Size exclusion characterization of the tBID/BCL-XL complex
To characterize the interactions between tBID and BCL-XL, we analyzed the free and mixed proteins using analytical size exclusion chromatography and solution NMR spectroscopy. All of the binding studies were carried out at pH 4 since human tBID has very limited solubility at pH values greater than 5. This reflects the dramatic changes in the physical properties of the protein that accompany caspase-8 cleavage: removal of residues 1-60 shifts the calculated pI from 5.3 to 9.3 and the net charge from -7 to +2, and exposes hydrophobic residues in helix h3, containing the BH3 domain and in the central core helices h6 and h7 (18
). The association of BCL-XL and BID with lipid bilayer membranes is promoted by acidic pH (31
) and involves a conformational change of the proteins (22
). Nevertheless, although acidic pH is important for membrane insertion, BCL-XL has been reported to retain its globular fold upon acidification from pH 7.4 to 4.9 (49
). We find that BID, like BCL-XL, is highly stable at pH 4, as evidenced by the 1
N HSQC NMR spectra which exhibit no evidence of unfolding compared to the spectra at pH 7 ().
1H/15N HSQC NMR spectra of 15N-labeled (A) BCL-XL and (B) BID at pH 7 (black) or pH 4 (red).
The data from analytical size exclusion chromatography () show that the free proteins elute at apparent molecular weights corresponding to those calculated from their respective amino acid sequences (BID: 23 kDa; BCL-XL: 21 kDa; tBID 15 kDa), reflecting their monomeric states in solution. A mixed sample of equimolar BID and BCL-XL elutes at an apparent molecular weight near 20 kDa, as observed for the individual components and, therefore, shows no evidence of complex formation. In contrast, a sample containing equimolar tBID and BCL-XL elutes at a distinctly higher molecular weight, close to the added molecular weights of the individual proteins (~36 kDa). This result is in agreement with the finding that tBID has a 10-fold greater binding affinity than BID for BCL-XL (9
) and demonstrates that tBID and BCL-XL form a long-lived complex in solution, while no stable interaction is observed between BID and BCL-XL.
Size exclusion chromatography showing formation of the tBID/BCL-XL complex in aqueous solution. The dotted lines indicate elution of (a) tBID, (b) BCL-XL, and (c) tBID+BCL-XL.
Structural characterization of the tBID/BCL-XL interaction
The 1H and 15N chemical shift frequencies in the HSQC NMR spectra of proteins are highly sensitive to the chemical environment and conformation of their corresponding15N-labeled protein site, and the peak line widths and shapes reflect protein backbone dynamics, conformational order, and aggregation state. In addition, the changes in chemical shifts can be used to monitor changes in protein structure and ligand binding. Although the CD spectrum of tBID in water shows that it adopts α-helical secondary structure like its intact precursor (), its 1H/15N HSQC spectrum exhibits the hallmark features of dynamically disordered proteins (, black). Most of the peaks cannot be detected in the spectrum, indicating that the protein adopts multiple conformations and undergoes dynamic conformational exchange in the intermediate range of the μsec-msec NMR time scale. Protein aggregation, which could also result in line broadening and degradation of the NMR spectrum, is ruled out by the size exclusion chromatography profile showing that tBID elutes as a single peak, at an apparent molecular weight corresponding to that of the monomeric protein. The few peaks that are visible in the HSQC spectrum can be assigned to residues in the unstructured N-terminal loop of the protein (), and their chemical shift frequencies reflect the highly dynamic random coil conformation of this segment.
Secondary structure and structural order of tBID and BID61-104. (A) CD spectra obtained at pH 4 or pH 7. (B) Predicted structural order for BID, tBID, BID61-104, BCL-XL, and BIM at pH 4 (●) or pH 7 (○).
HSQC NMR spectra of 15N-labeled tBID at pH 4, free (black), or bound to equimolar BCL-XL (red). Arrows point to examples of new peaks that appear in the spectrum of the complex.
Figure 6 HSQC NMR spectra of 15N-labeled tBID and BID61-104, free or bound to BCL-XL. (A) Free tBID (black) and free BID61-104 (red). Selected peaks from the loop and from the BH3 domain (boxes) are labeled. (B) BCL-XL-bound tBID (black) and BCL-XL-bound BID61-104 (more ...)
To see whether the tertiary structure of tBID could be stabilized by association with BCL-XL, we examined its NMR spectrum within the complex (, red). Overall, the association of tBID with BCL-XL produces little improvement in its NMR spectrum, however, binding BCL-XL does cause many peaks to appear anew in the spectrum. These peaks have dispersed 1H and 15N chemical shift frequencies consistent with stable secondary and tertiary structures of proteins, indicating that the structure of at least a region of tBID is indeed stabilized upon binding BCL-XL. To assign these peaks, and map the interaction of tBID with BCL-XL, we compared the spectra of tBID with those of a shorter polypeptide, BID61-104, spanning residues 61 to 104 which include part of the unstructured loop and the entire BH3 domain of the protein. In the NMR spectrum of free BID61-104 (, red) the peaks from residues in the N-terminal loop (e.g. N62, R63, H66, R68, E73, A74) perfectly overlap the peaks from the same residues in the spectrum of free tBID (, black). In contrast, peaks from the BH3 domain (e.g. S78, I82, A87, L90, A91, G94, D95, S96, L97, G104), which are clearly resolved in the spectrum of BID61-104, cannot be seen in the spectrum of tBID. In the spectrum of free BID61-104, peaks from the BH3 domain have chemical shifts that are consistent with random coil conformation, indicating that the peptide is unstructured in water. This is further supported by the CD spectra of BID61-104 showing that the polypeptide in water is a random coil at both pH 4 and pH 7, while intact tBID is α-helical ().
The addition of BCL-XL induces identical changes in the spectra of BID61-104
and tBID (). Resonance assignments of the spectra from free and bound BID61-104
() show that the peaks which experience frequency changes in the tBID/BCL-XL complex correspond to residues in the BH3 domain and in helix h3 identified in the solution NMR structure of full-length human BID (18
). In contrast, peaks from residues outside this region do not change in the spectrum of BID61-104
, and remain undetected in the spectrum of tBID, suggesting that binding to BCL-XL does not induce a major conformational change of tBID in water.
The distinct effects of BCL-XL on the spectra of tBID and BID61-104
reflect changes in both protein conformation and dynamics. The peaks that experience the largest frequency changes map to residues in helix h3, particularly, R84, A87, R88, L90, A91, and D95, which line up along the length of helix h3 in the structure of BID (). These residues are highly conserved in the BH3 domains of the pro-apoptotic BCL-2 proteins (50
), and are crucial for the interactions of the BH3 domain with pro-survival proteins (23
). For example, in the structure of a BAK BH3 peptide bound to BCL-XL, they make key contacts with residues in the hydrophobic binding groove of BCL-XL, including key hydrogen bonds between BAK D83 (corresponding to BID D95) and the BCL-XL R139 guanidinium group (23
). Our results demonstrate that the same BH3 domain residues are involved in the interaction of tBID with BCL-XL.
Figure 7 Mapping the effects of BCL-XL on tBID. (A) Total change in BID61-104 amide chemical shifts induced by BCL-XL. (B) Helicity of free (black) and BCL-XL-bound (red) BID61-104, measured as the difference between experimentally measured and random coil values (more ...)
The NMR data also show that BID61-104 undergoes a major conformational change upon binding BCL-XL, from random coil and highly dynamic in its free form, to helical and ordered when bound to BCL-XL (). Carbon chemical shifts are strongly dependent on backbone torsion angles and can be analyzed to obtain useful of indices of polypeptide secondary structure. Analysis of the CA and CB chemical shifts in the spectra of the free and bound peptide show that BCL-XL binding induces the formation of a well-defined α-helix between residues 78 to 97, which correspond to helix h3 in the solution NMR structure of BID.
The frequency changes and helix formation observed upon binding BCL-XL are accompanied by a sharp decrease in HSQC peak intensity, for residues 78 to 101, reflecting the change from a free highly dynamic conformation to a more rigid bound state (). This indicates that the entire 20-residue helical segment, and not just the 14-residue BH3 domain previously identified for the shorter BAK peptide, associates with the binding pocket of the pro-survival partner, consistent with the results reported for a short BID BH3 peptide bound to BCL-XL (29
) or to BCL-W (26
). Since the spectra of BCL-XL-bound BID61-104
and BCL-XL-bound tBID are fully overlapped in this region, the results in also mirror the interaction of the entire tBID molecule with BCL-XL. Taken together, the data show that the three-dimensional conformation of tBID is highly disordered in water compared to the well-defined globular structure of its full-length precursor, and that binding to BCL-XL induces localized but not global structural ordering of tBID.
To examine the binding site of tBID on BCL-XL we mapped the frequency changes caused by tBID on the 1
N HSQC spectrum of 15
N-labeled BCL-XL. Addition of tBID causes a subset of peaks in the spectrum of BCL-XL to shift position, while many others exhibited no or only minor changes, indicating that complex formation does not induce a major structural rearrangement of BCL-XL (). The largest changes are observed for peaks from residues in helices h2, h3, h4 and h5 (), and when these changes are mapped onto the structure of BCL-XL determined in complex with the BAK BH3 peptide (23
) they clearly highlight the same hydrophobic groove formed by the BH1, BH2, and BH3 regions of BCL-XL, that was previously identified as the BH3-binding site (). Short BH3 peptides from BAD and BAK bind BCL-XL in the same binding region and with similar helicities, and structural studies of 25- or 16-residue BAD BH3 peptides bound to BCL-XL show that the greater affinity of the longer peptide is attributed to the formation of additional interactions with BCL-XL, and to its increased helix propensity (23
). Furthermore, a short BIM BH3 peptide forms a C-terminally extended helix upon binding BCL-XL (25
Figure 8 Mapping the effects of tBID on BCL-XL. (A) HSQC NMR spectra of 15N-labeled BCL-XL without (black) or with (red) of unlabeled tBID. (B) Total change in BCL-XL amide chemical shifts induced by equimolar tBID. The helical regions identified in the solution (more ...)
The mode of association of tBID with BCL-XL shares similarities as well as subtle differences with those observed for the other BH3-only protein BIM. In both cases, binding to BCL-XL induces selective stabilization of the BH3 domain. BIM-L has an intrinsically unfolded random coil conformation in the absence of pro-survival binding partners, and its BH3 domain becomes α-helical when it binds pro-survival proteins while the rest of the molecule remains unstructured (30
). The situation for tBID is somewhat different because the protein maintains the α-helical secondary structure of its precursor, despite its dynamically disordered three-dimensional conformation; in this case, the structure of the BH3 domain is already helical but becomes ordered upon binding BCL-XL while the rest of the molecule remains disordered.
This difference between tBID and BIM is consistent with an analysis of their amino acid sequences based on normalized net charge and mean hydrophobicity (51
), indicating a higher degree of structural order for tBID compared to the natively unfolded BIM isoforms (). Like BIM, two other BH3-only proteins BAD and BMF are also predicted to be natively unstructured (30
), suggesting that lack of structure, structural disorder, and plasticity may be common features of the BH3-only proteins required for their activity and ability to bind multiple partners within and outside the BCL-2 family, and in both cytosolic and membrane-bound conformations. Indeed, the BIM BH3 domain is capable of binding not only BCL-XL but also BAX (28
), albeit at completely different sites of these two structurally similar BCL-2 proteins, indicating that a principal structural requirement for these interactions is the capability of helix formation by the BH3 domain.
Thermodynamics of tBID/BCL-XL association
To further characterize the interaction of tBID with BCL-XL, we performed ITC experiments by titrating both tBID and BID61-104 into BCL-XL. Representative titrations are shown in , and the associated thermodynamic parameters for binding are reported in .
Figure 9 (A-C) ITC titrations of tBID and BID61-104 into BCL-XL. Top panels show calorimetric data. Bottom panels show data obtained by integration of each calorimetric peak, after subtraction of blank titrations and concentration normalization. The red lines (more ...)
Dissociation constants and thermodynamic parameters, determined from ITC, for the polypeptides tBID and BID61-104 binding to BCL-XL at 303°K.
The binding affinity of tBID for BCL-XL at pH 4 (Kd
=27 nM) is only about 3.5 times weaker than that of BID61-104
at pH 4 (Kd
=7.25 nM), and very similar to that of BID61-104
at pH 7 (Kd
=30.58 nM). These values are also within the range of affinities reported in literature for short BH3 peptides binding to BCL-XL and other anti-apoptotic BCL-2 proteins (23
), indicating that the binding interaction of tBID with BCL-XL does not involve additional regions of tBID other than residues in and flanking the BH3 domain.
For both tBID and its short BH3 analog, BID61-104
, the association with BCL-XL is enthalpy-driven. The binding thermodynamics are characterized by very similar and opposing terms of ΔH and ΔS (), with favorable enthalpy (ΔH<0) and unfavorable entropy (ΔS<0), as previously observed for a series of short BH3 peptides binding the anti-apoptotic BCL-2 family protein MCL-1 (27
). The overall binding enthalpy (ΔH) includes a term for intrinsic enthalpy (ΔHi
) that reflects the selectivity of the specific interactions between ligand and target, terms associated with conformational change of the target (ΔHct
) or of the ligand (ΔHcl
) upon binding, and a term (ΔHH
) associated with protonation/deprotonation events (53
). The overall binding entropy (ΔS) includes a term for the hydrophobic effect (ΔSh
) that contributes an entropy increase through the generation of free water molecules, and terms associated with the loss of conformational flexibility of the target (ΔSct
) and of the ligand (ΔScl
) both of which contribute an entropy decrease.
Since both tBID and BID61-104 bind the same groove of BCL-XL through the same BH3 sequence, and induce a similar conformational change in BCL-XL, similar values of ΔHi, ΔHct, ΔSh and ΔSct are expected for their association with BCL-XL. Thus, any small differences between the affinities of tBID and BID61-104 would be due to differences in the thermodynamic terms associated with ligand conformation (ΔHcl, ΔScl). Protonation/deprotonation effects do not contribute for measurements made at the same pH, however, the slightly greater affinity observed for BID61-104 at pH 4 compared to pH 7 may be related to protonation strengthening the hydrogen bonds between the ligand and BCL-XL and/or promoting helix formation of the BID ligand. In the case of tBID, this effect may be countered by the overall greater unfavorable entropy associated with inducing conformational order in the bigger full-length molecule, resulting in similar thermodynamic parameters for tBID at pH 4 and BID61-104 at pH 7. It is possible that the BH3 domain of tBID retains some interactions with other regions of the protein, as it does in the precursor BID. In this case, binding the hydrophobic groove of BCL-XL would first require release of the tBID BH3 domain. The overall favorable free energy ΔG associated with tBID binding to BCL-XL indicates that BCL-XL-bound tBID is thermodynamically more stable than free tBID. Nevertheless, we note that the close similarity in the affinities of tBID and BID61-104 for BCL-XL overshadows any minor differences, and demonstrates that the interaction is limited to the tBID BH3 domain and the BCL-XL hydrophobic cleft.
Unlike full-length tBID, where the NMR and CD spectra reflect the dynamic exchange of conformations with α-helical secondary structure, BID61-104
and all of the small BH3 ligands are random coils in their free state, and adopt α-helical conformation only upon binding their pro-survival target. This is consistent with the finding that binding of BH3 domains to their pro-survival targets is favored by propensity toward helical structure (24
) and that short BH3 peptides chemically modified to promote α-helicity bind with greater affinity (29
). Although full-length tBID already has a significant amount of α-helical secondary structure in its free state, the NMR and ITC data show that its three-dimensional conformation is stabilized by binding BCL-XL.