Orlando, M. A. | Search bioRxiv

bioRxiv2026

Structure and conformational dynamics of the Pseudomonas CbrA transceptor

The CbrA protein is a central regulator of carbon metabolism, biofilm formation, and virulence in Pseudomonas species, but the molecular mechanisms by which CbrA links nutrient sensing to downstream signaling has remained unclear. CbrA is a rare "transceptor" that combines membrane transporter and histidine kinase domains into a single functional polypeptide. The structural basis for histidine recognition and membrane transport, as well as signaling through intracellular histidine kinase domains has remained elusive. Here we determined a cryo-EM structure of CbrA which provides key molecular details of the SLC5-STAC domains in this unusual system. Unexpectedly, the small peptide CbrX encoded upstream of CbrA formed a stable complex with the SLC5 transporter domain. The structure reveals how histidine binds within the transporter, and molecular dynamics simulations provide insight into proton gradient driven conformational changes that enable histidine transport. These findings define the molecular architecture of key CbrA functional domains, and pave a path toward developing a comprehensive understanding of coupling between membrane transport and downstream signaling pathways that guide essential physiological traits in Pseudomonas. Significance StatementCbrA is a key regulator of carbon-nitrogen metabolism in Pseudomonas and is essential for successful host infection. The molecular basis for CbrAs dual role in membrane transport and downstream signaling has remained elusive. Here we determined a cryo-EM structure that defines the organization of the CbrA SLC5 and STAC domains, and reveals that the small peptide CbrX encoded upstream of CbrA forms a stable complex with the SLC5 transporter region. A structure with histidine trapped in the binding cavity, together with molecular dynamics simulations, identifies protonation dependent transitions that guide the transport cycle. This work establishes a mechanistic foundation for understanding how CbrA and related transceptors integrate substrate sensing and transport with regulatory control of signaling.

biophysics↗

bioRxiv2025

Structure and Post-Translational Modification of the Prostaglandin Transporter

The prostaglandin transporter (PGT) is a member of the Organic Anion Transporting Polypeptide (OATP) family of membrane transporters. PGT mediates the uptake of prostaglandins from the extracellular environment to enable intracellular enzymatic degradation and termination of signaling. In addition to importing prostaglandins, PGT is also an essential core component of the Maxi-Cl channel, which facilitates cellular release of ATP and other small organic anions. Despite progress on understanding the (patho)physiological roles of PGT, and development of small molecules to inhibit this transporter, molecular details of the overall structure and transport mechanism remain elusive. Here we determined the cryo-EM structure of human PGT, which demonstrates an overall topology consistent with other OATPs despite possessing a dual transporter/channel functionality. We additionally investigated the role of eight potential disulfide bonds found in the extracellular loops of PGT and paralogous transporters. Through biochemical and functional characterization we demonstrate that six intra-loop disulfide bonds (C420-C511, C450-C470, C492-C474, C459-C507, C444-C494, C580-C594) are essential for proper N-glycosylation, plasma membrane trafficking, and prostaglandin import activity of PGT. In contrast, two inter-loop disulfides (C155-C587 and C143-C448) were found to restrict maximal prostaglandin uptake, suggesting a possible regulatory role in modulating PGT activity. In total, our studies provide a fresh molecular perspective on the structure, post-translational modification, and overall function of PGT. Significance StatementThe prostaglandin transporter (PGT) is essential for cellular uptake of prostaglandins and serves as a core component of the Maxi-Cl channel. Using cryo-EM we resolved the structure of human PGT, revealing a similar overall topology as other OATP transporters. Extensive site-directed mutagenesis and functional assays identified eight disulfide bonds in PGTs extracellular domain as key regulators of glycosylation, trafficking, and transport activity. These findings provide a structural basis for PGT function and lay a foundation to further explore substrate recognition and inhibitor design for this biologically significant transporter.

biochemistry↗