Structural insights of tBid, the caspase-8-activated Bid, and its BH3 domain

Abstract

The Bcl-2 family proteins regulate mitochondria-mediated apoptosis through intricate molecular mechanisms. One of the pro-apoptotic proteins, tBid, can induce apoptosis by promoting Bax activation, Bax homo-oligomerization, and mitochondrial outer membrane permeabilization. Association of tBid on the mitochondrial outer membrane is key to its biological function. Therefore knowing the conformation of tBid on the membrane will be the first step toward understanding its crucial role in triggering apoptosis. Here, we present NMR characterization of the structure and dynamics of human tBid in 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)] micelles. Our data showed that tBid is monomeric with six well defined α-helices in the micelles. Compared with the full-length Bid structure, a longer flexible loop between tBid helix α4 and α5 was observed. Helices in tBid do not pack into a compact-fold but form an extended structure with a C-shape configuration in the micelles. All six tBid helices were shown to interact with LPPG micelles, with helix α6 and α7 being more embedded. Of note, the BH3-containing helix α3, which was previously believed to be exposed above the membrane surface, is also membrane associated, suggesting an "on the membrane" binding mode for tBid interaction with Bax. Our data provided structural details on the membrane-associated state of tBid and the functional implications of its membrane-associated BH3 domain.

Keywords: Apoptosis; Bcl-2; LPPG Micelles; NMR; Protein Dynamics; Protein Structure; tBid.

FIGURE 1.

Secondary structures of human tBid in LPPG micelles. The secondary chemical shift index (¹³Cα, ¹³Cβ, ¹Hα, and ¹³CO) defined the presence of six α-helices. The chemical shift differences were calculated by subtracting the average random coil chemical shift from the assigned tBid chemical shift values. The consecutive large positive bars in ¹³Cα,¹³CO and negative bars in ¹³Cβ,¹Hα suggest the presence of the α-helical conformation. The defined α-helices were also indicated by the characteristic medium range inter-proton NOE connectivities of Hαⁱ to Hβⁱ⁺³ and strong NOE connectivities of H_Nⁱ to H_Nⁱ⁺¹. The thickness of bars used to show NOE connectivities corresponds to the relative NOE intensity. The missing NOEs within the α-helical regions are due to overlapped peaks. The secondary structures of human Bid reported previously were also shown for comparison (32). Residue numbering is kept the same as the full-length Bid.

FIGURE 2.

Backbone ¹⁵N relaxation dynamics of human tBid in LPPG micelles. tBid ¹⁵N T₁ (A) and T₂ (B) relaxation times at two magnetic fields of 600 MHz (red) and 800 MHz (black) and the fitted backbone order parameter S² using measured T₁ and T₂ data at both magnetic fields (C) are plotted as a function of residue number, indicating all six (α_3–8) helical regions were shown to be rigid. The shorter T₂ values (B) for both helices α₆ and α₇ suggested they are likely more embedded in the micelle. Error bar for the ¹⁵N T₁ and T₂ relaxation data were estimated as previously described (50). D, the residues undergoing microsecond-millisecond conformational exchange identified using the backbone ¹⁵N R₂-CPMG rate differences, ΔR₂, the difference between measurements at two different τ_cp values (ΔR₂ = R₂ (6 ms) − R₂ (0.3 ms)). The cutoff line corresponds to the ΔR₂ threshold of 1.5 times the S.D. The missing values are due to resonance peak overlap. Secondary structure elements of tBid are indicated at the top for reference.

FIGURE 3.

Measured PRE effects for human tBid cysteine mutants in LPPG micelles at three different sites. The experimental ¹H_N-Γ₂ values for S78C (A), Q136C (B), and Q180C (C) were plotted as the function of residue number. Residues experiencing significant PRE effects are mainly localized to residues that are close to the paramagnetic center. For the S78C sample (A), weak PRE effects for residues in the C terminus included helix α₈. C, albeit even weaker, similar weak PRE effects were also observed between the tBid N terminus and C terminus. For the Q136C sample (B), weak PRE effects were observed for residues in helices α₄ and α₄ as well as some residues in flexible N and C termini. Error bars were estimated as previously described (51). Secondary structure elements of tBid are indicated at the top for reference.

FIGURE 4.

Interactions between human tBid and LPPG micelles. A, illustration of inter-proton NOEs between the tBid BH₃ domain and LPPG micelles. A selection of the ¹⁵N-edited three-dimensional NOESY-HSQC strips of the tBid BH₃ domain, containing helix α₃ residues, showing the intermolecular NOEs to the LPPG acyl chain ¹H at a chemical shift of 1.26 ppm. Among these residues, Ile⁸⁶, Ala⁸⁷, Leu⁹⁰, Val⁹³, and Gly⁹⁴ showed obvious NOE cross-peaks to LPPG micelles. Conversely, these residues did not show an obvious cross-peaks signal to water (4.61 ppm). B, relative peak intensities are calculated by taking the ratios of measured NOE intensities between protein backbone amides (¹H_N) and the LPPG acyl chain methylene (CH₂) groups at 1.26 ppm against the diagonal peak intensities. These relative peak intensity ratios are used to quantify the strength of interaction between various sites in tBid and micelles. Residues showing obvious protein-lipid interactions are from all six tBid helices (α_3–8). C, the experimental solvent PRE measurement for tBid backbone amide protons. The measured ¹H_N-Γ₂ PRE values in the presence of a water-soluble paramagnetic probe showed obvious relaxation enhancement for residues that are water exposed and weak enhancement for residues that are embedded in the lipid. Secondary structure elements of tBid are indicated at the top for reference.

FIGURE 5.

Calculated human tBid structure shows a C-shape conformation. A, schematic representation of the 20 lowest energy structures of human tBid in LPPG micelles. The calculated tBid structures show six well defined helices α_3–8 and the superposition to the mean structure on tBid helical regions results in a backbone r.m.s. deviation of 3.64 Å. Both N-terminal (Gly⁶¹–Ser⁷⁸) and C-terminal (Ala¹⁹⁰–Asp¹⁹⁵) tails are unstructured and are omitted for clarity. Overall (B) tBid adopts a C-shape structure with both N and C termini in close conformation. C, tBid helices (α_3–8) are parallel to the putative membrane surface with helices α₆ and α₇ more embedded. For the membrane-associated tBid helix α₃, charged or polar residues (Asp⁸¹, Arg⁸⁴, Arg⁸⁸, Gln⁹², and Asp⁹⁵) are facing away from the membrane surface and are exposed for potential interactions with other proteins. C, on the opposite side of helix α₃ residues (Ile⁸⁶, Ala⁸⁷, Leu⁹⁰, Val⁹³, and Gly⁹⁴) with strong micelle interactions are facing toward the membrane surface. The flexible N terminus (Gly⁶¹–Ser⁷⁸) was omitted for clarity. D, electrostatic potential surface view of the side of the tBid structure facing the membrane shows dominant hydrophobic patches along the ordered tBid helices. E, the opposite side of the tBid surface shows strong charged areas along tBid helices with a continuous positive charged surface along the helices α₆-α₈. D and E, the flexible loop with a highly charged surface between helix α₄ and helix α₅ contains a number of charged residues.