(A) Alignment of the repeats of mouse cortactin (NP_031829). Amino acids are colored according to their chemical character: green, hydrophobic; red, negatively charged; cyan, positively charged; orange, glycines. Cysteine residues 112 and 246 are highlighted in yellow. (B) Proposed model of the repeat region of cortactin [Zhang et al. 2007]. Some inconsistencies of this model include: the presence of long a-helices within each repeat that are not supported by structure prediction or the CD spectrum (see Figure 1), the presence of charged amino acids buried in the core region (atom-type color coded), and the presence of hydrophobic amino acids at the surface (green). The acetylated lysine residues are shown (purple). Although these lysine residues were described as forming two opposite patches in the model, they appear relatively scattered. (C) Most of the cortactin molecule (except the C-terminal SH3 domain) is predicted to be disordered (blue and magenta traces) by the program PONDR [Romero et al. 2001]. However, the cortactin repeat (amino acids 80 to 325) is predicted to undergo a disorder-to-order transition upon binding to a target (red trace).
(D) Oblique representation of the sequences of each of the cortactin repeats, showing the distribution of clusters of hydrophobic amino acids as implemented in the program HCA [Callebaut et al. 1997]. Amino acids forming part of hydrophobic clusters are contoured and colored green. Symbols are: Gly, black diamond; Pro, red star; Thr, empty square; Ser, partially filled square. The region contoured by a black rectangle includes the conserved clusters of hydrophobic amino acids of the cortactin repeats. Although these clusters have the characteristic shape of a-helices, this region also contains four conserved glycine residues (black diamonds) and formation of α-helices will likely depend upon stabilization by interaction with a partner. Notice that the helices predicted by HCA occur at the N-terminus of each repeat, not the C-terminus as in the proposed model (part B of this figure).

(A) The endogenous cysteine residues 112 and 246 of the cortactin repeat were crosslinked and the ability of the crosslinked protein to bind F-actin was tested in a co-sedimentation assay (Materials and Methods). The pellet (P) and supernatant (S) were analyzed on 12% SDS-PAGE. Lanes 1 and 2, uncrosslinked cortactin; lanes 3 and 4, disulfide crosslinked cortactin (xl-cortactin); lanes 5 and 6, reduction of the disulfide crosslink with 10mM DTT.
(B) The ability of the NEM-labeled cortactin repeat (at residues C112 and C246) to bind to F-actin was assessed using a co-sedimentation assay (Materials and Methods). The pellet and supernatant were analyzed on 12% SDS-PAGE. Lanes 1 and 2, non-labeled cortactin; lanes 3 and 4, NEM-labeled cortactin.
(C) Crosslinking of cortactin repeat constructs containing pairs of cysteine residues at positions 83 and 306 (left), 83 and 246 (middle), and 83 and 112 (right). Prior to each crosslinking reaction, DTT was removed from the samples on a Sephadex G-50 spin column equilibrated with 20 mM HEPES pH 7.0, 0.1 M KCl, and 100μM PMSF. The crosslinking reactions were performed on ice and catalyzed by addition of 10μM CuSO₄. Each cysteine pair was crosslinked either directly by disulfide bond formation (zero length crosslink), or using the crosslinking reagents DBB (4.4 Å span) or MTS-6 (9.6 Å span).

Far-UV CD spectrum of the cortactin repeat. The CD spectrum was obtained at a protein concentration of 18 μM in 10 mM Tris-HCl pH 7.6, 300 mM KCl, 0.5 mM DTT, using a Jasco J-810 spectropolarimeter. Measurements were taken at 20° C. Note that the CD spectrum of the cortactin repeat presents a minimum at 205 nm, similar to the spectrum of the molten globule state of protein folding (Kelly and Price, 1997).

The Cortactin repeat alters inter-subunit contacts in F-actin. The cortactin repeat inhibits the formation of lateral (A) and longitudinal (B) crosslinks in the actin filament between C374 and mutant cysteine residues at position 265 and 41 of yeast actin, respectively. Note how the formation of higher molecular weight crosslinked species in the absence of cortactin is inhibited upon addition of the cortactin repeat. Conditions: DTT-free actin (10μM) was polymerized for 20 min at room temperature by addition of 3.0 mM MgCl₂. Cysteine residues 374 and 265 (lateral inter-subunit contact) and 374 and 41 (longitudinal inter-subunit contact) were crosslinked on ice in the presence or the absence of the cortactin repeat at a 1:1 molar ratio. These cysteine pairs were crosslinked directly, via disulfide bond formation catalyzed by addition of 5μM CuSO₄. Aliquots of the reactions were taken at selected time intervals and analyzed on non-reducing 7.5–10% SDS-PAGE. xl-actin, crosslinked actin.