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The repeat region of cortactin is intrinsically disordered in solution.

ABSTRACT: The multi-domain protein, cortactin, contains a 37-residue repeating motif that binds to actin filaments. This cortactin repeat region comprises 6½ similar copies of the motif and binds actin filaments. 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 find 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 (R g) 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.

SUBMITTER: Li X

PROVIDER: S-EPMC5711941 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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The repeat region of cortactin is intrinsically disordered in solution.

Li Xiaofeng X Tao Yeqing Y Murphy James W JW Scherer Alexander N AN Lam TuKiet T TT Marshall Alan G AG Koleske Anthony J AJ Boggon Titus J TJ

Scientific reports 20171201 1

The multi-domain protein, cortactin, contains a 37-residue repeating motif that binds to actin filaments. This cortactin repeat region comprises 6½ similar copies of the motif and binds actin filaments. 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-oli ...[more]

PMID: 29196701

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Project description:The TonB system actively transports vital nutrients across the unenergized outer membranes of the majority of Gram-negative bacteria. In this system, integral membrane proteins ExbB, ExbD, and TonB work together to transduce the proton motive force (PMF) of the inner membrane to customized active transporters in the outer membrane by direct and cyclic binding of TonB to the transporters. A PMF-dependent TonB-ExbD interaction is prevented by 10-residue deletions within a periplasmic disordered domain of ExbD adjacent to the cytoplasmic membrane. Here, we explored the function of the ExbD disordered domain in more detail. In vivo photo-cross-linking through sequential pBpa substitutions in the ExbD disordered domain captured five different ExbD complexes, some of which had been previously detected using in vivo formaldehyde cross-linking, a technique that lacks the residue-specific information that can be achieved through photo-cross-linking: two ExbB-ExbD heterodimers (one of which had not been detected previously), previously detected ExbD homodimers, previously detected PMF-dependent ExbD-TonB heterodimers, and for the first time, a predicted, ExbD-TonB PMF-independent interaction. The fact that multiple complexes were captured by the same pBpa substitution indicated the dynamic nature of ExbD interactions as the energy transduction cycle proceeded in vivo In this study, we also discovered that a conserved motif-V45, V47, L49, and P50-within the disordered domain was required for signal transduction to TonB and to the C-terminal domain of ExbD and was the source of motif essentiality.IMPORTANCE The TonB system is a virulence factor for Gram-negative pathogens. The mechanism by which cytoplasmic membrane proteins of the TonB system transduce an electrochemical gradient into mechanical energy is a long-standing mystery. TonB, ExbB, and ExbD primary amino acid sequences are characterized by regions of predicted intrinsic disorder, consistent with a proposed multiplicity of protein-protein contacts as TonB proceeds through an energy transduction cycle, a complex process that has yet to be recapitulated in vitro This study validates a region of intrinsic disorder near the ExbD transmembrane domain and identifies an essential conserved motif embedded within it that transduces signals to distal regions of ExbD suggested to configure TonB for productive interaction with outer membrane transporters.

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The repeat region of cortactin is intrinsically disordered in solution.

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The repeat region of cortactin is intrinsically disordered in solution.

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