Bad is a BH3 domain-containing protein that forms an inactivating dimer with Bcl-XL.

The Bcl-2 related protein Bad is a promoter of apoptosis and has been shown to dimerize with the anti-apoptotic proteins Bcl-2 and Bcl-XL. Overexpression of Bad in murine FL5.12 cells demonstrated that the protein not only could abrogate the protective capacity of coexpressed Bcl-XL but could accelerate the apoptotic response to a death signal when it was expressed in the absence of exogenous Bcl-XL. Using deletion analysis, we have identified the minimal domain in the murine Bad protein that can dimerize with Bcl-xL. A 26-amino-acid peptide within this domain, which showed significant homology to the alpha-helical BH3 domains of related apoptotic proteins like Bak and Bax, was found to be necessary and sufficient to bind Bcl-xL. To determine the role of dimerization in regulating the death-promoting activity of Bad and the death-inhibiting activity of Bcl-xL, mutations within the hydrophobic BH3-binding pocket in Bcl-xL that eliminated the ability of Bcl-xL to form a heterodimer with Bad were tested for the ability to promote cell survival in the presence of Bad. Several of these mutants retained the ability to impart protection against cell death regardless of the level of coexpressed Bad protein. These results suggest that BH3-containing proteins like Bad promote cell death by binding to antiapoptotic members of the Bcl-2 family and thus inhibiting their survival promoting functions.     

Dynamics of the BH3-Only Protein Binding Interface of Bcl-xL

The balance and interplay between pro-death and pro-survival members of the B-cell lymphoma-2 (Bcl-2) family proteins play key roles in regulation of the mitochondrial pathway of programmed cell death. Recent NMR and biochemical studies have revealed that binding of the proapoptotic BH3-only protein PUMA induces significant unfolding of antiapoptotic Bcl-xL at the interface, which in turn disrupts the Bcl-xL/p53 interaction to activate apoptosis. However, the molecular mechanism of such regulated unfolding of Bcl-xL is not fully understood. Analysis of the existing Protein Data Bank structures of Bcl-xL in both bound and unbound states reveal substantial intrinsic heterogeneity at its BH3-only protein binding interface. Large-scale atomistic simulations were performed in explicit solvent for six representative structures to further investigate the intrinsic conformational dynamics of Bcl-xL. The results support that the BH3-only protein binding interface of Bcl-xL is much more dynamic compared to the rest of the protein, both unbound and when bound to various BH3-only proteins. Such intrinsic interfacial conformational dynamics likely provides a physical basis that allows Bcl-xL to respond sensitively to detailed biophysical properties of the ligand. The ability of Bcl-xL to retain or even enhance dynamics at the interface in bound states could further facilitate the regulation of its interactions with various BH3-only proteins such as through posttranslational modifications.     

Bh3 induced conformational changes in Bcl‐Xl revealed by crystal structure and comparative analysis

Apoptosis or programmed cell death is a regulatory process in cells in response to stimuli perturbing physiological conditions. The Bcl‐2 family of proteins plays an important role in regulating homeostasis during apoptosis. In the process, the molecular interactions among the three members of this family, the pro‐apoptotic, anti‐apoptotic and BH3‐only proteins at the mitochondrial outer membrane define the fate of a cell. Here, we report the crystal structures of the human anti‐apoptotic protein Bcl‐X_L in complex with BH3‐only BID^BH3 and BIM^BH3 peptides determined at 2.0 Å and 1.5 Å resolution, respectively. The BH3 peptides bind to the canonical hydrophobic pocket in Bcl‐X_L and adopt an alpha helical conformation in the bound form. Despite a similar structural fold, a comparison with other BH3 complexes revealed structural differences due to their sequence variations. In the Bcl‐X_L‐BID^BH3 complex we observed a large pocket, in comparison with other BH3 complexes, lined by residues from helices α1, α2, α3, and α5 located adjacent to the canonical hydrophobic pocket. These results suggest that there are differences in the mode of interactions by the BH3 peptides that may translate into functional differences in apoptotic regulation. Proteins 2015; 83:1262–1272. © 2015 Wiley Periodicals, Inc.     

Tethering-facilitated DNA ‘opening’ and complementary roles of β-hairpin motifs in the Rad4/XPC DNA damage sensor protein

XPC/Rad4 initiates eukaryotic nucleotide excision repair on structurally diverse helix-destabilizing/distorting DNA lesions by selectively ‘opening’ these sites while rapidly diffusing along undamaged DNA. Previous structural studies showed that Rad4, when tethered to DNA, could also open undamaged DNA, suggesting a ‘kinetic gating’ mechanism whereby lesion discrimination relied on efficient opening versus diffusion. However, solution studies in support of such a mechanism were lacking and how ‘opening’ is brought about remained unclear. Here, we present crystal structures and fluorescence-based conformational analyses on tethered complexes, showing that Rad4 can indeed ‘open’ undamaged DNA in solution and that such ‘opening’ can largely occur without one or the other of the β-hairpin motifs in the BHD2 or BHD3 domains. Notably, the Rad4-bound ‘open’ DNA adopts multiple conformations in solution notwithstanding the DNA’s original structure or the β-hairpins. Molecular dynamics simulations reveal compensatory roles of the β-hairpins, which may render robustness in dealing with and opening diverse lesions. Our study showcases how fluorescence-based studies can be used to obtain information complementary to ensemble structural studies. The tethering-facilitated DNA ‘opening’ of undamaged sites and the dynamic nature of ‘open’ DNA may shed light on how the protein functions within and beyond nucleotide excision repair in cells.     

Plastid Proteomic Analysis in Tomato Fruit Development

To better understand the mechanism of plastid differentiation from chloroplast to chromoplast, we examined proteome and plastid changes over four distinct developmental stages of ‘Micro-Tom’ fruit. Additionally, to discover more about the relationship between fruit color and plastid differentiation, we also analyzed and compared ‘Micro-Tom’ results with those from two other varieties, ‘Black’ and ‘White Beauty’. We confirmed that proteins related to photosynthesis remain through the orange maturity stage of ‘Micro-Tom’, and also learned that thylakoids no longer exist at this stage. These results suggest that at a minimum there are changes in plastid morphology occurring before all related proteins change. We also compared ‘Micro-Tom’ fruits with ‘Black’ and ‘White Beauty’ using two-dimensional gel electrophoresis. We found a decrease of CHRC (plastid-lipid-associated protein) and HrBP1 (harpin binding protein-1) in the ‘Black’ and ‘White Beauty’ varieties. CHRC is involved in carotenoid accumulation and stabilization. HrBP1 in Arabidopsis has a sequence similar to proteins in the PAP/fibrillin family. These proteins have characteristics and functions similar to lipocalin, an example of which is the transport of hydrophobic molecules. We detected spots of TIL (temperature-induced lipocalin) in 2D-PAGE results, however the number of spots and their isoelectric points differed between ‘Micro-Tom’ and ‘Black’/‘White Beauty’. Lipocalin has various functions including those related to environmental stress response, apoptosis induction, membrane formation and fixation, regulation of immune response, cell growth, and metabolism adjustment. Lipocalin related proteins such as TIL and HrBP1 could be related to the accumulation of carotenoids, fruit color and the differentiation of chromoplast.     

Spliceosomal SL1 RNA binding to U1-70K: the role of the extended RRM

The RNA recognition motif (RRM) occurs widely in RNA-binding proteins, but does not always by itself support full binding. For example, it is known that binding of SL1 RNA to the protein U1-70K in the U1 spliceosomal particle is reduced when a region flanking the RRM is truncated. How the RRM flanking regions that together with the RRM make up an ‘extended RRM’ (eRRM) contribute to complex stability and structural organization is unknown. We study the U1-70K eRRM bound to SL1 RNA by thermal dissociation and laser temperature jump kinetics; long-time molecular dynamics simulations interpret the experiments with atomistic resolution. Truncation of the helix flanking the RRM on its N-terminal side, ‘N-helix,’ strongly reduces overall binding, which is further weakened under higher salt and temperature conditions. Truncating the disordered region flanking the RRM on the C-terminal side, ‘C-IDR’, affects the local binding site. Surprisingly, all-atom simulations show that protein truncation enhances base stacking interactions in the binding site and leaves the overall number of hydrogen bonds intact. Instead, the flanking regions of the eRRM act in a distributed fashion via collective interactions with the RNA when external stresses such as temperature or high salt mimicking osmotic imbalance are applied.     

Crystal structure of Bak bound to the BH3 domain of Bnip5, a noncanonical BH3 domain-containing protein

The activation or inactivation of B-cell lymphoma-2 (Bcl-2) antagonist/killer (Bak) is critical for controlling mitochondrial outer membrane permeabilization-dependent apoptosis. Its pro-apoptotic activity is controlled by intermolecular interactions with the Bcl-2 homology 3 (BH3) domain, which is accommodated in the hydrophobic pocket of Bak. Bcl-2-interacting protein 5 (Bnip5) is a noncanonical BH3 domain-containing protein that interacts with Bak. Bnip5 is characterized by its controversial effects on the regulation of the pro-apoptotic activity of Bak. In the present study, we determined the crystal structure of Bak bound to Bnip5 BH3. The intermolecular association appeared to be typical at first glance, but we found that it is maintained by tight hydrophobic interactions together with hydrogen/ionic bonds, which accounts for their high binding affinity with a dissociation constant of 775 nM. Structural analysis of the complex showed that Bnip5 interacts with Bak in a manner similar to that of the Bak-activating pro-apoptotic factor peroxisomal testis-enriched protein 1, particularly in the destabilization of the intramolecular electrostatic network of Bak. Our structure is considered to reflect the initial point of drastic and consecutive conformational and stoichiometric changes in Bak induced by Bnip5 BH3, which helps in explaining the effects of Bnip5 in regulating Bak-mediated apoptosis.     

'In-line attack' conformational effect plays a modest role in an enzyme-catalyzed RNA cleavage: a free energy simulation study

Since the proposal of ‘in-line attack’ conformation as a possibly important intermediate in RNA cleavage, its structure has been captured in various protein and RNA enzymes; these structures strengthen the belief that this conformation plays an essential role in the catalysis of RNA cleavage. As generally discussed, this intermediate structure can be involved in energy barrier reduction in two possible ways, e.g. through either conformational effect or electrostatic effect. In order to quantitatively elucidate the contribution of conformational effect in this type of enzyme catalysis, free energy simulations were performed on the RNA structures both in a splicing endonuclease complex and in the aqueous solution. Our free energy simulation results revealed that the ‘in-line attack’ conformational effect plays a modest role in facilitating the reaction rate enhancement (~12-fold) compared with the overall 10¹²-fold rate increase. The close agreement between the present computational estimation and an experimental measurement on the spontaneous RNA cleavage in an in vitro evolved ATP aptamer motives us to realize that the conformation distribution of an enzyme substrate prior to rather than after its binding determines the upper bound of the rate enhancement ability through the conformational strategy.     

Molecular mechanism underlying the di-uridylation activity of Arabidopsis TUTase URT1

In Arabidopsis, HESO1 and URT1 act cooperatively on unmethylated miRNA and mRNA uridylation to induce their degradation. Their collaboration significantly impacts RNA metabolism in plants. However, the molecular mechanism determining the functional difference and complementarity of these two enzymes remains unclear. We previously solved the three-dimensional structure of URT1 in the absence and presence of UTP. In this study, we further determined the structure of URT1 in complex with a 5′-AAAU-3′ RNA stretch that mimics the post-catalytic state of the mRNA poly(A) tail after the addition of the first uridine. Structural analysis and enzymatic assays revealed that L527 and Y592 endow URT1 with a preference to interact with purine over pyrimidine at the -1 RNA binding position, thus controlling the optimal number of uridine added to the 3′ extremity of poly(A) as two. In addition, we observed that a large-scale conformational rearrangement in URT1 occurs upon binding with RNA from an ‘open’ to a ‘closed’ state. Molecular dynamic simulation supports an open-closed conformational selection mechanism employed by URT1 to interact with RNA substrates and maintain distributive enzymatic activity. Based on the above results, a model regarding the catalytic cycle of URT1 is proposed to explain its di-uridylation activity.     

MCL1 nuclear translocation induces chemoresistance in colorectal carcinoma

Colorectal cancer (CRC) is one of the most common and deadliest forms of cancer. Myeloid Cell Leukemia 1 (MCL1), a pro-survival member of the Bcl-2 protein family is associated with chemo-resistance in CRC. The ability of MCL1 to inhibit apoptosis by binding to the BH3 domains of pro-apoptotic Bcl-2 family members is a well-studied means by which this protein confers resistance to multiple anti-cancer therapies. We found that specific DNA damaging chemotherapies promote nuclear MCL1 translocation in CRC models. In p53^null CRC, this process is associated with resistance to chemotherapeutic agents, the mechanism of which is distinct from the classical mitochondrial protection. We previously reported that MCL1 has a noncanonical chemoresistance capability, which requires a novel loop domain that is distinct from the BH3-binding domain associated with anti-apoptotic function. Herein we disclose that upon treatment with specific DNA-damaging chemotherapy, this loop domain binds directly to alpha-enolase which in turn binds to calmodulin; we further show these protein−protein interactions are critical in MCL1’s nuclear import and chemoresistance. We additionally observed that in chemotherapy-treated p53^−/− CRC models, MCL1 nuclear translocation confers sensitivity to Bcl-xL inhibitors, which has significant translational relevance given the co-expression of these proteins in CRC patient samples. Together these findings indicate that chemotherapy-induced MCL1 translocation represents a novel resistance mechanism in CRC, while also exposing an inherent and targetable Bcl-xL co-dependency in these cancers. The combination of chemotherapy and Bcl-xL inhibitors may thus represent a rational means of treating p53^−/− CRC via exploitation of this unique MCL1-based chemoresistance mechanism.Subject terms: Targeted therapies, Senescence     

The BCL-2 protein family,BH3-mimetics and cancer therapy

Escape from apoptosis is a key attribute of tumour cells and facilitates chemo-resistance. The ‘BCL-2-regulated'' or ‘intrinsic'' apoptotic pathway integrates stress and survival signalling to govern whether a cancer cell will live or die. Indeed, many pro-apoptotic members of the BCL-2 family have demonstrated tumour-suppression activity in mouse models of cancer and are lost or repressed in certain human cancers. Conversely, overexpression of pro-survival BCL-2 family members promotes tumorigenesis in humans and in mouse models. Many of the drugs currently used in the clinic mediate their therapeutic effects (at least in part) through the activation of the BCL-2-regulated apoptotic pathway. However, initiators of this apoptotic pathway, such as p53, are mutated, lost or silenced in many human cancers rendering them refractory to treatment. To counter such resistance mechanisms, a novel class of therapeutics, ‘BH3-mimetics'', has been developed. These drugs directly activate apoptosis by binding and inhibiting select antiapoptotic BCL-2 family members and thereby bypass the requirement for upstream initiators, such as p53. In this review, we discuss the role of the BCL-2 protein family in the development and treatment of cancer, with an emphasis on mechanistic studies using well-established mouse models of cancer, before describing the development and already recognised potential of the BH3-mimetic compounds.     

Ligand binding and membrane insertion compete with oligomerization of the BclXL apoptotic repressor

B-cell lymphoma extra large (BclXL) apoptotic repressor plays a central role in determining the fate of cells to live or die during physiological processes such as embryonic development and tissue homeostasis. Herein, using a myriad of biophysical techniques, we provide evidence that ligand binding and membrane insertion compete with oligomerization of BclXL in solution. Of particular importance is the observation that such oligomerization is driven by the intermolecular binding of its C-terminal transmembrane (TM) domain to the canonical hydrophobic groove in a domain-swapped trans fashion, whereby the TM domain of one monomer occupies the canonical hydrophobic groove within the other monomer and vice versa. Binding of BH3 ligands to the canonical hydrophobic groove displaces the TM domain in a competitive manner, allowing BclXL to dissociate into monomers upon hetero-association. Remarkably, spontaneous insertion of BclXL into DMPC/DHPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dihexanoyl-sn-glycero-3-phosphocholine) bicelles results in a dramatic conformational change such that it can no longer recognize the BH3 ligands in what has come to be known as the "hit-and-run" mechanism. Collectively, our data suggest that oligomerization of a key apoptotic repressor serves as an allosteric switch that fine-tunes its ligand binding and membrane insertion pertinent to the regulation of apoptotic machinery.     

Calcium Causes a Conformational Change in Lamin A Tail Domain that Promotes Farnesyl-Mediated Membrane Association

Lamin proteins contribute to nuclear structure and function, primarily at the inner nuclear membrane. The posttranslational processing pathway of lamin A includes farnesylation of the C-terminus, likely to increase membrane association, and subsequent proteolytic cleavage of the C-terminus. Hutchinson Gilford progeria syndrome is a premature aging disorder wherein a mutant version of lamin A, Δ50 lamin A, retains its farnesylation. We report here that membrane association of farnesylated Δ50 lamin A tail domains requires calcium. Experimental evidence and molecular dynamics simulations collectively suggest that the farnesyl group is sequestered within a hydrophobic region in the tail domain in the absence of calcium. Calcium binds to the tail domain with an affinity K_D ≈ 250 μM where it alters the structure of the Ig-fold and increases the solvent accessibility of the C-terminus. In 2 mM CaCl₂, the affinity of the farnesylated protein to a synthetic membrane is K_D ≈ 2 μM, as measured with surface plasmon resonance, but showed a combination of aggregation and binding. Membrane binding in the absence of calcium could not be detected. We suggest that a conformational change induced in Δ50 lamin A with divalent cations plays a regulatory role in the posttranslational processing of lamin A, which may be important in disease pathogenesis.     

The Free Energy Profile of Tubulin Straight-Bent Conformational Changes,with Implications for Microtubule Assembly and Drug Discovery

αβ-tubulin dimers need to convert between a ‘bent’ conformation observed for free dimers in solution and a ‘straight’ conformation required for incorporation into the microtubule lattice. Here, we investigate the free energy landscape of αβ-tubulin using molecular dynamics simulations, emphasizing implications for models of assembly, and modulation of the conformational landscape by colchicine, a tubulin-binding drug that inhibits microtubule polymerization. Specifically, we performed molecular dynamics, potential-of-mean force simulations to obtain the free energy profile for unpolymerized GDP-bound tubulin as a function of the ∼12° intradimer rotation differentiating the straight and bent conformers. Our results predict that the unassembled GDP-tubulin heterodimer exists in a continuum of conformations ranging between straight and bent, but, in agreement with existing structural data, suggests that an intermediate bent state has a lower free energy (by ∼1 kcal/mol) and thus dominates in solution. In agreement with predictions of the lattice model of microtubule assembly, lateral binding of two αβ-tubulins strongly shifts the conformational equilibrium towards the straight state, which is then ∼1 kcal/mol lower in free energy than the bent state. Finally, calculations of colchicine binding to a single αβ-tubulin dimer strongly shifts the equilibrium toward the bent states, and disfavors the straight state to the extent that it is no longer thermodynamically populated.     

In Silico Adoption of an Orphan Nuclear Receptor NR4A1

A 4.1μs molecular dynamics simulation of the NR4A1 (hNur77) apo-protein has been undertaken and a previously undetected druggable pocket has become apparent that is located remotely from the ‘traditional’ nuclear receptor ligand-binding site. A NR4A1/bis-indole ligand complex at this novel site has been found to be stable over 1 μs of simulation and to result in an interesting conformational transmission to a remote loop that has the capacity to communicate with a NBRE within a RXR-α/NR4A1 heterodimer. Several features of the simulations undertaken indicate how NR4A1 can be affected by alternate-site modulators.     

Identification of structural transitions in bacterial fatty acid binding proteins that permit ligand entry and exit at membranes

Fatty acid (FA) transfer proteins extract FA from membranes and sequester them to facilitate their movement through the cytosol. Detailed structural information is available for these soluble protein–FA complexes, but the structure of the protein conformation responsible for FA exchange at the membrane is unknown. Staphylococcus aureus FakB1 is a prototypical bacterial FA transfer protein that binds palmitate within a narrow, buried tunnel. Here, we define the conformational change from a “closed” FakB1 state to an “open” state that associates with the membrane and provides a path for entry and egress of the FA. Using NMR spectroscopy, we identified a conformationally flexible dynamic region in FakB1, and X-ray crystallography of FakB1 mutants captured the conformation of the open state. In addition, molecular dynamics simulations show that the new amphipathic α-helix formed in the open state inserts below the phosphate plane of the bilayer to create a diffusion channel for the hydrophobic FA tail to access the hydrocarbon core and place the carboxyl group at the phosphate layer. The membrane binding and catalytic properties of site-directed mutants were consistent with the proposed membrane docked structure predicted by our molecular dynamics simulations. Finally, the structure of the bilayer-associated conformation of FakB1 has local similarities with mammalian FA binding proteins and provides a conceptual framework for how these proteins interact with the membrane to create a diffusion channel from the FA location in the bilayer to the protein interior.     

High‐resolution analysis of the conformational transition of pro‐apoptotic Bak at the lipid membrane

Permeabilization of the outer mitochondrial membrane by pore‐forming Bcl2 proteins is a crucial step for the induction of apoptosis. Despite a large set of data suggesting global conformational changes within pro‐apoptotic Bak during pore formation, high‐resolution structural details in a membrane environment remain sparse. Here, we used NMR and HDX‐MS (Hydrogen deuterium exchange mass spectrometry) in lipid nanodiscs to gain important high‐resolution structural insights into the conformational changes of Bak at the membrane that are dependent on a direct activation by BH3‐only proteins. Furthermore, we determined the first high‐resolution structure of the Bak transmembrane helix. Upon activation, α‐helix 1 in the soluble domain of Bak dissociates from the protein and adopts an unfolded and dynamic potentially membrane‐bound state. In line with this finding, comparative protein folding experiments with Bak and anti‐apoptotic BclxL suggest that α‐helix 1 in Bak is a metastable structural element contributing to its pro‐apoptotic features. Consequently, mutagenesis experiments aimed at stabilizing α‐helix 1 yielded Bak variants with delayed pore‐forming activity. These insights will contribute to a better mechanistic understanding of Bak‐mediated membrane permeabilization.     

Polyethylene glycol binding alters human telomere G-quadruplex structure by conformational selection

Polyethylene glycols (PEGs) are widely used to perturb the conformations of nucleic acids, including G-quadruplexes. The mechanism by which PEG alters G-quadruplex conformation is poorly understood. We describe here studies designed to determine how PEG and other co-solutes affect the conformation of the human telomeric quadruplex. Osmotic stress studies using acetonitrile and ethylene glycol show that conversion of the ‘hybrid’ conformation to an all-parallel ‘propeller’ conformation is accompanied by the release of about 17 water molecules per quadruplex and is energetically unfavorable in pure aqueous solutions. Sedimentation velocity experiments show that the propeller form is hydrodynamically larger than hybrid forms, ruling out a crowding mechanism for the conversion by PEG. PEGs do not alter water activity sufficiently to perturb quadruplex hydration by osmotic stress. PEG titration experiments are most consistent with a conformational selection mechanism in which PEG binds more strongly to the propeller conformation, and binding is coupled to the conformational transition between forms. Molecular dynamics simulations show that PEG binding to the propeller form is sterically feasible and energetically favorable. We conclude that PEG does not act by crowding and is a poor mimic of the intranuclear environment, keeping open the question of the physiologically relevant quadruplex conformation.