Towards Understanding Mcl-1 Promiscuous and Specific Binding Mode

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Towards Understanding Mcl-1 Promiscuous and Specific Binding Mode. Jade Fogha, Bogdan Marekha, Marcella De Giorgi, Anne Sophie VoisinChiret, Sylvain Rault, Ronan Bureau, and Jana Sopková-de Oliveira Santos J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.7b00396 • Publication Date (Web): 10 Oct 2017 Downloaded from http://pubs.acs.org on October 13, 2017

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Journal of Chemical Information and Modeling

Towards Understanding Mcl-1 Promiscuous and Specific Binding Mode. Jade Fogha±,$,††, Bogdan Marekha±,$, , Marcella De Giorgi±’†, Anne Sophie Voisin-Chiret±, Sylvain Rault±, Ronan Bureau± and Jana Sopkova-de Oliveira Santos±,*

±

Normandie Univ, UNICAEN, CERMN, FR CNRS 3038 INC3M, SF 4206 ICORE bd Becquerel, F-14000 Caen, France

$

J.F. and B.M. contributed equally to this work and should be considered as joint first authors.

ACS Paragon Plus Environment

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ABSTRACT

Mcl-1, an anti-apoptotic member of the Bcl-2 protein family, is overexpressed in various cancers and promotes the aberrant survival of tumour cells. To inhibit Mcl-1, and initiate apoptosis, an interaction between BH3-only proteins and Mcl-1 anti-apoptotic protein is necessary. These protein-protein interactions exhibit some selectivity: Mcl-1 binds specifically to Noxa, whereas Bim and Puma bind strongly to all anti-apoptotic proteins. Even if the 3D structures of several Mcl-1/BH3-only complexes have been solved, the BH3-only binding specificity to Mcl-1 is still not completely understood. In this study, molecular dynamics simulations were used to elucidate the molecular basis of the interactions with Mcl-1. Our results corroborate the importance of four conserved hydrophobic residues and a conserved aspartic acid on BH3-only as a common binding pattern. Furthermore, our results highlight the contribution of the fifth hydrophobic residue in the C-terminal part and a negatively charged patch in the N-terminal of BH3-only peptides as important for their fixation to Mcl-1. We hypothesize that this negatively charged patch will be a Mcl-1 specific binding pattern.


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INTRODUCTION During the last several years, many anti-cancer therapies have converged in promoting apoptosis, a programmed cell death, via the Bcl-2 family proteins.1-4 Indeed, in mammalian cells, this family of proteins plays a major role in the intrinsic pathway of apoptosis defined by mitochondrial outer membrane permeabilization.5 The Bcl-2 family includes both anti-apoptotic and pro-apoptotic proteins, which regulate respectively the cellular life and death decision. Bcl-2 family proteins share a conserved domain, Bcl-2 Homology domain (BH), which allows their differentiation into three groups. Anti-apoptotic proteins such as Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and Bfl-1, have three or four BH domains (BH1-4). Among the pro-apoptotic members, there are both multidomain pro-apoptotic proteins (such as Bax, Bak, and Bok), which share also four BH domains (BH1-4)6, and proteins presenting only one BH3 domain (named BH3-only) which include Puma, Bim, Noxa, Bid, Bik, Bad, Hrk, and Bmf.7-9 Under cellular stress, BH3-only proteins initiate apoptosis by either blocking the activity of the anti-apoptotic members or directly activating the multidomain pro-apoptotic members such as Bax or Bak.10, 11 This allows for the homodimerization of Bax or Bak, which in its turn leads to the induction of caspase activation and cell death.12 In human cancers, anti-apoptotic proteins are overexpressed and favour cell proliferation and chemoresistance.13 The Mcl-1 gene, isolated from human myeloid leukemia cells, plays a critical role in cell development.14 The amplification of Mcl-1 is one of the most common genetic aberrations observed in human cancers.15,

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Thus, it is an important target in therapeutic research for

overcoming cancer and also chemoresistance.17-20 As an anti-apoptotic protein, Mcl-1 can be neutralized by its interaction with a BH3-only protein,12 which is governed by a binding specificity. Both in vitro and in vivo binding studies have shown that Mcl-1 binds to the BH3-


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only Puma and Bim with a high affinity, but these BH3-only proteins can also bind to other antiapoptotic proteins comparably well. Mcl-1 binds weakly to Bid, Bik, and Hrk. By contrast, Bad and Bmf preferentially interact with Bcl-2, Bcl-xL, Bcl-w, but not with Mcl-1 or Bfl-1. Noxa is the only BH3-only protein that engages Mcl-1 and Bfl-1, but not the other anti-apoptotic proteins.10 The resolution of 3D structures of numerous anti-apoptotic/BH3-only complexes21-26 yielded detailed information about the complex. They showed that it is ensured by an amphipatic helix of the BH3 domain which lies in a hydrophobic groove formed by BH1, BH2 and BH3 domains of the anti-apoptotic partner.27 The 3D structure analysis highlighted the crucial role of four conserved hydrophobic residues on the contact face of the BH3 helix and a conserved Asp (Aspc) of the BH3-only proteins.21-26 The four hydrophobic side chains corresponding to the i, i+4, i+7 and i+11 positions of BH3 amphipathic helix are projected into the hydrophobic cleft of the antiapoptotic partner and among them a fully conserved Leu (Leuc) at the i+4 position (Scheme 1) plays a pivotal role.28 On the opposite side of the BH3-only helix the complex coupling is completed by a conserved salt bridge between Aspc (i+9) of BH3-only proteins and Argc, fully conserved in all anti-apoptotic proteins. Even though several complexes with Mcl-1 have been solved and numerous biological studies have been carried out, the binding specificity of Mcl-1 towards the BH3-only proteins is still not completely understood. For example, regarding to the Mcl-1/Bim binding, several structural and limited mutagenesis studies have been performed and consensus result did not often appear.23, 29, 30 Despite some oppositions, those data suggested that the four conserved hydrophobic residues in Bim BH3 domains did not have the same contribution to the binding free energy in interaction with Mcl-1. As an illustration, Lee et al. have shown by saturation mutagenesis experiments that on the one hand29, Leuc (i+4) of Bim


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could only be replaced with any hydrophobic residue to retain measurable Mcl-1 binding while on the other hand, any side chain substitutions, even by a charged residue, at the i+11 position were tolerated by Mcl-1 contrary to Bcl-xL. To understand the binding specificity, crystal structures of some Bim mutants were solved and they showed that mutations had surprisingly modest effects on the complex stability, and that Mcl-1 can undergo small changes, via side chain rearrangements and/or backbone alterations, to accommodate the mutant ligands.23 For instance, the crystal structure of the mutated L62Y Bim peptide, which leads to a 60 fold decrease in affinity towards Mcl-1, showed only minor structural changes within the Leu binding pocket allowing to accommodate the bulkier Tyr residue.23 In the case of a large physicochemical mutation, Phe69 residue (i+11) substituted by Glu, reduced the affinity for BclxL and not for Mcl-1. The crystal structure of the Mcl-1/BimF69E complex showed a simple rotation of Glu at the i+11 site out of the hydrophobic groove. These results demonstrate that Mcl-1 is able to maintain high affinity for peptides with a broad variety of residues at these key hydrophobic sites.31 Nonetheless, these structural rearrangements observed in X-ray structures of the complexes are difficult to anticipate and make it challenging to predict whether individual BH3 peptides would bind to various Bcl-2 proteins.31 Contrary to the hydrophobic residues, many mutagenesis studies converged to the fact that Apsc mutations reduce binding to Mcl-1 and other anti-apoptotic proteins.32 For example, Lee et al. observed that Bim mutation of Aspc67 by alanine reduces the binding to Mcl-1 8-folds.32

Given the importance of Mcl-1 as a pathologic factor and a few Mcl-1 selective inhibitors in clinical trials33, we present in this paper an in silico strategy, based on molecular dynamics (MD) simulation, to elucidate the molecular basis of a selective interaction with Mcl-1. MD allows for


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a detailed analysis of structural and functional aspects of PPIs and thus provides valuable insights into PPI mechanisms and supports the design of PPI modulators (see for example reviews34,35). For this purpose, we explored protein-protein interactions between Mcl-1 and different BH3-only proteins in a comparative study with non-selective, particularly selective as well as non-binding ligands. In this study, we focused on human complexes Mcl-1/Bim, Mcl1/Noxa, Mcl-1/Puma and a hypothetical interaction between Bad and Mcl-1 since Bad does not bind Mcl-1 (see peptide sequences in Scheme 1).10

MATERIALS AND METHODS Proteins Three-dimensional (3D) structure of human Mcl-1 associated with human Bim (PDB ID: 2NL9)26 solved by X-ray crystallography was extracted from the Protein Data Bank (PDB).36,37

Homology modelling The human Mcl-1/Noxa, Mcl-1/Puma complexes absent in the PDB were built using the sequence homology module of Discovery Studio software version 3.0.38 The amino acid sequences of human Mcl-1 and human Noxa were retrieved from the Uni-Prot database (accession number: hMcl-1 Q07820, hNoxa Q13794) and aligned to two complex sequences as template: i) mouse NoxaA/Mcl-1 (PDB ID: 2ROD)25,78.1% identity, 88% similarity and ii) mouse NoxaB/mutated human Mcl-1 (PDB ID: 2NLA)26,81.2% identity or 87.4% similarity. The human Mcl-1/Puma complex was built using the human Mcl-1 and Puma sequences (accession number: hMcl-1 Q07820, hPuma Q9BXH1) and structural data of its mouse homologous available in PDB (PDB ID: 2ROC; 85.4% identity and 93.2% similarity25).


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The hypothetical Mcl-1/Bad complex was built using a multiple template homology modelling using human Mcl-1 and human Bad sequences (accession number: hMcl-1 Q07820, hBad Q92934) and the following 3D structures (PDB ID: 2NLA26, 2NL926, 2JM626, 2ROC25, 2ROD25, 3PK139, 2PQK31). Sequence identity and similarity was respectively: 76,8% and 82,1% for 2NLA; 74.7% and 78.9% for 2NL9; 75.8% and 84.7% for 2JM6; 76.3% and 88.4% for 2ROC; 74.7% and 85.3% for 2ROD; 75.3% and 80% for 3PK1; 80.4% and 82% for 2PQK. The sequence alignment was carried out using the multiple sequence alignment module of Discovery Studio software and the 3D structure of human complex was built by the MODELER program.38 The best model was selected according to the DOPE score of the MODELER program. The selected model was further evaluated by the Profiles-3D programs of Discovery Studio Software.38

Molecular dynamics simulations All simulations were carried out using NAMD 2.1240 with the all-atom CHARMM 36 force field.41 To simulate aqueous solvent environment, each system was surrounded by a rectangular box of TIP3P water molecules42 using CHARMmGUI solvator.43,

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The chosen box size

ensured, for each complex, that the simulated protein was at a minimum distance of 10 Å from the edge. Orthorhombic periodic boundary conditions were employed. Van der Waals interactions were truncated using a shift function with a cut-off distance of 12 Å and electrostatic ones using a force switching function, between 10 and 12 Å. The vacuum dielectric constant was used during all calculations. The systems underwent energy minimization in three stages. Water molecules were minimized first, then the protein-ligand complex and finally the whole system. Each minimization process consisted of 15000 conjugated gradient steps.


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Molecular dynamics simulations were performed using the Verlet algorithm and 2 fs integration step. The minimized systems were gently heated from 0 K to 300 K by 5 K jumps during 300 ps and then the dynamics were temperature-equilibrated during 1 ns under NPT ensemble. Langevin dynamics with a damping coefficient of 1 ps−1 was used to maintain the system temperature and Nosé-Hover Langevin piston method to control the pressure at 1 atm. Finally, the systems ran freely for 160 ns and 3200 frames were taken for subsequent analysis. Residue-wise decomposition of the interaction energy was performed within the MM-PBSA approach45 using CHARMM v.40b246 and home-made scripts. We employed single trajectory approach and the same CHARMM 36 force-field that was used in the dynamics simulations. The contributions of each BH3 peptide residue to the interaction energy was divided into polar ∆Gpolar and nonpolar ∆Gnonpolar parts. The polar interaction energy ∆Gpolar is calculated as a sum of the polar electrostatic solvation energy, ∆GPB, and the force-field Coulombic interaction energy, ∆Gelec. The former was calculated by solving the linear Poisson-Boltzmann equation with the static permittivity of solute and solvent set to 1 and 80 respectively and 0.3 Å grid. The nonpolar term ∆Gnonpolar was calculated from ∆Gnps which is the non-polar solvation term represented as γ SASA + β, SASA being solvent accessible surface area estimated with water probe size of 1.4 Å, γ = 0.00542 kcal·mol−1·Å−2 and β = 0.92 kcal·mol−1 and from ∆GvdW which stands for the residue contribution to the force-field vdW interaction energy.

RESULTS AND DISCUSSION As we were interested in the study of complexes of human origin, human Mcl-1/Bim26 complex was directly retrieved from the PDB and the others, unavailable in the PDB, were built


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using homologous complexes also extracted from the PDB.36,

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In order to investigate the

evolution of Mcl-1/BH3-only interaction, 160 ns all-atoms MD simulations were carried out on each complex, previously solvated and equilibrated to approach physiological conditions. The simulations were analysed in terms of averaged structures (Figure 1) as well as the dynamic evolution of the key interatomic distances. Afterward, key residues responsible for the complex formation were brought out based on the interaction energy contribution of each BH3-only residue with the whole Mcl-1 protein along the trajectory. For more insight, polar (electrostatic interaction and polar solvation) and nonpolar (van der Waals interaction and apolar solvation) energy contributions were calculated separately. Mean values per BH3-only residue in each complex calculated from the collected data are showed in Figure 2. MD simulations revealed that the studied complexes were overall stable, even the one with Bad. Indeed, the all-atom root mean square deviations (RMSDs) calculated on the stable parts of the Mcl-1 structure with respect to the average structures along the trajectories did not exceed 2 Å. The calculated Cα fluctuations during the simulations confirmed that the helical parts of the BH3-only peptides and of Mcl-1 protein were preserved and stable during MD simulations in all four complexes. Fluctuation values in helical parts did not exceed 1.5 Å (SI Figure S2). In some complexes an enhancement of α-helicity for the peptide ligands was observed, a phenomenon already described in molecular dynamic simulations by Zhao et al.47 In the four complexes, the most important flexibilities for Mcl-1 were observed in N and C-terminal parts and in the loop between α1 and α2 helices (SI Figure S2), in the same manner as in the simulations on Mcl1/Bim, Mcl-1/Bax and Mcl-1/Mcl-1 BH3 peptide complexes by Zhao and co-wokers47 but not in the Mcl-1/Bim simulations published by Yang et al.48 where this loop was omitted in simulations. Our fluctuations analysis showed, once more in agreement with the Zhao’s results,


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likewise elevated Mcl-1 flexibility in loop α3-α4. Zhao et al. proposed that the fluctuations in this loop are related to the structural adjustment of Mcl-1 α4 helix with different BH3 peptides.47 Nevertheless, the flexibility of this loop and of the α4-α5 loop was different in Mcl-1/Bad complex. That is probably due to the loss of the key interactions with Argc263 and Asn260 present in the latter loop in the Bad complex (see discussion below). We have noted as well somewhat enhanced flexibility of the Bad peptide even within the core part of the binding sequence thus suggesting lower stability of this virtual complex.

Bim and Puma, promiscuous partners of anti-apoptotic proteins. MD simulation results on Mcl-1/Bim complex are in good agreement with the literature data.47, 49

In fact, the strongest nonpolar contributions (|∆Gnonpolar| > 4 kcal·mol−1) were found for Ile58,

Leu62, Ile65 and Phe69 (Figure 1 and 2), which correspond to the four buried hydrophobic residues (i, i+4, i+7 and i+11). In addition to these four conserved residues, Bim residue Arg63 (i+5) contributes also significantly to the nonpolar interaction energy as well as Tyr73, which is the next residue (i+15) on the interacting BH3 helix face with respect to the four buried ones (Figure 1 and 2). The nonpolar contribution of the Tyr73 to the complex formation was already observed by Zhao et al.47 Our average dynamic structure showed that Tyr73 interacts through πstacking with Mcl-1 Phe319 and that Arg63 is surrounded principally by Mcl-1 residues Val253, Ser255, Asp256, Arg263 and His252.

Concerning the polar contributions, our simulation results on Mcl-1/Bim showed that Aspc67 of Bim represented the strongest interaction (i+9, |∆Gpolar| ~ 54 ± 5 kcal·mol−1) (Figure 2) and the calculated average complex structure showed that Aspc67 establishes a salt bridge with Arg263,


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as expected and as it was observed in previously published simulations of Mcl-1/Bim (Figure 3).47,48 Among the Bim peptide charged residues we also detected three others that contributed significantly to the Mcl-1/Bim electrostatic interaction energy: Glu55(i-3), Glu61(i+3) and Glu68 (i+10). Bim Glu55 according to the average structure establishes a salt bridge with Mcl-1 Arg248. This Glu55 is located in the beginning of the Bim BH3 helix and this salt bridge is stable throughout the simulation, an anchor point already highlighted in simulations of Zhao and co-wokers.47 The side chains of two other charged residues, Glu61 and Glu68, are very flexible along the dynamics and they establish most often an H-bond with Mcl-1 His224. Bim Arg63 whose nonpolar contribution was detected as an important one, seems to interact with many residues along the dynamics, namely His252, Ser255, Asp256, Arg263 (Figure 3), but the net effect is strongly destabilizing polar repulsion with Arg263. We can therefore conclude that in the helical part the most important Bim residue for the electrostatic interaction is the Aspc67 forming a salt bridge with Mcl-1 Arg263 with significant contributions originating as well from the Glu55, Glu61, and Glu68 via the salt bridge with Arg248 and H-bond with His224 of Mcl-1. The human Mcl-1/Puma complex was built using the sequence and structural data of its mouse homologue available in the PDB (PDB ID: 2ROC, 85.4% identity25). In the simulated human complex, the four buried hydrophobic residues Ile137 (i), Leu141 (i+4), Met144 (i+7) and Leu148 (i+11) once again contribute strongly to the vdW interaction energy of this complex (|∆Gnonpolar| > 4 kcal·mol−1) (Figure 1 and 2). In addition to these four residues an important nonpolar interaction energy contribution was also detected for Trp133 (i-4) and Tyr152 (i+15) which correspond to the preceding and following residues on the interacting BH3 helix face with respect to the four buried ones mentioned before. Once more, an important nonpolar energy was


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also detected for Arg142 (i+5) of Puma, the Leuc neighbouring residue as in the Mcl-1/Bim complex. In the helical region of Puma sequence, the main residue contributing strongly to the complex polar free energy along our simulations is Aspc146 (i+9) (Figure 2). The average structure from dynamics simulation has shown that Aspc146 forms a salt bridge with Argc263 as expected (Figure 3). Two highly flexible Arg154 and Arg155 residues in the Puma C-terminal interact occasionally with Mcl-1 Asp323 giving rise to attractive contributions to the polar interaction energy. An additional residue, Glu136 situated in the Puma helix, was also detected and along the trajectory it seems to be close to Lys234 but this interaction is essentially transient (see Figure 4). This H-bond is present only during 25% of the simulation time. Another important electrostatic interaction pattern was also found to involve Arg248 of Mcl-1. This Mcl-1 residue transiently interacts with a negative patch at the C-terminal of Puma sequence establishing an Hbond either with Glu130 or Glu131 (Figure 4). Due to the high flexibility of this region of Puma sequence (see Figure S2) the corresponding averaged contributions to the polar interaction energy are cancelled out. Recently, Campbell and coworkers carried out the mapping of the BH3 binding interface of anti-apoptotic proteins and demonstrated that Mcl-1 binding to Bim and Puma was perturbed by alanine substitution of four Mcl-1 residues, which are the same for the two ligands: Val220, Asn260, Arg263, and Phe319.50 From our simulations, the corresponding BH3-only partners of these four Mcl-1 hotspots have been determined and we have found that many of them are the binding determinants listed above, such as Aspc (i+9) and Tyr (i+15) (Figure 5). Unsurprisingly, Argc263/Aspc, interaction existed along the entire MD simulation in both Bim and Puma complexes (see SI Figure S3). Mcl-1 Asn260 was also pointed out in Ala scan as a crucial one. It


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is situated in the vicinity of Arg263 and it could also bind to Argc. In fact, in our simulations, the Asn260/Aspc interaction was present in both the Mcl-1/Bim (as in Zhao et al.47 simulations) and Mcl-1/Puma complexes throughout the whole trajectories (see SI Figure S4). This result confirmed the outcome of the Ala scan. The visualisation of the average dynamic structure has showed that Val220, pointed out in the mapping study by Campbell et al., is situated on the top between the Mcl-1 cavities welcoming the i+7 and i+11 residues, and it interacts in both complexes with the hydrophobic residues at the i+11 position that are always bulky ones (Leu148 in Puma and Phe69 in Bim) (Figure 5). The fourth important Mcl-1 residue revealed by the Ala scan, Phe319, according to the average structures interacts with the hydrophobic residue i+11 and establishes above all π-stacking with Bim Tyr73 or Puma Tyr152 residues situated on the hydrophobic interacting face of the BH3 helix at the i+15 position. In our simulation of both complexes, an electrostatic interaction with Mcl-1 Arg248 was detected; it interacts with Glu55 of Bim and with Glu130/Glu131 of Puma. We also observed an interaction with Mcl-1 His224 but it was relatively weak in both complexes.

Bim and Puma bind to Mcl-1 and other anti-apoptotic proteins with comparable high affinities. Our simulations confirmed that Puma and Bim interact principally through the common binding motif which confers the ability to bind to any anti-apoptotic protein. Mutation studies, mainly carried out on Bim peptide, have shown that some substitutions at the key four hydrophobic positions can be tolerated especially at i+11. Either a small hydrophobic residue (Ala)32 or a polar one (Glu, Gln)29, 30 at the i+11 position maintained the high affinity for Mcl-1 and lead to selective binding to Mcl-1 versus Bcl-xL since those substitutions considerably reduced the BclxL binding affinity. Considering the Mcl-1 hotspot residues, additional interactions could play an


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important role in Mcl-1 binding affinity according to our simulations: firstly, an interaction between Phe319 and an aromatic residue in the i+15 (Tyr) position was put ahead in our simulations. We observed that this Phe319 interacts also with a hydrophobic residue at the i+11 position (Leu of Puma and Phe of Bim). More so, this residue at the i+11 position established also hydrophobic contacts with Mcl-1 hotspot residue Val220. Our simulations have highlighted the contribution of other charged Mcl-1 residues, in addition to Aspc, to the binding of Puma and Bim: Arg248 and to a lesser extent His 224. The mapping of the binding site 50 did not reveal the necessity of Mcl-1 His224 to Bim binding but the importance of Arg248 has not been evaluated in that study. Interestingly, an Arg is present in this position only in Mcl-1, in Bcl-xL and Bcl-2 it is replaced by a Gln.

Noxa, a selective Mcl-1 partner. A homology model of human Mcl-1/Noxa was used for MD simulation. It was built using two complexes as templates: mouse Mcl-1/NoxaA complex (PDB ID 2ROD26: 78.1% identity) and a hybrid complex between mutated human Mcl-1 and mouse NoxaB (PDB ID 2NLA26: 81.2% identity). The visualisation of the Mcl-1/Noxa average structure showed that Bim, Puma and Noxa helices have globally similar position in these complexes. In the Noxa complex, the four buried hydrophobic residues Cys25 (i), Leu29 (i+4), Phe32 (i+7) and Leu36 (i+11) once more, strongly contributed to the nonpolar interaction energy (|∆Gnonpolar| > 4 kcal·mol−1) (Figure 1 and 2). Besides these four residues, an important nonpolar contribution was also detected for Arg30, the Leuc neighbouring residue as in the two complexes discussed above and for Gln40, the residue at the i+15 position (Figures 1 and 2). The detected nonpolar contribution to the interaction energy for Gln40 in Noxa is slightly weaker than in the previous complexes, where


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aromatic Tyr residues were present at the i+15 position and they interacted through π-π stacking with Phe319 of Mcl-1. A published alanine scan on the mouse NoxaA pointed once more the crucial role of the hydrophobic residues at the i+4, i+7 and i+11 positions for the Mcl-1 binding25 which seems to be consistent with our simulation results. Day and co-workers have also observed that the mutation of the i+15th residue (Trp) in mouse NoxaA, by alanine decreased Mcl-1 binding, but to a lesser extent compared to the three mentioned before. Nevertheless, the residue in the i+15 position in human Noxa is Gln, a non-aromatic one and therefore it is difficult to extrapolate the magnitude of this mutation in human Noxa.

Among the polar interactions in the Mcl-1/Noxa complex three amino acids were detected as significant ones: Glu22 (i-3), Glu24 (i-1), and Aspc34 (i+9) (Figure 2). In the average Mcl1/Noxa complex, the Aspc34 formed indeed a salt bridge with Argc263 as in Puma and Bim complexes. The mutation studies published by Day et al. on the mouse NoxaA are in the same sense, when Aspc was replaced by the alanine the NoxaA did not bind to the mouse Mcl-1.25 Important contributions were also detected for Glu22 (i-3) which interacts with Arg248 (Figure 4) and it is situated in the same position as Bim Glu55 which also interacts with this Arg248 during the entire simulation. According to the average structure Glu24 binds by a salt bridge to Lys234 and this interaction is repetitively present (~50% of the time) during the 160 ns simulation (Figure 4). During the determination of Mcl-1 hotspots, Campbell and coworkers did not consider Noxa.50 Nevertheless, according to our simulation results, Mcl-1 hotspot residues, highlighted in the interaction studies with Bim and Puma, Val220, Asn260, Arg263, Phe319, can be engaged by


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Noxa in a similar way (Figure 5). Aspc binds to Arg263 and also interacts with Asn260 during the whole simulation (see SI Figure S3 and S4). The residue i+11 also establishes nonpolar interactions with Val220 and Phe319. Gln40 (i+15) sets up a hydrophobic contact with Phe319, but it is not aromatic, so an absence of π-π stacking is detected in Mcl-1/Noxa complex.

In agreement with the previous published structural analysis, as for Puma and Bim, our study confirmed that the hydrophobic residues situated on the one face of the BH3 helix as well as the Aspc on the opposite face make significant energetic contributions to the anti-apoptotic/BH3only complex formation. We also observed that Noxa can interact with Mcl-1 hotspot residues but compared to Puma and Bim, there is no π-π stacking between the residue i+15 and Phe319 in the Mcl-1/Noxa complex. This difference can be related to the fact that Puma or Bim have better binding affinities to Mcl-1 than Noxa. Unlike Puma or Bim, Noxa does not bind to all antiapoptotic Bcl-2 proteins. Determinants of this specificity are not yet fully understood, although prior studies provided some insights. For example, Chen and coworkers proposed that the presence of an aromatic residue at the i+7 position (Phe32) as well as the presence of a positively charged residue at the i+10 position (Lys35) in Noxa inhibited its interaction with Bcl-xL and Bcl-w and thus had an effect on the Mcl-1 specificity.10 Along our results, Phe32 plays an important role in the hydrophobic interaction between Noxa and Mcl-1, it is the third strongest nonpolar contribution, but our study does not point out the importance of Lys35 which is exposed to the solvent in our average structure. Based on our simulations, we have been oriented in a different way to understand this specificity. Indeed, the Glu24 (i-1), one of the most important polar contributors to the complex formation, detected by our simulations, interacts with Mcl-1 Lys234. Interestingly a Lys is present in this position only in the Mcl-1 sequence,


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replaced by a Gln in Bcl-xL and Bcl-2 according to the sequence alignment. This interaction also exists in the 3D structure of the mouse complex Mcl-1/NoxaA25 and the mouse complex Mcl1/NoxaB26. In our simulation, Lys234/Glu24 interaction is repetitively present (50%) throughout the simulation of human Mcl-1/Noxa complex. As we can see in Figure 4, the oppositely charged Glu24 (salt bridge acceptor) and Lys234 (salt bridge donor) are spatially close for periods of up to 10 ns. This salt bridge is significantly less stable than the crucial Argc/Aspc one (see SI Figure S3). The Argc/Aspc salt bridge is preserved over the whole MD simulation in Noxa as well as in Puma and Bim complexes. Interestingly, a glutamic acid (Glu136) is in the same position as Noxa Glu24 in the Puma sequence. Analysis of the MD simulation showed that the salt bridge between this Glu136 and Mcl-1 Lys234 in Mcl-1/Puma complex is only observed during 25% of the time (Figure 4). The superposition of Puma and Noxa complexes showed that in Puma complex, the position of Mcl-1 helix α3 containing Lys234 is somewhat different with respect to the Noxa one. This helix in Puma complex is located far away from the Puma helix. One reason for this could be the presence of a bulkier residue, Ile, at the i position in Puma with respect to the Cys in Noxa. In Puma complex, the Ile shape pushes the α3 helix away in order to avoid steric clashes between Ile and the residues of the α3 helix.

Mutation studies have shown that Glu22 in mouse NoxaA, the equivalent Glu24 (i-1) residue in human Noxa, are not critical for mouse Noxa binding.25 This does not seem to be in contradiction with our simulation results. The latter have shown that a dual negatively charged patch exists in the Noxa N-terminus including Glu22 and Glu24. Glu24 binds directly to Mcl-1 Lys234 and the second interaction through Glu22 with Arg248 is also present. Thus, when one residue is replaced by alanine, the other will be able to resume the N-terminus fixation in Mcl-1.


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Interestingly, in the Bcl-2 and Bcl-xL sequences a glutamine is present instead of Arg248. Furthermore, one published mutagenesis study on Bim revealed an intriguing result which has not been developed.30 The mutation of Trp in i-1 position of Bim peptide by Ala or by Glu increased the binding affinity for Mcl-1 and the same mutations had either no effect on the affinity for Bcl-xL (mutation Ala) or decreased the affinity (mutation to Glu). From this result, it seems that Glu in i-1 position of Bim appears to be important for the affinity and specificity towards Mcl-1. Given the fact that the BH3 domain of Noxa binds selectively to Mcl-1, we can make the hypothesis that this Glu patch in the N-terminal extremity can play an important role in Noxa selectivity.

Bad, a non-partner for Mcl-1 The hypothetical Mcl-1/Bad complex was built using a multiple template homology modelling (PDB ID: 2NLA26, 2NL926, 2JM626, 2ROC25, 2ROD25, 3PK139, 2PQK31) with sequence identities always great than 74%. During the molecular dynamic simulations the Bad helix has somewhat moved from its initial position. The average structure of Mcl-1/Bad shows that its Nterminus is situated closer to the α4 helix of Mcl-1 and higher with respect to the BH3 binding grove in Mcl-1 compared to the other studied BH3-only peptides (Figure 1). The per residue analysis has shown that the hydrophobic interactions were preserved, the four buried hydrophobic residues Tyr110 (i), Leu114 (i+4), Met117 (i+7) and Phe121 (i+11) always contribute significantly to the nonpolar interaction free energy (|∆Gnonpolar| > 4 kcal·mol−1). Moreover, a new important hydrophobic contribution was observed for Phe125, a residue at the i+15 position, as in the other complexes (Figure 2). In a similar vein to previously discussed


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complexes, the nonpolar contribution of Arg115, the neighbouring Leuc residue, was also significant. By contrast, important differences have been detected in the electrostatic interaction energies. In particular, none of the Bad residues exhibited strong polar contribution to the binding free energy. These results seem in agreement with the experimentally observed no affinity of Bad for Mcl-1.10 There are only two residues that had significant electrostatic interaction energies in the complex: Arg115 (i + 5) and Aspc119 (i + 9). According to the average structure, Arg115 interacts with Mcl-1 Asp256 and Ser255 and Aspc with the conserved Arg263. We noted that the calculated polar contribution of conserved Aspc119 is much weaker than in three other studied BH3 peptides. Subsequently, the interaction between Mcl-1 Asn260 and Bad Apcc, which accompanied the Argc263/Aspc in the three other studied complexes, is completely lost (Figure S4). As Bad changed its position in the Mcl-1 binding groove during the simulation, consequently its C-terminal part with the conserved Aspc119 lies further away from the α4-α5 loop with Argc263. The Argc263/Aspc119 interaction, detected as the key one in the formation of the other three previous complexes is weak in the Mcl-1/Bad complex (Figure S3). In spite of the involvement of the four hydrophobic residues, the weakened Argc263/Aspc salt bridge and the absence of negatively charged residues at the N-terminus capable of engaging Mcl-1 via Arg248 and/or Lys234 seems to be enough to inhibit the complex formation. Considering the hotspot residues determined by Campbell et al.50, we also noticed that except the loosening of the Argc/Aspc interaction and the loss of the Asn260/Aspc interaction, the hypothetical Bad complex displays a quite similar interaction scheme compared to Puma and Bim complexes (Figure 5). Mcl-1 Phe 319 establishes π-π stacking with Phe125 (i+15) and hydrophobic interactions also exist between Mcl-1 Val220/Phe121 (i+11), Mcl-1 Phe319/Phe121 along our simulation. Thus,


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all these results emphasized the fact that the binding affinity is governed by a complex combination of various interactions and especially Argc263/Aspc plays the crucial role in Mcl-1 binding.

What is the origin of different position of Bad in Mcl-1? Bad does not interact with Mcl-1 but it binds to Bcl-2 and Bcl-xL with a good affinity (Kd of about 10 nM).10 Determinants of this specificity are not yet fully understood, although prior studies provide some insights. For instance, in the first buried position (i) of various BH3 peptides, Ile and other medium-sized hydrophobic residues are found to be able to bind to both Mcl-1 and Bcl-xL. However, for Bad this residue is Tyr. This observation led Day et al. to hypothesize that bulky residue in this position might disfavour Bad binding to Mcl-1 and mutation of Tyr to Ile in Bad improved its binding to Mcl-1 while retaining binding to Bcl-xL.25 Unexpectedly, when the first buried residue in human Bim (Ile) was mutated to Tyr, this peptide bound to Mcl-1 with higher affinity. Therefore, steric clashes at the position i do not provide a reliable single site rule to govern the specificity. Another explored track was positions i+7 and i+10, mentioned by Chen et al.: an aromatic residue at i+7 and/or a positively charged residue at i+10 inhibited Bcl-xL interactions.10 Just like Bim or Puma, Bad has a negatively charged residue (Glu) at i+10 and a non-aromatic residue (Met) at i+7. Our dynamic study has shown that the Bad BH3 helix position is different with respect to the three other studied BH3-only peptides, which leads to the weakening of the fully conserved Argc/Aspc tether and to the loss of interaction with Mcl-1 Asn260. Only two complexes with Bad were solved to date and they are both with Bcl-xL. The interaction between Argc/Aspc was detected in one of the structures (PDB ID: 2BZW51) while it was absent in the other (PDB ID:


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1G5J22). Of course, as Bad does not bind to Mcl-1 the mapping studies of Bad for Mcl-1 binding grove cannot be carried out. Contrariwise, the experimental mapping of the Bad binding for BH3 binding groove in Bcl-xL was performed and interestingly it has not identified equivalents of Argc263 or Asn260 as the critical residues for Bad binding to Bcl-xL. Campbell et al. have detected as crucial Bcl-xL residues: Phe97, Tyr101 and Leu130.50 The comparison of the BclxL/Bad X-ray structure (2BZW) with our average Mcl-1/Bad model showed two important differences (Figure 6): (i)

In front of Bad Tyr110, a bulky Met residue is in Mcl-1 with respect to the smaller Leu in Bcl-xL, which causes a different orientation of this Tyr side chain in the Mcl-1/Bad complex.

(ii)

In the C-terminal part, along the Bcl-xL/Bad X-ray structure an interaction through hydrogen bond exists between Bad Glu120, Met117 carbonyl and Bcl-xL Tyr101. Interestingly, the mutation studies, which were carried out on the Bcl-xL BH3 binding groove, pointed this Tyr101 as one of the most important residues for the complex formation.50 In our Mcl-1/Bad dynamic a presence of hydrogen bond was observed between Met117 carbonyl and Mcl-1 His224 (residue in equivalent position to Tyr101 in Bcl-xL) and occasionally the hydrogen bond was formed also between Glu120 and this His224, but for Glu120 the net effect taking into account the desolvation terms was weakly unfavourable. As the His residue is smaller compared to the Tyr one the Bad Cterminal part is attracted more in the Mcl-1/Bad complex towards the Mcl-1 α2 helix and the Bad Aspc on the opposite site is too far from Argc of the α4-α5 loop and it cannot establish a strong interaction. In our average structure it is Arg115 that interacts with this loop.


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The BH3-only Bad is not a natural inhibitor of Mcl-1. A theoretical binding between these two proteins have pointed out interesting results. The orientation of the Bad helix in Mcl-1 cavity is different compared to the other BH3-only Puma, Bim, and Noxa. Important interactions, described in the previous study of complexes Mcl-1/Puma and Mcl-1/Bim, such as the fifth hydrophobic interactions, and also interactions with Phe319 and Val220 Mcl-1 hotspot residues, are also detected in this theoretical complex. However, the main observations are (i) a significantly weakened Aspc/Arg263 interaction and related loss of the Aspc/Asn260 interaction and (ii) the absence of a strong electrostatic interaction at the N-terminal part with either Lys234 or Arg248.

CONCLUSION Previously published data had underlined the importance of the conserved hydrophobic residues on the BH3 helix face, buried in the anti-apoptotic partner interface, as well as the presence of BH3-only Aspc, interacting with anti-apoptotic Argc. Our molecular simulations carried out on different complexes with Mcl-1 have fully confirmed these data since these residues are always involved in the major energetic contribution for Mcl-1 complex formation. However, those BH3only key residues do not have the same impact. Above all, the Aspc is the primary determinant of the binding affinity. Then, among the hydrophobic conserved residues, we noticed that the position i+11 appears to be important for the binding affinity through contacts with Mcl-1 hotspots residues Val220 and Phe319. Additionally, our molecular simulations highlighted importance of another hydrophobic residue at the position i+15, which also interacts with Phe319. The π-π interaction at this site seems to contribute significantly to the binding affinity.


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Interestingly, simulations with the selective BH3-only Noxa revealed that the position i+15, with a nonaromatic motif, can also be important to achieve strong Mcl-1 binding. Indeed, at this site, unlike Puma and Bim complexes, there is no π-π stacking in Noxa complex. Moreover, our simulations have shown the existence of negatively charged residues in the BH3-only N-terminal end, interacting with Mcl-1 Arg248 and/or Lys234, which seem to be essential for binding to Mcl-1. These two positively charged residues are present only in Mcl-1. In all three complexes binding the BH3-only (Puma; Bim and Noxa) a stable salt bridge with Mcl-1 Arg 248 was established accompanied with an electrostatic interaction with Mcl1 Asn260. Among the studied complexes Noxa is the only one, which established relatively stable interaction with Lys234. Therefore, in regard of mutation studies30, we hypothesize that this interaction could be responsible for the Noxa selectivity versus Mcl-1. Thus, joining these elements together could be an interesting approach for the design of specific Mcl-1 inhibitors.


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FIGURES Figure 1. Common binding pattern with residue at (i+15) as revealed from the average structures of Mcl-1/Bim (A), Mcl-1/Puma (B), Mcl-1/Noxa (C) and Mcl-1/Bad (D) complexes. Mcl-1 is shown as grey surface and the conserved Arg is highlighted with blue. Bim is represented in purple, Puma in cyan, Noxa in green and Bad in yellow. The BH3-only conserved hydrophobic residues as well as conserved Asp are presented in stick. Figure 2. Histograms of the average nonpolar (top) and polar (bottom) contributions of BH3-only residues to Mcl-1/Noxa, Mcl-1/Bim, Mcl-1/Puma and Mcl-1/Bad free energies of interaction. Figure 3. MD average structures of Mcl-1/Noxa (A), Mcl-1/Bim (B), Mcl-1/Puma (C), and Mcl1/Bad (D) complexes. The observed strongly interacting residues are represented in sticks. Mcl-1 is shown in light grey, Bim is in purple, Puma is in cyan, Noxa is in green, Bad is in yellow. Figure 4. Distance evolution over 160 ns simulation between selected atoms of N-terminal Glu residues (Noxa, Puma, Bim) and Arg248 (Mcl-1) on the top and Glu(i-1) (Noxa, Puma) and Lys234 (Mcl-1) on the bottom. Figure 5. Hotspot Mcl-1 residues from Campbell et al.50 studies (Val220, Asn260, Arg263 and Phe319) in orange and their interacting residues around 4 Å represented in our MD average structures of the Mcl-1/Bim (A), Mcl-1/Puma (B), Mcl-1/Noxa (C) and Mcl-1/Bad (D) complexes. Figure 6. Principal structural difference between Mcl-1/Bad (average MD), in yellow and BclxL/Bad (crystal structure 2BZW), in green. The steric clash between Mcl-1 Met231 and Bad


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Tyr110 induces the different orientation of Bad N-terminus and the different shape of His224 and Tyr101 induces reorientation of Bad C-terminus and then, of the conserved Aspc119.


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A

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B

Aspc

Aspc i+15

i

i+4

i+15 i+7

i+4

i

C

i+11

i+7

i+11

D

Aspc

Aspc

i

i+15

i+4

i+11

i

i+15

i+4 i+7

i+7

i+11

Figure 1. Common binding pattern with residue at (i+15) as revealed from the average structures of Mcl-1/Bim (A), Mcl-1/Puma (B), Mcl-1/Noxa (C) and Mcl-1/Bad (D) complexes. Mcl-1 is shown as grey surface and the conserved Arg is highlighted with blue. Bim is represented in purple, Puma in cyan, Noxa in green and Bad in yellow. The BH3-only conserved hydrophobic residues as well as conserved Asp are presented in stick.


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Figure 2. Histograms of the average nonpolar (top) and polar (bottom) contributions of BH3-only residues to Mcl-1/Bim, Mcl-1/Puma, Mcl-1/Bad and Mcl-1/Noxa free energies of interaction.


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B

A D25 6

D256

R248

R154 R155

R263

E130

R263

D146

R248

D67

D323 E136

E131 E68

E55 E61

H224

K234

D

C

D256 S255

D256

R263

R263 R248

R248

R115

D34 E22

K234

D119

E24

K234

Figure 3. MD average structures of Mcl-1/Noxa (A), Mcl-1/Bim (B), Mcl-1/Puma (C), and Mcl1/Bad (D) complexes. The observed strongly interacting residues are represented in sticks. Mcl-1 is shown in light grey, Bim is in purple, Puma is in cyan, Noxa is in green, Bad is in yellow.


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Figure 4. Distance evolution over 160 ns simulation between selected atoms of N-terminal Glu residues (Noxa, Puma, Bim) and Arg248 (Mcl-1) on the top and Glu(i-1) (Noxa, Puma) and Lys234 (Mcl-1) on the bottom.


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A

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B

N149

R63

R142

D67 N70 G66

Y73 F69

L62

D146 A145 L14 8

E153

Y152

I65

C

D

N44 D34 R30

D119 R115

G33 L36

Q40

V122 S114 F121

F125

Figure 5. Hotspot Mcl-1 residues from Campbell et al.50 studies (Val220, Asn260, Arg263 and Phe319) in orange and their interacting residues around 4 Å represented in our MD average structures of the Mcl1/Bim (A), Mcl-1/Puma (B), Mcl-1/Noxa (C) and Mcl-1/Bad (D) complexes.


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Dc 119

Y110

Bcl-xL L108

E120 Mcl-1 M231

Mcl-1 H224

Bcl-xL Y101

Figure 6. Principal structural difference between Mcl-1/Bad (average MD), in yellow and BclxL/Bad (crystal structure 2BZW), in green. The steric clash between Mcl-1 Met231 and Bad Tyr110 induces the different orientation of Bad C-terminus and the different shape of His224 and Tyr101 induces reorientation of Bad c-terminus and then, of the conserved Aspc119.


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SCHEMES

. Scheme 1. Alignment of BH3-only sequences used in this study. The key i, i+4, i+7, and i+11 hydrophobic residues are highlighted in blue and the conserved Asp is in red


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KEYWORDS: Apoptosis • Protein-Protein Interaction • BH3-only/Mcl-1 complexes • Molecular dynamics • Binding specificity ASSOCIATED CONTENT Supporting Information. RMSD and fluctuation evolution are included as Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author Jana Sopkova-de Oliveira Santos Normandie Univ, UNICAEN, CERMN, FR CNRS 3038 INC3M, SF 4206 ICORE bd Becquerel, F-14000 Caen, France Phone: (33)2-31-56-68-21 Fax: (33)2-31-56-68-03 Email: [email protected] Present Addresses † Institut de Chimie Organique et Analytique - ICOA UMR7311, Pôle de chimie, rue de Chartres - BP 6759, 45067 Orléans Cedex 2, France †† Laboratoire de Conception et Application de Molécules Bioactives, Equipe de BioVectorologie, UMR 7199 – CNRS/Université de Strasbourg, Faculté de Pharmacie, 74 route du Rhin, BP 60024, 67401 Illkirch Cedex, France


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Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources We acknowledge financial support from the Région Basse-Normandie.. ACKNOWLEDGMENT We thank the CRIANN (Centre Régional Informatique et d’Applications Numériques de Normandie) and the European Community (FEDER) for the molecular modelling software. We acknowledge financial support from the Région Basse-Normandie.


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Insert Table of Contents Graphic and Synopsis Here

Mcl-1/BH3-only

R263

R248

i+11 i

i+4

i+7

i+15

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Towards Understanding Mcl-1 Promiscuous and Specific Binding Mode

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