A major aim of our research group is to understand what molecular features qualify a membrane protein as a substrate for intramembrane proteolysis. Answering this question is not only essential to elucidate the physiological function of a protease and evaluate its potential as a drug target but also to understand how a protease recognizes its substrates and cleaves them mechanistically and to elucidate how a protease differs from related proteases. In order to address this question it is necessary to identify the membrane proteins that are cleaved by a protease (substrates) and those membrane proteins that are not cleaved (non-substrates).

Combining methods from biochemistry (such as domain swap experiments, together with P4 and P6), bioinformatics (sequence comparisons, together with P7) and biophysics (analyzing structural parameters, together with P5 and P8) will then allow elucidating the molecular characteristics that qualify a membrane protein as a protease substrate. Among the four major families of intramembrane proteases – rhomboids, SPP/SPPLs, g-secretase and S2Ps – we focus on three for substrate identification and mechanistic analysis, namely PARL, the SPPL2 subfamily and γ-secretase.

Recent data suggest that intramembrane proteolysis is a surprisingly slow process whose overall kinetics is composed of the kinetics of several steps. These steps may include:

• substrate recognition by an exosite on the protease,
• subsequent conformational changes of the substrate/enzyme complex to expose the scissile amide bonds to the catalytic residues,
• formation of the tetrahedral intermediate leading to hydrolysis, and
• product release

It is currently unclear which of the above step(s) limit the rate of proteolysis, how the ratelimiting step(s) depend on the primary structures and/or the conformational dynamics of the substrates, how disease-related point mutations affect these properties and which step(s) are relevant for substrate/non-substrate discrimination. Furthermore, we do not know whether all intramembrane proteases employ exosites for substrate binding, where they are located, and whether substrates are recognized and/or cleaved as monomers or multimers.

Although no structure of a substrate/enzyme complex is currently available, it is informative to study the intrinsic dynamics of isolated substrates. This is justified by the observation that an isolated protein already exhibits the types of structural changes that may occur after binding to other proteins. Helices are indeed quite adaptable as almost half of the TM domains within crystallized membrane proteins contain non-canonical Elements and exhibit different curvatures.

 A combined experimental and computational effort will be directed at unravelling the fine details of TM helix flexibility for two paradigmatic substrates, C99 and PINK1.

Several backbone NMR structures have been published for C99  while PINK1 is completely uncharted territory.

In sum, this will show the commonalities and the differences in structural dynamics of substrate TM helices that are proteolyzed by different enzymes i) by providing descriptors of the global TM domain dynamics, i.e., bending, twisting and stretching modes location of hinges, and ii) by characterizing local features like side-chain packing, hydrogen-bond occupancies, local hydration and cooperative local unfolding at cleavage sites. Importantly, these analysis will also reveal the structural and dynamical impact of mutations that alter proteolytic processing by linking them to experiments in Aim 2. This will allow us to extract those conformational and dynamical features that are relevant at different steps of substrate proteolysis!

Project P7: Christina Scharnagl, Dmitrij Frishman

Sequence requirements, conformational flexibility, and functional networks of intramembrane protease substrates

Project P8: Burkhard Luy, Daniel Huster

Investigation of Molecular Dynamics of Substrate Transmembrane α-Helices by Solution and Solid-State NMR Spectroscopy