Protein assemblies adopt both transient and permanent complex structures that can vary strongly in overall architecture. Complexes that adopt open helical symmetries often switch dynamically between assembly and disassembly and thus require factors that regulate complex formation. Structural studies on such systems benefit from an integrated methodological approach to gain insights into the relevant mechanistic and physiological features of the complex. This thesis focuses on two proteins: one directly modulates and regulates the assembly of microtubules in plant cells, while the other assembles to functional filaments that are involved in a signalosome pathway of the human innate immune response. Microtubules are protein assemblies that are essential for cell proliferation, growth and transport of cargo in eukaryotic cells. In plants, the cortical microtubule network directs the synthesis of cellulose, a fundamental component of the cell wall, which provides the bulk of plant biomass. Microtubule-associated proteins (MAPs) play an important role in maintaining the organization, structure and dynamics of the microtubule array. The MAP companion of cellulose synthase (CC) supports cellulose synthesis during salt stress by promoting the formation of a microtubule array with increased stress tolerance and by regulating cellulose synthase localization in Arabidopsis thaliana. Strikingly, the cytosolic N-terminus of CC1 (CC1∆C223) is sufficient to facilitate both microtubule reassembly and cellulose synthesis during salt stress. This thesis outlines the molecular mechanism for how CC1∆C223 binds to and bundles microtubules to sustain cellulose synthesis under conditions of high salinity. Solution-state nuclear magnetic resonance (NMR) spectroscopy was employed to characerize the structural features of CC1∆C223 and its interaction with microtubules. Chemical shifts of backbone carbon, nitrogen and protons were assigned by combining 3D and 4D triple-resonance NMR experiments with non-uniform sampling (NUS). Free CC1∆C223 in solution is intrinsically disordered but the carbon chemical shifts hint at several regions with enhanced propensity for β -strand secondary structure. The addition of microtubules to isotopically-enriched CC1∆C223 resulted in reversible and residue-specific line broadening effects, stemming from the reversible association of CC1∆C223 to the microtubule surface, where it experiences fast transverse relaxation due to the long rotational correlation time of the complex. Interestingly, the results showed that CC1∆C223 binds with four hydrophobic and conserved linear motifs that are connected by flexible linker regions. Peptides that each contained one microtubule-binding motif retained microtubule-binding activity and STD-NMR experiments indicated strong contributions of aromatic side chains to the overall binding. The mutation of two key tyrosine residues in the N-terminal binding region reduced the binding affinity in vitro and resulted in a salt-sensitive phenotype in vivo. Electron microscopy analysis showed that CC1∆C223 induces bundling of microtubules in a concentration-dependent manner, and fluorescence microscopy revealed that CC1∆C223 can diffuse on microtubules bidirectionally. Cross-linking of CC1∆C223 and tubulin dimers combined with mass spectrometry analysis suggested binding of the protein at the protomer interfaces along the microtubule lattice and the hydrophobic pocket longitudinally between tubulin dimers. The microtubule-binding behaviour of CC1∆C223 is reminiscent of that of the neuropathology- related and non-homologous protein Tau, which also bundles and diffuses on microtubules in a highly dynamic manner. The microtubule-binding motifs of CC1∆C223 share some remarkable similarities in hydrophobicity, size, sequence and spacing with the microtubule- binding regions of Tau. Hence, CC1 sustains microtubule organization and cellulose synthase localization during salt stress via a Tau-like mechanism that may have evolved independently. Signalosomes are higher-order intracellular protein assemblies that play important roles in several signalling cascades of the innate immune system. The filamentous core of signalosomes typically consists of proteins containing death domains like CARD, PYD or DD that, through assembly of the filament, link the upstream danger signal to the downstream enzyme-driven pathway. The second part of the thesis focuses on assemblies formed by the CARD domain of the adaptor protein RIP2 that are initiated by the cytosolic receptor NOD2. Since RIP2 forms insoluble filaments via its CARD domain, solid-state NMR spectroscopy was employed to study the structure of the RIP2CARD assembly. To obtain the backbone resonance assignments, proton-detected experiments on 2H, 13C, 15N-labelled and 100 % back-exchanged RIP2CARD samples were acquired at 60 kHz magic angle spinning (MAS). These data were evaluated together with carbon-detected 13C-13C DARR correlations on protonated samples that were either uniformly 13C-labelled or selectively [2-13C]- or [1,3- 13C]-glycerol labelled, yielding the assignment of backbone and side-chain resonances. The chemical shifts of the assigned residues of filamentous RIP2CARD closely matched the chemical shifts of monomeric RIP2CARD in solution, showing that the overall conformation is maintained upon filament formation. The solid-state MAS NMR data yielded no signals from the C-terminal segment of the protein, which typically contains a helix in the CARD fold. This result corroborates the lack of ordered structure in this region reported already by the structure of the monomeric RIP2CARD domain and indicates that the local disorder is retained in filamentous RIP2CARD. The structure of the RIP2CARD filament, solved by cryogenic electron microscopy (cryo- EM), has a helical configuration that is similar to other CARD filaments of the innate immune system. The most significant chemical shift differences between RIP2CARD in solution and within the filament map to the subunit interfaces of the assembly structure. These chemical shift differences report on local conformational changes due to packing effects in the filament and therefore independently confirm the overall architecture of the assembly structure. The results give important structural insights into the NOD2-RIP2 pathway and highlight the importance of RIP2 polymerization for the signalling mechanism. Moreover, the work paves the way for future research on the structural aspects of the NOD2CARDs-RIP2CARD interface and the regulation of filament formation.
