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Manipulating folding, assembly and disassembly of protein complexes - from molecule to disease

Final Report Summary - MANIFOLD (Manipulating folding, assembly and disassembly of protein complexes - from molecule to disease)

ITN-IDP ManiFold
http://www.manifold-idp.nl

Protein folding and misfolding
- We demonstrated that Hsp90 interacts with CFTR. He has show that this interaction is directed towards the CFTR NBD1 domain and promotes its correct folding. These results resulted from the implementation of an immunoprecipitation method of the chaperone Hsp90 with CFTR. ESR3 also characterized the folding pathway of NBD1, by developing an in-vitro translation system coupled to protease susceptibility assay that allows to probe for conformational states and the impact of Cystic Fibrosis disease-causing mutations, such as ΔF508, in the folding of NBD1. This research has impact on the field as this assay has been used in collaboration with the company ProQR, to determine the mode of action of some of their therapeutical molecules.
- The action of several promising novel oligomer modulators was tested in model membrane systems. Anle145c, identified as a potential amyloid inhibitor, acts effectively on IAPP aggregation both in the absence and presence of membranes. The inhibitor targets another oligomeric species in the presence of membranes than in the absence of membranes. The mechanism in the presence of membranes is reminiscent of in-vivo findings, suggesting that membranes play an important role in the mode of action of inhibitors.
- A new purification protocol for Hsp90, which allows quick and effective purification Hsp90 and results in a stable sample. Codon optimised Tau gene was designed, which makes possible to make other constructs (eg. mutants) with only subcloning. Successful cloning of codon optimised Tau into pSUMO vector was performed. We cloned and purified the co-chaperones that regulate the ATPase cycle and also link the chaperone machinery to the proteasome.
- Fluorescence experiments, titrating Hsp90 with its natural substrate Tau and the next step is to compare this Tau affinity with designed mutants in the binding interface, and the variation that they can produce in the binding site. We established a novel concept for the mechanism of action of the Hsp70-Hsp90 protein folding cascade.
- We showed that Hsp90 is the key factor to complete folding downstream from the folding path. The folding process of substrates is assisted by chaperones in early stages of the folding reaction. Consequently, substrates evolve towards the native state on their own afterwards.

Complex multi-protein assemblies
- By combining mass spectrometric techniques and HADDOCK modeling, the Heck lab recently investigated the structure of the KaiBC complex, elucidating a 6:6 stoichiometry and defining the interacting regions of the proteins (Snijder et al., PNAS, 2014). The KaiABC complex was initially investigated with native MS. Subsequently, we probed the complex with cross-linking/MS, applying classical amine-reactive cross-linking as well as the recently developed concept of acidic residue cross-linking at physiological pH. Additionally, we are preforming cryo-EM experiments to obtain high resolution structural models of the KaiBC and KaiABC complexes.
- We used Flock House Virus and its VLPs as a model system, Studying RNA packaging and gamma-protein release of non-enveloped virus by mass spectrometry. We have results constitute one of the first native MS studies on wild-type infectious virions, thus representing an important step forward in application of native MS to biological systems. We also investigate the architecture of an assembled terminase complex, which is involved in viral genome packaging process. Using the S-terminase: L-terminase complex of the Salmonella phage P22 as an example, we present the first direct visualization of a purified viral terminase complex analyzed in the absence of DNA and procapsid.
- Interactions between the C-terminal acidic domain of histone chaperone and DNA repair factor APLF with core histone complexes were studied by microscale thermophoresis (MST) and isothermal titration calorimetry (ITC) to characterise the interactions. MST results point at two binding sites on APLF for both histone dimer and tetramer with similar affinities. ITC results point at one binding site on the histone dimer and two binding sites on the histone tetramer with different affinities for APLF. The two individual binding sites of APLF were mapped by NMR spectroscopy titration experiments. Currently, NMR experiments are being performed to test for structural changes in APLF upon binding the histone complexes.

Folding and assembly of membrane proteins
- Grp94 is an ER-resident chaperone of the Hsp90 family and is involved in protein folding, quality control, stress response, embryo and muscle development, antigen presentation and calcium buffering. Identification of its mechanism of action and its possible co-chaperones, such as pERps, will help in the design of new drugs that could be used for several protein folding diseases. Due to systematic study of Grp94, we have strong indications about how Grp94 acts exactly.
- The full-length ectodomain of Lrp1 (about 600 kDa) was expressed and purified. MALLS confirmed the molecular size of this very large protein. EM negative stain images of Lrp1 ectodomain were obtained showing different conformations due to high flexibility of the receptor. SPR studies showed high affinity binding of Lrp1 to prototypically ligands RAP and coagulation proteins. SAXS studies performed on 'free' Lrp1 vs. Lrp1-ligand complexes at medium and low pH revealed different receptor states that may be related to uptake of ligand cargo from the surrounding medium and release of cargo in the acidic endosomes of the cell.
- All proteins involved in the Sortilin/SorCS2 complexes were successfully cloned, expressed and purified. Structures were obtained for part of the complexes which helped to gain insight into the signalization through these receptors. Complementary techniques are being used for further understanding of the involved mechanisms. Structure based mutants have been designed and produced to confirm the supposed interaction mechanism. The combination of these structural and biophysical techniques with cellular assays will provide molecular understanding of Sortilin and SorCS2 receptor signaling.
- KcsA was isolated in native nanodiscs, novel lipid–protein particles that are stabilized by an amphipathic co-polymer of styrene and maleic acid (SMA). Applying SMA for solubilisation yields more stable protein compared with conventional solubilisation approaches using detergents. Further, the lipid composition of the immediate membrane environment was directly determined. It was also shown that this method can be exploited to reconstitute functional protein into artificial membranes without ever exposing it to detergent.