Abstract
The sodium-dependent citrate transporter of Klebsiella pneumoniae (KpCitS) belongs to the 2-hydroxycarboxylate transporter (2-HCT) family and allows the cell to use citrate as sole carbon and energy source in anaerobic conditions. Here we present crystal structures of KpCitS in citrate-bound outward-facing, citrate-bound asymmetric, and citrate-free inward-facing state. The structures reveal that the KpCitS dimerization domain remains stationary throughout the transport cycle due to a hydrogen bond network as well as extensive hydrophobic interactions. In contrast, its transport domain undergoes a ~35° rigid-body rotation and a ~17 Å translocation perpendicular to the membrane to expose the substrate-binding site alternately to either side of the membrane. Furthermore, homology models of two other 2-HCT proteins based on the KpCitS structure offer structural insights into their differences in substrate specificity at a molecular level. On the basis of our results and previous biochemical data, we propose that the activity of the 2-HCT CitS involves an elevator-like movement in which the transport domain itself traverses the lipid bilayer, carrying the substrate into the cell in a sodium-dependent manner.
Highlights
The 2-hydroxycarboxylate transporter (2-HCT) family of transporters generally translocate molecules with a 2-hydroxycarboxylate motif (HO-CR1R2-COO−), such as citrate, malate and lactate across the plasma membrane, and activity is tightly coupled to energy provided by a sodium or proton gradient as a form of secondary active transport[1, 2]
We have studied the molecular mechanism of CitS in the 2-HCT family by determining multiple crystal structures along the transport cycle
Our results show that the KpCitS has a homodimeric structure in which its dimerization domain remains fixed during the transport cycle due to a hydrogen bond network as well as extensive hydrophobic interactions, while the substrate-loaded transport domain undergoes an elevator-type translocation across the membrane
Summary
2-HCT family in more detail, we sought to determine the KpCitS structure by X-ray crystallography in different functional states. In the outward-facing state, the hairpin loops are located close to the membrane/periplasm interface, which appears to favor the entry of external substrate and ions (Fig. 3a, left) This is consistent with mutational and cross-linking data showing that the hairpin loops, which contain conserved GGxG motives, are positioned close together to form the binding sites for the citrate and sodium ions[24, 25]. S405 of KpCitS is changed to threonine at positions 402 and 384 of LmCitP and LlMleP, respectively, where it mediates the invariant hydrogen bonding with the hydroxyl group of the 2-hydroxycarboxylate of citrate (Fig. 4) This is consistent with data, showing that removal of the hydroxyl group reduces the affinity for substrate of these proteins[1]. M173 of LlMleP is located in a position such that it can directly collide with the 3-carboxylate group of citrate, which accounts for why the latter is a substrate of LmCitP but not of LlMleP (Fig. 4, right)[39]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
