Biophysical properties of intrinsically disordered p130Cas substrate domain--implication in mechanosensing.
Mechanical stretch-induced tyrosine phosphorylation in the proline-rich 306-residue substrate domain (CasSD) of p130Cas (or BCAR1) has eluded an experimentally validated structural understanding. Cellular p130Cas tyrosine phosphorylation is shown to function in areas without internal actomyosin contractility, sensing force at the leading edge of cell migration. Circular dichroism shows CasSD is intrinsically disordered with dominant polyproline type II conformations. Strongly conserved in placental mammals, the proline-rich sequence exhibits a pseudo-repeat unit with variation hotspots 2-9 residues before substrate tyrosine residues. Atomic-force microscopy pulling experiments show CasSD requires minimal extension force and exhibits infrequent, random regions of weak stability. Proteolysis, light scattering and ultracentrifugation results show that a monomeric intrinsically disordered form persists for CasSD in solution with an expanded hydrodynamic radius. All-atom 3D conformer sampling with the TraDES package yields ensembles in agreement with experiment when coil-biased sampling is used, matching the experimental radius of gyration. Increasing β-sampling propensities increases the number of prolate conformers. Combining the results, we conclude that CasSD has no stable compact structure and is unlikely to efficiently autoinhibit phosphorylation. Taking into consideration the structural propensity of CasSD and the fact that it is known to bind to LIM domains, we propose a model of how CasSD and LIM domain family of transcription factor proteins may function together to regulate phosphorylation of CasSD and effect machanosensing.
Department of Biological Sciences, National University of Singapore, Singapore; School of Biosciences, The University of Nottingham Malaysia Campus, Selangor, Malaysia.
Kinya
K
Hotta
Department of Biological Sciences, National University of Singapore, Singapore; Mechanobiology Institute, National University of Singapore, Singapore.
Soumya
S
Ranganathan
Mechanobiology Institute, National University of Singapore, Singapore; Department of Physics, National University of Singapore, Singapore.
Ruchuan
R
Liu
Department of Physics, National University of Singapore, Singapore.
Fei
F
Wu
Department of Biological Sciences, National University of Singapore, Singapore; Mechanobiology Institute, National University of Singapore, Singapore.
Hiroaki
H
Machiyama
Department of Biological Sciences, National University of Singapore, Singapore; Mechanobiology Institute, National University of Singapore, Singapore.
Rong
R
Gao
Mechanobiology Institute, National University of Singapore, Singapore.
Hiroaki
H
Hirata
Indian Institute of Science Education and Research, Pune, India.
Neelesh
N
Soni
Locomotive Syndrome Research Institute, Nadogaya Hospital, Kashiwa, Japan.
Takashi
T
Ohe
Department of Biological Sciences, National University of Singapore, Singapore; Mechanobiology Institute, National University of Singapore, Singapore.
Christopher W V
CW
Hogue
Department of Biological Sciences, National University of Singapore, Singapore; Indian Institute of Science Education and Research, Pune, India; Bioinformatics Institute, Singapore.
M S
MS
Madhusudhan
Department of Biological Sciences, National University of Singapore, Singapore; Mechanobiology Institute, National University of Singapore, Singapore; Locomotive Syndrome Research Institute, Nadogaya Hospital, Kashiwa, Japan.
Yasuhiro
Y
Sawada
D001703:Biophysics; D050719:Crk-Associated Substrate Protein / Q000737:chemistry*; D064267:Intrinsically Disordered Proteins / Q000737:chemistry*; D040542:Mechanotransduction, Cellular; D018625:Microscopy, Atomic Force; D058767:Protein Unfolding
D050719:Crk-Associated Substrate Protein; D064267:Intrinsically Disordered Proteins