Coil 2 of intermediate filaments: its complete structure and impact of desminopathy-related mutations.

The eukaryotic cytoskeleton consists of three major filamentous networks. The thinnest filament network of about 6 nm diameter is made up of globular actin subunits, whereas the thicker microtubule network of about 22 nm diameter contains 13 protofilament subunits of globular [alpha]- and [beta]-tubulin arranged in a helical manner. This thesis deals with intermediate filaments (IFs) of about 15 nm diameter. IFs have a long [alpha]-helical rod domain flanked by variable in length and non-helical N-terminal ‘head’ and C-terminal ‘tail’ domains. The elementary dimer forming the rod domain has an underlying heptad repeat sequence characteristic for left-handed coiled-coils. This heptad repeat, however, discontinues at three positions called linker L1, L12 and L2. The linker L1 divides the coil 1 into 1A and 1B subdomains and the linker L2 the coil 2 into 2A and 2B. The linker L12 is found between coil 1 and coil 2. In contrast to microfilaments and microtubules, IFs like vimentin build up from elongated fibrous protein dimers. These dimers laterally assemble into tetramers and further into unit-length filaments (ULFs). ULFs then anneal longitudinally, and after a radial compaction step form mature IFs filaments.
Within this thesis, the dimer structure of vimentin was further investigated using a ‘divide-and-conquer’ approach (Strelkov, 2001). For this, seven different vimentin (D1-D7) fragments covering the predicted linkers L12 and L2 were expressed in E. coli and screened towards crystallization. One of them, the fragment D3, yields crystals suitable for X-ray diffraction. The resulting 2.4Å structure covers the regions of the vimentin molecule formerly designated as coil 2A, linker L2 and the beginning of coil 2B. We show that this region forms a single, contiguous [alpha]-helix and thus, there is no linker L2. Further, the N-terminal part of this fragment starting with Pro263 and ending with Ala302 is not twisted and should be designated as a parallel [alpha]-helical bundle rather than a coiled-coil. The rest of coil 2 is a regular left-handed coiled-coil with the exception of a small unwound region, the stutter, at position 351. The considerable overall sequence similarity of D3 to other IFs (80% for NFL and 60% for keratin types and nuclear lamins) clearly suggests a conserved structural feature to all IF proteins.
We also investigate the impact of some myopathic point mutations in the IF desmin. For this, desmin wild-type (wt) and six mutant fragments (L345P, A360P, L370P, L385P, deltaN366 and E401S) homologous to the previously crystallized vimentin cys2 and lamin A lam1 fragments were expressed in E. coli. With analytical ultracentrifugation and gel-filtration profiles, we show that wt and L345P mutant fragment form dimers in solution, whereas the mutant fragments A360P and E401S are mostly monomeric. Further, we show by circular dichroism that all our fragments were [alpha]-helical. Since our attempts to crystallize these fragments were unsuccessful, we decided to investigate the desmin wt assembly versus desmin myopathic mutants by small angle X-ray scattering (SAXS). For this, we triggered full-length desmin assembly by adding salts to a low ionic strength buffer known to prevent desmin polymerization. Our SAXS measurements in low ionic strength buffer show that almost all mutants are similar to desmin wt, but exhibit more variability with increased ionic strength buffers. In high ionic strength buffer, about half of our samples turned into gel and could not be analyzed by SAXS. Based on SAXS measurements, we were able to classify our mutant into three groups; one group of desmin mutants (L345P, R406W, R350P, A360P, deltaN366, A377P, N342D) behaves as the wt, a second mutant group (E413K, A357P, deltaE359-S361, L385P, E245D, A213V, D399Y, I451M) has an increased assembly upon ionic strength compared to desmin wt and a third group is composed of two outlier mutants (Q389P forming a gel in a low ionic strength buffer and L370P having a broader cross-section diameter than wt already in tetramer buffer).