytochelatin 2 (PC2) at resolutions of 3.2 and 3.1 Å, respectively. The overall folding of the two structures resembles the inward-facing apo state but with less separation between the two halves of the transporter. Two GSH molecules are symmetrically bound to the Cd(II) ion in a bent conformation, with the central cysteine protruding towards the metal. The N-terminal glutamate and C-terminal glycine of GSH do not directly interact with Cd(II) but contribute to neutralizing positive charges of the binding cavity by forming hydrogen bonds and van der Waals interactions with nearby residues. In the presence of PC2, Cd(II) binding to ABCB6 is similar to that observed with GSH, except that two cysteine residues of each PC2 molecule participate in Cd(II) coordination to form a tetrathiolate. Structural comparison of human ABCB6 and its homologous Atm-type transporters indicate that their distinct substrate specificity might be attributed to variations in the capping residues situated at the top of the substrate-binding cavity.|Part II: Crystal structure of γ-carbonic anhydrase from the polyextremophilic bacterium Aeribacillus pallidus The polyextremophilic bacterium Aeribacillus pallidus produces a thermo- and alkali-stable γ-carbonic anhydrase (γ-apCA), a homotrimeric metalloenzyme containing a zinc ion in its active site that catalyzes the reversible hydration of carbon dioxide (CO2). In this study, I determined the first crystal structure of γ-apCA at 1.7 Å resolution, revealing two trimers in the asymmetric unit. The overall structure is consistent with other γ-CAs, where each monomer adopts a prism-like structure consisting of an N-terminal left-handed β-helix and a C-terminal α-helix. The active site, located at the interface between two monomers, coordinates the zinc ion with three histidine residues (H65, H82, and H87) and a water molecule in a tetrahedral configuration. Structural comparison indicates that the amino acid composition at the active site of γ-apCA differs significantly from the prototypic γ-CA from Methanosarcina thermophila. This variation likely accounts for the lack of measurable CO2 hydration activity in γ-apCA. Additionally, the structure reveals non-catalytic zinc and sulfate ions trapped at the trimer core and trimer-trimer non-crystallographic interfaces. These may contribute to stabilizing enzyme assembly and promoting crystal packing.