Characterization of the Dynein-Dynactin Interaction
by Peggy Findeisen
Date of Examination:2014-08-01
Date of issue:2015-02-13
Advisor:PD Dr. Martin Kollmar
Referee:Prof. Dr. Reinhard Lührmann
Referee:Dr. Hans Dieter Schmitt
Persistent Address:
http://dx.doi.org/10.53846/goediss-4851
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Abstract
English
In eukaryotic cells many biological processes like cell division, the distribution of vesicles and organelles require the dynamic rearrangement of molecules and cellular components. These rearrangements are controlled by three superfamilies of motor proteins, which move along the cytoskeletal tracks. One of these motors is the cytoplasmic dynein complex, which exclusively moves along microtubules towards the minus-end thereby transporting cargo from the cell periphery back to the cell centre (retrograde transport). In order to adapt to the diverse cargoes dynein interacts with various proteins. One of them is dynactin, a large complex that is involved in almost every dynein function. The interaction of both complexes occurs between the N-terminus of two dynein intermediate chains (IC) and a coiled-coil region in the dynactin DCTN1 dimer. For the characterization of the dynein-dynactin interaction chicken DCTN1412-533 and human N-terminal IC were expressed in tandem in E. coli. Several constructs were designed in which the IC length varied between 47 and 84 amino acids. Crystallization trials were performed and a condition was found for growing large crystals with edge lengths of 100-200 µm. X-ray measurements were performed at PETRA III (DESY, Hamburg) and the best native crystal diffracted to 2.9 A. Phases were obtained at 3.4 A using the single anomalous diffraction approach and crystals consisting of selenomethionine-incorporated protein. A model of DCTN1412-533 was built by iterative cycles of manual model building and refinement of the model against the diffraction data. Surprisingly, the asymmetric unit of the crystal only contained DCTN1412-533 in coiled-coil conformation without bound IC molecules. The DCTN1412-533 structure belongs to the canonical parallel left-handed coiled-coil. Its surface is mainly negatively charged suggesting an electrostatical interaction with IC, which exhibits an alkaline pI. Analyses of the N-terminal IC sequence supported an assumption that IC might not bind as a coiled-coil but rather as separate alpha-helices onto DCTN1. Cross-linking studies were performed with BS3 to narrow down the IC binding site on DCTN1. Two binding regions could be determined, which are mainly negatively charged. This is the first structural insight into the DCTN1412-533 coiled-coil. Based on the crosslinking experiments and the assumption of two single ICs binding onto DCTN1 several binding models were determined and analysed. A possible model is the interaction of two IC molecules, which bind parallel to both DCTN1 bindings sites. A parallel/antiparallel interaction of the IC molecules is also conceivable. The localization of the dynein-dynactin interaction could be further refined compared to previous studies.
Keywords: Dynactin; Dynein; Crystallisation; Coiled-coil; Cross-linking
