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, on timescales
from nanoseconds to milliseconds. Several backbone NMR structures have been published for C99 while PINK1 is completely uncharted territory. Here, we will mimic the situation of substrate/enzyme interaction by analysis in membranes and the state of a substrate in an enzyme interior by low-polarity isotropic solvent.
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!
The main objective of the proposal is to achieve a better mechanistic understanding of intramembrane protease substrates and to extend the spectrum of known substrates by theoretical predictions. To this end we will investigate the structural and dynamical requirements of a substrate transmembrane domain and relate them to sequence using in silico modeling and bioinformatics. We thus hope to uncover the code that links protein properties to cleavability.
In Goal 1 we will use molecular dynamics simulations in order to characterize the local and global dynamics of the transmembrane domain of known g-secretase substrates. We will characterize site-specific dynamics by flexibility profiles which allow the identification of key dynamical motifs. Further, we will investigate whether gamma-secretase substrates share a common pattern of large-scale backbone dynamics and whether or not mutations affecting cleavage interfere with these global motions. The backbone dynamics of gamma-secretase substrates will be compared to that of the rhomboid substrate PINK1 and the substrates of SPPL proteases. The crucial questions to answer will be whether flexibility profiles discriminate between enzyme binding sites, cleavage sites, and hinges and how the structural dynamics of substrate transmembrane domains compares to the dynamics of non-substrate transmembrane domains to be identified by this consortium.
Goal 2 is the sequence-based prediction of structurally flexibible regions. We will develop a machine learning approach to predict structural flexibility from sequence based on two types of data: crystallographic B-factors derived from known 3D structures of transmembrane proteins and flexibility profiles generated by molecular dynamics simulations.
In Goal 3 novel substrates of intramembrane proteases will be predicted by sequence analysis and machine learning using the expanded set of substrate sequences to be determined by this consortium as well as the growing number of substrates determined by other researchers, in particular for gamma-secretase. Beyond mere sequence motifs we will exploit a broad spectrum of structural features pertaining to TM regions, including the flexibility profiles. Furthermore, we will exploit various types of genomic context, such as co-expression of substrates with their cognate proteases as well as the topology of the molecular interaction network, to uncover additional candidate substrates that do not necessarily contain recognizable cleavage site motifs.
Intramembrane proteolysis is an important mechanism of membrane protein processing with immediate relevance for the development of diseases. How this process takes place in the lipophilic environment and which details of the sequence or dynamic features of the substrates are essential for proteolysis are questions that are currently under debate.
In this project we investigate the structural and dynamical requirements of selected substrate TM helices by liquid and solid-state NMR spectroscopy, currently the only experimental techniques to study dynamics of biomolecules at atomic detail. We will begin with two model TM helices: APP, a well-studied γ-secretase, and PINK1, substrate of the rhomboid protease PARL.
In Goal 1 we will concentrate on the conformational plasticity of the TM domains in solution and membrane-mimicking environments focusing in particular on deviations from the canonical helical structure and searching for lowly populated states in exchange with the main conformation as well as the related dynamical parameters. We will directly measure dynamical parameters characterizing the TM domains such as local exchange rates and motional amplitudes for individual interatomic vectors in a range of membrane models.
In Goal 2 we will investigate the impact of mutations of the two TM domains.. Some of these mutations are already known to impair proteolysis. We will clarify how they alter TM helix flexibility.
In Goal 3 we will study how the membrane influences TM helix structural and dynamic properties. The expected results will be compared with molecular dynamics simulations conducted in P7 and show if the TM domain dynamics is a relevant factor for the protease recognition and/or processing. Depending on progress in goals 1-3, protocols and experiments will be applied on selected novel substrates identified in P1/P2/P3 to judge the general validity of these studies.
The proteomic platform will be an integral part of the research consortium and performs the numerous quantitative mass spectrometry measurements required for identification and validation of novel intramembrane protease substrates for projects P1, P2 and P3. The results of the mass spec analyses will be required for all projects in the consortium, e.g. for the comparative bioinformatic analyses in P7.
The platform has two high resolution Orbitrap mass spectrometers plus associated equipment, including nanoLCs, and has expertise with the required methods, such as label free quantification and different isotope label-containing methods. This includes ‘stable isotope labelling with amino acids in cell culture’ (SILAC) analyses in vitro and in vivo.