Bcl 2 Human (minus BH3 domain)

What is the structural organization of human Bcl-2 and how does the BH3 domain contribute to its architecture?

Human Bcl-2 contains four conserved Bcl-2 homology domains (BH1, BH2, BH3, and BH4) arranged in a globular structure with several α-helices. The BH3-BH1-BH2 region forms an extended binding groove that recognizes the BH3-death domain motifs of pro-apoptotic proteins like Bax . The protein also contains a flexible loop domain (FLD) connecting the BH4 and BH3 domains, which contains multiple glycine residues that provide flexibility and can sense cytosolic signals .

The C-terminal region contains a transmembrane domain that anchors Bcl-2 to the mitochondrial outer membrane, making it insoluble and membrane-associated, unlike some other family members like Bax which can transition between soluble and membrane-associated states . This structural arrangement enables Bcl-2 to sequester pro-apoptotic proteins, preventing mitochondrial outer membrane permeabilization and subsequent cytochrome c release.

How does removal of the BH3 domain alter the structural properties of human Bcl-2?

Deletion of the BH3 domain significantly disrupts the binding groove formed by the BH3-BH1-BH2 regions that is critical for recognizing and inhibiting pro-apoptotic proteins . NMR studies have shown that different domains of Bcl-2 respond individually to the presence of BH3 domain peptides through chemical shift perturbations, indicating domain-specific roles in recognizing apoptotic proteins .

Without its own BH3 domain, these domain-specific responses would be altered, potentially affecting how the remaining domains coordinate their functions. The flexible loop domain (FLD) that connects to the BH3 domain would likely adopt a different conformation without its C-terminal anchor point, potentially affecting the regulatory phosphorylation sites in this region that control Bcl-2 activity . The absence of BH3 would likely impact the stability of neighboring structural elements and potentially create new solvent-exposed surfaces that could affect protein solubility and aggregation propensity.

What specific molecular interactions are lost when the BH3 domain is removed from human Bcl-2?

When the BH3 domain is removed from human Bcl-2, several critical molecular interactions are compromised:

• Contribution to the hydrophobic groove that binds pro-apoptotic proteins: The loss of this structural component would impair Bcl-2's ability to sequester and inhibit proteins like Bax .

• Domain-domain communication: NMR studies have shown that binding of BH3 domains causes perturbations across multiple Bcl-2 domains, suggesting an integrated communication network . Removal of the BH3 domain would disrupt this network.

• Potential homodimerization capacity: While BH3 peptides don't significantly inhibit Bcl-2 homodimerization , the domain itself could still contribute to the structural stability of homodimers.

Research has shown that BH3 domains are "critically important for Bax/Bcl-2 heterodimerization" . Interestingly, peptide binding studies show that Bcl-2 BH3 peptide itself was inactive toward Bax/Bcl-2 heterodimerization and only weakly affected Bax/Bcl-x(L) interactions , suggesting that the context of the BH3 domain within the full protein structure is important for its function.

How does BH3 domain deletion impact Bcl-2's anti-apoptotic function in cancer cells?

The deletion of the BH3 domain would likely significantly compromise the anti-apoptotic function of human Bcl-2. Since Bcl-2 inhibits apoptosis primarily by binding and sequestering pro-apoptotic molecules like Bax through its binding groove (which includes the BH3 domain) , the loss of this domain would impair this critical function.

In cancer contexts, this could potentially reduce the ability of Bcl-2 to protect cancer cells from apoptosis. Research indicates that Bax BH3 peptide can induce cytochrome c release from mitochondria even in Bcl-2-overexpressing cells , highlighting how disruption of these interactions can overcome Bcl-2's protective effects. A Bcl-2 protein lacking its BH3 domain would potentially mimic this disrupted state, being unable to properly sequester pro-apoptotic factors.

What expression systems and purification strategies are most effective for producing recombinant human Bcl-2 lacking the BH3 domain?

Producing functional human Bcl-2 lacking the BH3 domain requires specialized expression and purification strategies:

For structural studies using NMR, isotope-labeled Bcl-2 variants can be produced using bacterial expression systems with specialized media containing 15N and/or 13C sources . The search results mention successful production of "13C and/or 15N isotope-labeled variants of intact human Bcl-2 protein" for NMR studies .

When expressing membrane proteins like Bcl-2, inclusion body formation in bacteria is common. Refolding protocols using detergents like DPC (dodecylphosphocholine) micelles can help obtain properly folded protein . Specific buffer conditions that have proven effective include "5 mM DPC micelles, 20 mM NaPi, 20 mM NaCl, 2 mM TCEP at pH 6.0" .

For functional studies, mammalian expression systems may provide proper folding and post-translational modifications, though with lower yields. Jurkat T leukemic cells have been used for studying Bcl-2 overexpression , suggesting they could also express modified versions of Bcl-2.

To verify proper folding and function, binding assays with known interaction partners should be performed, including in vitro interaction assays to assess heterodimerization between Bcl-2 and pro-apoptotic proteins .

What NMR spectroscopy approaches can effectively analyze structural changes in Bcl-2 when the BH3 domain is removed?

Several NMR approaches have been successfully applied to study full-length Bcl-2 and can be adapted to analyze BH3-deleted variants:

• TROSY-HSQC experiments: "1H-15N-TROSY-HSQC spectra" have been used for monitoring Bcl-2 and its interactions with BH3 peptides . This technique is particularly valuable for larger proteins and would allow researchers to compare chemical shift patterns between wild-type and BH3-deleted Bcl-2.

• Glycine-specific monitoring: This approach uses glycine residues as structural probes distributed across the protein. As reported: "By identifying all individual glycine residues distributed across the entire Bcl-2 protein in the corresponding NMR spectra, we were able to trace each glycine residue and protein domain individually" . This would be particularly useful for detecting structural changes resulting from BH3 deletion.

• Chemical shift perturbation (CSP) analysis: The research shows that "concentration- and residue-specific changes were used to determine individual glycine residue-specific affinity constants and to identify which part of the protein that is most effected upon binding" . Similar analysis could reveal how BH3 deletion affects the structural organization of the remaining domains.

• Membrane-mimicking conditions: Performing NMR in membrane-mimicking environments like DPC micelles is critical since Bcl-2 is normally membrane-associated . The specific conditions used include "5 mM DPC micelles, 20 mM NaPi, 20 mM NaCl, 2 mM TCEP at pH 6.0" .

How can computational modeling predict the structural reorganization when the BH3 domain is removed from human Bcl-2?

Computational modeling offers several approaches to predict structural reorganization after BH3 domain deletion:

• Molecular dynamics (MD) simulations: MD can model the dynamic behavior of proteins over time in membrane-mimicking environments similar to the DPC micelles mentioned in the research . These simulations could reveal how the remaining domains reorganize and how protein flexibility changes, identifying stable intermediate states during conformational transitions.

• Homology modeling: If experimental structures of BH3-deleted Bcl-2 are unavailable, homology models could be constructed based on related Bcl-2 family proteins with known structures .

• Protein-protein docking: Computational docking could predict how BH3 domain deletion affects binding to partner proteins like Bax. The research discusses the importance of these interactions , and docking studies could reveal altered binding interfaces and affinities.

• Normal mode analysis (NMA): This approach can identify large-scale collective motions in proteins, revealing how domain removal affects the major conformational states accessible to the protein.

• Network analysis: Analyzing the network of non-covalent interactions within Bcl-2 could identify how BH3 domain deletion propagates structural changes throughout the protein, particularly relevant given the observed domain-specific responses to BH3 peptide binding mentioned in the research .

What complementary structural biology techniques beyond NMR provide insights into BH3-deleted Bcl-2 conformation?

While NMR has been effectively used to study full-length Bcl-2 in membrane environments , several complementary techniques can provide additional insights:

These techniques could complement the NMR approaches described in the research to provide a comprehensive structural understanding of how BH3 domain deletion affects Bcl-2 conformation.

What quantitative methods best characterize the altered binding profile of Bcl-2 lacking the BH3 domain?

Several quantitative methods can effectively characterize the altered binding profile of BH3-deleted Bcl-2:

• In vitro interaction assays: Research mentions using these assays to assess heterodimerization between Bcl-2 and pro-apoptotic proteins . Similar approaches could compare wild-type and BH3-deleted Bcl-2 binding to partners like Bax.

• Surface plasmon resonance (SPR): This label-free technique directly measures binding kinetics and affinities in real-time, providing both association and dissociation rate constants (kon and koff) and equilibrium dissociation constants (KD).

• Isothermal titration calorimetry (ITC): ITC measures heat changes during binding interactions, providing thermodynamic parameters (ΔH, ΔS, ΔG) and stoichiometry in addition to binding affinities.

• NMR titration experiments: The research describes using chemical shift perturbations in NMR to monitor Bcl-2's response to BH3 peptides . Similar experiments could compare how wild-type and BH3-deleted Bcl-2 respond to binding partners, revealing residue-specific affinity changes.

• Fluorescence-based techniques: Methods like fluorescence polarization can monitor binding interactions. The research describes measuring IC50 values for peptide inhibition of protein-protein interactions , suggesting similar approaches could be used.

Research specifically mentions determining "individual glycine residue-specific affinity constants" using NMR chemical shift perturbations , suggesting this approach could be particularly valuable for detailed binding characterization of BH3-deleted Bcl-2.

How do interactions between Bcl-2 lacking the BH3 domain and various BH3-only proteins differ from wild-type interactions?

The interactions between BH3-deleted Bcl-2 and BH3-only proteins would likely differ significantly from wild-type interactions:

Based on the research, we know that the BH3-BH1-BH2 region in Bcl-2 forms an extended binding groove that recognizes the BH3-death domain motifs of other proteins . The BH3 domain contributes to this groove, so its deletion would alter the binding surface.

Research indicates that BH3 domains are "critically important for Bax/Bcl-2 heterodimerization" . This suggests that removing the BH3 domain from Bcl-2 could disrupt or weaken these interactions, potentially releasing pro-apoptotic BH3-only proteins from inhibition.

Different BH3 domains have distinct effects on Bcl-2 family interactions. For example, "Bad BH3 peptide, while potently inducing cytochrome c release in wild-type Jurkat cells, only partially overcame the effects of Bcl-2 overexpression" . This suggests that BH3-deleted Bcl-2 might retain some selectivity in its interactions with different BH3-only proteins.

To study these altered interactions, several approaches could be employed, including binding assays comparing wild-type and BH3-deleted Bcl-2, NMR studies monitoring glycine residues across Bcl-2 in response to BH3 peptides , and functional assays such as cytochrome c release assays in cells expressing BH3-deleted Bcl-2.

How can studying Bcl-2 lacking the BH3 domain advance the development of cancer therapeutics targeting Bcl-2 family proteins?

Studying Bcl-2 lacking the BH3 domain can advance cancer therapeutics in several ways:

• Refined understanding of binding interactions: Research indicates that "Bcl-2 inhibits apoptosis by binding and inhibiting pro-apoptotic molecules such as Bax" and that "agents that disrupt the ability of Bcl-2, or other anti-apoptotic molecules, to bind to pro-apoptotic molecules may have therapeutic value" . By studying how BH3 domain deletion affects the binding groove, researchers could identify critical structural features for designing more selective BH3 mimetics.

• Identification of allosteric sites: BH3-deleted Bcl-2 might reveal alternative binding pockets or conformational states. Research mentions that BH3 peptide binding causes chemical shift perturbations across multiple domains of Bcl-2 , suggesting interconnected allosteric networks that could be therapeutically exploited.

• Differential targeting of Bcl-2 family members: Research notes that "Bax BH3 peptide potently inhibited both Bax/Bcl-2 and Bax/Bcl-x(L) interactions" while "Bad BH3 peptide was slightly more potent than Bax BH3 at inhibiting Bax/Bcl-x(L) but failed to disrupt Bax/Bcl-2" . Understanding these differential effects could guide development of mimetics with optimized selectivity profiles.

• Structure-based drug design: Detailed structural information from studying BH3-deleted Bcl-2 could directly inform computational and rational drug design approaches for developing next-generation BH3 mimetics with improved potency, selectivity, and pharmacological properties.

Research highlights that "agents based on the Bax BH3 domain may have therapeutic value in cancers overexpressing Bcl-2, while agents based on the BH3 domain of Bad may be more useful for tumors overexpressing Bcl-x(L)" , demonstrating the therapeutic relevance of understanding these domain-specific interactions.

What methods can determine if BH3 mimetics maintain efficacy against Bcl-2 variants lacking the BH3 domain?

To determine if BH3 mimetics maintain efficacy against BH3-deleted Bcl-2 variants, several methodological approaches can be employed:

• Direct binding assays: Techniques like surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or fluorescence polarization can quantify binding affinity of BH3 mimetics to wild-type versus BH3-deleted Bcl-2, revealing potential differences in binding kinetics and thermodynamics.

• Structural studies: NMR chemical shift perturbation analysis, as described in the research , can map the binding interface of BH3 mimetics on BH3-deleted Bcl-2 at residue-level resolution, identifying potential compensatory binding interactions.

• Competitive displacement assays: These can assess whether BH3 mimetics can still displace pro-apoptotic proteins from BH3-deleted Bcl-2. Research shows that "Bax BH3 peptide (20-amino acids) potently inhibited both Bax/Bcl-2 and Bax/Bcl-x(L) interactions, exhibiting IC50 values of 15 and 9.5 microM, respectively" . Similar assays could determine if mimetics maintain this ability with BH3-deleted Bcl-2.

• Cellular assays: Cytochrome c release assays and apoptosis measurements in cells expressing BH3-deleted Bcl-2 could determine if BH3 mimetics retain functional efficacy. Research demonstrates that "Bax BH3 peptide was able to overcome Bcl-2 overexpression and induce cytochrome c release" . Similar functional readouts would assess mimetic efficacy against BH3-deleted variants.

• Molecular dynamics simulations: Computational approaches can model mimetic binding to BH3-deleted Bcl-2, predicting potential binding mode alterations and suggesting structural modifications to maintain or improve efficacy.

These methods would provide comprehensive insights into whether current BH3 mimetics remain effective against Bcl-2 lacking the BH3 domain or if new design strategies are needed.

How does deletion of the BH3 domain affect Bcl-2's interaction with mitochondrial membranes?

The deletion of the BH3 domain from Bcl-2 could significantly impact its interactions with mitochondrial membranes in several ways:

• Altered membrane insertion: While the C-terminal transmembrane domain is primarily responsible for anchoring Bcl-2 to the mitochondrial outer membrane , the BH3 domain may influence the orientation and depth of membrane insertion. Without the BH3 domain, the remaining protein might adopt a different orientation relative to the membrane surface.

• Changed lipid preferences: Research indicates that Bcl-2 is "insoluble and located at the MOM (mitochondrial outer membrane)" . The BH3 domain may contribute to specific lipid interactions, and its removal could alter Bcl-2's preference for different membrane microdomains or lipid compositions.

• Modified membrane topology: The flexible loop domain (FLD) that connects the BH4 and BH3 domains contains multiple glycine residues that provide flexibility . Without the BH3 domain, this loop region might adopt a different conformation relative to the membrane, potentially affecting how it senses cytosolic signals.

• Impact on membrane permeabilization: The BH3 domain contributes to the binding of pro-apoptotic proteins that can induce mitochondrial outer membrane permeabilization. Its deletion might alter how Bcl-2 regulates this process at the membrane interface.

Studying these effects would require specialized membrane mimetics as described in the research, including DPC micelles for structural studies and potentially more complex systems like liposomes or nanodiscs for functional membrane interaction studies.

What membrane-mimicking environments best preserve the native-like properties of BH3-deleted Bcl-2 for structural and functional studies?

Based on the research and the membrane-associated nature of Bcl-2, several membrane-mimicking environments are suitable for studying BH3-deleted Bcl-2:

• Detergent micelles: The research specifically mentions using "DPC (dodecylphosphocholine) micelles" at a concentration of "5 mM" for NMR studies of Bcl-2 . This system appears effective for structural studies, allowing identification of glycine residues and monitoring of their responses to BH3 peptide binding.

• Lipid nanodiscs: These provide a more native-like bilayer environment than detergent micelles. For Bcl-2, which normally resides in the mitochondrial outer membrane, nanodiscs composed of POPC/POPE mixtures or cardiolipin-containing formulations would better approximate the mitochondrial lipid environment.

• Liposomes: For functional studies rather than high-resolution structural analysis, liposomes could be valuable. Large unilamellar vesicles (LUVs) could be used for binding and permeabilization assays, while giant unilamellar vesicles (GUVs) would be suitable for microscopy-based studies of membrane association.

• Bicelles: These lipid-detergent mixtures form disc-like structures and can be suitable for both NMR and crystallography studies of membrane proteins like Bcl-2.

The research emphasizes the importance of studying Bcl-2 in membrane environments, noting that "apoptotic events occurring at the MOM require protein with BH3 domains to interact with the hydrophobic grooves of other Bcl-2 members" . This highlights the need for membrane mimetics that accurately recapitulate the native environment for functional relevance when studying BH3-deleted Bcl-2.

How do post-translational modifications of Bcl-2 change when the BH3 domain is absent?

Post-translational modifications (PTMs) of Bcl-2 likely undergo significant changes when the BH3 domain is absent:

• Altered phosphorylation patterns: The research mentions that the flexible loop domain (FLD) of Bcl-2 "can sense cytosol signals and become phosphorylated at multiple sites for controlling the activity of Bcl-2" . This region connects the BH4 and BH3 domains, suggesting that BH3 domain deletion might significantly alter how this regulatory region is accessed by kinases and phosphatases.

• Modified ubiquitination: The removal of the BH3 domain might expose or conceal lysine residues that are targets for ubiquitination, potentially affecting Bcl-2's turnover and degradation rate.

• Changed accessibility to modifying enzymes: The global conformational changes resulting from BH3 domain deletion could alter the accessibility of various sites to modifying enzymes, creating new modification patterns not seen in the wild-type protein.

Investigating these changes requires sophisticated approaches including mass spectrometry-based PTM mapping, particularly phosphoproteomics to identify phosphorylation sites in the regulatory loop domain. Comparative analysis between wild-type and BH3-deleted Bcl-2 would reveal differential modification patterns that might contribute to functional differences.

Enzymatic assays could identify regulatory kinases/phosphatases with altered activity toward BH3-deleted Bcl-2, while proximity labeling approaches could identify differential interaction partners, including potential modifying enzymes.

What domain-specific labeling approaches can reveal the conformational dynamics of Bcl-2 lacking the BH3 domain?

Domain-specific labeling approaches offer powerful tools to study conformational dynamics of BH3-deleted Bcl-2:

• Site-specific isotopic labeling: The research describes using "15N-labeled Bcl-2 protein" for NMR studies . For BH3-deleted Bcl-2, selective isotopic labeling of specific domains could reveal domain-specific dynamics. This could be achieved through segmental isotopic labeling using protein trans-splicing or residue-specific labeling using auxotrophic bacterial strains.

• Fluorescence spectroscopy approaches: Strategic placement of fluorophores can provide information about conformational changes and dynamics. FRET (Förster Resonance Energy Transfer) pairs at domain interfaces can monitor interdomain distance changes, while environmentally sensitive fluorophores at key positions can detect changes in local environment.

• Spin labeling for EPR/DEER: Electron paramagnetic resonance techniques with site-specific spin labels can measure distances between labeled sites. Labeling residues at domain boundaries would monitor domain reorganization after BH3 deletion.

• Unnatural amino acid incorporation: Site-specific incorporation of unnatural amino acids with special properties like photocrosslinking groups or IR probes can provide site-specific information about local environments and interaction partners.

The research describes monitoring "glycine residues across all functional domains of the Bcl-2 protein" and their "residue-specific individual response" . Similar approaches focusing on strategic residues in the remaining domains could reveal how BH3 deletion affects the dynamics and interactions of the entire protein.