The role of unstructured highly charged regions on the stability and specificity of dimerization of two-stranded alpha-helical coiled-coils: analysis of the neck-hinge region of the kinesin-like motor protein Kif3A.
We investigated the folding, stability, and specificity of dimerization of the neck-hinge region (residues 356-416) of the kinesin-like protein Kif3A. We showed that the predicted coiled-coil on its own (residues 356-377) will fold autonomously in solution. We then explored the ability of oppositely charged regions to specify heterodimer formation in coiled-coils by synthesizing analogs of the neck coiled-coil region with and without various negatively and positively charged extensions to the C-terminus of the neck coiled-coil and characterizing these analogs by circular dichroism spectroscopy. The charged region alone (residues 378-416) adopted a random-coil structure and this region remained unfolded in the presence of the coiled-coil. Redox experiments demonstrated that oppositely charged regions specified the formation of a hetero-two-stranded coiled-coil. Denaturation studies with urea demonstrated a decrease in coiled-coil stability with the addition of negatively charged residues in the homostranded coiled-coil; conversely, the addition of the positively charged region (residues 403-416) of Kif3A C-terminally to the neck coiled-coil did not affect coiled-coil stability. Overall, our results suggest that electrostatic attractions drive the specificity of heterodimerization of the coiled-coil, not the removal of positive or negative charge-charge repulsions, while maintaining the stability of the heterodimer compared to that of the stablest homodimer.
Chana
Mundeep
M
Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Tripet
Brian P
BP
Mant
Colin T
CT
Hodges
Robert S
RS
D000595:Amino Acid Sequence; D001703:Biophysics / Q000379:methods*; D002942:Circular Dichroism; D019281:Dimerization; D016547:Kinesins / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D013997:Time Factors
D010455:Peptides; D016547:Kinesins