Kristallographische Untersuchungen zur Schweren Kette von Dynein und dem Capping-Protein Cap32/34

Abstract

Intracellular movements are an essential prerequisite for material transport, cell division and food ingestion. They can be mediated through two different ways. Firstly, through motor proteins, like dynein, kinesin and myosin, which transform the energy stored in ATP into directed movement to, for instance, facilitate organelle transport along microtubule- and actin-filaments. Secondly, via ATP-dependent polymerization of actin and tubulin into polarized filaments and, thereby, generating directed movement. Therefore, the precise regulation of the cytoskeleton, for example, of F-actin through actin-binding proteins, such as capping protein (CP), is of central significance. The first part of this work focused on the structural characterization of the heavy chain of dynein (DHC), a microtubule-associated motor protein complex, from human and Dictyostelium discoideum by means of X-ray-crystallography. With a molecular mass of around 520 kDa, the DHC belongs to one of the largest proteins in eukaryotic cells. Due to its size, so far, only a small part of the DHC has been solved at atomic resolution. In order to obtain protein in amounts sufficient for structural studies, the DHC was systematically subdivided into smaller sections and the constructs were subsequently expressed in the lower eukaryote Dictyostelium discoideum. Altogether, numerous sections of the DHC could be produced in large amounts. For two constructs, each of which included domains essential for dynein force generation, crystals were obtained. However, although extensive optimization attempts were performed, the crystals diffracted to only low resolution and, thus, were not suitable for atomic structure solution. In the second part of this work, the crystal structure of Cap32/34 from Dictyostelium discoideum - the first high resolution structure of a cytoplasmic capping protein - is reported. The structure was solved at 2.2 Å via the method of molecular replacement. The heterodimer has the shape of a mushroom, comprising a stem and a cap. Despite sharing only low sequence-identity, the two subunits have very similar secondary- and tertiary-structures, resulting in a pseudo two-fold rotational axis of the Cap32/34 molecule. Comparisons with the crystal structure of muscle-specific CP (GgCapZ) revealed two marked differences between the isoforms, whose overall structures turned out to be strongly conserved. While a basic loop region could not be integrated into the structure of Cap32/34, due to a high degree of flexibility, the corresponding segment is highly ordered in GgCapZ. Therefore, it can be assumed that this part of the molecule might be involved in the interaction with negatively charged binding partners in Cap32/34 and possibly in every cytoplasmic capping protein. Possible candidates are F-actin and phospholipids. Moreover, the CP-variants greatly differ in another loop-region, not only in structural regard, but also in terms of flexibility. In GgCapZ, this segment is characterized by a substantially higher temperature factor, and - since it is solvent-exposed - it might be involved in the interaction with the Z-disc of the sarcomere, where the protein is specifically located at.