Engineering Dimer Mutants of Human Geranylgeranyl Pyrophosphate Synthase for Crystallographic and Functional Insights

Sean Ezekiel
PhD Student 
Department of Biochemistry

Date: January 13, 2025
Time: 1:00 p.m. to 2:00 p.m. 
Room: CSF 1302

Abstract:

The human geranylgeranyl pyrophosphate synthase (GGPPS) produces geranylgeranyl pyrophosphate, a crucial isoprenoid involved in cellular processes such as signal transduction and cell cycle regulation. Due to its role in multiple forms of cancer as well as Alzheimer’s disease, GGPPS has been studied extensively. However, drug discovery efforts have been largely unsuccessful, partly due to challenges related to the enzyme's hexameric structure, which complicates structural and drug screening studies. My work focuses on engineering dimeric forms of human GGPPS to enable high-resolution crystallographic analysis and facilitate drug screening. To this end, dimeric mutants were designed to disrupt the inter-dimer interactions critical for hexamer assembly. All mutants retained catalytic activity comparable to the wild-type enzyme under various substrate concentrations. Thermal stability analysis revealed ~10 °C lower melting temperatures in mutants, correlating with their dimeric forms. The dimeric form consistently yielded diffraction-quality crystals, enabling determination of its structure at 2.1 Å resolution. This structure provided the first visualization of isopentenyl pyrophosphate binding in human GGPPS. We also observed an unexpected consequence of the engineered mutation: intermolecular disulfide bonds formed during crystallization, potentially enhancing crystallizability but introducing artifacts that may affect inhibitor-binding studies. These findings demonstrate the potential of dimeric GGPPS mutants as structural surrogates, providing a simplified platform for drug discovery and mechanistic exploration.