Molecular Mechanism of STIL Coiled-Coil Domain Oligomerization

Mai Shamir; Freddie J O Martin; Derek N Woolfson; Assaf Friedler

doi:10.3390/ijms241914616

Molecular Mechanism of STIL Coiled-Coil Domain Oligomerization

Int J Mol Sci. 2023 Sep 27;24(19):14616. doi: 10.3390/ijms241914616.

Authors

Mai Shamir¹, Freddie J O Martin², Derek N Woolfson^{2

3

4}, Assaf Friedler¹

Affiliations

¹ Institute of Chemistry, The Hebrew University of Jerusalem, Safra Campus Givat Ram, Jerusalem 91904, Israel.
² School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 1TS, UK.
³ School of Biochemistry, University of Bristol, Biomedical Sciences Building, University Walk, Bristol BS8 1TD, UK.
⁴ Bristol BioDesign Institute, University of Bristol, Cantock's Close, Bristol BS8 1TS, UK.

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 (STIL_400-700) and a structured CCD (STIL CCD_718-749). 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, (K_D = 8 ± 2 µM) and tetramerization (K_D = 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.

Keywords: STIL; coiled coil domains; oligomerization; peptides; protein interactions.

MeSH terms

Biophysical Phenomena
Circular Dichroism
Dimerization
Humans
Intracellular Signaling Peptides and Proteins* / chemistry
Peptides / chemistry
Protein Domains
Proteins

Substances

Peptides
Proteins
STIL protein, human
Intracellular Signaling Peptides and Proteins

Grants and funding

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.