Structural Requirements and Dynamics of Mitosin-Kinetochore Interaction in M Phase

Mitosin is a 350-kDa human nuclear protein which transiently associates with centromeres and spindle poles in M phase. Ultrastructure studies reveal that it is located at the outer kinetochore plate. In this work, we explored the detailed structural basis and dynamics of the mitosin-kinetochore interaction. Two major regions important for targeting to centromeres were identified by analyzing different deletion mutants expressed in CHO cells: (i) the “core region” between amino acids 2792 and 2887, which was essential for the centromere localization of mitosin; and (ii) the internal repeats between residues 2094 and 2487, which cooperated with the core region to achieve strong mitosin-kinetochore interaction. The core region is characteristic of two leucine zipper motifs. Deletion of either motif abolished the centromere localization activity. In addition, Cys²⁸⁶⁴, adjacent to the second motif, was also essential for the activity of the core region. In contrast, the internal repeats alone were insufficient for centromere localization. We propose that this region may serve as a regulatory domain to facilitate interaction of the core region with the kinetochore. We showed that mitosin molecules entering nuclei after nuclear envelope breakdown (NEBD) were not assembled onto kinetochores efficiently, suggesting that the mitosin-kinetochore interaction is stabilized prior to NEBD. This result supports the idea of an ordered process for kinetochore assembly. Our data also suggest that mitosin might interact with chromatin in interphase. Evidence for coordinated regulation between the centromere-targeting and the putative chromatin-binding activities is also provided.     

A major determinant of hnRNP C protein binding to RNA is a novel bZIP-like RNA binding domain.

The C protein tetramer of hnRNP complexes binds approximately 150-230 nt of RNA with high cooperativity (McAfee J et al., 1996, Biochemistry 35:1212-1222). Three contiguously bound tetramers fold 700-nt lengths of RNA into a 19S triangular intermediate that nucleates 40S hnRNP assembly in vitro (Huang M et al., 1994, Mol Cell Biol 14:518-533). Although it has been assumed that the consensus RNA recognition motif (RRM) of C protein (residues 8-87) is the primary determinant of RNA binding, we report here that a recombinant construct containing residues 1-115 has very low affinity for RNA at physiological ionic strength (100 mM NaCl). Moreover, we demonstrate that an N-terminal deletion construct lacking the consensus RRM but containing residues 140-290 binds RNA with an affinity sufficient to account for the total free energy change observed for the binding of intact protein. Like native C protein, the 140-290 construct is a tetramer in solution and binds RNA stoichiometrically in a salt-resistant manner in 100-300 mM NaCl. Residues 140-179 of the N-terminal truncated variant contain 11 basic and 2 acidic residues, whereas residues 180-207 specify a leucine zipper motif that directs dimer assembly. Elements within the 50-residue carboxy terminus of C protein are required for tetramer assembly. A basic region followed by a leucine zipper is identical to the domain organization of the basic-leucine zipper (bZIP) class of DNA binding proteins. Sequence homologies with other proteins containing RRMs and the bZIP motif suggest that residues 140-207 represent a conserved bZIP-like RNA binding motif (designated bZLM). The steric orientation of four high-affinity RNA binding sites about rigid leucine zipper domains may explain in part C protein''s asymmetry, its large occluded site size, and its RNA folding activity.     

DNA-induced increase in the alpha-helical content of C/EBP and GCN4

Leucine zipper proteins comprise a recently identified class of DNA binding proteins that contain a bipartite structural motif consisting of a "leucine zipper" dimerization domain and a segment rich in basic residues responsible for DNA interaction. Protein fragments encompassing the zipper plus basic region domains (bZip) have previously been used to determine the conformational and dynamic properties of this motif. In the absence of DNA, the coiled-coil portion is alpha-helical and dimeric, whereas the basic region is flexible and partially disordered. Addition of DNA containing a specific recognition sequence induces a fully helical conformation in the basic regions of these fragments. However, the question remained whether the same conformational change would be observed in native bZip proteins where the basic regions might be stabilized in an alpha-helical conformation even in the absence of DNA, through interactions with portions of the protein not included in the bZip motif. We have now examined the DNA-induced conformational transition for an intact bZip protein, GCN4, and for the bZip fragment of C/EBP with two enhancers that are differentially symmetric. Our results are consistent with the induced helical fork model wherein the basic regions are largely flexible in the absence of DNA and become fully helical in the presence of the specific DNA recognition sequence.