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Figure 4.
Figure 4. The WASP-Homology Domain 2 of MIM and IRSp53
(A) Comparison of a classical WH2 (represented by WASP,
Wiskott-Aldrich syndrome protein) with the WH2s of MIM, ABBA,
and IRSp53. Red, blue, green, and yellow correspond to
negatively charged, positively charged, hydrophobic, and small
(Thr, Val, Ser, Ala) conserved amino acids, respectively. The
diagram above the sequences represents a secondary structure
assignment based on the structure determined here (cylinder, α
helix; arrow, β strand). Accession numbers are as in Figure 1,
and WASP_HUMAN, P42768. Red arrows point to noncanonical amino
acids present in the WH2 of IRSp53.
(B) Structure of the
WH2 of MIM (red ribbon) bound to actin (gray surface). Numbers
1–4 indicate actin's four subdomains. The side chains of some
of the amino acids involved in interactions with actin are shown
(green, hydrophobic; blue, positively charged).
(C) Binding
of the WH2 of IRSp53 to actin measured by ITC. The upper graph
corresponds to the heat evolved upon repeated 10 μl injections
of a 100 μM solution of the WH2 peptide into a 10 μM solution
of actin in G buffer. The lower graph shows the binding isotherm
produced by integration of the heat for each injection. The line
represents a nonlinear least squares fit to the data using a
single-site binding model. The following thermodynamic
parameters were determined from the fitting: dissociation
constant K[d] = 0.28 ± 0.04 μM; molar enthalpy ΔH =
−7.2 ± 0.1 kcal.mol^−1; and stoichiometry n = 0.9.
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