Uncovering the Binding and Specificity of Î²-Wrapins for Amyloid-Î² and

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Uncovering the Binding and Specificity of #Wrapins for Amyloid-# and #-Synuclein Asuka Autumn Orr, Michael M. Wördehoff, Wolfgang Hoyer, and Phanourios Tamamis J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b08485 • Publication Date (Web): 29 Nov 2016 Downloaded from http://pubs.acs.org on November 30, 2016

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The Journal of Physical Chemistry

Uncovering the Binding and Specificity of β-Wrapins for Amyloid-β and α-Synuclein Asuka A. Orr 1, Michael M. Wördehoff 2, Wolfgang Hoyer 2,3, Phanourios Tamamis 1* 1

Artie McFerrin Department of Chemical Engineering, Texas A&M University, TX 77843-3122,

College Station, U.S.A. 2

Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, 40204 Düsseldorf,

Germany. 3

Institute of Structural Biochemistry (ICS-6), Research Centre Jülich, 52425, Jülich, Germany.

*Corresponding Author Email: [email protected]

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ABSTRACT: Amyloidogenic proteins amyloid-β peptide (Aβ) and α-synuclein (α-syn) selfassemble into fibrillar amyloid deposits, senile plaques and Lewy bodies, pathological features of Alzheimer’s and Parkinson’s respectively. Interestingly, half of Alzheimer’s disease cases also exhibit aggregation of α-syn into Lewy bodies, and growing evidence also suggests that Αβ and α-syn oligomers are toxic. Therefore, the simultaneous inhibition through sequestration of the two amyloidogenic proteins may constitute a promising therapeutic strategy. Recently discovered β-wrapin proteins pave the way toward this direction as they can inhibit the aggregation and toxicity of both Αβ and α-syn. Here, we used computational methods, primarily molecular dynamics simulations and free energy calculations, to shed light into the key interaction-based commonalities leading to the dual binding properties of β-wrapins for both amyloidogenic proteins, to identify which interactions act as switches diminishing β-wrapins’ binding activity for Aβ/α-syn, and to examine the binding properties of the current most potent β-wrapin for Aβ. Our analysis provides insights into the distinct role of the key determinants leading to β-wrapin binding to Αβ and α-syn, and reveals that the Aβ

18VFFAED23

and α-syn

38LYVGSK43

are key

domains determining the binding specificity of a β-wrapin. Our findings can potentially lead to the discovery of novel therapeutics for Alzheimer’s and Parkinson’s disease.


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Introduction Amyloidogenic proteins amyloid-β peptide (Aβ), α-synuclein (α-syn), and islet amyloid polypeptide (IAPP or amylin) self-assemble into β-sheet rich amyloid states, pathological features of Alzheimer’s disease, Parkinson’s disease, and type 2 diabetes respectively. In Alzheimer’s disease, senile plaques consist of Αβ proteins; in Parkinson’s disease, Lewy bodies consist of α-syn proteins; in types 2 diabetes, the pancreatic islet amyloid consist of IAPP proteins1-4. The amyloid states formed by amyloidogenic proteins share similar self-assembly and structural cross β-spine properties, containing β-strands that are oriented perpendicular to the fibril axis5-8. Successful strategies to prevent protein aggregation and amyloid formation have been reported, which among others include (i) inhibition of aggregation by sequestering individual amyloid monomers, (ii) inhibition of aggregation through small-molecules, and (iii) inhibition of amyloid growth through peptide-based inhibitors (reviewed in ref.9). Inhibition of Aβ aggregation by sequestering amyloidogenic monomers is achievable with ΖΑβ3, an Αβbinding affibody protein homodimer with a disulfide bridge between the Cys28 residues of the two β-wrapin subunits10,11 , hereinafter referred to as subunit 1 and subunit 2. ZAβ3 stabilizes a β-hairpin conformation of Αβ. Thereby it prohibits the initial aggregation of Αβ monomers into toxic forms, and also functions to dissociate the pre-formed oligomeric aggregates11,12. In addition to the β-sheet related similarities in their fibrilar states, Aβ, α-syn, and IAPP share homologous molecular recognition motifs. β-wrapins (β-wrap proteins), derived from ZAβ3 by combinatorial protein engineering, were recently discovered with the ability to recognize and form β-hairpin conformations with the homologous motifs of the amyloidogenic proteins11,13-15. The introduction of between one and four amino acid exchanges in ZAβ3 resulted in β-wrapins AS9, AS10, AS34, AS60, and AS69 which display different affinities for Aβ and α-syn (Figure 1A)14. According to experiments, AS10 binds to Aβ, α-syn, and IAPP with sub-micromolar affinity and inhibits aggregation and toxicity, while AS9 and AS34 bind somewhat stronger to Aβ than to α-syn, AS60 and AS69 bind significantly stronger to α-syn than to Aβ, and ZAβ3 binds significantly stronger to Aβ than to α-syn (Figure 1A)14. The discovery of β-wrapins with different potency and specificity for Αβ and α-syn could allow the discovery of dual/multi-specific binding agents that can serve in therapeutic applications for 3 ACS Paragon Plus Environment


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Alzheimer’s and Parkinson’s disease. Such agents can be of vital therapeutic importance, especially when considering the growing evidence showing that up to 50% of Alzheimer’s disease cases exhibit – in addition to Aβ senile plaques and tau neurofibrillary tangles - a third prevalent neuropathology which is associated with the aggregation of α-syn into Lewy bodies, as is the case in Parkinson’s disease16,17. Notably, the existence of Lewy bodies in Alzheimer’s disease is related to an aggressive disease course and more pronounced cognitive dysfunction than patients with pure Alzheimer’s disease. Experimental data support that Αβ, α-syn, and tau interact in vivo, prompting the aggregation and accumulation of each other thus accelerating cognitive dysfunction18. Experiments also support the contention that Aβ interacts with α-syn, stabilizing the formation of hybrid nanopores that change neuronal activity and possibly contributing to the mechanisms of neurodegeneration in Alzheimer’s disease and Parkinson’s disease19. Furthermore, recent findings show that Αβ and α-syn oligomers are neurotoxins20-25. Thus, β-wrapins with the capacity to sequester Αβ and α-syn may additionally eliminate the negative effects of oligomers by disrupting Αβ and α-syn monomers from self-assembling into oligomers. The NMR structures depicting how ZAβ3 and AS69 bind to Aβ11 and α-syn13, respectively, provide the grounds to computationally investigate a series of β-wrapins in complex with Aβ and α-syn. Shedding light into the molecular interactions associated with the dual- or multi- binding properties of β-wrapins is of biomedical importance. In this study, we utilized computational methods, primarily molecular dynamics (MD) simulations and free energy calculations, to investigate the binding and specificity for both Αβ and α-syn of previously published β-wrapins whose affinities to both Aβ and α-syn have been experimentally evaluated14. Computational methods can be introduced in thought experiments investigating the binding of inactive binding proteins in comparison to active, and elucidating the key structural and physicochemical features leading to binding and specificity26,27. Motivated by this, here, we uncovered the binding and specificity of β-wrapins for sequestering and inhibiting amyloid formation by amyloidogenic proteins Αβ and α-syn. Through our investigation of β-wrapins with experimentally evaluated binding affinities for amyloidogenic proteins Αβ and α-syn14, we produced four key findings. Our results (i) indicate the key commonalities leading to the dual Αβ/α-syn binding properties of specific β-wrapins, (ii) reveal the key interactions which act as switches diminishing β-wrapins’ 4 ACS Paragon Plus Environment

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binding activity for Aβ/α-syn, (iii) delineate the specific and distinct role of the key energetic driving determinants leading to β-wrapin binding and specificity for Αβ and α-syn, and (iv) provide insights into the binding of the current most active β-wrapin in complex with Aβ. The results of this study may enhance the development of novel improved β-wrapins as potential therapeutic agents for Alzheimer’s and Parkinson’s disease.



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Methods Initial Computational Modeling of the β-Wrapin : Aβ/α-Syn Complexes: The first set of coordinates from the NMR ensemble of structures for ZAβ3 : Αβ (PDB ID: 2OTK11) and AS69 : α-syn (PDB ID: 4BXL13) were used as initial structural templates for the MD simulations. In accordance with the NMR structures11,13 , we investigated the structural and physicochemical properties of the experimentally resolved residues 16 through 40 of Aβ (sequence: KLVFFAEDVGSNKGAIIGLMVGGVV),

residues

35

to

56

of

α-syn

(sequence:

EGVLYVGSKTKEGVVHGVATVA) in complex with β-wrapin residues 12 to 56. The β-wrapin sequence length investigated was identical in both β-wrapin : Aβ and β-wrapin : α-syn complexes for a fair comparison between β-wrapins’ binding to the two amyloidogenic proteins. The importance of residue position 13 in β-wrapins’ affinity for α-syn led us to additionally model residue 12, which was not resolved in either of the two NMR structures11,13; the backbone elongation of residue 12 was performed in CHARMM and was guided by the dihedral backbone angles of β-wrapin residue 13 in reference13. Modeling of the β-wrapins’ mutated/introduced side-chains was performed using SCWRL4.028. The Cys28-Cys28 disulfide bridge between the two subunits of all the β-wrapins was maintained using a disulfide patch from the CHARMM topology file29. With the exception of the Lys7Thr mutation in AS60 which is part of the unstructured and experimentally unresolved β-wrapin N-terminal domain, all of the mutations across all β-wrapins are part of the experimentally resolved β-wrapin domains, and are explicitly investigated in this study (Figure 1A). The N- and C- termini of the β-wrapins and amyloidogenic proteins were acetylated and amidated so as to alleviate any artifacts of artificially placing positively and negatively charged groups, respectively, at the backbone termini of the truncated biomolecules.

MD Simulations Investigating the Conformational Properties of β-Wrapins in Complex with Aβ/α-Syn: We investigated β-wrapins ZAβ3, AS9, AS10, AS34, AS60, and AS69 in complex with Αβ and α-syn. All MD simulations were performed using the CHARMM36 topology and parameters29. Each complex was solvated in a 71 Å cubic explicit water box. The potassium chloride concentration in each water box was set to 0.15 M. Additional potassium and chloride ions were introduced to neutralize the charge of the systems. The ions were placed through 2,000 steps of Monte Carlo simulations30,31. Solvent molecules were subsequently 6 ACS Paragon Plus Environment

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minimized through 50 steps of steepest descent minimization and 50 steps of Adopted Basis Newton-Raphson minimization. Periodic boundary conditions were applied in each simulation. An additional 50 steps of steepest descent minimization and 50 steps of Adopted Basis NewtonRaphson minimization were performed on the system with all backbone atoms constrained under 1.0 kcal/mol/Å2 harmonic constraints and side-chain atoms under 0.1 kcal/mol/Å2 harmonic constraints. The systems were equilibrated in stages, analogously to ref.32. During solvation-equilibration stages, we rigorously solvated the biomolecular complexes, modeled the mutated residues and introduced β-wrapin residues, as well as aimed to alleviate any artifacts on both the β-wrapins’ and amyloidogenic proteins’ truncated domains. In the first solvation stage, each complex was simulated for 1.0 ns with 1.0 kcal/mol/Å2 harmonic constraints on backbone atoms and 0.1 kcal/mol/Å2 harmonic constraints on side-chain atoms. In the second stage, all constraints were released, and the systems were simulated for 10 ns in order to allow the modeled and mutated residues of the β-wrapins to adopt improved conformations and interactions. At this stage, we visually ensured that no artificial structural deformations of the truncated termini occurred; structural deformation was observed only once and the specific equilibration was repeated successfully with different initial velocities. Finally, we performed six different equilibration runs, according to which the final refined-equilibrated structure of the second stage was subjected to an additional 0.25 ns, 0.5 ns, 0.75 ns, 1.0 ns, 1.25 ns, and 1.5 ns simulation with light constraints to allow the simulated systems to further equilibrate. At this stage, the velocities were reinitialized and the duration of the simulations was different so as to produce six independent runs for each complex. Subsequently, all constraints were released, and the systems entered the final production stage, where they were simulated for 24 ns with frames extracted every 20 ps. The simulations were performed using the Leap-frog Verlet algorithm under isobaric and isothermal conditions with the pressure set to 1.0 atm and the temperature held at 300 K using the Hoover thermostat. Fast table lookup routines for non-bonded interactions33 were applied, and the SHAKE algorithm was implemented to constrain the bond lengths to hydrogen atoms34. The simulations were performed using CHARMM, version c39b235. Six independent runs for each β-wrapin : amyloidogenic protein system were performed to check reproducibility of our results. The 24 ns duration of the MD simulation production runs was sufficient to 7 ACS Paragon Plus Environment


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investigate the conformational properties of ZAβ3 : Αβ and AS69 : α-syn complexes, of which their structural integrity was preserved during the simulations with respect to NMR studies11,13 , and proved to be adequate to observe conformational changes in the minimally active or inactive β-wrapins in complex with amyloidogenic protein complexes. Upon the completion of the MD simulation runs, water molecules and ions were stripped from the trajectories and the sextuplicate 24 ns simulation snapshots were analyzed as follows. RMSD Calculations: For each β-wrapin : amyloidogenic protein complex, we merged the six independent trajectories into one unified trajectory, and calculated the root mean square deviation (RMSD) of the β-wrapin complexes with respect to the average structure over all six MD simulation runs using Visual Molecular Dynamics (VMD)48,49. In addition, we calculated the RMSD of the ZAβ3 : Αβ and AS69 : α-syn simulated complexes with respect to the first set of coordinates from the NMR ensemble of structures for ZAβ3 : Αβ (PDB ID: 2OTK11) and AS69 : α-syn (PDB ID: 4BXL13). The RMSD calculations were performed using the backbone (N, Cα, C) atoms. Residue Pairwise Interaction Free Energy Calculations: The residue pairwise interaction free energies for each individual production run were calculated for the full 24 ns in increments of 200 ps using CHARMM35, Wordom36,37, and in-house FORTRAN programs. Eq. 1 was introduced to calculate the interaction free energy between all possible pairs of interacting residues, as in refs.38-45.

inte ∆GRR' =

1 nf

  Elec GB vdW   E + E + E + γ ∆ SASA ∑ ∑ ∑ ∑ ( ij ∑ ( ij ) ∑ ∑ ij i )  k∈n m∈ f  i∈R j∈R' i∈R j∈R' i∈R,R'

Eq. 1

The first, second and third components of the equation above represent the polar, van der Waals and non-polar solvation interactions free energies between R and R' respectively.

For

interactions between subunit 1 of the β-wrapins and the amyloidogenic proteins, R corresponds to a given residue in subunit 1 of the β-wrapins and R' corresponds to the Αβ or α-syn monomer. For interactions between subunit 2 of the β-wrapins and the amyloidogenic proteins, R corresponds to a given residue in subunit 2 of the β-wrapins and R' corresponds to the Αβ or α8 ACS Paragon Plus Environment

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syn monomer. For interactions between subunits 1 and 2 of the β-wrapins, R refers to a given residue in subunit 1 and R' corresponds to a given residue in subunit 2. The interaction free energies of k = 1 to n (=6) simulations and m = 1 to f (=120) frames were summed and averaged. inte inte = GRR' − GR' − GR ) is comprised of The polar component of the total interaction free energy ( ∆GRR'

electrostatic interaction ( EijElec ) and generalized-Born ( EijGB ) energy contributions between residues R and R'. The polar component represents the interaction between residues R and R' and the interaction between residue R and the solvent polarization potential induced by R'. The nonpolar component (sum of the second and third term) consists of the van der Waals interactions

( EijvdW ) between the two residues and the change in the non-polar solvation free energy due to binding ( γ ⋅ ∆SASAi ). The non-polar interaction free energy term represents the non-polar interactions with the surrounding solvent and cavity contributions. The solvation terms were determined using the GBSW generalized-Born model46. These calculations were executed with the non-polar surface tension coefficient, γ, set to the default value of 0.03 kcal/mol/Å2. The generalized-Born energy contribution ( EijGB ) and solvent accessible surface area ( ∆SASAi ) are affected by the location of R and R' in the complex47. To compute the EijGB term in Eq. 1, all atoms were included, and the charges of atoms outside the inte groups RR', R', and R were set to zero in each calculation of the terms GRR' , GR ' , and GR ,

respectively. The

∆SASAi term expresses the difference in solvent accessible surface areas of

residues R and R' within the complex and in unbound states. The non-polar interaction free energy term represents the creation of a cavity in the surrounding solvent to accommodate molecules. For these calculations, we used infinite cutoff values. The standard deviation value for each pairwise interaction in each β-wrapin : amyloidogenic protein complex was calculated based on the average corresponding interaction free energy values from the six independent simulation runs. The average and standard deviation values of the residue pairwise interaction free energies for AS10 : Aβ/α-syn complexes are presented in Figure S1. The residue pairwise interaction free energies for the rest of the β-wrapin : Aβ/α-syn complexes are presented in Figure S2 in comparison to AS10 : Aβ/α-syn complexes. 9 ACS Paragon Plus Environment


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Structural Superposition, Sequence Alignment, and Determination of Corresponding Interactions: To determine the corresponding pairwise interactions across all β-wrapin : amyloidogenic protein complexes, the interactions were projected in accordance with structurebased sequence alignment of the bound α-syn to the bound Aβ as shown in Figure 1B. The structure-based sequence alignment (Figure 1B) was determined by superimposing the AS10 : Aβ/α-syn complexes’ backbone atoms using VMD48,49 and was also validated via the residue pairwise interaction free energies (Figure S1 and Figure S2). Interestingly, this alignment does not coincide with a local sequence alignment obtained using Clustal Omega50,51 (Figure 1B).

Determination of β-Wrapin: β-Wrapin and β-Wrapin: Aβ/α-Syn Interactions Acting as Switches Diminishing β-Wrapins’ Binding Activity for Aβ/α-Syn: The average pairwise interaction free energies between residues in subunit 1 or subunit 2 and Aβ or α-syn, or between residues in the two different subunits, calculated through Eq. 1, can be used to uncover the residue pairwise interactions that act as switches diminishing a β-wrapin’s binding activity for Αβ or α-syn. To identify these switches, we used AS10 as a basis (see Results) and, through Eq. 2, we calculated independently the polar and non-polar residue pairwise interaction free energy differences of all β-wrapins : Aβ/α-syn complexes with respect to the AS10 : Aβ/α-syn complex. inte, polar inte, polar inte, polar inte, non-polar inte, non-polar inte, non-polar ∆∆GRR' = ∆GRR',AS10 − ∆GRR', = ∆GRR',AS10 − ∆GRR', β -wrapin ; ∆∆GRR' β -wrapin

Eq. 2

As in Eq. 1, for residue-pairwise interactions between subunit 1 of the β-wrapins and the amyloidogenic proteins, R corresponds to a given residue in subunit 1 of the β-wrapins and R' corresponds to the Αβ or α-syn monomer; for interactions between subunit 2 of the β-wrapins and the amyloidogenic proteins, R corresponds to a given residue in subunit 2 of the β-wrapins and R' corresponds to the Αβ or α-syn monomer; for interactions between subunits 1 and 2 of the β-wrapins, R refers to a given residue in subunit 1 and R' corresponds to a given residue in subunit 2. Interactions between residues in the two subunits were also investigated as possible switches since the weakening of interactions formed between β-wrapin and amyloidogenic 10 ACS Paragon Plus Environment

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protein residues may indirectly result in the destabilization of specific interactions formed between residues in different subunits. For β-wrapin : Αβ complexes, polar or non-polar interactions acting as switches diminishing the activity of β-wrapins AS69 and AS60 in complex with Αβ are expected to possess unfavorable (e.g., negative) ∆∆G interaction free energy in both or any of the two complexes, and negligible or marginally higher ∆∆G interaction free energy in the rest of the β-wrapin : Αβ complexes. Thus, residue pairs for which the polar or non-polar ∆∆G residue pairwise interaction free energy is negative (e.g., at least less than -0.1 kcal/mol) in any of the AS69 : Aβ and AS60 : Aβ complexes were collected and their ∆∆G interaction free energy was compared with respect to the corresponding interaction free energies in the ΖΑβ3, AS9, AS34 : Aβ complexes. Analogously, for β-wrapin : α-syn complexes, polar or non-polar interactions acting as switches diminishing the activity of β-wrapin ΖΑβ3 in complex with α-syn are expected to possess unfavorable (e.g., negative) ∆∆G interaction free energy in the ΖΑβ3 : α-syn complex, and negligible or marginally higher ∆∆G interaction free energy in the rest of the β-wrapin : α-syn complexes. Thus, residue pairs for which the polar or non-polar ∆∆G residue pairwise interaction free energy is negative (e.g., at least less than -0.1 kcal/mol) in the ΖΑβ3 : α-syn complex were collected and their interaction free energy was compared against the corresponding interaction free energies in the AS9, AS34, AS60, AS69 : α-syn complexes. According to a visual inspection of the simulation trajectories and the ∆∆G residue pairwise interaction free energy comparisons across all complexes, we selected the most notable polar and non-polar interactions that constitute switches diminishing β-wrapins’ activity in AS60 : Αβ, AS69 : α-syn, and ΖΑβ3 : α-syn complexes, and listed them in Tables 1 and 2, respectively. Notably, for all interactions listed in Table 1, the corresponding inte, polar inte, polar inte, polar ∆∆ GRR = ∆GRR ' ', AS10 or ZΑβ 3 orAS9 or AS34 − ∆ G RR ', AS60 or AS69

or

inte, non -polar inte, non -polar inte, non -polar ∆∆ GRR = ∆G RR ' ',AS10 or Z Αβ 3 orAS9 or AS34 − ∆ G RR ',AS60 or AS69

values are at least less than -0.6 kcal/mol, irrespective of the β-wrapin used as a basis (AS10 or ZAβ3 or AS9 or AS34), indicating that identified interactions between residue pairs in AS60 or AS69 : Aβ complexes that constitute switches diminishing the β-wrapins’ activity for Αβ, 11 ACS Paragon Plus Environment


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differentiate AS60 or AS69 : Aβ complexes with respect to all other β-wrapin : Αβ complexes (see Table 1). Similarly, for all interactions listed in Table 2, the corresponding inte, polar inte, polar inte, polar ∆∆G RR = ∆G RR ' ', AS10 or AS60 orAS69 orAS9 or AS34 − ∆G RR ', Z Αβ 3

or

inte, non -polar inte, non -polar inte, non -polar ∆∆ G RR = ∆G RR ' ', AS10 or AS60 orAS69 orAS9 or AS34 − ∆G RR ', Z Αβ 3

values, irrespective of the β-wrapin used as a basis (AS10 or AS60 or AS69 or AS9 or AS34), are always at least less than -0.6 kcal/mol, indicating that the identified interactions between residue pairs in ΖAβ3 : α-syn complex that constitute switches diminishing the ΖAβ3 activity for α-syn, differentiate the ΖAβ3 : α-syn complex with respect to all other β-wrapin : α-syn complexes (see Table 2). With the exception of one interaction, the ∆∆G standard deviation values shaded in gray in both Tables 1 and 2 are significantly low, indicating a statistical convergence of the reported interaction free energy values.

MM GBSA Association Free Energy Calculations– Differentiating Between Active Versus Minimally Active/Inactive β-Wrapins in Complex with Aβ/α-Syn: The Molecular Mechanics Generalized Born Surface Area (MM GBSA) approximation52-54 has been successfully introduced in several studies to differentiate between active and inactive binding proteins26,27, to estimate the binding affinity of designed peptides in complex with protein receptors39, as well as to rank MD simulated docking poses38,41-45. Here, we used the MM GBSA approximation, to assess the association free energy of β-wrapins in complex with Αβ and α-syn using Eq. 3:

∆G = GWP − GW − GP

Eq. 3

where GWP, GW, and GP denote the energies of the β-wrapin : amyloidogenic protein complex, βwrapin, and amyloidogenic monomer respectively. The individual free energies were estimated using the MM GBSA approximation and the following equation55:

G = E Bonded + E

Elec

+ E GB + E vdW + γ ⋅ SASA

Eq. 4


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Bonded Elec GB vdW where E , E , E , E , and SASA represent the bonded energy, electrostatic

interaction energy, generalized-Born energy, van der Waals energy, and solvent-accessible surface area of the system respectively. The sum of electrostatic interaction energy and generalized-Born energy terms represent the polar contribution to the total MM GBSA association free energy. The sum of the van der Waals energy and solvent-accessible surface area terms represent the nonpolar contribution to the total MM GBSA association free energy. These terms were calculated using the GBSW generalized-Born model46 with the non-polar surface tension coefficient, γ, set to the default value (0.03 kcal/mol/Å2). The cutoffs used for these calculations were infinite. In the MM GBSA approximation, we utilized the one-trajectory approximation53,54 , according to which the free state of the β-wrapins and the amyloidogenic proteins adopt the same conformation as when they are bound26,27,38,39,41-45,56, and thus, the bonded energy term is subtracted out in the total association free energy calculation (Eq. 3); while this assumption omits the effect of entropy due to structural relaxation, it also eliminates intramolecular contributions, which may introduce large variability in the calculations as these terms subtract out in the total association free energy calculation (Eq. 3)39. Solvation effects are taken into account implicitly in conjunction with a molecular mechanics energy function and solute conformational entropy is evaluated in an approximate manner, as in refs.26,27,39. According to the calculations, the MM GBSA association free energy (see Table 3 in Results) can differentiate which β-wrapins are inactive or minimally active from active β-wrapins and correlates well with the experimentally measured binding affinities. Driven by this result, we decomposed the total MM GBSA association free energies into non-polar and polar components to shed light into the specific and distinct role of the key energetic driving determinants leading to β-wrapins’ binding and specificity for Αβ and α-syn.



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Results We conducted six independent explicit-water simulations of each β-wrapin : amyloidogenic protein (Aβ/α-syn) complex with individual production lengths of 24 ns in order to gain an atomistic view of their structures and interactions. The β-wrapins investigated in this study are ZAβ3, AS9, AS10, AS34, AS60, and AS69 as their affinities for both Aβ and α-syn are known14 (Figure 1A) and our primary goal was to uncover the binding and specificity of β-wrapins for both amyloidogenic proteins. Experimentally resolved structures of the ZAβ3 : Aβ complex (PDB ID: 2OTK11) and the AS69 : α-syn complex (PDB ID : 4BXL13) were used as templates to build all β-wrapin : Aβ and β-wrapin : α-syn complexes, respectively. The procedures followed for the simulations are detailed in the Methods section. Convergence and Preservation in the MD Simulations: The structural convergence of the MD simulations was confirmed by calculating the RMSD of the β-wrapin complexes with respect to their average structure over all six MD simulation runs, for the full 24 ns in 20 ps increments using VMD48,49. The average RMSDs for each complex using the backbone atoms of each complex do not exceed 1.7 Å, and thus, the structure of biomolecular complexes is well conserved throughout the simulation runs. As expected, N-terminal and C-terminal ends of the βwrapin complexes experience slightly higher fluctuations compared to the rest of the complex in all simulations. The structural convergence was additionally verified by visual inspection of the simulation trajectories. The RMSD of ZAβ3 : Αβ and AS69 : α-syn simulated complexes with respect to the corresponding NMR structures11,13 (see Methods) is equal to 1.5 ± 0.2 Å and 1.7 ± 0.2 Å, respectively; the two values are indicative of the preservation of the backbone conformations throughout the two simulations with respect to the experimentally derived initial conformations used in the simulations11,13. Additionally, the energetic sampling convergence of the MD simulations was verified by splitting the six simulation runs of each β-wrapin : amyloidogenic protein complex investigated into two sets of three simulation runs: Set 1 consisted of the runs with the three smallest equilibration duration times, and Set 2 consisted of the runs with the three largest equilibration duration times. In both Set 1 and Set 2 independently, a decent linear correlation between the total MM GBSA association free energies and experimentally measured affinities was observed, reminiscent of the correlation observed when all runs were considered together in the statistics (see Results). 14 ACS Paragon Plus Environment

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Conformational Properties of β-Wrapins in Complex with Aβ and α-Syn: According to MD simulations, the conformational properties of all investigated β-wrapins in complex with Aβ and α-syn are nearly identical, with specific exceptions. These conformational properties are analyzed in detail below. In accordance with the experimentally resolved structures, the two covalently bonded β-wrapin subunits, bonded through a Cys28-Cys28 disulfide bridge, synergistically “lock” both domains Αβ(16-40) and α-syn(35-56) into β-hairpin conformations; the numbers in parentheses denote residue domains. The bound conformations are stabilized by intermolecular β-sheet interactions between specific residue moieties of the two proteins and the two subunits as well as the tight packing of the Aβ and α-syn β-hairpins against the hydrophobic core of the α-helices. Additionally, the formation of inter-helical contacts on one side proximal to the disulfide bridge and hydrophobic contacts on the other side of the two subunits contribute to the stability of the binding conformations (Figure 2). Upon structural superposition of the complex structures, the corresponding domains of both proteins (Figure 1B) are different from the corresponding domains based on local sequence alignment (Figure 1B). The two bound βhairpin-like protein domains Αβ(16-40) and α-syn(35-56) differ with regard to the residue length of their loops, equal to 6 (VGSNKG) and 4 (TKEG) residues in Αβ(16-40) and α-syn(35-56) respectively. Common Interactions Between β-Wrapin ΑS10 and Aβ and α-Syn: As AS10 (i) is the most potent β-wrapin for both proteins, (ii) has the same order of magnitude binding affinity for both proteins, and (iii) is also a potent binder of IAPP14, it will serve as our reference point to provide an in-depth analysis of the interactions occurring in a bound conformation consisting of two AS10 subunits in complex with the two amyloidogenic protein domains, Αβ(16-40) and αsyn(35-56). It is worth noting that the choice of a reference β-wrapin does not have an effect on the results drawn regarding the interactions that act as switches leading to minimal activity/inactivity (see Methods). The average residue pairwise intermolecular interactions between subunit 1 : amyloidogenic protein, subunit 2 : amyloidogenic protein, and subunit 1 : subunit 2 in the AS10 : Αβ and AS10 : α-syn complexes are decomposed into polar and nonpolar contributions; the corresponding maps are presented in Figure S1 and Figure S2.



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The residue moieties

15EIVYL19

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of AS10 subunits 1 and 2 form antiparallel extended β-sheets

with Αβ(19-23, 32-36) and α-syn(39-43, 50-54). These polar β-sheet interactions are the key determinants for the two protein domains Αβ(16-40) and α-syn(35-56) to adopt a β-hairpin conformation. The hydrogen bonding β-sheet network is facilitated by the formation a predominantly hydrophobic surface, having solvent-excluded interfaces between β-sheet strands as well as the β-sheet and the hydrophobic core of the β-wrapin helices, occurring as a consequence of strong non-polar interactions formed between the 14GEIVYL19 fragments of the two AS10 subunits and parts of Αβ and α-syn fragments (Figure 2C, D, E, F). Both the polar and non-polar interactions associated with the β-sheet formation are expected to contribute significantly to the dual binding properties of AS10. Interestingly, the majority of these fragments are known to be highly amyloidogenic and possess amyloid propensities outside the context of the entire proteins. Specifically, in complex with Αβ, fragment 14GEIVYL19 of ΑS10 subunits 1 and 2 interacts partly with Αβ’s amyloidogenic fragments AIIGLMV57 and KLVFFAE58, respectively; in complex with α-syn, fragment 14GEIVYL19 of ΑS10 subunits 1 and 2 interacts partly with α-syn’s fragments GVLYVGS and GVATVA59, respectively. Apart from the intermolecular β-sheet interactions formed between AS10 subunits 1, 2, and the amyloidogenic proteins, a series of polar and primarily non-polar interactions between the subunits and the proteins contribute to the dual binding properties and the stabilization of the bound structures. Interestingly, a significant portion of the non-polar interactions involve interactions between pairs of subunit residues and corresponding residues of the two amyloidogenic proteins according to structural superposition (Figure 1B), abbreviated here as CR; interactions between AS10 residues and corresponding residues across all different binding proteins may, in addition to the common β-sheet interactions mentioned above, contribute significantly to the dual binding properties of AS10. According to Figure S1 and Figure S2, the AS10 subunit 1 : amyloidogenic proteins’ (Aβ/α-syn) CR pairs involved in important non-polar interactions in all systems are Ile16:Phe20/Val40, Tyr18:Phe20/Val40, Leu27:Ile32/His50, Phe30:Leu34/Val52,

Phe31:Leu34/Val52,

Phe31:Phe20/Tyr39,

Val34:Leu17/Val37,

Ser41:Leu17/Val37, Leu45:Leu17/Val37, Leu45:Leu34/Val52, and Leu45:Val36/Thr54 (Figure 3). According to Figure S1 and Figure S2, the AS10 subunit 2 : amyloidogenic proteins’ (Aβ/αsyn) CR pairs involved in important non-polar interactions are Leu27:Phe19/Tyr39, 16 ACS Paragon Plus Environment

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Phe31:Ile32/His50, Leu45:Ala21/Gly41,

Val34:Ala30/Val48, Leu45:Asp23/Lys43

Ser41:Gly29/Gly47, and

Leu45:Ala30/Val48

Ser41:Ala30/Val48, (Figure

3).

The

aforementioned pairs of β-wrapins and Αβ/α-syn CR consist of mainly hydrophobic residues, and in some cases aromatic (italicized) residues only, a fact emphasizing that hydrophobic CR interactions across the two systems play a significant role for the β-wrapin’s binding to the two amyloidogenic proteins. Polar interactions between AS10 subunits 1 or 2 and amyloidogenic protein residues outside the β-sheet cores mainly correspond to hydrogen bond formations. A portion of these polar interactions involve AS10 subunit 1, 2 : amyloidogenic proteins’ (Aβ/α-syn) CR pairs, or residue pairs involving amyloidogenic protein residues which are not strictly CR based on the structural superposition but are formed between AS10 subunit 1,2 residues and nearest neighbor residues of the amyloidogenic proteins’ (Aβ/α-syn) CR. Both types of interactions are expected to correlate with the dual Αβ/α-syn binding properties of AS10 and include hydrogen bonds between (i) Tyr18 OH of AS10 subunit 1 with Glu22 OE1/OE2 of Αβ and Ser42 OH of α-syn, (ii) Ser41 OH of AS10 subunit 1 with the backbone carboxyl or amide groups of Glu35/Gly36 of α-syn, (iii) Ser41 OH of AS10 subunit 2 with the backbone carboxyl or amide groups of Asn27/Gly29 of Αβ and Gly47/Val48 of α-syn, (iv) the backbone carboxyl group of Pro38 of AS10 subunit 2 with the backbone amide group of Gly29 of Αβ and Gly47 οf α-syn. Additionally, salt bridges formed between AS10 subunit 2 Glu15 and Lys43 of α-syn, may enhance the binding properties of AS10 to α-syn.

Common Interactions Between the Two β-Wrapin Subunits of AS10 in Complex with Aβ and α-Syn: Interactions between the two subunits of β-wrapin ΑS10 are also expected to contribute significantly to the binding and the stability of the complex formation. Previous experiments investigating ΖAβ3 binding to Αβ showed that the Cys28 disulfide linkage generates a dimer interface stabilized by interactions between the subunits which greatly increases thermal stability of the disulfide-linked ΖAβ3 dimer compared to the ΖAβ3 monomer and head-to-tail dimeric constructs10. According to the residue pairwise interaction free energy analysis between subunits 1 and 2 (Figure S1 and Figure S2), the key stabilizing non-covalent intermolecular driving forces between the two subunits comprise of inter-helical non-polar contacts between 17 ACS Paragon Plus Environment


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residues in moieties 24-35. It is worth noting that the non-polar interactions formed between the two subunits in complex with the two amyloidogenic proteins (Aβ/α-syn) are nearly identical. The formation of inter-helical contacts is significantly facilitated by the presence of the Cys28Cys28 disulfide bridge (Figure 2A, B). In addition to the formation of inter-helical contacts on one side of the complex, on the opposite side of the AS10 : Aβ/α-syn, a hydrophobic cluster comprising Ile16 and Tyr18 residues participating in intra- (subunit 1 : subunit 1, subunit 2 : subunit 2) and inter- (subunit 1/2 : subunit 2/1) molecular interactions is expected to further stabilize the complex formation and act as a “molecular gate” locking and wrapping Aβ/α-syn (Figure 2A, B, C, D); this is observed in the experimentally resolved structures11,13 as well as in simulations of other active β-wrapin : amyloidogenic protein complexes. Thus, “gate-keeper” residues Ile16 and Tyr18 possess a key role in intramolecular β-wrapin interactions, and in intermolecular interactions with both amyloidogenic proteins and the different β-wrapin subunits.

β-Wrapin : Aβ/α-Syn and β-Wrapin : β-Wrapin Interactions Acting as Switches Diminishing β-Wrapins’ Binding Activity for Aβ/α-Syn: We calculated the average pairwise interaction free energies between β-wrapin subunit residues and Aβ or α-syn residues, as well as between β-wrapin residues of opposite subunits in all complexes investigated. Through a comparative analysis we identified specific β-wrapin: β-wrapin and β-wrapin: Aβ/α-syn interactions acting as switches diminishing β-wrapins’ binding activity for Aβ/α-syn (see Methods). Our analysis indicates the interactions that act as “switches” and have a negative impact on βwrapins AS60 or AS69 binding to Αβ as well as on β-wrapin ZAβ3 binding to α-syn. The interactions acting as switches diminishing the binding activity of β-wrapins AS60 and AS69 for Aβ are summarized in Table 1, and a portion of these interactions are presented in molecular graphics images through purple dotted lines (Figure 4B) in comparison to the interactions in an active β-wrapin (AS10) in complex with Αβ, which are encapsulated in purple surfaces (Figure 4A). Likewise, the interactions acting as switches diminishing the binding activity of β-wrapin ZAβ3 for α-syn are summarized in Table 2, and the majority of these are presented in molecular graphics images through purple dotted lines (Figure 4D) in comparison to the interactions in an 18 ACS Paragon Plus Environment

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active β-wrapin (AS10) in complex with α-syn, which are encapsulated in purple surfaces (Figure 4C). For a detailed description of these interactions please refer to the Supporting Information. All interactions diminishing AS60 or AS69’s binding to Aβ listed in Table 1 and ZAβ3’s binding to α-syn listed in Table 2 acquire notably lower ∆∆G interaction free energy values in the aforementioned complexes compared to the rest of the β-wrapin : Aβ and β-wrapin : α-syn complexes, respectively; the specific ∆∆G interaction free energy values are shaded in gray and reflect the weakening or destabilization of certain interactions mentioned above. The reported average ∆∆G residue pairwise interaction free energy values were calculated with respect to the corresponding AS10 : amyloidogenic protein complexes. Interestingly, the identified β-wrapin : amyloidogenic protein or β-wrapin (subunit 1) : β-wrapin (subunit 2) interactions that act as switches diminishing a β-wrapin’s inhibitory-sequestering activity for Aβ and α-syn, are distinct in the two systems under investigation. Therefore, at least for the β-wrapins investigated here, the uncovered interaction-based switches diminishing activity are different across the two βwrapin : amyloidogenic protein complexes.

MM GBSA Association Free Energy Differentiating Minimally Active and Inactive βWrapins from Active β-Wrapins: The total MM GBSA association free energy can distinguish between active versus inactive and minimally active β-wrapins in complex with a specific amyloidogenic protein, Aβ or α-syn (Table 3); AS60 : Aβ, AS69 : Aβ, and ΖΑβ3 : α-syn complexes acquire the highest association free energies in comparison to the rest of the β-wrapin : Aβ and β-wrapin : α-syn complexes, respectively. Additionally, there is a decent linear correlation between the total MM GBSA association free energy and experimentally measured affinities14, shown in Figure 5. The decomposition of the MM GBSA association free energies into non-polar and polar components, which is presented in Table 3, indicates that the non-polar association free energy component contributes most in differentiating between active versus inactive and minimally active β-wrapins, underlining the key role of van der Waals interactions in constituting a key energetic determinant for β-wrapins’ binding as well as specificity for Aβ/αsyn. Nevertheless, it is worth noting that the ΖΑβ3 : Αβ complex acquires the lowest polar association free energy component across all β-wrapin : Αβ complexes, and also, the AS60 : α19 ACS Paragon Plus Environment


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syn and AS69 : α-syn complexes acquire the lowest polar association free energy components across all β-wrapin : α-syn complexes; thus, a significantly low polar association free energy in a β-wrapin : Αβ/α-syn complex is indicative of a highly active β-wrapin, given that the non-polar association free energy component is reasonably favorable too. Additional simulations of ZSYM73 in complex with Aβ, a recently discovered β-wrapin with a 50-fold improvement in affinity to Aβ with respect to ZAβ360, also support our findings. ZSYM73 : Aβ and ZSYM73 : αsyn complexes were generated by introducing the mutations Ile16Arg, Pro24Ala, His32Arg, and His35Glu to the equilibrated structure of the ZAβ3 : Aβ and

ZAβ3 : α-syn complexes

respectively, and 24 ns sextuplicate runs (see Methods) of both complexes were investigated. The total MM GBSA association free energy of the ZSYM73 : Aβ complex is -178.3 ± 5.4 kcal/mol with the non-polar and polar components contributing -154.1 ± 4.5 kcal/mol and -24.2 ± 1.2 kcal/mol, respectively. The decomposition of the total association free energy of the ZSYM73 : Aβ reveals a polar association free energy which is interestingly approximately two times lower than that of the ZAβ3 : Αβ complex, strengthening our assertion that a low polar association free energy is a key characteristic of a highly active β-wrapin for a specific amyloidogenic protein. The total MM GBSA association free energy of the ZSYM73 : α-syn complex is -143.6 ± 2.4 kcal/mol with the non-polar and polar components contributing -135.2 ± 3.2 kcal/mol and -8.5 ± 2.3 kcal/mol respectively. The fact that the ZSYM73 : α-syn complex has a higher total association free energy and non-polar component than all other β-wrapin : α-syn complexes (see Table 3) suggests that ZSYM73 is likely inactive for α-syn.

β-Wrapin : Aβ and β-Wrapin : β-Wrapin Interactions Potentially Contributing to the Enhanced Binding of ZSYM73 in Complex with Aβ: The ZSYM73 : Aβ complex was investigated in detail to uncover the key interactions potentially contributing the enhanced binding affinity of ZSYM73 in complex with Aβ60. The Ile16Arg mutation from ZAβ3 to ZSYM73 results in a salt-bridge between Arg16 in subunit 2 of ZSYM73 and Glu22 of Aβ (Figure 6A). This mutation also increases the strength of the “gate-keeper” residues allowing the formation of hydrogen bonds between Arg16 and Tyr18 while retaining favorable hydrophobic interactions (Figure 6A). Additionally, the His32Arg, His35Glu, and Pro24Ala mutations contribute to the stabilization of the β-wrapin monomer conformations as well as the stabilization of the β-wrapin dimer conformations. As for the former, an intramolecular salt-bridge between 20 ACS Paragon Plus Environment

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Arg32 : Glu35, formed primarily in one of the two subunits. As for the latter, our simulations reveal the formation of salt-bridges formed concurrently between Arg32 of subunit 1/2 and Asp25 of subunit 2/1, as well as the formation of hydrogen bonds formed between the charged group of Glu35 of subunit 1/2 and the backbone amide group of Ala24 of subunit 2/1. The intermolecular subunit 1 : subunit 2 interactions formed between residue pairs Arg32 : Asp25, Glu35 : Ala24, contribute to a “zipper-like” formation between the two subunits, shown in Figure 6B, and should contribute to a firm stabilization of the β-wrapin dimer, as they are stronger than the corresponding inter-subunit interactions occurring in ZAβ3 (Figure S3 and Figure S4). All aforementioned interactions occur only in the ZSYM73 : Aβ complex in comparison with the other β-wrapin : amyloidogenic protein complexes investigated in this study, and highlight the importance of optimized polar interactions stabilizing the β-wrapin monomer, the β-wrapin dimer, and the β-wrapin in complex with an amyloidogenic protein.



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Discussion Computational methods are increasingly becoming powerful tools in the investigation of selfassembly and the formation of amyloid oligomers61-73. Of particular interest is the use of MD simulations to provide atomic-detail insights into oligomers formed by combination of amyloidogenic proteins (e.g., Aβ and IAPP74-76 or Aβ and α-syn77). Additionally, computational methods have been introduced to elucidate the mechanism of self-assembly inhibition, discover novel self-assembly inhibitors78-81, and provide insights into β-wrapins in complex with amyloidogenic proteins82. Here, we introduced computational methods to investigate the binding and specificity of βwrapins for sequestering and inhibiting amyloid formation by amyloidogenic proteins Aβ and αsyn; we investigated the binding of inactive β-wrapins in comparison to active β-wrapins and shed light onto the key structural and physicochemical properties leading to binding and specificity. Recently, Wang et al.82 introduced molecular dynamics simulations to investigate the binding of ZAβ3 in complex with Aβ and identified the key interacting ZAβ3 residues with regard to interactions with Aβ. While the findings of our study are, in general, in line with the study of Wang et al., at least for the ZAβ3 : Aβ complex, our study provides additional novel insights into (i) the investigation of interactions between pairs of ZAβ3 : Aβ residues, as well as (ii) the investigation of interactions between pairs of ZAβ3 (subunit 1) : ZAβ3 (subunit 2). Both are of significant importance since they encompass the information on the role of individual ZAβ3 : Aβ interactions, and shed light into the important interactions between the two subunits that contribute to the stability of the ZAβ3 dimer. To the best of our knowledge, our study examines for the first time in detail the binding of a series of β-wrapins in complex with Αβ and α-syn, for which their binding affinities have already been experimentally investigated13,14. Our simulations and energy calculations suggest the significant role of van der Waals complementarity between the β-wrapins and the two amyloidogenic proteins, and interestingly reveal an abundance of hydrophobic/aromatic interactions. These hydrophobic/aromatic interactions, as a network formed between AS10 : Αβ and AS10 : α-syn CR pairs, contribute significantly to the dual binding properties of AS10. The aforementioned interactions along with primarily the polar intermolecular β-sheet hydrogen 22 ACS Paragon Plus Environment

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bonding interactions comprise the key commonalities contributing to the dual Αβ/α-syn binding properties of AS10. Undoubtedly, the presence of common molecular recognition motifs between the amyloidogenic proteins is a key factor for AS10 dual Aβ/α-syn binding properties. Based on this, α-syn fragments GVLYVGS, GVATVA and Aβ fragments KLVFFAE, AIIGLMV are respectively common molecular recognition motifs for dual-binding β-wrapins. Apart from the aforementioned commonalities in the β-wrapin : amyloidogenic protein (Aβ/αsyn) complexes, our analysis also reveals the molecular switches that differentiate β-wrapin : amyloidogenic protein (Aβ/α-syn) complexes and lead to minimal activity and inactivity of βwrapins AS60/AS69 for Aβ and ZAβ3 for α-syn. In the AS60/AS69 : Aβ complexes, the Gly13Asp and Val17Phe mutations in β-wrapins AS60 and AS69 (with respect to all other βwrapins in this study) synergistically destabilize specific non-polar and polar interactions between residue pairs between subunits 1/2 and Aβ, as well as interactions between residue pairs between subunit 1 and subunit 2 in the AS60/AS69 : Aβ complexes. In the ZAβ3 : α-syn complexes, the Phe31Ile mutation of β-wrapin ZΑβ3 (with respect to all other β-wrapins) primarily destabilizes hydrophobic/aromatic cluster within the ZAβ3 : α-syn complex. Notably, the identified pairwise interactions acting as switches diminishing the activity are different in βwrapin : Αβ and β-wrapin : α-syn complexes; nevertheless, they involve structurally aligned residue pairs (e.g., Val18, Phe19 in Αβ, and Leu38, Tyr39 in α-syn). Despite the commonalities between Αβ and α-syn that were examined here within the framework of β-wrapins’ binding, the detection of residue pairwise switches between β-wrapins and Aβ or α-syn residues diminishing activity undoubtedly shed light into the key differences between Aβ and α-syn. The β-wrapin : Αβ/α-syn switches indicate that, at least for the amyloidogenic domains investigated here, the key differences between Αβ versus α-syn primarily involve Αβ/α-syn residues Val18/Leu38, Phe19/Tyr39, Glu22/Ser42, and Asp23/Lys43. Thus, the Aβ 38LYVGSK43

18VFFAED23

and α-syn

are suggested to be key residue domains that determine a β-wrapin’s binding

specificity. This is further supported by our simulations investigating the newly discovered βwrapin, ZSYM73, which is highly active for Αβ60 and also predicted to be inactive for α-syn. The engineered Arg16 residue of subunit 2 in ZSYM73 in complex with Aβ and α-syn, respectively, forms a salt-bridge with Glu22, and is strongly repulsed by Lys43; the former may be among the key interactions leading to the enhanced binding of ZSYM73 in complex with Aβ, 23 ACS Paragon Plus Environment


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whereas the latter may be among the key unfavorable interactions contributing to ZSYM73’s predicted inactivity for α-syn. We suggest that these key differences can be exploited to tune the specificity of novel designed β-wrapins, and in general, novel discovered molecules targeting Αβ and α-syn. Both non-polar and polar interactions synergistically contribute as driving forces leading to the binding affinity, and consequently the inhibitory-sequestering activity of a β-wrapin in complex with Αβ/α-syn. Our results show that the MM GBSA association free energy can provide evidence for the minimal activity or inactivity of β-wrapins AS60 and AS69 for Aβ and β-wrapin ZAβ3 for α-syn. The decomposition of the total MM GBSA association free energy into polar and non-polar components indicates the key role of the non-polar energy component in binding as well as in differentiating between active versus inactive or minimally active β-wrapins in complex with both amyloidogenic proteins. This is supported by the abundance of non-polar switches diminishing the activity of β-wrapins in complex with both Αβ and α-syn. Yet, a significantly low polar association free energy is indicative of high activity, given a reasonably favorable non-polar association free energy. The key energetic determinants identified leading to a β-wrapin’s binding and specificity for Aβ and α-syn are independent of the amyloidogenic protein, and thus, it is possible that the same determinants will also govern β-wrapins’ binding specificity to IAPP. Our simulations depict the formation of additional key interactions in the ZSYM73 : Aβ complex which are not present in the ΖΑβ3 : Aβ complex, and thus most likely contribute to ZSYM73’s significantly higher affinity for Aβ. In addition to the interactions predicted by Lindberg et al.60, here we identified for the first time the existence of polar interactions which contribute to the stabilization of the ZSYM73 dimer, and which include, among others, salt-bridges formed concurrently between Arg32 of subunit 1/2 and Asp25 of subunit 2/1. These interactions along with the Cys28 disulfide bridge contribute to the formation of a “zipper” between the two ZSYM73 subunits, which is not present in any of the other β-wrapins investigated here. This suggests the importance of inter-subunit stabilizing interactions, in addition to β-wrapin : Aβ interactions, in the design of optimized novel β-wrapins.


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Conclusions The insights from our study can be used for the design of highly potent β-wrapins specific for Aβ or α-syn as well as β-wrapins multi-targeting Aβ and α-syn. As our simulations indicate that the Aβ

18VFFAED23

and α-syn

38LYVGSK43

are key residue domains determining a β-wrapin’s

binding specificity and that β-wrapin complexes acquiring significantly favorable polar association free energies are indicative of a highly active β-wrapin, we propose that engineered β-wrapins with the capacity to form improved interactions with Glu22 and Asp23 of Αβ can potentially be highly specific and potent for Aβ, and similarly, engineered β-wrapins with the capacity to form improved interactions with Lys43 of α-syn can potentially be highly specific and potent for α-syn. Additionally, in this study we identified a common network of hydrophobic interactions occurring in both AS10 : Aβ and AS10 : α-syn complexes contributing to AS10’s enhanced dual binding properties for the two amyloidogenic proteins; thus, novel engineered dual-targeted β-wrapins may be designed by optimizing this network of interactions. Our findings are expected to provide impetus for the investigation of β-wrapins’ binding to IAPP and for the design of novel specific, highly active β-wrapins for Αβ or α-syn only, as well as dual-binding, highly active β-wrapins sequestering both Αβ and α-syn. The two latter directions are promising for the discovery of novel potential specific or dual therapeutics for the two diseases. Notably, the latter may be of utmost importance to treat Alzheimer’s disease patients exhibiting a third prevalent neuropathology, which is also associated with the aggregation of αsyn into Lewy bodies16,17. The recently engineered β-wrapin ZSYM73, with an affinity of 300 pM for Aβ60, demonstrates that the design of β-wrapins for the sequestration of amyloidogenic proteins is indeed a promising and feasible therapeutic direction83. While the blood brain barrier may limit the delivery of β-wrapins towards treatments directed at Alzheimer’s disease and Parkinson’s disease, there are several options to circumvent this challenge, including the temporary disruption of the blood-brain barrier to allow the passage of β-wrapins through the blood-brain barrier84.



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Supporting Information Detailed description of β-wrapin : Aβ/α-syn and β-wrapin : β-wrapin interactions acting as switches diminishing β-wrapin’s binding in complex with Aβ/α-syn. Figure S1: Favorable average and standard deviation non-polar and polar interaction free energy map for AS10 in complex with Aβ; Figure S2: Favorable average and standard deviation non-polar and polar interaction free energy map for AS10 in complex with α-syn; Figure S3: Favorable average and standard deviation non-polar and polar interaction free energy map for ZAβ3 in complex with Aβ; Figure S4: Favorable average and standard deviation non-polar and polar interaction free energy map for ZSYM73 in complex with Aβ.

Acknowledgements All MD simulations and free energy calculations were performed on the Ada supercomputing cluster at the Texas A&M High Performance Research Computing Facility. This study is supported by the Texas A&M University Graduate Diversity Fellowship from the TAMU Office of Graduate and Professional Studies awarded to AAO, and by startup funding from the Artie McFerrin Department of Chemical Engineering at Texas A&M University awarded to PT.


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Table 1: Residue pairwise interaction free energy ∆∆G differences (kcal/mol) between Αβ : subunit 1, Αβ : subunit 2, and subunit 1 : subunit 2 residues in β-wrapin : Aβ complexes which act as switches diminishing β-wrapins’ AS60 and AS69 activity for Aβ. AS9

AS34

AS60

AS69

ZAβ3

‡

0.2 ± 0.4

-0.2 ± 0.4

-0.9 ± 0.4

-0.7 ± 0.4

0.5 ± 0.4

Αβ

Subunit 1

V39

A12

Αβ

Subunit 2

V18

P20

‡

0.1 ± 0.1

0.1 ± 0.1

-1.2 ± 0.2

-0.6 ± 0.2

0.4 ± 0.0

F19

Y18

§

0.2 ± 0.1

0.1 ± 0.1

-0.6 ± 0.2

-0.1 ± 0.1

0.2 ± 0.0

F19

L19

§

0.1 ± 0.1

0.0 ± 0.1

-1.8 ± 0.4

-0.9 ± 0.2

0.4 ± 0.1

F19

L27

‡

-0.1 ± 0.1

-0.1 ± 0.1

-1.5 ± 0.1

-0.8 ± 0.2

0.2 ± 0.1

E22

G13AS10,AS9,AS34,ZAβ3

§

-0.1 ± 0.1

0.3 ± 0.3 -0.7 ± 0.1

-0.7 ± 0.1

-0.8 ± 0.2

-0.3 ± 0.2

D13AS60,AS69

0.0 ± 0.1

E22

G14

‡

-0.2 ± 0.2

0.1 ± 0.2

D23

G13AS10,AS9,AS34,ZAβ3

§

0.1 ± 0.3

0.2 ± 0.2 -1.1 ± 0.1

-1.0 ± 0.1

§

-0.1 ± 0.4

0.0 ± 0.3

-0.8 ± 0.2

-1.2 ± 0.2

-0.3 ± 0.3

D13AS60,AS69

-0.1 ± 0.2 0.1 ± 0.2

D23

G14

Subunit 1

Subunit 2

H32

P24

‡

-0.5 ± 0.3

-0.2 ± 0.3

-2.2 ± 0.2

-2.1 ± 0.2

-0.6 ± 0.2

H32

D25

‡

-0.3 ± 0.2

-0.3 ± 0.2

-1.3 ± 0.1

-1.2 ± 0.2

-0.5 ± 0.1

H35

P24

‡

-0.3 ± 0.2

-0.2 ± 0.2

-2.0 ± 0.2

-1.7 ± 0.2

-0.1 ± 0.2

Residue pairwise interaction ∆∆G free energy (kcal/mol) differences in the AS9 : Αβ, ΑS34 : Αβ, AS60 : Αβ, AS69 : Αβ and ΖΑβ3 : Αβ complexes with respect to the AS10 : Αβ complex, calculated via Eq. 2, for the residue pairs in that were identified as switches diminishing β-wrapins’ AS60 and/or AS69 activity for Αβ (shaded in gray). The table consists of three panels; the top panel lists interactions between residues in Aβ (left), and β-wrapin subunit 1 (right), the middle panel lists interactions between residues in Aβ (left), and β-wrapin subunit 2 (right), and the bottom panel lists interactions between residues in β-wrapin subunit 1 (left), and β-wrapin subunit 2 (right). The βwrapin’s name is shown in subscript next to mutated β-wrapin residues. Pairwise interactions marked with ‘‡’ and ‘§’ were according to Eq.2 identified as non-polar and polar interacting switches, respectively.



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Table 2: Residue pairwise interaction free energy differences between α-syn : subunit 1, and subunit 1 : subunit 2 residues in β-wrapin : α-syn complexes which act as switches diminishing β-wrapin’s ΖΑβ3 activity for α-syn.

α-syn

Subunit 1

L38

F31AS10,AS9,AS34,AS60,AS69

‡

AS9

AS34

AS60

AS69

-0.2 ± 0.1

0.4 ± 0.1

0.3 ± 0.1

0.8 ± 0.2

I31ZAβ3 Y39

-1.1 ± 0.1


‡

-0.2 ± 0.1

-0.4 ± 0.1

-0.5 ± 0.1

-0.2 ± 0.1

I31ZAβ3

-1.3 ± 0.0

H50

L19

§

-0.1 ± 0.2

0.0 ± 0.5

0.0 ± 0.3

0.4 ± 0.1

V52


‡

0.2 ± 0.1

0.2 ± 0.1

0.2 ± 0.1

0.2 ± 0.1

I31ZAβ3 Subunit 2

P24


‡

-0.1 ± 0.1

-0.1 ± 0.1

0.2 ± 0.1

0.0 ± 0.1

I31ZAβ3

-0.8 ± 0.1


‡

-0.5 ± 0.1

0.0 ± 0.1

0.0 ± 0.1

-0.2 ± 0.1

‡

0.1 ± 0.1

0.2 ± 0.1

0.0 ± 0.1

0.1 ± 0.1

‡

0.2 ± 0.1

-0.0 ± 0.1

-0.2 ± 0.1

-0.2 ± 0.1

I31ZAβ3 C28

-1.1 ± 0.6 -0.8 ± 0.1

Subunit 1

L27

ZAβ3

-1.2 ± 0.0

F31AS10,AS9,AS34,AS60,AS69 I31ZAβ3

-0.8 ± 0.0


F31 AS10,AS9,AS34,AS60,AS69

I31ZAβ3

I31ZAβ3

-0.8 ± 0.1

Residue pairwise interaction ∆∆G free energy (kcal/mol) differences in the AS9 : α-syn, ΑS34 : α-syn, AS60 : α-syn, AS69 : α-syn and ΖΑβ3 : α-syn complexes with respect to the AS10 : α-syn complex, calculated via Eq. 2, for the residue pairs that were identified as switches diminishing β-wrapin’s ZAβ3 activity for α-syn (shaded in gray). The table consists of two panels; the top panel lists interactions between residues in α-syn (left), and β-wrapin subunit 1 (right), and the bottom panel lists interactions between residues in β-wrapin subunit 1 (left), and β-wrapin subunit 2 (right). The β-wrapin’s name is shown in subscript next to mutated β-wrapin residues. Pairwise interactions marked with ‘‡’ and ‘§’ were according to Eq. 2 identified as non-polar and polar interacting switches, respectively.


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Table 3: Average MM GBSA association free energies (kcal/mol) decomposed into non-polar and polar components for β-wrapin : Αβ/α-syn complexes. AS10

AS9

AS34

AS60

AS69

ZAβ3

β-wrapin : Αβ Total MM GBSA Free Energy

-170.2 ± 4.2

-167.5 ± 3.5

-173.9 ± 2.2

-165.3 ± 3.3

-162.2 ± 6.8

-173.6 ± 3.6

Non-polar Component

-160.3 ± 4.1

-157.1 ± 3.9

-162.8 ± 1.6

-153.9 ± 2.7

-153.3 ± 4.4

-159.9 ± 2.9

-9.9 ± 0.8

-10.4 ± 1.6

-11.1 ± 1.5

-11.4 ± 1.4

-8.9 ± 2.7

-13.7 ± 0.8

Polar Component β-wrapin : α-syn Total MM GBSA Free Energy

-159.2 ± 2.9

-155.4 ± 2.8

-163.0 ± 5.8

-161.5 ± 4.1

-160.5 ± 3.3

-147.2 ± 7.6

Non-polar Component

-151.4 ± 3.4

-149.5 ± 2.3

-155.2 ± 5.2

-147.7 ± 2.9

-150.0 ± 2.9

-138.3 ± 6.9

-7.8 ± 1.7

-5.8 ± 1.0

-7.7 ± 2.0

-13.8 ± 2.2

-10.5 ± 1.9

-8.9 ± 1.5

Polar Component

Average MM GBSA association free energies of β-wrapin : Aβ (top panel), and α-syn (bottom panel) complexes. The total association free energies are decomposed into non-polar and polar components. These energies were calculated via Eq. 3 and Eq. 4. Energy values of β-wrapins with experimentally measured minimal or diminished activity (AS60 and AS69 : Aβ for the top panel, ZAβ3 : α-syn for the bottom panel) are shaded gray.



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Figure 1: Overview of β-wrapin variants investigated and local sequence alignment of α-syn to Aβ. (Panel A) Sequences of β-wrapins aligned to AS10 and their affinities for Aβ and α-syn reported in Mirecka et al.16 and Shaykhalishahi et. al.17. Affinities of ZAβ3 and AS60 to α-syn and Aβ, respectively, were not detected (n.d.). (Panel B) Local algorithm-based (Clustal Omega) and structure-based sequence alignments of α-syn to Aβ. Interestingly, the structural sequence alignment of the bound α-syn to Aβ differs from the local sequence alignment. Structurally aligned Aβ and α-syn residue pairs are considered corresponding residues (CR) for the purposes of this study.The residue loop for the Aβ peptide consist of 6 residues (VGSNKG) and the residue loop for the α-syn consist of 4 residues (TKEG) when bound by a β-wrapin.


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Figure 2: Molecular graphics images of AS10 in complex with Aβ (Panels A, C, E) and α-syn (Panels B, D, F), shown in orthographic view. Inter-helical non-polar contacts (in surface representation) are facilitated by the disulfide bridges formed by Cys28 of subunits 1 (in red) and 2 (in orange) in both AS10 : Aβ and AS10 : αsyn (Panels A and B). Tyr18 and Ile16 of subunits 1 and 2 act as molecular gates (in surface representation), locking the amyloidogenic proteins in AS10 (Panels A, B, C, D), β-sheets (in new cartoon representation) are formed between residues 14-19 of the β-wrapins (in red and orange) and 19-23 (in cyan), 32-36 (in blue) of Αβ and 39-43 (in cyan), 50-54 (in blue) of α-syn. The hydrophobic surface (in surface representation) facilitates the formation of these β-sheets (Panels C, D, E, F). Red, orange, and blue/cyan labels indicate residues of subunit 1 of the β-wrapin, subunit 2 of the β-wrapin, and the amyloidogenic protein monomer (Aβ or α-syn) respectively.



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Figure 3: Molecular graphics images of the common hydrophobic interactions of AS10 in complex with Aβ (panel A) and α-syn (Panel B), shown in orthographic view. These non-polar interactions contribute significantly to the ability of AS10 to sequester both Aβ and α-syn. β-wrapin subunits 1 and 2 are shown in red and orange tube representation, respectively, and the amyloidogenic proteins, Aβ and α-syn, are shown in blue tube representation. Red, orange, and blue labels indicate residues of subunit 1 of the β-wrapin, subunit 2 of the β-wrapin, and the amyloidogenic protein monomer (Aβ or α-syn) respectively.


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Figure 4: Molecular graphics images of interactions that act as switches diminishing a β-wrapin’s ability to sequester Aβ and α-syn, shown in perspective view. Favorable interactions occurring in AS10 : Aβ and AS10 : α-syn complexes are encapsulated in purple color (Panels A and C respectively). Unfavorable or significantly weaker interactions occurring in AS69 : Aβ (Panel B) and ZAβ3 : α-syn (Panel D) complexes are indicated with purple dashed lines. The unfavorable and weaker interactions shown for the AS69 : Aβ complex are also encountered in the AS60 : Aβ complex. β-wrapin subunits 1 and 2 are shown in red and orange tube representation, respectively, and the amyloidogenic proteins, Aβ and α-syn, are shown in blue tube representation. Red, orange, and blue labels indicate residues of subunit 1 of the β-wrapin, subunit 2 of the βwrapin, and the amyloidogenic protein monomer (Aβ or α-syn) respectively.



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Figure 5: Total MM GBSA association free energy (in kcal/mol) plotted against experimentally determined affinities13,14 (in nM) for β-wrapins in complex with Aβ (Panel A) and α-syn (Panel B).


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Figure 6: Molecular graphics images of the key interactions occurring in the ZSYM73 : Aβ complex , and are not present in the ZSYM73 : Aβ complex, shown in orthographic view. The formation of the salt bridge between ZSYM73’s residue Arg16 of subunit 2 and Glu22 of Aβ and the strengthening of the “gate-keeper” residue interactions between Arg16 and Tyr18 of ZSYM73 are shown in panel A. A “zipper-like” conformation comprising the salt-bridges between Arg32 of subunit 1/2 and Asp25 of subunit 2/1, the hydrogen bond between Glu35 of subunit 1/2 and Ala24 of subunit 2/1 are shown in panel B. β-wrapin subunits 1 and 2 are shown in red and orange tube representation, respectively, and Aβ is shown in blue tube representation. Red, orange, and blue labels indicate residues of subunit 1 of the β-wrapin, subunit 2 of the βwrapin, and Aβ respectively.



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