Abstract
: Coiled-coil domains (CCDs) play key roles in regulating both healthy cellular processes
and the pathogenesis of various diseases by controlling protein self-association and protein–protein
interactions. Here, we probe the mechanism of oligomerization of a peptide representing the CCD of
the STIL protein, a tetrameric multi-domain protein that is over-expressed in several cancers and associated with metastatic spread. STIL tetramerization is mediated both by an intrinsically disordered
domain (STIL400–700) and a structured CCD (STIL CCD718–749). Disrupting STIL oligomerization
via the CCD inhibits its activity in vivo. We describe a comprehensive biophysical and structural
characterization of the concentration-dependent oligomerization of STIL CCD peptide. We combine
analytical ultracentrifugation, fluorescence and circular dichroism spectroscopy to probe the STIL
CCD peptide assembly in solution and determine dissociation constants of both the dimerization, (KD
= 8 ± 2 µM) and tetramerization (KD = 68 ± 2 µM) of the WT STIL CCD peptide. The higher-order
oligomers result in increased thermal stability and cooperativity of association. We suggest that this
complex oligomerization mechanism regulates the activated levels of STIL in the cell and during
centriole duplication. In addition, we present X-ray crystal structures for the CCD containing destabilising (L736E) and stabilising (Q729L) mutations, which reveal dimeric and tetrameric antiparallel
coiled-coil structures, respectively. Overall, this study offers a basis for understanding the structural
molecular biology of the STIL protein, and how it might be targeted to discover anti-cancer reagents.
and the pathogenesis of various diseases by controlling protein self-association and protein–protein
interactions. Here, we probe the mechanism of oligomerization of a peptide representing the CCD of
the STIL protein, a tetrameric multi-domain protein that is over-expressed in several cancers and associated with metastatic spread. STIL tetramerization is mediated both by an intrinsically disordered
domain (STIL400–700) and a structured CCD (STIL CCD718–749). Disrupting STIL oligomerization
via the CCD inhibits its activity in vivo. We describe a comprehensive biophysical and structural
characterization of the concentration-dependent oligomerization of STIL CCD peptide. We combine
analytical ultracentrifugation, fluorescence and circular dichroism spectroscopy to probe the STIL
CCD peptide assembly in solution and determine dissociation constants of both the dimerization, (KD
= 8 ± 2 µM) and tetramerization (KD = 68 ± 2 µM) of the WT STIL CCD peptide. The higher-order
oligomers result in increased thermal stability and cooperativity of association. We suggest that this
complex oligomerization mechanism regulates the activated levels of STIL in the cell and during
centriole duplication. In addition, we present X-ray crystal structures for the CCD containing destabilising (L736E) and stabilising (Q729L) mutations, which reveal dimeric and tetrameric antiparallel
coiled-coil structures, respectively. Overall, this study offers a basis for understanding the structural
molecular biology of the STIL protein, and how it might be targeted to discover anti-cancer reagents.
Original language | English |
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Article number | 14616 |
Number of pages | 14 |
Journal | International Journal of Molecular Sciences |
Volume | 24 |
Issue number | 19 |
DOIs | |
Publication status | Published - 27 Sept 2023 |
Bibliographical note
Funding Information:A.F. was supported by a grant from the ISF (Israel Science Foundation) and by the Minerva Center for bio-hybrid complex systems. A.F. thanks the Saerree K. and Louis P. Fiedler Chair in Chemistry. F.J.O.M. and D.N.W. were funded by a BBSRC grant (BB/R00661X/1) to D.N.W. F.J.O.M. was also supported by the Bristol Chemical Synthesis Centre for Doctoral Training funded through the EPSRC (EP/G036764). We would like to thank the CCP4/DLS workshop (2019) and all the staff at Diamond Light Source for their for training and expertise with macromolecular crystallography.
Publisher Copyright:
© 2023 by the authors.
Research Groups and Themes
- Bristol BioDesign Institute