Conformational Transition of Key Structural Features Involved in

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Conformational Transition of Key Structural Features Involved in Activation of ALK Induced by Two Neuroblastoma Mutations and ATP Binding: Insight from Accelerated Molecular Dynamics Simulations Muyang He, Weikang Li, Qingchuan Zheng, and Hong-Xing Zhang ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00105 • Publication Date (Web): 11 Apr 2018 Downloaded from http://pubs.acs.org on April 11, 2018

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ACS Chemical Neuroscience

Conformational Transition of Key Structural Features Involved

in

Activation

of

ALK

Induced

by

two

Neuroblastoma Mutations and ATP Binding: Insight from Accelerated Molecular Dynamics Simulations

Mu-Yang He,2 Wei-Kang Li,2Qing-Chuan Zheng1,2*Hong-Xing Zhang2*

1. Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, College of Life Science, Jilin University, Changchun 130012, People’s Republic of China

2. Laboratory of Theoretical and Computational Chemistry, Institute of Theoretical Chemistry, International Joint Research Laboratory of Nano-Micro Architecture Chemistry, Jilin University, Changchun 130023, People’s Republic of China

Correspondence to:

Qing-Chuan Zheng, Institute of Theoretical Chemistry, Jilin University, Changchun, 130023, People’s Republic of China.

Fax: (+86) 431-8849-8966, E-mail address: [email protected]

Hong-Xing Zhang, Institute of Theoretical Chemistry, Jilin University, Changchun, 130023, People’s Republic of China.

Fax:

(+86) 431-8849-8962, E-mail address: [email protected]

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ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Deregulated kinase activity of Anaplastic Lymphoma Kinase (ALK) has been observed to be implicated in developing of tumor progress. The activation mechanism of ALK is proposed to be similar with other receptor tyrosine kinase (RTK), but the distinct static X-ray crystal conformation of ALK suggests its unique conformational transition. Herein, we have illustrated the dynamic conformational property of wild type ALK as well as the kinase activation equilibrium variation induced by two neuroblastoma mutations (R1275Q and Y1278S) and ATP binding by performed enhanced sampling accelerated Molecular Dynamics (aMD) simulations. The results suggested that the wild type ALK is mostly favored in the inactive state, whereas the mutations and ATP binding promoted clear shift toward the active-like conformation. The R1275Q mutant stabilize the active conformation by rigidified the αC-in conformation. The Y1278S mutant promotes the activation at the expense of a π-stacking hydrophobic cluster which plays critical role in the stabilization of inactive conformation of native ALK. ATP produces a more compact active site and thereby facilitates the activation of ALK. Taken together, these findings not only elucidate the diverse conformations in different ALKs, but also can shed light on new strategies for protein engineering and structural based drug design for ALK.

KEY WORDS: ALK, Kinase Activation, Neuroblastoma mutation, ATP, aMD


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INTRODUCTION Anaplastic lymphoma kinase (ALK) is a member of the receptor tyrosine kinase (RTK) superfamily, which is postulated to play a pivotal role in normal development and function of the nervous system in embryos.

1, 2

Elevated activities of ALK are

associated with the development and progression of multiple human malignancies.2 Structurally, ALK is composed of an extracellular receptor, a transmembrane region, and an intracellular tyrosine kinase domain.2 The kinase domain of ALK adopts the canonical kinase-fold, which is built of a smaller N-terminal lobe and an α-helical C-terminal lobe.3 The ATP binding-site is at the interlobe cleft referred as the active site of ALK. A common activation mechanism is believed to exist for members of this superfamily which mainly controlled by conformational changes in two conserved structural motifs: the αC-helix and the activation loop (A-loop) (Figure 1). 4-8

On one hand, the active state is characterized by an “αC-in” conformation which is

proper for the catalytic salt bridge formation between conserved lysine and glutamic acid in N-terminal lobe. On the other hand, the A-loop assumes an extended conformation to make adequate space for peptide substrate binding. The X-ray crystal structures of dormant ALK reveal a unique partially inactive tyrosine kinase conformation which lack the complete repertoire of negative regulatory structural elements that exist in the inactive RTKs.3, 9, 10 In dormant ALK, αC-helix adopts an “in” conformation, and A-loop exhibits a semi-closed conformation, which is in an intermediate position between the inactive and active RTKs. It may highlight the fact that the activation of ALK is rather a unique mechanism which makes it both



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interesting yet elusive. Substantial studies have demonstrated that mutations and ligand binding could play significant role as allosteric modulators in the activation of kinase.11-13 In ALK, mutations caused missense substitution of single amino acid in the kinase domain could promote constitutive, ligand independent tyrosine kinase activation.2, 14, 15 Many somatic and germline mutations of ALK arouse clinical notice on account of their activating or drug-resistance effects in many diseases, including neuroblastoma,16 non small cell lung cancer (NSCLC),17 and inflammatory myofibroblastic tumor (IMT).18 The hot spot mutation R1275Q is the single most common mutation

(approximately

43% of all mutations) detected in neuroblastoma,19 a pediatric disease that arises in the sympathetic nervous system, representing the most frequently diagnosed malignant tumor in the first year of life.16,

20

Point mutation is also observed in

Y1278S from the patients of neuroblastoma, although less frequently.21 Y1278 is the first tyrosine undergoes autocatalytic phosphorylation in A-loop, and its substitution also reported to promotes constitutive kinase activity of ALK.21,

22

To date, the

biological effects of these activating drivers are clear,21, 23, 24 whereas the molecular origins and modulating modes in the activation process of ALK have not been rationalized. In addition, recent studies state that ATP can serve not only as an energy provider but also as an allosteric modulator that regulates the process of kinase activation.25-28 In the ALK involved cellular signal transduction, ATP serves as substrate and provides phosphate group in a number of signal transduction cascades.23, 29-31

In contrast with the well understood role in phosphorylation, the allosteric


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behaviors of ATP in ALK are rarely illuminated. As ALK is highly expressed only in the nervous system during embryogenesis, it is difficult to implement experiments that pursue its mechanism of activation.2, 32, 33 Molecular dynamics (MD) simulations are increasingly being applied to study the structural biology of biomacromolecules.34-37 The computational study of ALK mainly focus on the drug resistance mechanisms and specific inhibitor design. Abundance of computational investigations have in-sighted both the first and second inhibitors drug resistance mechanisms at atomic-level induced by ALK mutations.38-41 Hou et.al have rationally designed a novel kind of inhibitors with excellent antiproliferation activity against ALK-positive cancer cells, 42-44 which have provided a new strategy in rational design of potent and selective ALK inhibitors to conquer drug resistance. Despite the plenty research about drug recognition of ALK, the activation properties of ALK have not been elaborated.

Thanks to the development of accelerated molecular dynamics

(aMD) simulation, the states transition of protein can be clarified at atomic level.45-48 Thus, we performed aMD simulations on the apo wild type, R1275Q mutant, Y1278S mutant, and ATP-bound forms of ALK to explore the potential activation mechanisms in this study. Specifically, the introduction of free energy landscape method figures out the dynamical consequences of two neuroblastoma mutants and ATP binding on the conformational alterations of the key structural features associated with the activation of ALK. These insights on the ALK structural features and conformation dynamic can provide clues to understand the detailed activation mechanisms of ALK on atomistic level.



RESULTS AND DISCUSSION Conformational Free Energy Landscape of Apo-WT ALK. To study the conformational states and structural dynamics of ALKs, 50 ns conventional MD (cMD) followed by 500 ns aMD were performed for each system. As shown in Figure S1, it is clear that the RMSD values are convergent for both the cMD and aMD simulations. The protein shows no significant deviation from the initial structure in the preliminary 50 ns cMD simulations. In the subsequent aMD simulations, diverse conformations were sampled, thus provided the opportunity for the capture of activation process of ALK. Free energy profiles of the ALKs are explored utilizing aMD simulations in terms of the formation of catalytic salt bridge, and the A-loop extension, both of which have been noted previously to be critical for ALK activation. The free energy profiles were projected along two collective variables (CV1 and CV2) for the four systems. CV1 measures the deviation of A-loop from the

X-ray crystal structure (PDB code 3L9P), which is able to characterize

the open and closed conformation of A-loop. The deviation of A-loop is measured by the average deviation of the residues in the distal of A-loop, including Y1283 to M1290. These residues approaching to the active site would block the peptide binding sites, a conformation that regarded as inactive kinase state. On the contrary, the distal residues move outward would shape an open A-loop conformation which is favorable for the peptide substrate binding. The negative value denotes deviation of A-loop approaching the active site, and the positive value is on behalf of the outward


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extension of A-loop. CV2 is defined as the distance of the salt-bridge-forming couple: K1150 and E1167, which is able to characterize the displacement of the functionally important component αC-helix of the N lobe in its transition between the “αC-in” and “αC-out” conformation. The value of CV2 less than 4 Å represents the formation of the salt bridge.49 The two dimensional free energy profiles of the Apo-WT ALK system is shown in Figure 2A. For the deviation of A-loop, the value sampled in this system mainly distributes between - 3Å to 5Å (with free energy less than 1 kcal/mol). Notably, no A-loop open conformation is sampled in Apo-WT ALK system. As shown in Figure S2, the conformation of A-loop with 5Å outward deviation is not full extended and is not able to provide sufficient space for the peptide binding. The 3Å inward deviation of A-loop obstructs the not only the peptide binding site but also the ATP binding site, which is very similar to the close conformation of A-loop observed in other inactive RTKs.50-52 These results demonstrate that the wild-type kinase domain on its own is indeed in an autoinhibited state. For the conformation of αC-helix, in the majority of simulation the distance drops below the value required for salt bridge to form between K1150 and E1167, representing an αC-in conformation. Remarkably, in the second low energy basin, both the distance below and more than 4 Å are sampled. The fairly little energy penalty suggests the toilless transition between αC-in and αC-out conformation in the Apo-WT ALK system. Apo-WT ALK Kinase Domain is Intrinsically Autoinhibited. The deepest free-energy minimum of Apo-WT corresponds to partial inactive



conformation with structural features similar to the X-ray crystal ALK structure (PDB code 3L9P),3 where the salt bridge between K1150 and E1167 is well maintained and the A-loop adopts a partial extended solvent exposed conformation (Figure 2A and 2B). In spite of the similarity, the aMD simulations provide more atomic details about sustain of the partial inactive conformation. As shown in Figure 3A, polar residues adorning the polar face of αC-helix and αAL (the first helix of A-loop) conduce to stabilize their relative position in the partial inactive conformation of ALK. The Aspartic acid in αC-helix (D1163) forms two hydrogen bonds with two positively charged residues in αAL (R1275 and R1279), which serve as the key factors that stabilize the αC-in conformation in this partial inactive state. For the conformation of A-loop, a series of hydrogen bonds set up the semi-closed architecture (listed in Table S1). As shown in Figure 3A, the hydrogen bond formed between Q1159 of αC-helix and the third A-loop tyrosine Y1283 constructs a sharp U-turn at the end of αAL that prevents A-loop shapes neither the full extension nor the closed conformation, which is regarded as the principle factor that stabilized the semi-closed A-loop conformation. The hydrogen bond formed between the main chain of D1276 and the first side chain nitrogen atom of R1284 further steady this U-turn. In addition, electrostatic and hydrophobic interactions among the distal part and the tail end of A-loop lend further stability to the semi-closed A-loop conformation. In the distal part of A-loop, a hydrogen bond between C1288 and L1291 is observed in this partial extended conformation, which may further restrain the adaptability of A-loop. The tail end residue of A-loop V1293 is fastened adjacent to αG-helix by a hydrophobic cluster


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consisted of residue M1296, F1301, and L1339. As shown in Figure 3A, both of the hydrogen bond and the hydrophobic cluster mentioned above can contribute to the stability of a hydrophobic stem consisted of the residue M1273, L1291, and P1292. This hydrophobic stem has been observed in the crystal structure of ALK and considered to play a critical role in holding the semi-closed A-loop conformation. Besides the most populated partial inactive conformation, a conformation that matches the common criteria of inactive RTK is sampled. The ensembles in the second low energy basin are clustered into four clusters. One of which represents the value of CV1 is no more than -3 Å, and CV2 is more than 4 Å. The center structure of this cluster is chosen as the representative structure of inactive-like Apo-WT. In this inactive-like conformation, the αC-helix is rotated outward and displaced from the active site, and the A-loop adopts a closed conformation that obstruct both the ATP and the peptide binding sites (Figure 1D and Figure 3B). As expected, the interactions among these two activation relative motifs altered from the partial inactive conformation. In the inactive-like conformation, the charged side chain of D1163 in αC-helix makes a hydrogen bond with Y1278 who located in the end of αAL instead of with the two middle situated arginine. Another negative charged residue in αC-helix E1167 interacts with R1275 and R1279 leading to the deflection of the side chain of E1167. Regarding to the A-loop conformation, the hydrogen bond network are distinct with that emerged in the semi-closed state are listed in Table S2. One of the significant alteration is the disruption of hydrogen bond between Y1283 and Q1159. In the A-loop closed state, the side chain of Y1283 is hauled approaching the



active site through the hydrogen bond chains consisted of E1167, R1279 and Y1283. Moreover, a second short helix after the αAL is observed in the forepart of A-loop. It is noteworthy that this second helical segment is commonly presented in the inactive conformations of other RTKs, such as EGFR,50 Src kinase,52 and is considered to be important in the formation of close A-loop conformation. In Apo-WT ALK, this helix is established by a network of hydrogen bonds between the main chains of residues S1281, Y1282, Y1283, R1284, and K1285. Indeed, the hydrogen bond network in this helix further reins the outward motion of residues in the middle part of A-loop. Notably, the residue M1273, L1291 and P1292 no longer construct the hydrophobic stem in this A-loop closed conformation. In contrast, M1290 packing against L1291 and P1292 forming a new hydrophobic stem that is close to the P-loop and αD-helix thus occupies both the ATP and peptide substrate binding pockets (Figure 2B). As shown in Figure 3C, L1291 and P1292 are held at the proline enrichment region by hydrophobic effect provided by P1329 and P1331, thus further consolidate the new hydrophobic stem. In addition, the terminal A-loop residue V1293 no longer stuck around αG-helix in the inactive state. As shown in Figure 3D, V1293 participates in a new hydrophobic cluster and sustains the closed conformation of A-loop. These hydrophobic interactions restrain the conformational dynamics of the A-loop and thus retain the active site in a “full-closed” state. In summary, the Apo-WT ALK kinase domain is intrinsically autoinhibited and the transition to its active state is hard to achieve. First of all, the negligible energy penalty for disruption of the K-E salt bridge indicates the unsteadiness of this catalytic


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bridge in the Apo-WT system. In addition, the conformation of A-loop in the Apo-WT system can transform between the initial semi-closed and the closed conformations, both of which are incompatible with substrate binding. R1275Q Maintains the Stability of K-E Salt Bridge. In contrast to the Apo-WT, the mutants populate to some extent the active conformations, and generate new minima in different locations of the FEL. The conformational FEL of the R1275Q ALK system is shown in Figure 2A. The most stable state of R1275Q corresponds to an ensemble of structures which are in similar with the partial inactive conformation, where the A-loop is folded and assumes a semi-closed conformation and the αC-helix closely interacts with β3-strand (Figure 2C). For the distribution of CV1, the value sampled in this system mainly distributes between - 4Å to 3Å (with free energy less than 1 kcal/mol).The range of A-loop deviation in R1275Q represents the conformational variation between closed and semi-closed status, which is in the same way of the Apo-WT system. For the distribution of CV2, the main population is in the range less than 4 Å. In another word, the conformation adopted by αC-helix in R1275Q appears to result more from an energetic advantage for the “in” conformation rather than an energetic disadvantage for an alternate “out” one. Thus, it is reasonable to infer that R1275Q mutation is expected to destabilize the inactive conformation by maintain the formation of the K-E salt bridge thus keep the αC-in pre-active conformation. To unravel the molecular origins of this phenomenon, we analysis the structural variations between the native and R1275Q mutated ALKs. In the Apo-WT



system, the side chain orientation of glutamic acid 1167 who engages in the catalytic salt bridge formation exhibits high flexibility. In quite a few frames, E1167 turns outward and makes electrostatic interactions with the negative charged arginine R1275 in αAL (Figure 4A). The shift of E1167 directivity leads to the departure from its salt bridge formation partner K1150, which impacts the formation of the important salt bridge and thus drives αC-helix switching to the inactive conformations. On account of the hydrogen bond formed between E1167 and arginine in αAL, the side chain of E1167 is constraint around the charged R1275 and hence the disruption of the K-E salt bridge is hard to recover. Upon introduction of the R1275Q mutation with an uncharged and rather shorter side chain, the chance to hold E1167 to face outward the active site is reduced significantly. In another word, residue E1167 is more favor to make polar interaction with K1159 and maintains the αC-in conformation in the R1275Q system. Moreover, another hydrogen bond is observed between E1167 and F1269 (the phenylalanine belongs to the DFG motif) in the active site of R1275Q (Figure 4B). This hydrogen bond then produces a force, pulling αC-helix inward, which further prevents the outward shift of E1167 and thus assists the formation of the K-E salt bridge between αC-helix and β3-strand. In summary, the global effect of R1275Q mutation is to stabilize the K-E salt bridge and to elevate the free energy barriers for the αC-helix transition from the αC-in to αC-out conformation. R1275Q mutation decreases the outward shift of E1167 and enhancing its interaction with K1150 to keep a αC-in conformation of ALK, thereby protect the indispensable structural prerequisite for the kinase


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activating process. Y1278S Shifts the Equilibrium to Activation State. In the case of the Y1278S mutant, the changes in conformational FEL is more visible and the equilibrium is shifted toward the active conformation (Figure 2A). The typical inactive conformation observed in Apo-WT has disappeared in Y1278S. The distance between the two atoms that forms the catalytic salt bridge sampled in Y1278S system is mainly populated less than 4Å. In analogue to the Apo-WT, the second populated energy minima incorporates the structures with both the K-E salt bridge formation and disruption conformations, indicating that the Y1278S mutation has little effect on the conformational switching of the αC-helix. For the conformation of A-loop, the free-energy landscape exhibits remarkable differences with that of Apo-WT. In the deepest free energy minimum, the deviation of A-loop in Y1278S distributes in the range between 3 Å to 10 Å, which is on behalf of the transition between the semi-closed and open conformations. When the representative structure of Y1278S is compared with the Apo-WT structures, it is observed the Y1278S mutation conduces the increased extension of A-loop (Figure 2B and 2D). In the most populated conformation of Apo-WT, the side chain aromatic ring of native Y1278 is favoring the establishment of a π-stacking hydrophobic cluster by packing against other three adjacent aromatic rings of phenylalanine (Figure 5A), including the phenylalanine F1174 at the base of αC-helix, the F1269 belonging the DFG motif, and F1245 situated right below the αC-helix. This hydrophobic cluster contributes the π-stacking interactions leading to an



extended aromatic box，which may take considerable effect on anchoring the relative positions of the aromatic side chains of these involving residues, thus maintains the relatively stationary position of αC-helix and αAL in Apo-WT system. Substitution of Y1278 with serine is expected to destabilize this hydrophobic cluster, as serine cannot be favorable accommodated in the π-stacking hydrophobic pocket occupied by Y1278. Evidently, the shorter side chain substitution S1278 obtains additional mobile space and thus increases the fluctuation of the end of αAL accordingly. The increased flexibility of the initial part of A-loop then gains the opportunity to leads to long-range process reorientation of the A-loop in Y1278S. It is worth noting that the increased flexibility induced by Y1278S mutation promotes the A-loop to a more open conformation rather than a more closed one. The underlying molecular mechanisms responsible for the directed conformational alteration of A-loop may origin from the structural unsuitability of the mutated S1278 in establishing the typical inactive-like conformation. In the typical inactive-like state of Apo-WT, residue Y1278 adopts the conformation by hydrogen bonded with the αC-helix aspartic acid D1163 (Figure 3B). The electrostatic interaction is considered to be one of the forceful factor that maintain the relative positions of αC-helix and initial part of A-loop and keeps their proper conformations accommodated in the inactive kinase conformation. Substituted S1278 with a shorter side chain is incompetent to form the hydrogen bond with D1163, thus will be unfavorable for stabilization of the inactive-like conformation. In addition, the steric flexible S1278 induces concomitant interaction changes which could further disrupt the mutual


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stabilization of the closed A-loop conformation. As shown in Figure 5B, the Y1278S mutation promotes the fluctuation of the neighboring “RAS” sequence motif, which has been predicted as a controlling unit for the activation process of ALK.53 In the A-loop closed conformation, R1279, A1280, and S1281 hydrogen bonded with residues situated distal part of A-loop, including Y1283, R1284, and K1285, respectively (Figure 5C). These hydrogen bonds help construct the second A-loop helical segment and thus restraint these distal residues of A-loop in the position obstructed the peptide substrate binding pocket. In Y1278S, the set of hydrogen bonds have dismantled with the fluctuation of “RAS” motif (Figure 5D). Without the electrostatic attraction, the distal residues are able to shift outward by little energy penalty, thus leading to an extended A-loop conformation. In addition, it is rationed to speculate that the less restraint S1278 is more easily to exposure in the solvent, which could increase the possibility of the phosphorylation of S1278. Hence, the activation of ALK may be more facile. Overall, the Y1278S substitution is readily accommodated in the active form of the kinase. The substitution of tyrosine with serine could disrupt these connections favored for the ALK inactive state, thereby promotes the activation balance to shift toward active state. Allosteric Effects of ATP Promotes Activation of ALK. Many allosteric effects on the kinase activation on account of ATP binding has been reported.27, 28 Herein, the ATP induced allosteric effects are observed directly at an atomic level by the accelerated MD simulations. As shown in Figure 2A, two energy



basins can be found in the free energy landscape of ATP-bound system. The first minima represents the ensembles that correspond to the partial inactive conformation which is similar with that emerged in Apo-WT system. The second minima which is the also the most populated one represents the ensembles subjected to the allosteric effect induced by ATP, which is regarded as the activate-like conformation of ALK with the αC-in and A-loop open conformation (Figure 2E). Compared with the representative structures of Apo-WT, we can suggest that ATP binding may bring the allosteric effect on promoting the connection between the active site and the two activated related elements, αC-helix and A-loop, thus establish a more compact active site in the ALK kinase domain. The allosteric details are mainly reflected in two aspect, one is the e αC-helix approaching to the active site, the other is the 4 Å shift of αAL approaching to the active site. As shown in Figure 6A, the triphosphate moiety of ATP engages in the electrostatic interactions with K1150 who participates in the catalytic salt bridge. The favorable electrostatic attraction anchors the side chain of K1150 orientated to the salt bridge partner E1167, thus protects the stability of the catalytic salt bridge. Another crucial connection is the hydrogen bond formed between the triphosphate moiety of ATP and the αAL arginine R1275. As mentioned above, the positive charged R1275 plays important role in the connection between αC-helix and A-loop by forming hydrogen bond with the αC-helix aspartic acid D1063. Herein, the interactions among ATP, R1275, and D1063 construct a hydrogen bonds chain (ATP-R1275-D1063), which further pulls the αC-helix approaching to the active site.


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In addition to the αC-helix, aMD simulations shows that A-loop undergoes a conformational rearrangement when ATP binds to ALK. In the ATP-bound system, the A-loop closed conformation has no longer been sampled, whereas the A-loop prefer to assume an open conformation where the sharp U-turn at the end of αAL has vanished (Figure 2E). In this open conformation, A-loop is fully extended to make space favor for the peptide substrate binding, which is regarded as a determinant step for the kinase activation.8,

10

This observation also suggested that the outward motion of

A-loop could be coupled to the intrinsic interactional changes in the regions of the active site and the A-loop (Figure 6A and 6B). In the Apo-WT, D1276 perfectly construct the hydrogen bond with the first side chain nitrogen atom of R1284, thereby generates the sharp U-turn of the A-loop, a structural controlling instrument for shaping the closed or semi-closed A-loop conformations (Figure 7A and 7B). By contrast, the αAL moves nearly 4 Å forward due to the pulling of ATP through the hydrogen bond between ATP and R1275 (Figure 6B). Due to spatial mismatch, main chain oxygen atom of D1276 losses the opportunity to make the crucial hydrogen bond with the first side chain nitrogen atom of R1284, which leads to the destruction of constrained U-turn and long-range process of reorientation of the A-loop into an open conformation (Figure 7C). In addition to the disruption of the U-turn, some structural features in favor of the open conformation of A-loop are observed in the ATP-bound system. As shown in Figure 7D, the A-loop distal residues C1288 and K1285 form a hydrogen bond, thus moor the distal residues in a position that no longer blocks the substrate binding site in ATP-bound ALK. Besides, residue M1273



at the initial part of A-loop packs against with P1292 and V1293 to form a hydrophobic stem in ATP-bound ALK (Figure 7D), which is similar to the hydrophobic stem in Apo-WT. Notably, the residues participate in this hydrophobic stem are more departure from the active site than the residues involve in the hydrophobic stem in Apo-WT system. This structural feature has been observed in active RTKs to firming the open A-loop conformation.8 As shown in Figure 7D, P1292 and V1293 participate hydrophobic interactions with L1291, P1298, F1301, and L1339, thereupon render the terminal part of A-loop adjacent to αG helix to consolidate the open conformation. Altogether, the allosteric performance of ATP in ALK is the induction of a more compact active site. This allosteric effect of ATP on the activation of ALK is twofold: one is that it stabilizes the αC-in conformation; the other is that it destabilizes the U-turn structure of A-loop and promote it to an open conformation. CONCLUSIONS Anaplastic Lymphoma Kinase (ALK) has been established as a therapeutic molecular target in neuroblastoma

19, 22

. It has been suggested that alterations in the equilibrium

between its inactive and active conformations are coupled with its oncogenic potential.19 To understand the mechanism of activation, herein, the structural features and conformation dynamics of the different forms of ALK are explored through accelerate MD (aMD) enhanced sampling. Our results indicate that the active state is rarely visited in the wild type ALK (Apo-WT). This phenomenon is supposed to attribute to the low catalytic activity,54


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which is similar with the behavior of EGFR monomer.55, 56 The energy minimum state identified form aMD of Apo-WT is partially inactive which is in similar with the X-ray structure of ALK.3 Additionally, the simulation of Apo-WT also revealed new low-energy state corresponding to the typical receptor tyrosine kinase (RTK) inactive conformation. Regarding to the conformational stabilization in each conformation, the hydrogen bond networks play a crucial role in the construction of the conformational framework, while the hydrophobic interactions further cemented the respective structure. In the case of mutations and ATP-bound ALK, the equilibriums are shifted to more active state to different extents. Substitution of R1275 with glutamine is expected to secure αC-helix assuming a “αC-in” conformation, which is favored for the formation of structural important K-E salt bridge. For Y1278S, the foremost variation is the disruption of the π-stacking hydrophobic box. The substitution of tyrosine with serine releases the terminal of αAL helix, and therefore disrupts the U-turn which is critical to maintain the closed and semi-closed conformation of A-loop, and hence shift the A-loop to an open conformation. For the ATP-bound ALK, the conformation equilibrium is shifted to the active state by the allosteric effects induced by ATP. Interacting with R1275 situated in αAL, ATP promotes a more compact active site which is favoring for the αC-helix assuming the “in” conformation. In addition, binding of ATP may force considerable migration of αAL, which is accommodated in the reorientation of A-loop to an open conformation. The complex changes seen in the conformational transition of the mutants and ATP-bound systems are attributed to both the local alterations in the network of hydrogen bonding



and hydrophobic interactions and to long-range allosteric couplings. Collectively, the aMD simulations present novel ALK conformations which is corresponding to the typical inactive and active state of RTKs, and gave rational explanations for the activation molecular origins induced by R1275Q, Y1275S mutations, as well as by ATP binding. The gained atomistic insight may provide valuable information for further atomistic and mechanistic studies of the oncogenic properties of ALK and as a background in designing more effective inhibitors as potential drugs.


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METHODS Simulation Systems Preparation. The initial structure for native ALK kinase domain was obtained from the Protein Data Bank (PDB), with PDB code 3L9P.3 Based on the WT structure, we generated the starting structures of R1275Q and Y1278S mutants by Discovery studio (DS) (Discovery Studio 4.1, Accelrys, San Diego, CA). The starting point for ATP-bound system was obtained by superposition of a homologous ATP-containing protein kinase (insulin receptor kinase in complex with Mg2+ and ATP; PDB code 3BU5 )57 on the ADP-bound ALK structure (PDB code 3LCT).3 Thus, the ATP molecule coupling with Mg2+ was optimally located in the nucleotide-binding site of ALK. All missing hydrogen atoms were added to ALKs using the Leap program form AMBER16 package.58 Sodium ions (Na+) were added to each system based on a coulomb potential grip in order to keep the whole system neutral. Each system was then solvated with the TIP3P water box model in truncated octahedron box in which protein atom was at least a 10 Å distance away from the nearest edge of the box. Molecular Dynamics Simulation. Four independent conventional MD (cMD) simulations were initially performed for each starting system, wild type apo form ALK (Apo-WT), R1275Q mutant, Y1278S mutant, and bound by ATP (ATP-bound), using AMBER16 software package employed the AMBER ff14SB force field.59 The particle Mesh Ewald (PME) algorithm was applied for long-range electrostatics interactions calculation, and a non-bonded cutoff of 10 Å was used for real space interactions. The SHAKE



algorithm was utilized to restrain all covalent bonds involving hydrogen atoms. The time step was set to 2 fs. Firstly, two steps of minimizations were exerted prior to cMD simulations, including a 5000 steps of the steepest descent (SD) method and a further 5000 steps of conjugate gradient minimization. Secondly, each system was gently heated from 0 to 300 K in 300 ps by Langevin dynamics and then followed by 500 ps of constant pressure equilibration with everything released in NVT conditions. Finally, 50 ns cMD production runs for all four systems were then performed under NPT ensemble condition with periodic boundary conditions. Accelerated Molecular Dynamics. Dual-boost aMD is a good enhanced sampling approach in studying the conformational transition of proteins, which has the capability to facilitate more sampling of the intermediate states and possibly even conversion between inactive and active states.46, 60 In this study, four dual- boost aMD simulations of ALK were initiate from the final structure of the corresponding cMD simulations. In dual-boost aMD, two sets of parameters are need to be specified are Etotal (the inverse strength boost factor for the total potential energy), αtotal (average total potential energy threshold), Edihed (average dihedral energy threshold); αdihed (inverse strength boost factor for the dihedral energy). The input parameters for aMD are as follows:61 Etotal=Etotal_avg + Natoms × 0.16

(1)

αtotal= Natoms × 0.16

(2)

Edihed=Edihed_avg+Nresdues ×4

(3)


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αdihed=Nresdues ×4/5

(4)

where Natoms and Nresdues are the total number of atoms and residues, respectively, Etotal_avg and Edihed_avg are the average total and dihedral energies calculated form the conventional MD simulations, respectively. In all of the four ALK systems, 500 ns aMD simulations were performed after the cMD simulations. Potential of Mean Force Calculation. The potential of mean force (PMF)

62

was used to generate the free energy landscape

(FEL) to study the free energetic change that accompanied the conformational change of ALK. The free energy landscape is calculated from the simulation trajectories using the following equation: ∆G (x, y) = kBT ln g(x, y) Where kB is Boltzmann constant, T is the simulation temperature, and g(x, y) is the normalized joint probability distribution. The minimum energy is set to zero. In this work, we use K1150-E1167 distance and A-loop deviation as the two reaction coordinates. The free energy landscapes were generated using 50 bins in both x and y directions. The free energy landscape includes the entire aMD trajectory data set. Clustering method isperformed to obtain a representative structure of each energy basin. In the most populated cluster of each basin, the central structure, i.e., the structure with smallest distance to all the other members of the cluster, was selected as representative of the basin.



ASSOCIATED CONTENT Supporting Information Figure S1: Comparison of active structure of insulin receptor (A) and inactive structure of insulin receptor (B) and partial inactive structure of ALK (C). Figure S2: Deviation of A-loop in Apo-WT ALK system. Table S1-S2: summary of key hydrogen bonds in partial inactive and typical inactive-like Apo-WT ALK. (PDF)

AUTHOR INFORMATION Corresponding Author [email protected] Notes The authors declare no competing financial interest.

Author Contributions Qing-Chuan Zheng and Hong-Xing Zhang conceived the project. Mu-Yang He and Wei-Kang Li designed and carried out the experiments. All of the authors analyzed the data and discussed the results. The manuscript was written through contributions of all of the authors. All of the authors read and approved the final manuscript.

ACKNOWLEDGMENTS This work is supported by Natural Science Foundation of China (Grant Nos.21273095


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and Grant Nos.21773084).



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For Table of Contents Use Only

Conformational Transition of Key Structural Features Involved

in

Activation

of

ALK

Induced

by

two

Neuroblastoma Mutations and ATP Binding: Insight from Accelerated Molecular Dynamics Simulations

Mu-Yang He,2 Wei-Kang Li,2Qing-Chuan Zheng1,2*Hong-Xing Zhang2*



Figure 1. Comparison of active structure of insulin receptor (A) and inactive structure of insulin receptor (B) and partial inactive structure of ALK (C).Key structural elements are colored in yellow (αC-helix) and purple (A-loop). Conserved residues form the catalytic salt bridge are shown in sticks. (D) ATP and peptide substrate binding site in the kinase domain of ALK. ATP binding site is shown as red mesh, peptide substrate binding site is shown in slate mesh. 165x52mm (300 x 300 DPI)


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Figure 2. Free energy landscape and representative structure of Apo-WT ALK and two mutants and ATPbound ALK. (A) FEL of four system as a function of CV1 and CV2 in Å. (B) Representative structure of ApoWT. (C) Representative structure of R1275Q. (D) Representative structure of Y1278S. (D) Representative structure of ATP-bound ALK. In the representative structures shown below the FEL the αC-helix is colored in yellow, the A-loop is colored in purple. 165x83mm (300 x 300 DPI)



Figure 3. Representative structures of Apo-WT system. Residues involve in conformation stationary interactions are shown in sticks. The αC-helix is colored in yellow and the A-loop is colored in purple. Hydrogen bonds are shown in yellow dash. Residues involve in the hydrophobic stem is colored in orange. Residues involve in the local hydrophobic cluster is colored in cyan. (A) Internal interactions of the partial inactive conformation of Apo-WT. (B) Internal interactions of the typical inactive-like conformation of ApoWT. (C) Hydrophobic cluster around L1291 and P1292 in the typical inactive-like conformation of Apo-WT. (D) Hydrophobic cluster around V1239 in the typical inactive-like conformation of Apo-WT. 165x80mm (300 x 300 DPI)


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Figure 4. K-E salt bridge comparison among the typical Apo-WT ALK and R1275Q mutant. The hydrogen bonds are shown in yellow dash. The salt bridge is shown in red dash. The αC-helix is colored in yellow. (A) Interaction between E1167 and R1275 pulls the side chain of E1167 departure from K1150 in typical ApoWT ALK. (B) Interaction between E1167 and F1269 pulls the side chain of E1167 approaching K1150 in R1275Q. 165x60mm (300 x 300 DPI)



Figure 5. Conformational variation induced by Y1278S. In all of the representative structures, the αC-helix is colored in yellow and the A-loop is colored in purple. (A) The π-stacking hydrophobic cluster in representative structure of Apo-WT system. The residues involving this cluster are shown in sticks and colored in green. (B) Deviation of “RAS” sequence motif in Y1278S. “RAS” residues in typical inactive-like Apo-WT and Y1278S are colored in white and slate, respectively. (C) Interactions between ”RAS“ with the residues in the distal part of A-loop in typical inactive-like Apo-WT. Hydrogen bonds are shown in yellow dash. (D) Shift of the A-loop distal part residues in Y1278S. In all of the representative structures above, the αC-helix is colored in yellow and the A-loop is colored in purple. 82x71mm (300 x 300 DPI)


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Figure 6. ATP induces a more compact active site. In ATP-bound ALK representative structure, the αC-helix is colored in yellow and the A-loop is colored in purple. The representative structure of typical inactive-like Apo-WT are colored in white. Hydrogen bonds are shown in yellow dash. (A) Inward shift of αC-helix in ATPbound system. (B) Migration of the αAL in ATP-bound ALK. 82x36mm (300 x 300 DPI)



Figure 7. Disruption of the U-turn and extension of A-loop in the ATP-bound ALK. A-loop is colored in purple in each structures. Hydrogen bonds are shown in yellow dash. Residues participate in structure stabilization are shown in white sticks. (A) Formation of U-turn in the partial inactive Apo-WT. (B) Formation of U-turn in the typical inactive-like Apo-WT. (C) Disruption of the U-turn in the ATP-bound ALK. (D) Open A-loop conformation of ATP-bound ALK. Residues involve in hydrophobic stem are colored in orange. 82x56mm (300 x 300 DPI)


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Conformational Transition of Key Structural Features Involved in

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