Abstract 1344 Role of GXXXG Dimerization Motif and Lipid-Protein Interactions in the Functional Modulation of Glutamate Carboxypeptidase II

Glutamate Carboxypeptidase II (GCPII) is a transmembrane, zinc metallopeptidase found in many organisms including humans. Human GCPII (hGCPII) is expressed as five paralogs in multiple tissues including the brain, prostate, and intestine, and exhibits tissue-specific hydrolase activities. GCPII dimerization on the plasma membrane is essential for the reported enzyme activities. Tissue-specific functional modulation and mechanisms of enzymatic and other functions, such as cell signaling, remain unknown. Comparative amino acid sequence analyses reveal the presence of a GXXXG dimerization motif, where "X" is a hydrophobic residue, in all the transmembrane domain (TMD) of the hGCPII paralogs. We hypothesize that tissue-specific functions of GCPII are modulated via GXXXG motif-mediated protein dimerization through tissue-specific lipid-protein interactions. Computational and Förster Resonance Energy Transfer (FRET)-based molecular dynamic studies of the transmembrane domains of hGCPII paralogs in lipid environment have been carried out to characterize these interactions. Ab initio density functional theory calculations show the nature of the GXXXG amino acid sequence and the GXXXG motif-flanking amino acid sequence influence dimer stability through altered hydrogen bonding networks. Data analyses reveal that unique combinations of lipid-protein and protein-protein intermolecular interactions are necessary for the formation of stable dimers. Overexpressed GCPII-TMDs, containing GFLFG motif and Superfolder mTurquoise2ox-mNeonGreen FRET pair (sfTq2ox-mNG), were incorporated into lipid nanodisc to study the lipid-protein and protein-protein interactions and the influence of GXXXG motif on GCPII dimerization. Collectively, both computational and nanodisc-based studies proved to be useful methods for understanding the molecular factors controlling the GXXXG motif mediated protein dimerization in GCPII and its role in functional modulation.