Chereau et al. 10.1073/pnas.0507021102. |
Fig. 5. Superimposition of the structures of the Wiskott–Aldrich syndrome protein (WASP) homology 2 domain (WH2) domains of WASP (yellow), WASP-family verprolin homologous protein (WAVE) (magenta) and WASP-interacting protein (WIP) (green), obtained as ternary complexes with actin–DNase I (gray–cyan). Notice that the C terminus of the WH2 domain of WIP binds near the binding site of DNase I, although the two proteins do not interact directly.
Fig. 6. Binding to actin of the Wiskott–Aldrich syndrome protein (WASP) homology 2 domain (WH2) domain of WASP by isothermal titration calorimetry. (A) Heat evolved upon repeated 10-ml injections of a 50 mM solution of the WASP peptide into a 5 mM solution of actin in G-buffer. (B) 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 results of the fit are presented in Table 3.
Fig. 7. Structure of a complex of actin (gray) with gelsolin domain 1 (yellow), showing part of the linker that connects to gelsolin domain 2 (PDB ID code 1P8Z). Like the Wiskott–Aldrich syndrome protein (WASP) homology 2 domain (WH2) domain gelsolin presents a a-helix (red) that binds in the cleft between actin subdomains 1 and 3 and an LKKT-related sequence (red) in the linker between domains 1 and 2. However, these two structural elements are disconnected in gelsolin, whose a-helix runs in the opposite direction than the a-helices of the WH2 and thymosin b domains.
Table 1. Crystallographic data and refinement statistics
Actin-DNase I | WASP | WAVE | WIP | |
Diffraction statistics | ||||
Space group | C2 | C2 | P21 | P212121 |
Cell parameters | ||||
a /b/c, Å | 152.7/41.5/117.9 | 153.3/41.8/119.4 | 118.7/41.6/153.1 | 41.8/77.1/222.3 |
a /b/c, ° | 90/109.28/90 | 90/108.68/90 | 90/108.79/90 | 90/90/90 |
Resolution, Å | 39-1.85 (1.92-1.85) | 40.1-2.1 (2.14-2.1) | 47.5-1.8 (1.85-1.8) | 45-2.6 (2.69-2.6) |
Completeness, % | 97.4 (81.9) | 99.7 (97.3) | 80.6 (69) | 96.1 (85.4) |
Redundancy | 6.9 (4.4) | 4.8 (4.2) | 5.4 (3.4) | 5.8 (4.4) |
Rmerge, % | 7.9 (37.3) | 9.4 (37.9) | 9.6 (28.3) | 8.1 (30.2) |
Average I/ s | 20.2 (3.4) | 15.8 (3.4) | 19.9 (4.0) | 21.8 (5.0) |
Refinement statistics | ||||
Resolution, Å | 39–1.85 | 40.1–2.1 | 47.5–1.8 | 45–2.6 |
Rfactor,, % | 16.1 | 15.3 | 16.6 | 16 |
Rfree, % | 19.9 | 21 | 21.5 | 21.9 |
rms deviations | ||||
Bond length, Å | 0.012 | 0.015 | 0.013 | 0.011 |
Bond angles, ° | 1.59 | 1.85 | 1.46 | 1.373 |
Average B-factor | ||||
Protein | 43.5 | 32.6 | 29.8 | 34.7 |
Solvent | 39.8 | 41.1 | 39.5 | 35.3 |
Ramachandran | ||||
Most Favored, % | 93.8 | 92.4 | 92.9 | 90.7 |
Additionally allowed, % | 6 | 7.4 | 6.9 | 9 |
Generously allowed, % | 0.2 | 0.2 | 0.2 | 0.3 |
PDB accession code | 2A42 | 2A3Z | 2A40 | 2A41 |
R
merge = S(I–<I>)/ S I; I is the intensity of an individual reflection and <I> the mean value of all the measurements for that reflection. Rfactor= S |Fo – Fc|/S|Fo|; Fo and Fc are respectively observed and calculated structure factors. Rfree is Rfactor calculated for a randomly selected subset of reflections (5%) that were omitted during the refinement. Values in parentheses correspond to highest resolution shell. WASP, Wiskott–Aldrich syndrome protein; WAVE, WASP-family verprolin homologous protein WIP, WASP-interacting protein .Table 2. Thermodynamic parameters of Wiskott–Aldrich syndrome protein (WASP) homology domain 2 (WH2) binding determined by isothermal titration calorimetry
K d, mM | Δ H, kcal•mol–1 | ΔS, eu | |
Tb domain | |||
T b4 (2–44) | 0.76 ± 0.12 | –8.8 ± 0.2 | –1.5 |
T b4 (2–33) | 3.1 ± 0.9 | –5.6 ± 0.5 | 6.4 |
T b4 (18–44) | n.b. | ||
Ciboulot (10–43) | 21.6 ± 3.8 | –16.6 ± 1.7 | –34.3 |
Ciboulot (49–81)* | 53 ± 9 | –28.2 ± 3.4 | –75.3 |
Ciboulot (87–119) | 6.25 ± 0.11 | –16.2 ± 1.3 | –30.4 |
WH2 domain | |||
MIM (724–755) | 0.23 ± 0.04 | –21.0 ± 0.5 | –39.9 |
WIP (29–60) | 0.16 ± 0.01 | –19.3 ± 0.1 | –33.5 |
WIP (29–46) | 2.2 ± 0.5 | –6.1 ± 0.2 | 5.3 |
WIP (46–63) | >>100 | ||
WASP (430–458) | 0.25 ± 0.02 | –13.4 ± 0.1 | –14.7 |
WAVE (433–464) | 0.052 ± 0.003 | –18.5 ± 0.1 | –28.6 |
WAVE (450–464) | 27 ± 15 | –2.0 ± 0.5 | 14.1 |
Using a one-site binding model, the dissociation constant, Kd, and molar entha
lpy, ΔH for the binding of each peptide was determined by a nonlinear least squares fit to the data. The entropy, S, was calculated from Kd and ΔH. The binding energetics of most of the peptides is dominated by favorable enthalpy, with an unfavorable contribution from entropy, in part the result of the unbound peptide being disordered in solution, becoming helical upon binding. Analysis by circular dichroism suggests that the peptides with a favorable entropic contribution to binding have substantial helical content in the unbound state (data not shown). Tb, thymosin b domain; WIP, Wasp-interacting protein; WAVE, WASP-family verprolin homologous protein.*The stoichiometry was fixed to one for this peptide.
Table 3. Dissociation constants (Kd, in mM) of Wiskott–Aldrich syndrome protein (WASP) homology 2 domain (WH2) complexed with acrylodan-labeled actin
WH2 domains | ATP–Ac-actin | ATP–Ac-actin–DNaseI | ADP–Ac-actin |
Short type | |||
WASP (430-458) | 1.65 ± 0.13 | 1.83 ± 0.09 | 7.98 ± 1.68 |
WAVE (433-464) | 0.69 ± 0.07 | 0.65 ± 0.03 | 1.69 ± 0.09 |
Long type | |||
WIP (29-60) | 0.010 ± 0.002 | 0.040 ± 0.004 | 0.083 ± 0.009 |
MIM (724-755) | 0.009 ± 0.004 | 0.018 ± 0.003 | 0.037 ± 0.015 |
Equilibrium titrations of acrylodan–G-actin (40 or 200 nM) with WH2 peptides were carried out in 96-well plates at room temperature in 2 mM Tris, pH 7.5/0.2 mM ATP or ADP/0.2 mM CaCl2/0.2 mM DTT/0.2 mg/ml bovine serum albumin. DNAse I:actin molar ratio was 2:1. The fluorescence intensity at 487 nm (excitation at 399 nm) was measured with a Cary Eclipse spectrofluorometer. The data were fitted with the equation: F = Fo+ΔFn, where Fo is the initial fluorescence, ΔF is the maximal change in fluorescence, and n is the fraction of the acrylodan–G-actin bound to a peptide calculated as a root of the quadratic equation: nAn2–(Kd + nA + B)n + B = 0, where A and B are the total concentrations of acrylodan–G-actin and the peptide, respectively, n is the apparent stoichiometry, and Kd is the dissociation constant.