Orientation and oligomerization specificity of the Bcr coiled-coil oligomerization domain

Abstract

The Bcr oligomerization domain, from the Bcr-Abl oncoprotein, is an attractive therapeutic target for treating leukemias because it is required for cellular transformation. The domain homodimerizes via an antiparallel coiled coil with an adjacent short, helical swap domain. Inspection of the coiled-coil sequence does not reveal obvious determinants of helix-orientation specificity, raising the possibility that the antiparallel orientation preference and/or the dimeric oligomerization state are due to interactions of the swap domains. To better understand how structural specificity is encoded in Bcr, coiled-coil constructs containing either an N- or C-terminal cysteine were synthesized without the swap domain. When cross-linked to adopt exclusively parallel or antiparallel orientations, these showed similar circular dichroism spectra. Both constructs formed coiled-coil dimers, but the antiparallel construct was approximately 16 degrees C more stable than the parallel to thermal denaturation. Equilibrium disulfide-exchange studies confirmed that the isolated coiled-coil homodimer shows a very strong preference for the antiparallel orientation. We conclude that the orientation and oligomerization preferences of Bcr are not caused by the presence of the swap domains, but rather are directly encoded in the coiled-coil sequence. We further explored possible determinants of structural specificity by mutating residues in the d position of the coiled-coil core. Some of the mutations caused a change in orientation specificity, and all of the mutations led to the formation of higher-order oligomers. In the absence of the swap domain, these residues play an important role in disfavoring alternate states and are especially important for encoding dimeric oligomerization specificity.

FIGURE 1

(A) The Bcr oligomerization domain, PDB ID 1K1F (51). (B) View of one of the homodimers that make up the tetramer.

FIGURE 2

Helical-wheel diagram of Bcr in an antiparallel (A) and parallel (B) orientation. Mutated positions are shown in bold with an asterisk. (A) Solid lines represent interhelical salt bridges that always form in the crystal structure. Dashed lines represent potential interhelical salt bridges that form in half of the copies in the asymmetric unit. (B) Dashed lines represent potential g to e’ salt bridges that could form in a parallel dimer. (C) Peptides used in this study. C-BCR and BCR-C have the same sequence for residues 30–65 as was used in the crystal structure. Mutated residues are shown in bold and underlined.

FIGURE 3

(A) Circular dichroism spectra of BCR-C^P (parallel, *) and BCR^AP (antiparallel, ●) (25 μM peptide concentration, 50 mM sodium phosphate, 150 mM NaCl, pH 7.2) at 25 °C. (B) Thermal denaturation of BCR^AP (antiparallel, ●) and BCR^P (parallel, *) monitored at 222 nm in 33 mM sodium phosphate, 100 mM NaCl, 2 M GdnHCl, pH 7.2. Melting temperatures indicate that the antiparallel conformation is more stable than the parallel by ~16 °C.

FIGURE 4

Analytical ultracentrifugation data for Bcr coiled coils. Global fits to data collected at three speeds and three concentrations are shown with representative experimental traces and residuals to the fit. Data shown were collected at 17,000 rpm with 50 μM total monomer concentration. (A) BCR^AP and (B) BCR^P are both two-stranded coiled coils at 25 μM, in 50 mM sodium phosphate, 150 mM NaCl, pH 7.2; (C) cap-C-BCRE52L is a single-species three-stranded coiled coil at 50 μM in 50 mM sodium phosphate, 150 mM NaCl, pH 7.2. All data were fit with WinNonLin.

FIGURE 5

Equilibrium disulfide-exchange experiment used to determine the helix orientation of the Bcr coiled coil. (A) N-terminal (C-BCR) and C-terminal (BCR-C) cysteine peptides are represented by cylinders containing arrows that run from N to C terminus. Combinations of disulfide-bonded and reduced peptides were mixed together and allowed to equilibrate at 25 °C, then the reaction was quenched with acetic acid and run on reverse phase HPLC. (B) & (C) Disulfide-exchange reactions were initiated from two conditions. The concentration of different species was monitored as a function of time by HPLC. Both experiments gave similar K_eq values, 1.3x10⁻³ in (B) and 1.5x10⁻³ in (C), confirming that the reaction had reached an equilibrium strongly favoring the antiparallel species. Integrated peak areas are provided as supporting information in Table S2.

FIGURE 6

Helix orientation of Bcr mutants determined as in Figure 5. Representative disulfide-exchange data in 50 mM sodium phosphate, 150 mM NaCl, 1 mM EDTA pH 7.2, 25 °C. (A) BCRA38L-C^P (12.5 μM) and C-BCRA38L (25 μM); K_eq ≈ 10⁻⁴. (B) C-BCRE52L^P (12.5 μM) and BCRE52L-C (25 μM); K_eq ≈ 2.2. Integrated peak areas are provided as supporting information in Table S2.

FIGURE 7

(A) Circular dichroism spectra of BCRA38L^AP (□), BCRA38L-C-cap (+), cap-C-BCRE52L (○), compared to BCR^AP (●). The peptide concentration was 50 μM for alkylated peptides and 25 μM for disulfide bonded peptides in 50 mM sodium phosphate, 150 mM NaCl, pH 7.2 at 25 °C. (B) Thermal melt of BCRA38L^AP (□), BCRA38L-C-cap (+), cap-C-BCRE52L (○), compared to BCR^AP (●) monitored at 222 nm. BCR^AP was in 33 mM sodium phosphate, 100 mM NaCl, 2 M GdnHCl, pH 7.2. All other peptides were in 50 mM sodium phosphate, 150 mM NaCl, pH 7.2 at 25 °C.