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Lanosterol disrupts aggregation of human #D-crystallin by binding to the hydrophobic dimerization interface Hongsuk Kang, Zaixing Yang, and Ruhong Zhou J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b03065 • Publication Date (Web): 19 Jun 2018 Downloaded from http://pubs.acs.org on June 19, 2018

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Journal of the American Chemical Society

Lanosterol disrupts aggregation of human γD-crystallin by binding to the hydrophobic dimerization interface Hongsuk Kang1,2, Zaixing Yang3, Ruhong Zhou1,2,* 1. Institute of Quantitative Biology, Department of Physics, Zhejiang University, Hangzhou, 310027, China 2. Computational Biology Center, IBM Thomas J. Watson Research Center, Yorktown Heights, NY 10598, USA 3. State Key Laboratory of Radiation Medicine and Protection, School for Radiological and interdisciplinary Sciences (RAD-X) and Collaborative Innovation Centre of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou 215123, China

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Abstract Cataracts are a leading cause of vision impairment 1, which stem from the misfolding and aggregation of crystallins in the eye lens. Despite its prevalence and severity, the detailed mechanism by which misfolded crystallins aggregate into cataracts remains elusive. Recently, in vitro and in vivo experiments demonstrated

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that lanosterol, a steroid-type compound found in

human and animal eyes, not only can prevent cataract formation but also reverse the formation. Inspired by these experimental observations, we investigate the preventive activity of lanosterol in the aggregate formation of human γD-crystallins (HγD-Crys) using all atom molecular dynamics (MD) simulation and free energy perturbation (FEP) techniques. Our results reveal that lanosterol preferentially binds to the HγD-Crys hydrophobic dimerization interface, in particular, to the structured C-terminus (near residues 135-165) with a stronger binding affinity than the unfolded N-terminus. Furthermore, we observe that the C-terminal binding is more favorable than lanosterol self-aggregation, further attesting to lanosterol efficacy. Finally, we compare the binding free energy of lanosterol with cholesterol using alchemical transformation and discuss the possible correlation of the molecular geometry of steroids with binding affinity.

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Introduction Cataracts are the leading cause of blindness and second leading cause of vision impairment globally, representing a major health concern1. It is well-established that the cataract formation is closely related to the aggregation of human α, β and γ-crystallins in the eye lenses. This precipitation of insoluble opaque aggregates reduces the transparency in the eye lens, and eventually leads to the impairment of vision by blocking the passage of light into the retina 3. Among the three crystallin families, α-crystallin is a chaperone-like protein resembling a small heat-shock protein whose functional role is to prevent aggregation of proteins, whereas both β and γ-crystallins are structural and functional entities that maintain the transparency and high refractive index of the lens 4. Human crystallins are extremely stable for their function and, in general, there is no further protein turnover after birth, which means that crystallins expressed at the embryo stage will be retained until the end of the life-span 3. Therefore, once crystallins have misfolded, they are hardly degraded. Consequently, any structural damages that accumulate in the eye lens can persist as the eyes deteriorate with the decreasing function of the chaperones 5. There are many pathways linked to protein misfolding that result in cataract formation. For example, UV-radiation may oxidize tryptophan (Trp) in human γD-crystallin (HγD-Crys) and transform Trp into a kynurenine (KN). It has been shown that such Trp-KN transition or Trp-Gln mutation mimicking Trp-KN transition facilitates the unfolding of HγD-Crys in silico and in vitro 6-7. Furthermore, a number of experimental studies demonstrated that just a single or a few amino acid mutations are sufficient to cause loss of transparency of the lens crystallin solution 715

, albeit without evidence that relates the specific mutation to the stability of the wild-type

crystallin.

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Despite the long history of crystallin aggregation study, no atomic level structure of cataracts consisting of the crystallin aggregates has been yielded by X-ray crystallography or NMR, and, consequently, the underlying mechanism of the cataract formation is still elusive. Inspired by neurodegenerative disease progression and experimental evidences, it was proposed that the misfolded crystallins may exist in amyloid fibrillary form characterized by intermolecular crossbeta-sheet formations and relatively ordered morphologies

13, 16

. Several experimental studies,

however, reported that in vitro crystallin aggregate may adopt alternative amorphous forms rather than the amyloid fibrils 8, 17-18. Domain-swapped aggregation is one proposed aggregation mechanism for cataract structures 19-21

. It is based on the observation that the N-terminal and C-terminal domains of HγD-Crys

exhibit different stability – there exist intermediate states at which the N-terminal domain is partially or fully unfolded while the C-terminal retains its native structure

19-21

. Subsequently,

amorphous aggregates may grow by the interaction between the unfolded N-terminal domain with relatively stable C-terminal domain of another monomer, or vice versa. The possibility of this domain-swapped aggregation pathway was substantiated by a computational study where thermally unfolded HγD-Crys monomers form domain-swapped dimers

22

. This is further

supported by a single molecule unfolding experiment using atomic force microscopy (AFM) with the wild-type HγD-Crys

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, and NMR study with the P23T HγD-Crys where insoluble

aggregates seem to retain its native fold, at least, partially 8. Nonetheless, there is another possibility that the amorphous protein complex is composed of native HγD-Crys with enhanced surface-surface interactions and irregular morphology 8. Strikingly, a recent experimental study reveals that the lanosterol, a natural steroid-type compound found in human lens, not only prevents mutated crystallin from aggregating but also

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reverses the cataractous lens into a transparent one in animals 2. In contrast, the same study showed that cholesterol is deficient in such therapeutic activity despite its molecular similarity to lanosterol. Another independent study revealed that a cholesterol-like compound has similar effects on αB crystallin with better solubility 24. Although these experiments provide evidence to the efficacy of steroid-like compounds for cataract treatment, the underlying molecular mechanism by which lanosterol (and other steroids) inhibits and reverses aggregation is still largely unknown. Moreover, structural information on the crystallin aggregate is very limited, so it has been challenging to understand the crucial lanosterol-protein interactions in detail. However, a better understanding of the lanosterol-crystallin interactions is crucial for the design of possible therapeutics based on lanosterol and/or other steroids. Therefore, a computational study of lanosterol binding may well complement experimental observations by providing atomic-detailed structural information as well as generating thermodynamic insights into the steroid-crystallin binding process. Here, we investigate the effect of lanosterol to reverse cataract formation by studying the lanosterol interaction with human γD-crystallin. We aim to reveal the lanosterol binding process using all-atom molecular dynamics (MD) simulations and to predict the binding affinity using free energy perturbation (FEP) calculations. We find that lanosterol displays a strong binding affinity to a hydrophobic interfacial region near residues 135-165 on the C-terminal domain. This region is crucial for HγD-Crys domain-swapping dimerization, which indicates that lanosterol binding likely disrupts the HγD-Crys dimerization. Furthermore, our FEP calculations reveal that cholesterol is less selective for this region than lanosterol, which might explain why lanosterol has much stronger preventive effects on cataract formation than cholesterol.

Results

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Lanosterol robustly binds to the hydrophobic dimerization interface of HγD crystallin To investigate the lanosterol effect on HγD-Crys dimerization, the first step to cataract formation, we started from partially unfolded monomer conformations taken from our previous work on domain-swapped dimers. Three representative domain-swapped dimer structures were selected, with each then separated into two monomers, resulting in a total of six partially unfolded monomer conformations. These monomers are then solvated in separate water boxes, with lanosterol molecules introduced into each of them to investigate their binding to HγD-Crys. Details of the simulations can be found in Methods and SI.

Fig. 1A illustrates lanosterol binding regions for six different HγD-Crys conformations. These regions, colored in red, are lanosterol contact probabilities for each residue in one representative trajectory obtained from each conformation. Here, a contact is counted if the distance between the center of mass of a residue in HγD-Crys and the center of mass of lanosterol aromatic rings is less than or equal to 8Å. For reference, next to each binding map, we show the structures highlighting the key protein domains: the N-terminal region (tan) and the C-terminal dimerization interface (shown in blue). As depicted in Fig. 1, one region of high lanosterol contact probability consists of residues 135-165, which was previously identified as a key region for the crystallin dimerization process

22

. Based on the domain-swapping theory of HγD-Crys

dimerization, the N-terminal region of one monomer (largely unfolded) interacts with the Cterminal region (largely intact) of another monomer. From our simulations, we observe that lanosterol would substantially disrupt this dimerization model given its strong propensity to bind to this key region of the largely intact C-terminus and the unfolded N-terminus suspected at the dimer interface (more below). Occupation of one or both domain-swapping sites by lanosterol would greatly reduce the dimerization capability. As mentioned above already, the other high

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contact probability area happens to be the N-terminal domain (residues 1-86), particularly its unfolded/extended conformations (e.g. conformers 1a, 1b and 3b in Fig. 1A), which were suggested to be the binding conformations in the domain-swapped mechanism

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. Thus, the

strong lanosterol binding to these extended N-terminus conformations would also contribute to the dimerization interface disruption. It is also interesting to note that the lanosterol binding is not like a typical drug molecule, and its binding site can be quite diffusive (i.e. not localized to a specific binding pocket), over several different regions on the molecular surface. As presented in Fig. 1C, we find that lanosterol binding is largely dependent on the protein residue type. Fig. 1C shows the lanosterol contact probabilities for each amino acid type, with a summation of probabilities equal to one. Most notably, lanosterol exhibits strong contact preference for the hydrophobic leucine. Another key amino acid is tyrosine which is moderately hydrophobic as well as aromatic, capable of forming ߨ − ߨ stacking interactions. In contrast to the hydrophobic residues, lanosterol shows little contact preference for both polar and charged residues. The highest contact probability among the hydrophilic residues is arginine (the most abundant amino acid in HγD-Crys), which has a relatively large planar guanidinium group that can form strong van der Waals interactions with lanosterol (more below). From the computed HγD-Crys contact probabilities, it is clear that lanosterol prefers interacting with hydrophobic residues.

Lanosterol binding modes and their binding affinity Next, we further explore the binding processes and binding modes by extending the simulation time of promising representative binding poses. We discovered that although lanosterol interacts most frequently with hydrophobic residues, the interaction strength could differ by the shape and composition of the crystallin binding conformations. From the above analyses, particular

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attention was paid to the binding modes where the ligand was bound to the C-terminal interface region (residues 135-165) or the N-terminal region (residues 1-86). Specifically, from the 30 final conformations obtained from the brief 30ns simulations for each, we chose two configurations binding to the C-terminal interface region and two to the N-terminal region to extend the unbiased MD simulations for an additional 270ns (Fig. S1). Interestingly, two of these four selected binding poses (1 C-terminal interface region and 1 N-terminal region and) remained robustly stable for the extended 270ns; the remaining two conformations displayed some fluctuation early on but stabilized over time. In fact, in one of the configurations, the lanosterol initially bound to the N-terminal region detached and rebound to the C-terminal interface for 230ns (1a.3 in Fig. S1A and Fig. S1B). Such an unbinding-rebinding event suggests that some binding poses may be transient rather than persistent. To comprehensively examine our predicted binding modes, we next computed the binding free energies for three promising conformations selected from the extended simulations: lanosterol bound to the C-terminal interface, lanosterol bound to the N-terminal half-open binding site and lanosterol bound to the N-terminal open binding site. (Fig. 2A, B, C). Here, we used the free energy perturbation (FEP) method to calculate the binding affinity. FEP is a rigorous method for estimating the absolute binding affinity with relatively high accuracy of a protein-ligand complex in silico

25-26

. During the course of FEP calculation, the relative position of the ligand to its

binding partner was restrained to avoid numerical instability due to wandering molecules when van der Waals interaction is nearly turned off

25

. FEP calculation details and results are

summarized in Fig. 2, Fig. S2 and Table S1. Notably, the C-terminal interface binding pose is the strongest with an absolute binding free energy ∆‫ ܩ‬of -10.31 kcal/mol. The N-terminal conformations are weaker, with the N-terminal open conformation having an affinity ∆‫ ܩ‬of -

8


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1.58kcal/mol and the N-terminal half-open binding free energy ∆‫ ܩ‬being -6.15 kcal/mol. Hence, based on these FEP calculations, the binding affinity between lanosterol and the C-terminal interface region is stronger than lanosterol with the N-terminal binding sites. The stronger affinity for the C-terminal region also helps explain why the transient N-terminal binder in the extended 300ns simulations eventually bound to the C-terminal region (Fig. S1). Interestingly, we also computed the binding free energy of lanosterol self-aggregation and compared with its binding affinity to the protein. It turns out that the self-aggregation strength, -7.68kcal/mol, lies in-between its binding to the N-term region (-6.15kcal/mol) and C-term region (-10.31kcal/mol). The binding strength to the C-term region is significantly higher than the self-aggregation. Thus, we believe although the N-term binding mode is somewhat weaker than the self-aggregation, the C-term binding outcompetes the self-aggregation significantly.

Lanosterol binding is mostly van der Waals interaction driven Intrigued by the relatively strong binding affinity of lanosterol to the hydrophobic residues, we analyzed the enthalpy change upon binding to further characterize the major driving force of lanosterol binding. We computed both electrostatics (coulomb) and van der waals (vdW) interaction energies, according to the equation ‫ܧ‬௜௡௧ ≡ ‫ܧ‬௉ା௅ − ‫ܧ‬௉ − ‫ܧ‬௅ , where ‫ܧ‬௉ା௅ , ‫ܧ‬௉ and ‫ܧ‬௅ are the coulomb/vdW energy of the lanosterol-HγD-Crys complex, HγD-Crys and the lanosterol, respectively. Not surprisingly, the proximate contact of the lanosterol with HγD-Crys strongly correlates with the plunge of vdW energy. Fig. 3 illustrates a typical time-series of the interaction energy during our simulation runs, showing the mostly vdW-energy-driven binding. As the lanosterol approaches to make a contact with HγD-Crys, the vdW energy starts to decrease sharply and remains stable after binding. By contrast, the electrostatic energy seems

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almost unaffected by the protein-ligand interaction, indicating that binding is not driven by electrostatics, consistent with the above amino acid contact probability analysis in Fig. 1C.

Lanosterol binding to HγD-Crys competes with its self-aggregation Because of its hydrophobic nature, multiple lanosterols tend to aggregate in water. Thus, lanosterol-HγD-Crys binding will compete with its self-aggregation. In experiments, the therapeutic activity of lanosterol was observed at relatively high concentration (25mM) 2, providing lanosterols with a self-aggregation-prone environment. Therefore, it is of interest to compare the binding free energy of lanosterol self-aggregation with that of the lanosterol-HγDCrys complex. To this effect, we examined HγD-Crys binding in the presence of multiple lanosterols. The simulations started from a single lanosterol-bound conformation (Fig. 2A, B and C). Then, an extra lanosterol molecule was introduced to the simulation box, and the simulations were run for at least 30ns or until the newly added ligand was either stably bound to the protein or another lanosterol. Following this same protocol, we introduced additional lanosterols one by one until the total lanosterol count reached four. Simulations of multiple lanosterols revealed that they bound to HγD-Crys either as a monomer or as oligomer. We found that additional lanosterol molecules could favorably adsorb onto the hydrophobic surfaces throughout the protein, similar to the single lanosterol discussed in Fig. 1, effectively covering the exposed hydrophobic surfaces in both N-terminal and C-terminal domains (Fig. 4A). In some other cases, however, the newly inserted lanosterol stuck onto other lanosterols already bound to HγD-Crys, forming a loosely packed aggregate as depicted in Fig. 4B. To understand the lanosterol self-aggregation affinity, we computed the lanosterol-lanosterol binding affinity, ∆‫ܩ‬ୢ୧ୱୱ୭ୡ , in a tetramer, using FEP calculations. Our results revealed a lanosterol-lanosterol binding affinity of -7.68kcal/mol, which is substantially weaker than the

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binding affinity of lanosterol for the C-terminal interface, -10.31kcal/mol. At the same time, selfaggregation is still more favorable compared to the N-terminal binding (up to -6.15kcal/mol). These findings imply that lanosterol still prefers the C-terminal region most.

Cholesterol exhibits less selective binding to the C-terminal interfacial region than lanosterol. One of the key findings in reference 2 is that only lanosterol can prevent and reverse cataract formation, while the similar cholesterol molecule has little efficacy. Since cholesterol is similarly hydrophobic to lanosterol, one would expect that it equally binds to the exposed hydrophobic patches of HγD-Crys (with similar self-aggregation propensity). The surprising experimental observation 2 indicates that not only the hydrophobicity but also the specific molecular geometry of steroids must be in play in the binding affinity. To resolve this mystery, we conducted FEP ୐ୟ୬ିେ୦୭୪ calculations for ∆∆‫ܩ‬ୠ୧୬ୢ୧୬୥ , i.e., the free energy change of transforming a lanosterol-HγD-Crys

complex to a cholesterol-HγD-Crys complex, to explore the binding difference between cholesterol and lanosterol. As previously described, the binding affinity of lanosterol to HγDCrys depends on its specific binding mode. Thus, to calculate the ∆∆‫ ܩ‬using FEP simulations, we selected three representative binding conformations similarly: ligand bound to the C-terminal interface, ligand bound to the N-terminal half-open binding site and ligand bound to the N୐ୟ୬ିେ୦୭୪ terminal open binding site. Details of FEP calculations as well as results of ∆∆‫ܩ‬ୠ୧୬ୢ୧୬୥ for each

binding mode are summarized in Table S2. Astonishingly, our FEP results demonstrate dramatic changes in the binding affinity, ୐ୟ୬ିେ୦୭୪ ୐ୟ୬ିେ୦୭୪ ∆∆‫ܩ‬ୠ୧୬ୢ୧୬୥ for different binding-sites. For example, ∆∆‫ܩ‬ୠ୧୬ୢ୧୬୥ for the C-terminal interfacial

regions is 2.61kcal/mol, indicating a stronger binding affinity for lanosterol than cholesterol. In

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୐ୟ୬ିେ୦୭୪ for half-open N-terminus binding can be as large as -3.60kcal/mol, which contrast, ∆∆‫ܩ‬ୠ୧୬ୢ୧୬୥

implies that the half-open binding site can favor cholesterol than lanosterol at this site. However, since the unfolded N-terminus is more flexible (transient) and less well-defined, and its binding to lanosterol is much weaker to start with than the C-terminal interfacial region, we believe the C-terminal interfacial region is way more important as the dominant binding site (more below). We also notice that the binding poses of the sterols change during FEP transformations. For the C-terminal binding pose, the ligand seems to flip directions as the lanosterol is transformed to the cholesterol (Fig. 5A). The free energy for half-open N-terminal binding is moderately perturbed by the change of the ligand (Fig. 5B). The most dramatic change was observed in the N-terminal open binding case where the ligand was detached from the protein and bound to another hydrophobic surface of the crystallin during FEP calculation (Fig. 5C). Our FEP calculations support that only lanosterol but not cholesterol exhibits preferential binding affinity to the dominant binding site at the C-terminal interfacial region. As discussed in our previous study

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, the native-like C-terminal interfacial region (residues

135-165) plays a crucial role in aggregation. The N-terminal region is unstructured in aggregation intermediates and any part of N-terminal domain may interact with C-terminal interfacial region to form domain-swapped dimers. This indicates that coverage of either Nterminal domain or C-terminal interface may provide effective protection from the aggregate formation. However, it should be noted that N-terminal region is larger than the C-terminal region and mostly unstructured. Thus, the surface area to be covered for the prevention of the dimerization will be much larger for N-terminal binding than for C-terminal binding. As cholesterol holds preferential affinity for the N-terminal domain, more cholesterol molecules (greater concentration) will be needed to disrupt the dimerization. As such, even if cholesterol

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may have the potential for the cataract treatment, our results indicate that the concentration required for treatment might be much higher than the concentration for lanosterol, which may partly explain why only little efficacy was observed experimentally.

Discussion In this paper, we studied the molecular mechanisms of lanosterol binding with HγD-Crys in the context of experimentally observed cataract reversal 2. We ran unbiased all-atom MD simulations and found that lanosterol preferentially binds to the hydrophobic regions of the HγDCrys, blocking domain-swapping interfacial regions, which may provide protection from aggregation and cataract formation. More importantly, from our binding free energy calculations, we found that lanosterol binds to the C-terminal interfacial region with higher affinity than lanosterol self-aggregation. Therefore, we expect that the protective effects of lanosterol against crystallin aggregation is mainly ascribed to its binding to the relatively stable C-terminal interfacial region, disrupting HγD-Crys dimerization. Lanosterol binding to the unfolded Nterminal region may also be partly responsible for the prevention of further aggregation. As substantiated by bis-ANS binding assays, hydrophobic pockets are exposed in wild-type HγDCrys aggregates21 and mutant9,

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, and thus the interaction of lanosterols with hydrophobic

residues in N-terminal region might contribute to the reduction in the aggregation rate to a certain extent. However, since lanosterol is poorly solvated in water, the binding of lanosterol to N-terminal region should outcompete self-aggregation to be effective. And our computations revealed that it is less favorable than the self-aggregation. Hence, we speculate that C-terminal binding is more influential in the anti-aggregation mechanism by lanosterol. Another interesting observation is that lanosterol displays stronger binding affinity to HγDCrys when compared to cholesterol. Since cholesterol is also insoluble in water (solubility:

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1.8×10-6 mg/mL at 30°C

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), it’s expected that its effectiveness should be comparable to

lanosterol if hydrophobicity is the only driving force for the lanosterol-protein interactions. However, this expectation is in contradiction to the experimental observations. Our free energy perturbation calculations indicate that molecular structure may play a major role in the favorable lanosterol binding affinity. Makley et al. recently reported that a sterol-derivative resembling cholesterol demonstrates compelling therapeutic effects selectively on the αB crystallin. In contrast, lanosterols seem to be effective on the full gamut of crystallins: αA, αB, γC and γD 2. The increased efficacy of lanosterol over cholesterol observed experimentally seems to be attributed to the two methyl groups protruding from the phenyl group (C28 and C29 in ref which are absent in both cholesterol and compound 29 in reference

24

29

),

. There are two possible

reasons for this enhancement. First, the two out-of-plane methyl groups may provide the molecule with an enlarged hydrophobic surface leading to the enhanced binding affinity at the hydrophobic interface. Second, as seen in Fig. 5A, the hydrocarbon branches may anchor at the hydrophobic pockets, reducing the mobility of the ligand and enhancing its binding stability. In contrast, other lanosterol-binding proteins such as cytochrome P450 seem less selective to the sterol-type 30-31 because their binding pocket is deeply buried, unlike the C-terminal region of the HγD-Crys. Since the entrance of the pocket is narrow, once a ligand enters the pocket there is less propensity to unbind. Furthermore, chemical interactions such as hydrogen bonds may also stabilize the binding. By contrast, we were not able to identify persistent hydrogen bonding between lanosterol and the crystallin in our simulations, and we attributed the binding to be mostly vdW-driven. In summary, through unbiased MD and FEP calculations, we showed that lanosterol preferentially binds to a potential dimerization interface in the C-terminal domain of HγD-Crys,

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which may provide protection from aggregation and cataract formation. Our FEP results and analyses suggest that the C-terminal binding of lanosterols is stronger than self-aggregation, and that such domain-specificity is only observed for lanosterol but not cholesterol. This further hints that the preventive effectiveness of lanosterol stems from preferentially covering dimeric interfaces. We believe that our theoretical study has shed light on the molecular-level role of sterols in cataract treatment, which would prompt the design of chemical compounds with improved activity and properties. Finally, it should be mentioned that FEP can converge slowly particularly on systems where the environments of the target alchemical modification undergoes slow response fluctuations; for this purpose, various advanced sampling strategies, such as Orthogonal Space Random Walk (OSRW), were developed32. It would be also desirable to study the dissociation of HγD-Crys aggregates in the future with more advanced sampling techniques, such as replica exchange33, replica exchange with solute-tempering

34

and metadynamics

35

to

help reduce the enormous computational resources required (it takes days in experiment to dissolve the HγD-Crys aggregates even with very high lanosterol concentration), and better understand the underlying mechanism of the therapeutic effects of lanosterols.

Materials and Methods Atomic coordinates of HγD-Crys were taken from the final snapshots of three previous dimerization simulations

22

. We separated the HγD-Crys dimers (1,2,3) into six monomers

(1A,1B,2A,2B,3A,3B) and placed them at the center of a simulation box with a 20Å margin surrounding the protein. A lanosterol molecule was inserted in one (randomly-chosen) corner of the simulation box. The HγD-Crys-lanosterol system was then solvated with TIP3P water and without ions. After standard energy minimization, we equilibrated the system at 310K and 1atm for 5ns under the NPT condition and then ran 30ns production runs. We generated 5 trajectories

15



per protein conformation and thus sampled a total of 30 different binding modes. After 30ns runs, we investigated that the binding pose persists for at least 10ns until the end of simulations to rule out the transient binding. If its persistent time is shorter than 10ns (i.e., the lanosterol molecule wanders off the protein), we extended simulation time up to 100ns to see if we can observe a stable binding mode lasting longer than 10ns. Among the 28 out 30 stable binding modes, we chose 4 representative ones and extended the simulations to 300ns to test the binding stability of each selected binding mode. For multiple lanosterol simulations, we started from a final conformation of the above single lanosterol simulation, removed all water molecules from the system, and inserted a lanosterol into the simulation box at the corner. Then, we solvated the simulation box with TIP3P water and carried out 5ns equilibration and 30ns production runs. This procedure was repeated until four lanosterols were simulated. The final concentration with four lanosterol molecules in our current simulation box is equivalent to ~11mM, which is comparable to the experimental concentration of 25mM in dissociation. 2 FEP calculations for absolute binding affinity were conducted similarly to ref25. Specifically, we applied harmonic restraints on distances and angles between the lanosterol and protein to prevent the ligand from escaping the binding pocket, which leads to numerical instability. The details of the harmonic restraints are described in Fig. S1. After the relative position of the ligand was fixed, electrostatic and van der Waals interactions were gradually turned off. At each step, the simulation was run for 500ps under NPT conditions at 310K and 1atm. Finally, the ligand restraints were analytically removed as proposed in ref 25. FEP for the alchemical transformation of lanosterol to cholesterol was done with the same procedure without distance and angle restraints. Errors for FEP results were estimated by block-averaging scheme implemented in

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GROMACS analysis tools36. For each window, the trajectory was divided into five consecutive blocks and the errors were calculated from the standard deviation of averages for each block. CHARMM36

37

was chosen for the protein force field while topology and parameters for

lanosterol were adopted from literature

29

. During the simulations, the protein backbones were

restrained with 1000pN force to maintain its original backbone structures while sidechains were free. Newtonian dynamics equation of motion was integrated with 2fs timestep for MD whereas stochastic dynamics with 1fs timestep was used for FEP. All simulations were performed with GROMACS 5.1.4 package 38. All graphics of proteins and ligands were rendered by VMD 1.9.3 39

.

Associated Content Supporting Information. Figure S1. Minimum distance of lanosterols from HγD-Crys for four selected binding poses as a function of time and snapshots of unbinding and rebinding of lanosterols to HγD-Crys. Figure S2. Initial configurations of FEP calculations for C-terminus, N-terminus (half-open) and N-terminus (open) binding poses and parameters for the distance and angle constraints for FEP. Table S1. Binding free energy values of lanosterols to HγD-Crys in FEP calculation for all λ values. Table S2. Binding free energy values for transforming lanosterol to cholesterol in FEP calculation for all λ values.

Author Information Corresponding Author *

[email protected]

Acknowledgments 17



We would like to thank David Bell, Leili Zhang, Tien Huynh and Binquan Luan for their various help with this work. RZ acknowledges the support of the IBM Blue Gene Science Program (W1258591, W1464125, W1464164).

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Fig. 1: Contact probability of lanosterols for 6 different conformations of partially unfolded human γD-crystallin (HγD-Crys). (A) Each conformation of the crystallin is colored by contact probability on the left side and protein domain on the right side for comparison. Contact probability is based on one representative trajectory for each conformation. (B) Residue contact probability across the 6 binding conformations, revealing the diversity of lanosterol binding sites on HγD-Crys. (C) Contact frequency by residue type (contact preference) over all 6 HγD-Crys conformations. Leucine has the highest contact preference followed by tyrosine, attesting to the propensity of lanosterol to bind hydrophobic residues. Fig. 2: Absolute binding affinity of the lanosterol-crystallin complex. Three different binding modes were selected for free energy perturbation (FEP): (A) C-terminal interface, (B) Half-open N-terminal, and (C) Open N-terminal binding. The protein insets are colored according to residue type: white is hydrophobic, green is polar non-charged, red is negatively charged and blue is positively charged. The binding free energy, ∆‫ܩ‬, is calculated by FEP with harmonic constraint. (D) Presents the accumulated ∆‫ ܩ‬for decoupling as the control variable λ increases. From λ=1 to 10, the harmonic constraints were turned on. From λ=11 to 14 and from λ=15 to 29, both electrostatic and van der Waals interactions were switched off. ∆‫ ܩ‬for binding was 10.31kcal/mol, -6.15kcal/mol and -1.59kcal/mol with ∆‫୳ܩ‬୬୰ୣୱ =-7.50kcal/mol, -8.38kcal/mol and -8.12kcal/mol and ∆‫ܩ‬ୢୣୱ୭୪୴ = -2.24kcal/mol for C-terminal interface, half-open and open Nterminal binding, respectively. ∆‫୳ܩ‬୬୰ୣୱ is the free energy change by relaxing harmonic restraints between the protein and the ligand and ∆‫ܩ‬ୢୣୱ୭୪୴ is desolvation free energy of the ligand. Errorbars were calculated by block-averaging with 5 blocks (and their sizes are comparable to or smaller than the size of the symbols). Fig. 3: Interaction energy change for a representative lanosterol-HγD-Crys binding conformation illustrating van der Waals-driven binding. Interaction energies are shown as a function of time with significant binding events illustrated at selected time points. Coulomb (red) and van der Waals (blue) interaction energies are defined by ‫ܧ‬୧୬୲ = ‫ܧ‬ୡ୭୫୮୪ୣ୶ − ‫ܧ‬୮୰୭୲ୣ୧୬ − ‫ܧ‬୪୧୥ୟ୬ୢ . The protein is colored according to secondary structure with yellow as beta sheet, cyan as turn, white as coil, purple as alpha helix, and blue as 3/10 helix.

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Fig. 4: Multi-lanosterol binding. Two competing configurations are illustrated: (left) increased lanosterol-protein contacts and (right) lanosterol self-aggregation. Although self-aggregation is stable, FEP calculations reveal that C-terminal binding is more favorable than self-aggregation while N-terminal binding is comparable to self-aggregation. Protein is colored the same as Figure 3. Fig. 5: Change in HγD-Crys binding affinity for the transformation of lanosterol to cholesterol, ∆∆‫ܩ‬୪ୟ୬ିୡ୦୭୪ . Initial (lanosterol) and final (cholesterol) configurations are depicted for (A) Cterminal domain binding, (B) half-open N-terminal domain binding and (C) open N-terminal domain binding. Lanosterols are colored in orange and cholesterols in pink; the two methyl groups that disappear in cholesterol are highlighted with large cyan spheres. The protein is colored similarly to Figure 2 insets. Binding conformations are substantially shifted for C-term (A) and half-open N-term (B) sites, with severe disruption for the open N-term site (C) where the ligand detaches and re-binds to another surface during FEP calculation. (D) Presents the accumulated ∆∆‫ܩ‬୪ୟ୬ିୡ୦୭୪ with increasing λ. The ∆∆‫ܩ‬୪ୟ୬ିୡ୦୭୪ shows considerable destabilization of C-terminal binding upon changing from lanosterol to cholesterol, indicating favorable lanosterol binding specificity is likely attributed to C-terminal binding. Errorbars were calculated by block-averaging with 5 blocks (and their sizes are comparable to or smaller than the size of symbols).

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Fig. 1.

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Fig. 2.

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Fig. 3.

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Fig. 4.

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Fig. 5.

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Lanosterol Disrupts Aggregation of Human Î³D ... - ACS Publications

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