| Popis: |
Oligomerization is a key biological control mechanism in protein function. Homo-dimerization versus homo-tetramerization transforms cellular function for different members of a protein superfamily. Case in point, the homotetrameric organization of Kinesin-5 proteins, via a ∼500 amino acid coiled-coil stalk, underlies their ability to organize the mitotic spindle. Little is known about the rules that govern homo-oligomerization of Kinesin-5 proteins. Our goal is to uncover the trigger sequence, or the element indispensable for tetramer formation, in Kinesin-5. Herein, sequence profiling tools show that the first 150 - 200 residues of the stalk is the only region, common to Kinesin-5 and Kinesin-1 proteins, that is calculated to form coiled-coils; the remaining ∼350 residues in Kinesin-5 exhibit variability in expected coiled-coil formation and do not parallel predictions for the dimeric Kinesin-1 proteins. In two protein constructs containing the first 141 residues of the stalk, in vitro tetramerization of the coiled-coil peptide and the truncated form of the human Kinesin-5 protein was observable by size-exclusion chromatography, static light scattering, and macro-ion mobility spectrometry. We also determined that the human Kinesin-5 and a chimeric protein consisting of the Kinesin-5 motor domain and Kinesin-1 stalk can crosslink and move more than one microtubule in situ. Our data support that, for more than one kinesin family member, the N-terminal portion of the stalk permits tetramerization. Regulation of the dimer-to-tetramer transition may require the sequence of flanking necklinker and C-terminal stalk domains. |
