Marco Evangelista

Structural basis of Bcl-xL recognition by a BH3-mimetic α/β-peptide generated by sequence-based design.

The crystal structure of a complex between the prosurvival protein Bcl‐xL and an α/β‐peptide 21‐mer is described. The α/β‐peptide contains six β‐amino acid residues distributed periodically throughout the sequence and adopts an α‐helix‐like conformation that mimics the bioactive shape of the Puma BH3 domain. The α/β‐peptide forms all of the noncovalent contacts that have previously been identified as necessary for recognition of the prosurvival protein by an authentic BH3 domain. Comparison of our α/β‐peptide:Bcl‐xL structure with structures of complexes between native BH3 domains and Bcl‐2 family proteins reveals how subtle adjustments, including variations in helix radius and helix bowing, allow the α/β‐peptide to engage Bcl‐xL with high affinity. Geometric comparisons of the BH3‐mimetic α/β‐peptide with α/β‐peptides in helix‐bundle assemblies provide insight on the conformational plasticity of backbones that contain combinations of α‐ and β‐amino acid residues. The BH3‐mimetic α/β‐peptide displays prosurvival protein‐binding preferences distinct from those of Puma BH3 itself, even though these two oligomers have identical side‐chain sequences. Our results suggest origins for this backbone‐dependent change in selectivity.

Structure-guided rational design of α/β-peptide foldamers with high affinity for BCL-2 family prosurvival proteins.

We have used computational methods to improve the affinity of a foldamer ligand for its target protein. The effort began with a previously reported α/β‐peptide based on the BH3 domain of the proapoptotic protein Puma; this foldamer binds tightly to Bcl‐xL but weakly to Mcl‐1. The crystal structure of the Puma‐derived α/β‐peptide complexed to Bcl‐xL was used as the basis for computational design of variants intended to display improved binding to Mcl‐1. Molecular modelling suggested modification of three α residues of the original α/β backbone. Individually, each substitution caused only a modest (4‐ to 15‐fold) gain in affinity; however, together the three substitutions led to a 250‐fold increase in binding to Mcl‐1. These modifications had very little effect on affinity for Bcl‐xL. Crystal structures of a number of the new α/β‐peptides bound to either Mcl‐1 or Bcl‐xL validated the selection of each substitution. Overall, our findings demonstrate that structure‐guided rational design can be used to improve affinity and alter partner selectivity of peptidic ligands with unnatural backbones that bind to specific protein partners.

Bid chimeras indicate that most BH3-only proteins can directly activate Bak and Bax, and show no preference for Bak versus Bax

The mitochondrial pathway of apoptosis is initiated by Bcl-2 homology region 3 (BH3)-only members of the Bcl-2 protein family. On upregulation or activation, certain BH3-only proteins can directly bind and activate Bak and Bax to induce conformation change, oligomerization and pore formation in mitochondria. BH3-only proteins, with the exception of Bid, are intrinsically disordered and therefore, functional studies often utilize peptides based on just their BH3 domains. However, these reagents do not possess the hydrophobic membrane targeting domains found on the native BH3-only molecule. To generate each BH3-only protein as a recombinant protein that could efficiently target mitochondria, we developed recombinant Bid chimeras in which the BH3 domain was replaced with that of other BH3-only proteins (Bim, Puma, Noxa, Bad, Bmf, Bik and Hrk). The chimeras were stable following purification, and each immunoprecipitated with full-length Bcl-xL according to the specificity reported for the related BH3 peptide. When tested for activation of Bak and Bax in mitochondrial permeabilization assays, Bid chimeras were ~1000-fold more effective than the related BH3 peptides. BH3 sequences from Bid and Bim were the strongest activators, followed by Puma, Hrk, Bmf and Bik, while Bad and Noxa were not activators. Notably, chimeras and peptides showed no apparent preference for activating Bak or Bax. In addition, within the BH3 domain, the h0 position recently found to be important for Bax activation, was important also for Bak activation. Together, our data with full-length proteins indicate that most BH3-only proteins can directly activate both Bak and Bax.

Crystal structure of a BCL-W domain-swapped dimer: implications for the function of BCL-2 family proteins.

The prosurvival and proapoptotic proteins of the BCL-2 family share a similar three-dimensional fold despite their opposing functions. However, many biochemical studies highlight the requirement for conformational changes for the functioning of both types of proteins, although structural data to support such changes remain elusive. Here, we describe the X-ray structure of dimeric BCL-W that reveals a major conformational change involving helices α3 and α4 hinging away from the core of the protein. Biochemical and functional studies reveal that the α4-α5 hinge region is required for dimerization of BCL-W, and functioning of both pro- and antiapoptotic BCL-2 proteins. Hence, this structure reveals a conformational flexibility not seen in previous BCL-2 protein structures and provides insights into how these regulators of apoptosis can change conformation to exert their function.

The Functional Differences between Pro-survival and Pro-apoptotic B Cell Lymphoma 2 (Bcl-2) Proteins Depend on Structural Differences in Their Bcl-2 Homology 3 (BH3) Domains

Background: Anti- and pro-apoptotic B cell lymphoma 2 (Bcl-2) proteins possess Bcl-2 homology 3 (BH3) domains generally associated with cell death induction. Results: Features of anti-apoptotic BH3 domains were identified that limit their killing activity but are important for protein stability. Conclusion: Pro- and anti-apoptotic BH3 domains are distinct, which affects their function. Significance: Differences in BH3 domains have significant consequences in apoptotic signaling. Bcl-2 homology 3 (BH3) domains are short sequence motifs that mediate nearly all protein-protein interactions between B cell lymphoma 2 (Bcl-2) family proteins in the intrinsic apoptotic cell death pathway. These sequences are found on both pro-survival and pro-apoptotic members, although their primary function is believed to be associated with induction of cell death. Here, we identify critical features of the BH3 domains of pro-survival proteins that distinguish them functionally from their pro-apoptotic counterparts. Biochemical and x-ray crystallographic studies demonstrate that these differences reduce the capacity of most pro-survival proteins to form high affinity “BH3-in-groove” complexes that are critical for cell death induction. Switching these residues for the corresponding residues in Bcl-2 homologous antagonist/killer (Bak) increases the binding affinity of isolated BH3 domains for pro-survival proteins; however, their exchange in the context of the parental protein causes rapid proteasomal degradation due to protein destabilization. This is supported by further x-ray crystallographic studies that capture elements of this destabilization in one pro-survival protein, Bcl-w. In pro-apoptotic Bak, we demonstrate that the corresponding distinguishing residues are important for its cell-killing capacity and antagonism by pro-survival proteins.

Physiological restraint of Bak by Bcl-xL is essential for cell survival

Due to the myriad interactions between prosurvival and proapoptotic members of the Bcl-2 family of proteins, establishing the mechanisms that regulate the intrinsic apoptotic pathway has proven challenging. Mechanistic insights have primarily been gleaned from in vitro studies because genetic approaches in mammals that produce unambiguous data are difficult to design. Here we describe a mutation in mouse and human Bak that specifically disrupts its interaction with the prosurvival protein Bcl-xL Substitution of Glu75 in mBak (hBAK Q77) for leucine does not affect the three-dimensional structure of Bak or killing activity but reduces its affinity for Bcl-xL via loss of a single hydrogen bond. Using this mutant, we investigated the requirement for physical restraint of Bak by Bcl-xL in apoptotic regulation. In vitro, Bak(Q75L) cells were significantly more sensitive to various apoptotic stimuli. In vivo, loss of Bcl-xL binding to Bak led to significant defects in T-cell and blood platelet survival. Thus, we provide the first definitive in vivo evidence that prosurvival proteins maintain cellular viability by interacting with and inhibiting Bak.

The BECN1 N-terminal domain is intrinsically disordered

ABSTRACT BECN1/Beclin 1 has a critical role in the early stages of autophagosome formation. Recently, structures of its central and C-terminal domains were reported, however, little structural information is available on the N-terminal domain, comprising a third of the protein. This lack of structural information largely stems from the inability to produce this region in a purified form. Here, we describe the expression and purification of the N-terminal domain of BECN1 (residues 1 to 150) and detailed biophysical characterization, including NMR spectroscopy. Combined, our studies demonstrated at the atomic level that the BECN1 N-terminal domain is intrinsically disordered, and apart from the BH3 subdomain, remains disordered following interaction with a binding partner, BCL2L1/BCL-XL. In addition, the BH3 domain α-helix induced upon interaction with BCL2L1 reverts to a disordered state when the complex is dissociated by exposure to a competitive inhibitor. No significant interactions between N- and C-terminal domains were detected.

Characterisation of the conformational preference and dynamics of the intrinsically disordered N-terminal region of Beclin 1 by NMR spectroscopy

Beclin 1 is a 450 amino acid protein that plays critical roles in the early stages of autophagosome formation. We recently reported the successful expression, purification and structural characterisation of the entire N-terminal region of Beclin 1 (residues 1-150), including its backbone NMR chemical shift assignments. Based on assigned backbone NMR chemical shifts, it has been established that the N-terminal region of Beclin 1 (1-150), including the BH3 domain (112-123), is intrinsically disordered in the absence of its interaction partners. Here, a detailed study of its conformational preference and backbone dynamics obtained from an analysis of its secondary structure populations using the δ2D method, and the measurements of effective hydrodynamic radius as well as (1)H temperature coefficients, (1)H solvent exchange rates, and (15)N relaxation parameters of backbone amides using NMR spectroscopy is reported. These data provide further evidence for the intrinsically disordered nature of the N-terminal region of Beclin 1 and support the view that the helical conformation adopted by the Beclin 1 BH3 domain upon interaction with binding partners such as BCL-2 pro-survival proteins is likely induced rather than pre-existing.

A transgenic mouse model to inducibly target prosurvival Bcl2 proteins with selective BH3 peptides in vivo.

A transgenic mouse model to inducibly target prosurvival Bcl2 proteins with selective BH3 peptides in vivo