Braza, M. K. E. | Search bioRxiv

bioRxiv2025

Conformational dynamics and activation of membrane-associated human Group IVA cytosolic phospholipase A2 (cPLA2)

Cytosolic phospholipase A2 (cPLA2) associates with membranes where it hydrolyzes phospholipids containing arachidonic acid to initiate an inflammatory cascade. All-atom molecular dynamics simulations were employed to understand the activation process when cPLA2 associates with the endoplasmic reticulum (ER) membrane of macrophages where it acts. We found that membrane association causes the lid region of cPLA2 to undergo a closed-to-open state transition that is accompanied by the sideways movement of loop 495-540, allowing the exposure of a cluster of lysine residues (K488, K541, K543, and K544), which binds the allosteric activator PIP2 in the membrane. The active site of the open form of cPLA2, containing the catalytic dyad residues S228 and D549, exhibited a three-fold larger cavity than the closed form of cPLA2 in aqueous solution. These findings provide mechanistic insight as to how cPLA2 ER membrane association promotes major transitions between conformational states critical to allosteric activation and enzymatic phospholipid hydrolysis.

biophysics↗

bioRxiv2024

An in vitro platform for the enzymatic characterization of the rhomboid protease RHBDL4

Rhomboid proteases are ubiquitous intramembrane serine proteases that can cleave transmembrane substrates within lipid bilayers. They exhibit many and diverse functions, such as but not limited to, growth factor signaling, immune and inflammatory response, protein quality control, and parasitic invasion. Human rhomboid protease RHBDL4 has been demonstrated to play a critical role in removing misfolded proteins from the Endoplasmic Reticulum and is implicated in severe diseases such as various cancers and Alzheimers disease. Therefore, RHBDL4 is expected to constitute an important therapeutic target for such devastating diseases. Despite its critical role in many biological processes, the enzymatic properties of RHBDL4 remain largely unknown. To enable a comprehensive characterization of RHBDL4s kinetics, catalytic parameters, substrate specificity, and binding modality we expressed and purified recombinant RHBDL4, and employed it in a Forster Resonance Energy Transfer-based cleavage assay. Until now, kinetic studies have been limited mostly to bacterial rhomboid proteases. Our in vitro platform offers a new method for studying RHBDL4s enzymatic function and substrate preferences. Furthermore, we developed and tested potential inhibitors using our assay and successfully identified peptidyl -ketoamide inhibitors of RHBDL4 that are highly effective against recombinant RHBDL4. We utilize ensemble docking and molecular dynamics (MD) simulations to explore the binding modality of substrate-derived peptides bound to RHBDL4. Our analysis focused on key interactions and dynamic movements within RHBDL4s active site that contributed to binding stability, offering valuable insights for optimizing the non-prime side of RHBDL4 ketoamide inhibitors. In summary, our study offers fundamental insights into RHBDL4s catalytic activities and substrate preferences, laying the foundation for downstream applications such as drug inhibitor screenings and structure-function studies, which will enable the identification of lead drug compounds for RHBDL4.

biochemistry↗