Hydrophobic membrane proteins perform a variety of functions in the cell, but their structures are notoriously difficult to solve. Thus, new strategies to obtain crystals of membrane proteins for structure determination are critical. The objectives of this proposal are to develop a toolbox of chaperones and use them to crystallize and solve the de novo, high resolution structure of two signal peptide peptidases (SPPs), which use catalytic aspartates to conduct hydrolysis within the lipid membrane. In contrast to work employing affinity reagents specific to the membrane protein of interest, our potentialy transformative aproach uses hypercrystallizable single chain antibody fragments (scFvs). Our chaperones are engineered for tight binding to a short epitope that can be inserted into any membrane protein. We expect that our tightly bound scFv chaperone will immobilize an SPP loop and provide a stable crystal lattice, leading to better diffracting crystals. SPPs trim signal peptides (SPs) to liberate them from the endoplasmic reticulum membrane. SPP substrates include SPs remnants derived from new histocompatibility complex 1b (MHC-1b) molecules. As a part of innate immunity, these processed peptides are presented on cell surfaces for recognition by Natural Killer cells to indicate that the cell is healthy. In addition, SPP substrates include SPs from proteins involved in immune response and muscle contraction. SPP is also hijacked by the Hepatitis C virus (HCV) for replication, and is related to presenilin, which uses similar chemistry to generate amyloidogenic peptides in Alzheimer Disease. SPP and presenilin comprise one of just three superfamilies of intramembrane proteases. 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, however, remain largely a mystery. At least 5 SPP variants have been sequenced, located in different regions of ER, and SPPs are conserved throughput biology, but there is no crystal structure yet. We will start by solving the structure an archeal homolog in complex with our chaperones as proof-of- principle, and then expand to a eukaryotic SPP, whose biomedical relevant activity is known. To date, we have engineered our first chaperone and isolated an affinity complex with SPP by gel filtration. Independently, we have grown crystals of the chaperone and SPP. However, the crystals of SPP do not diffract well enough for structure determination, and thus the cocrystalllization technology is critical. The expected outcomes are a toolbox of crystallization chaperones as well as the first molecular picture of SPP, including the location of the active site and substrate-docking patches. Taken together, this project will contribute not only to the biology of immunoregulation and intramembrane proteolysis, but also broaden our knowledge of membrane proteins and enable other membrane protein structures to be solved.
The integral membrane protein protease, SPP, is an essential protein implicated in a wide variety of diseases, including Alzheimer's and Hepatitis C virus. The work proposed here aims to develop a suite of novel technologies to aid in determination of the thre-dimensional structure of SPP and other membrane proteins of pharmacological interest. An SPP structure will help to understand its role in disease and aid in development of specific inhibitors as potential Alzheimer's and Hepatitis C therapies.
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