Biology subjects

Orlando, B. J.

Publications and source records attributed to Orlando, B. J..

6 recordsLinked to original sources

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↗

bioRxiv2026

Conformation Driven Enhancement of Neurolysin Activity in Presence of a Small Molecule Activator

Neurolysin (Nln) is an M3 metallopeptidase that regulates neuropeptide concentration in the central nervous system. It has emerged as a therapeutic target for mitigating post-ischemic injury by hydrolyzing and inactivating several neuropeptides. It has been recently shown that small molecule activators, such as pyridine piperazine (Py-Pip) derivatives, can enhance Nln catalytic activity, facilitating hydrolysis of Nln substrate peptides. However, binding sites of these molecules and the mechanism of action remain unclear due to the dynamic nature of Nln. Here, we use molecular dynamics (MD) simulation of the apo and activator-bound Nln systems along with Markov state modeling to identify three binding sites of Py-Pip, and quantify their effects on the Nln conformational landscape and kinetics. Two of these sites have opposing functional outcomes: Binding inside the interhelical channel favors closed conformations by slowing down channel opening, thus allowing the substrates to orient favorably for the catalytic reaction, while binding at the exterior region of the channel stabilizes open states, potentially facilitating both substrate entry and cleaved product release. These findings suggest a possibility of a multi-site allosteric activation model in which distinct binding locations selectively modulate different steps within a catalytic cycle. This framework provides a structural and kinetic model for the action of small molecule Nln activators, offering insight into the design of cerebro-protective therapeutics that reduce neuropeptide accumulation after ischemic injury.

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↗

bioRxiv2024

Predicting Inhibitors of OATP1B1 via Heterogeneous OATP-Ligand Interaction Graph Neural Network (HOLI-GNN)

Organic anion transporting polypeptides (OATPs) are membrane transporters crucial for drug uptake and distribution in the human body. OATPs can mediate drug-drug interactions (DDIs) in which the interaction of one drug with an OATP impairs the uptake of another drug, resulting in potentially fatal pharmacological effects. Predicting OATP-mediated DDIs is challenging, due to limited information on OATP inhibition mechanisms and inconsistent experimental OATP inhibition data across different studies. This study introduces Heterogeneous OATP-Ligand Interaction Graph Neural Network (HOLIgraph), a novel computational model that integrates molecular modeling with a graph neural network to enhance the prediction of drug-induced OATP inhibition. By combining ligand (i.e., drug) molecular features with protein-ligand interaction data from rigorous docking simulations, HOLIgraph outperforms traditional DDI prediction models which rely solely on ligand molecular features. HOLIgraph achieved a median balanced accuracy of over 90 percent when predicting inhibitors for OATP1B1, significantly outperforming purely ligand-based models. Beyond improving inhibition prediction, the data used to train HOLIgraph can enable the characterization of protein residues involved in inhibitory drug-OATP interactions. We identified certain OATP1B1 residues that preferentially interact with inhibitors, including I46 and K49. We anticipate such interaction information will be valuable to future structural and mechanistic investigations of OATP1B1. Scientific ContributionHOLIgraph introduces a new paradigm for DDI prediction by incorporating protein-ligand interactions derived from docking simulations into a graph neural net framework. This approach, enabled by recent structural breakthroughs for OATP1B1, represents a significant departure from traditional models that rely only on ligand features. By achieving high predictive accuracy and uncovering mechanistic insights, HOLIgraph sets a new trajectory for computational tools in drug design and DDI prediction.

bioengineering↗

bioRxiv2022

Development, Structure, and Mechanism of Synthetic Antibodies that Target Claudin and Clostridium perfringens Enterotoxin Complexes

Strains of the Gram-positive bacterium Clostridium perfringens produce a two-domain enterotoxin (CpE) that afflict millions of humans and domesticated animals annually by causing prevalent gastrointestinal illnesses. CpEs C-terminal domain (cCpE) binds cell surface receptors then its N-terminal domain restructures to form a membrane-penetrating {beta}-barrel pore, which is toxic to epithelial cells of the gut. The claudin family of membrane proteins are the receptors for CpE, and also control the architecture and function of cell/cell contacts called tight junctions that create barriers to intercellular transport of solutes. CpE binding disables claudin and tight junction assembly and induces cytotoxicity via {beta}-pore formation, disrupting gut homeostasis. Here, we aimed to develop probes of claudin/CpE assembly using a phage display library encoding synthetic antigen-binding fragments (sFabs) and discovered two that bound complexes between human claudin-4 and cCpE. We established each sFabs unique modes of molecular recognition, their binding affinities and kinetics, and determined structures for each sFab bound to ~35 kDa claudin-4/cCpE in three-protein comprised complexes using cryogenic electron microscopy (cryoEM). The structures reveal a recognition epitope common to both sFabs but also that each sFab distinctly conforms to bind their antigen, which explain their unique binding equilibria. Mutagenesis of antigen/sFab interfaces observed therein result in further binding changes. Together, these findings validate the structures and uncover the mechanism of targeting claudin-4/cCpE complexes by these sFabs. Based on these structural insights we generate a model for CpEs cytotoxic claudin-bound {beta}-pore that predicted that these two sFabs would not prevent CpE cytotoxicity, which we verify in vivo with a cell-based assay. This work demonstrates the development and targeting mechanisms of sFabs against claudin/cCpE that enable rapid structural elucidation of these small membrane protein complexes using a cryoEM workflow. It further provides a structure-based framework and therapeutic strategies for utilizing these sFabs as molecular templates to target claudin/CpE assemblies, obstruct CpE cytotoxicity, and treat CpE-linked gastrointestinal diseases that cause substantial economic and quality of life losses throughout the world.

biophysics↗

bioRxiv2019

Structure and function of a 9.6 megadalton bacterial iron storage compartment

Iron storage proteins are essential for maintaining intracellular iron homeostasis and redox balance. Iron is generally stored in a soluble and bioavailable form inside ferritin protein compartments. However, some organisms do not encode ferritins and thus rely on alternative storage strategies. Encapsulins, a class of protein-based organelles, have recently been implicated in microbial iron and redox metabolism. Here, we report the structural and mechanistic characterization of a 42 nm two-component encapsulin-based iron storage compartment from Quasibacillus thermotolerans. Using cryo-electron microscopy and x-ray crystallography, we reveal the assembly principles of a thermostable T = 4 shell topology and its catalytic ferroxidase cargo. We show that the cargo-loaded compartment has an exceptionally large iron storage capacity storing over 23,000 iron atoms. These results form the basis for understanding alternate microbial strategies for dealing with the essential element iron.

biochemistry↗