Enzyme-driven catalysis controls most metabolic pathways in all living organisms, yet is poorly understood. Enzymatic failures lead to genetic disease, such as phenylketonuria, and cancers such as xeroderma pigmentosum. Although much progress has been made in small molecule and antibody catalysis, drug design efforts have been impeded by lack of understanding of the remarkable specificity and catalytic power of naturally occurring enzymes.
We aim to develop an entirely new structural technique based on the world's first X-ray free electron laser (xFEL), to track enzyme conformational changes with unprecedented structural resolution for non-crystalline samples. The xFEL, which has been developed at the SLAC National Accelerator lab as the Linac Coherent Light Source, or LCLS delivers pulse of x-rays which last a few tens of femtoseconds (10-15 seconds) and deliver about 2mJ of energy in a single pulse. The laser has already shown its ability to obtain terabytes of structural information on nano crystals of biomolecules. The present proposal is to do measurements on droplets of solution of biomolecules in non-crystalline form in which enzyme molecules can go through their catalytic cycles under close to physiological conditions. Rapid mixing of enzyme and substrate will be followed by injection into the x-ray laser beam giving a series of snapshots of changes in conformation of the enzyme which will help in design of drug molecules which can intervene in the functioning of the enzyme. By combining the measurements with computer simulations of the structural intermediates which are activated during the enzymatic cycle, we will be able to build a roadmap of the catalytic function that will provide a free energy landscape to guide enzyme engineering and drug design efforts. This will enable us to obtain previously inaccessible information on the function of Cyclophilin A (CypA), an enzyme central to protein folding, signal transduction, trafficking, receptor assembly, cell cycle regulation and stress response. Two of CypA's most important roles in human health are in controlling immunosuppression and viral infection. CypA is the target of the widely used immunosuppressive cyclosporine. The HIV virus has been shown to use human cyclophilin during its final stages of viral replication.
Our Project will enable new detailed understanding of the mechanism of action of enzymes of importance to human health. Our Principal target will be cyclophilin A. The Use of the x-ray laser will enable of a series of snapshots of the functioning of cyclophilin A that will help in design of new drugs against HIV.
|Mendez, Derek; Lane, Thomas J; Sung, Jongmin et al. (2014) Observation of correlated X-ray scattering at atomic resolution. Philos Trans R Soc Lond B Biol Sci 369:20130315|