As a part of innate immunity, the eukaryotic cell reports on the fidelity of its protein folding machinery by presenting specific peptides on its cell surface that are recognized by Natural Killer cells. These peptides are remnants of signal peptides (SPs) derived from newly synthesized histocompatibility complex 1b (MHC-1b) molecules, and signal to NK cells that protein synthesis is normal. The enzyme responsible for trimming signal peptides to liberate them from the ER membrane is a membrane-embedded aspartyl protease, signal peptide peptidase (SPP). In addition MHC Class 1b, human SPP substrates include SPs from proteins involved in immune response and muscle contraction. SPP is also hijacked by the Hepatitis c virus for replication, and is related to presenilin, which uses similar chemistry to generate amyloidogenic peptides in Alzheimer Disease. SPP and presenilin form one of just three superfamilies of intramembrane proteases. The objective of this proposal is to solve the first high resolution X-ray crystal structure of SPP, providing the first snapshot of an intramembrane aspartyl protease (IAP). The details of regulated intramembrane proteolysis, from cell biological signaling to active site chemistry, are of both fundamental biochemical importance and potential therapeutic application. How substrates are presented and hydrolyzed within the confines of the hydrophobic space of the lipid membrane remain largely a mystery. The structure of SPP will contribute not only to the biology of proteostasis and intramembrane proteolysis, but also to our knowledge of membrane proteins, few structures of which are known to high resolution. We will solve the structure of SPP as a complex with a single chain antibody fragment (scFv) chaperone. The scFv will be engineered for tight binding to a peptide epitope while retaining the scaffold of an scFv known to use crystal contacts remote from the epitope binding site. We expect that our tightly bound crystallization chaperone will immobilize an SPP loop and provide a stable crystal lattice, leading to better diffracting crystals. In contrast to previous work with crystallization chaperones, which employed affinity reagents specific to the membrane protein of interest, this technology is potentially transformative as the epitope can be incorporated into any membrane protein crystallization target. The expected outcome is the molecular architecture of IAPs, including the location of the active site and any substrate-docking patches. The SPP structure will provide insight into biochemistry and function for comparison with well-studied soluble aspartyl proteases and a basis for directed biochemical studies to evaluate key residues that modulate catalysis. In the long-term this knowledge is likely to facilitate drug development for immune modulation or agents against the Hepatitis C virus.
Signal peptide peptidase is an essential lipid-submerged enzyme involved in innate and adaptive immunity, and is implicated in a variety of diseases, including Alzheimer's and Hepatitis C viral infection. The work proposed here aims to determine the first molecular structure of SPP in order to understand its role in health and disease, and aid in development of specific inhibitors as potential Alzheimer's and Hepatitis C therapies.
