Epistatic mutations in PUMA BH3 drive an alternate binding mode to potently and selectively inhibit anti-apoptotic Bfl-1
- Massachusetts Institute of Technology, United States
- Dana-Farber Cancer Institute, United States
Figures
Figure 1 with 3 supplements
Computational design of a library of PUMA BH3 variants selective for Bfl-1.
(A) PUMA BH3 is pan-selective; the design objective was a peptide that binds tightly only to Bfl-1. (B) Sequence of PUMA BH3 showing the heptad numbering convention used in this paper. (C) Overview …
Figure 1—figure supplement 1
Affinities of BIM point mutants for different Bcl-2 proteins are predicted to be strongly correlated.
Larger scores predict tighter binding. Red points represent wt PUMA BH3. (A) PSSMSPOT scores for binding to Bcl-xL versus Bfl-1, (B) PSSMSPOT scores for binding to Mcl-1 versus Bfl-1, (C) STATIUM …
Figure 1—figure supplement 2
Composition of the Bcl-xL, Mcl-1 and Bfl-1-targeted libraries.
https://doi.org/10.7554/eLife.25541.004
Figure 1—figure supplement 3
Scores for members of three libraries designed to target Bfl-1 (blue), Mcl-1 (green) or Bcl-xL (red).
Scores show predicted affinities of peptides in each library for each target. Larger scores predict tighter binding. (A) PSSMSPOT scores for binding to Bcl-xL versus Bfl-1, (B) PSSMSPOT scores for …
Figure 2 with 5 supplements
Experimental library screening for Bfl-1 affinity and selectivity.
(A) Yeast-surface display configuration. BH3 peptides were expressed as fusions to Aga2; HA tag expression was detected with APC and Bfl-1 binding was detected with PE. (B) FACS analysis showed that …
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Figure 2—source data 1
Data collected from competition fluorescence polarization experiments.
- https://doi.org/10.7554/eLife.25541.007
Figure 2—figure supplement 1
The PUMA BH3 library was screened to enrich for selective binders of Bfl-1.
Several positive, negative and competition screens were used to identify tight and selective Bfl-1 binders. Competition screens included unlabeled competitors at the indicated concentrations. In …
Figure 2—figure supplement 2
FACS analysis of the designed libraries after first two rounds of sorting (FL2, see Figure 2—figure supplement 1).
Plots show binding of library peptides to 100 nM Myc-tagged Bfl-1 in the presence of excess unlabeled competitor (Mcl-1, Bcl-2, Bcl-w and Bcl-xL; 1 μM each) when encoded in (A) a BIM background or (B…
Figure 2—figure supplement 3
Conventionally sequenced clones from pool FL6’.
https://doi.org/10.7554/eLife.25541.010
Figure 2—figure supplement 4
Peptide affinities for Bfl-1, Bcl-xL, Mcl-1, Bcl-2 and Bcl-w.
Ki obtained from competition assays with fluoresceinated BIM peptide. Data are mean ± SD of three replicates.
Figure 2—figure supplement 5
FS2 mutations made in a BIM background generate a weak binder of Bfl-1.
Fluorescence anisotropy competition experiments for unlabeled BIM (blue) vs. BIM including mutations from FS2 (BIM-FS2; E2bV, A2eA, E2gG, I3dA, F4aV, Y4eV; red). BIM-FS2 (Ki = 720 ± 110 nM) binds >10…
Figure 3
Evaluation of the library design.
(A) Sequences from the Bfl-1 library were preferentially enriched during sorting. Sequences with no more than one amino-acid mutation from the Bfl-1 (blue), Mcl-1 (green), or Bcl-xL (red) targeted …
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Figure 3—source code 1
Deep sequencing data analysis.
- https://doi.org/10.7554/eLife.25541.014
Figure 4 with 1 supplement
Epistatic mutations in PUMA confer Bfl-1 binding specificity.
(A) Sequence logo of unique peptide sequences in the final sorted pool from the Bfl-1 targeted library. (B) Location of mutated sites in FS1, FS2 and FS3. Mutations at positions 2a and 2e are in red …
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Figure 4—source data 1
Data collected from competition fluorescence polarization experiments.
- https://doi.org/10.7554/eLife.25541.016
Figure 4—figure supplement 1
Affinities of FS2 chimeric proteins binding to Bfl-1, Bcl-xL, Mcl-1, Bcl-2 and Bcl-w.
Ki obtained from competition assays with fluoresceinated BIM peptide. Data are mean ± SD of three replicates.
Figure 5 with 4 supplements
High-resolution structures of PUMA and FS2 bound to human Bfl-1.
(A) Binding groove of Bfl-1 (gray, surface) with PUMA (yellow) and FS2 (purple). (B) Cα- Cα shifts between FS2 and PUMA. Sites with larger/smaller residues in FS2 are indicated in red/blue. (C) The …
Figure 5—figure supplement 1
Comparison of PUMA and FS2 binding geometry with that in other crystal structures of BH3:Bfl-1 complexes deposited in the PDB.
All Bfl-1:peptide structures were aligned to Bfl-1 and the mean C-alpha positions were calculated for each BH3 peptide position. Mean distances were calculated separately for all complexes in the …
Figure 5—figure supplement 2
FACS analysis of cells displaying FS2 or FS2 with single point mutants at position 3f.
FACS profiles for mutants are nearly indistinguishable from that of FS2. Data were collected the same day with the same settings, and all plots use the same scale of arbitrary units.
Figure 5—figure supplement 3
Multiple-sequence alignment of helices 2–4 of human anti-apoptotic Bfl-1 homologs.
Bfl-1 has an amino-acid insertion that may contribute to the widened binding groove between helices 3 and 4 (dotted box). Additionally, there is an asparagine that is unique to Bfl-1 at the elbow …
Figure 5—figure supplement 4
Residues in FS2 are not readily accommodated in the PUMA -binding geometry.
Modeling on a fixed backbone indicates that all valine rotamers would clash (red spheres) with Bfl-1 (green) when modeled in to position 2a of PUMA (gray). Shown here is the most preferred rotamer. …
Figure 6 with 1 supplement
Crystal structure of FS2 bound to human Mcl-1.
(A) Binding groove of Mcl-1 (blue, surface) with BIM (yellow, 2PQK [Fire et al., 2010]) and FS2 (purple). (B) Cα- Cα shifts between FS2 and BIM when bound to Mcl-1. (C) The canonical Bfl-1:BH3 salt …
Figure 6—figure supplement 1
Alignment of all crystal structures in the PDB of Bfl-1/Mcl-1 bound to BH3 peptides.
Helix three in Bfl-1 is shifted relative to Mcl-1, resulting in a widened binding groove.
Figure 7 with 2 supplements
Designed Bfl-1 inhibitors selectively induce MOMP in Bfl-1 dependent cells.
(A) The BH3 profiling assay detects MOMP by monitoring JC-1 fluorescence in permeabilized cells treated with different peptides. (B–C) Depolarization of mitochondria induced by designed peptides in …
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Figure 7—source data 1
Data collected from BH3 profiling and cytochrome c release assays.
- https://doi.org/10.7554/eLife.25541.026
Figure 7—figure supplement 1
At low concentrations, FS1, FS2 and FS3 selectively induced cytochrome c release only in Bfl-1 dependent cell lines. iBH3 was performed on highly primed cells of known anti-apoptotic protein dependency.
Data are mean ± SD of 3 or more independent measurements.
Figure 7—figure supplement 2
iBH3 was performed on unprimed cells to test for activation function.
These cells require a BAK/BAX activator to release cytochrome c. Cytochrome c release is stimulated by the known activator peptides BIM and PUMA BH3. Truncated PUMA (1e-4c), PUMAsh and FS3 have …
Figure 8 with 4 supplements
An electrophilic variant of FS2 reacts covalently with Bfl-1.
(A) C55 in Bfl-1 is close to the BH3 binding groove in BIM:Bfl-1 structure 2VM6 (Herman et al., 2008). (B–C) Modeling suggested two ways in which an N-terminal acrylamide group could be incorporated …
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Figure 8—source data 1
Data collected from gel shift assays.
- https://doi.org/10.7554/eLife.25541.030
Figure 8—figure supplement 1
Conventionally sequenced clones from pool FL6 and their frequencies.
https://doi.org/10.7554/eLife.25541.031
Figure 8—figure supplement 2
Library members bind covalently to Bfl-1 cysteine 55.
FACS analysis of yeast cells displaying (A) PUMA in the presence of Bfl-1, (B) PUMA in the presence of the cysteine-to-serine point mutant Bfl-1 C55S, (C) the FL6 library pool in the presence of …
Figure 8—figure supplement 3
Kinetics of the reaction of Bfl-1 with electrophilic peptides.
(A) There is a time-dependent shift in apparent molecular weight, as assessed by SDS-PAGE, when Bfl-1 is incubated with FS2_1fX but not when Bfl-1 C55S one is incubated with FS2_1gX, consistent with …
Figure 8—figure supplement 4
Depolarization of mitochondria induced by designed peptides, including covalent inhibitor FS2_1gX, in four cell lines that depend on ectopic expression of Mcl-1, Bcl-2, Bcl-xL or Bfl-1 for survival.
Data are mean ± SD of three or more independent measurements.
Additional files
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Supplementary file 1
Summary of X-ray data collection and refinement statistics.
- https://doi.org/10.7554/eLife.25541.035
