SCientifiC RepoRTS | 7: 16696 | DOI:10.1038/s41598-017-16959-1
The repeat region of cortactin is
intrinsically disordered in solution
, Yeqing Tao
, James W. Murphy
, Alexander N. Scherer
, TuKiet T. Lam
Alan G. Marshall
, Anthony J. Koleske
& Titus J. Boggon
The multi-domain protein, cortactin, contains a 37-residue repeating motif that binds to actin
laments. This cortactin repeat region comprises 6½ similar copies of the motif and binds actin
laments. To better understand this region of cortactin, and its fold, we conducted extensive
biophysical analysis. Size exclusion chromatography with multi-angle light scattering (SEC-MALS)
reveals that neither constructs of the cortactin repeats alone or together with the adjacent helical
region homo-oligomerize. Using circular dichroism (CD) we nd that in solution the cortactin repeats
resemble a coil-like intrinsically disordered protein. Small-angle X-ray scattering (SAXS) also indicates
that the cortactin repeats are intrinsically unfolded, and the experimentally observed radius of gyration
) is coincidental to that calculated by the program Flexible-Meccano for an unfolded peptide of this
length. Finally, hydrogen-deuterium exchange mass spectrometry (HDX-MS) indicates that the domain
contains limited hydrophobic core regions. These experiments therefore provide evidence that in
solution the cortactin repeat region of cortactin is intrinsically disordered.
Cortactin is an actin-binding protein and activator of actin branch nucleation by the Arp2/3 complex
, it also
interacts with the nonreceptor tyrosine kinase Arg to regulate actin lament stability and promote actin-based
protrusions in a variety of contexts
. ese functions of Arg and cortactin are important for normal stabiliza-
tion of dendritic spines. Defects in spine stability are associated with psychiatric disorders such as depression and
schizophrenia, and neurodegenerative diseases such as Alzheimer’s Disease
. A better understanding of the
structure and function of actin binding proteins (e.g. cortactin, Arg, Arp2/3) should facilitate a deeper under-
standing of how they regulate actin in dendritic spines and other biological contexts.
Cortactin is a multi-domain protein that contains an N-terminal acidic (NTA) domain, 6½ cortactin repeats,
a helical domain that is sometimes referred to as a coiled coil domain, a exible region that is tyrosine phospho-
rylated, and a C-terminal SH3 domain
(Fig.1A). e cortactin repeat region is necessary and sucient to bind
, however, how this region folds still remains largely unknown. A series of studies have suggested that cort-
actin is either folded and globular
, or unfolded and extended
. ese studies propose that cortactin binding
to actin laments either induces cortactin folding
, or does not change its secondary structure
. erefore, to
better understand the molecular function of cortactin an improved understanding of its solution state structure
Here, we have conducted a series of biophysical analyses of cortactin. Size exclusion chromatography with
multi-angle light scattering (SEC-MALS) shows that neither constructs of the cortactin repeats alone or the cort-
actin repeats together with the adjacent helical domain are able to homo-oligomerize. Circular dichroism suggests
that cortactin repeats fold as a coil-like intrinsically disordered domain. Small-angle X-ray scattering indicates
that the cortactin repeats are intrinsically disordered, and hydrogen-deuterium exchange mass spectrometry
(HDX-MS) suggests that they contain only minimal hydrophobic core regions. Together, these experiments pro-
vide a comprehensive description of solution-state folding for the cortactin repeat domain, and allow us to dene
this as a coil-like intrinsically disordered domain.
Department of Pharmacology, Yale University School of Medicine, New Haven, CT, 06520, USA.
of Chemistry, Florida State University, 600 W., College Avenue, Tallahassee, FL, 32306, USA.
Department of Cell
Biology, Yale University School of Medicine, New Haven, CT, 06520, USA.
Department of Molecular Biophysics
and Biochemistry, Yale University, New Haven, CT, 06520, USA.
Yale MS & Proteomics Resource, Yale University,
New Haven, CT, 06520, USA.
Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, 1800 E.
Paul Dirac Dr., Tallahassee, FL, 32310, USA.
Present address: Department of Pathology, Yale University School of
Medicine, New Haven, CT, 06520, USA.
Present address: Biopharmaceutical Analytical Sciences, Biopharm R&D,
GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA, 19406, USA. Correspondence and requests for materials
should be addressed to T.J.B. (email: firstname.lastname@example.org)
Received: 18 August 2017
Accepted: 19 November 2017
Published: xx xx xxxx