Conformational and binding mode assessment of the human IL‐3 recognition by its alpha receptor

Protein–protein interactions (PPIs) are attractive targets as they are critical in a variety of biological processes and pathologies. As an illustration, the interleukin 3 (IL‐3) and its α subunit receptor (IL‐3Rα) are two proteins belonging to the cytokine or receptor βc family and are involved in several disorders like inflammatory diseases or hematological malignancies. This PPI exhibits a low binding affinity and a complex formed by a mutant form of IL‐3 (superkine) and IL‐3Rα have emerged from the literature, with an increase of the affinity. Therefore, in this study, we performed molecular dynamics simulations and binding energy calculation in order to evaluate protein dynamics and to characterize the main interactions between IL‐3 and IL‐3Rα, considering both wild‐type and mutant. First, in the case of IL‐3Rα/IL‐3 wild‐type complex, IL‐3Rα can adopt three different conformations essentially driven by NTD domain, including the open and closed conformations, previously observed in crystal structures. Additionally, our results reveal a third conformation that has a distinct interaction profile that the others. Interestingly, these conformational changes are attenuated in IL‐3Rα/IL‐3 mutant complex. Then, we highlighted the contribution of different residues which interact principally with IL‐3 or IL‐3Rα conserved region. As for the mutated residue at position 135 of IL‐3, other residues such as IL‐3 E138, IL‐3 D40, IL‐3Rα Y279, IL‐3Rα K235, or IL‐3Rα R277 seem important for a low or a high binding affinity. Altogether these findings yield new information that could be exploited in a drug discovery process.

activation or survival factor. Beyond the immune system, these cellular functions can be extended to other biological systems like the nervous and vascular ones. 4 As a cytokine, IL-3 biological activities are mediated by the presence of its receptor, which is expressed on the target cell surface. The IL-3 receptor comprises two different subunits: IL-3 receptor α (IL-3Rα or CD123) and the so-called common β (βc or CD131) receptor. 5 The presence of two distinct subunits indicated a complex signalization process. In fact, IL-3 firstly binds to IL-3Rα before the recruitment of the βc dimer. These consecutive steps form a multimeric complex, which activate a downstream signalization through different pathways like Janus tyrosine kinase (JAK)-Signal transducers of activated T (STAT) pathway. 6 IL-3 and IL-3Rα are members of the βc cytokine family and receptor cytokine family respectively. 7,8 The βc cytokine family is a subfamily of the type I cytokine glycoprotein family, characterized by four helical bundle, namely α-helix A to D (see Figure 1B). It includes also interleukin-5 (IL-5) and granulocyte-macrophage colony stimulating factor (GM-CSF). The associated βc receptor comprises heterodimeric glycoproteins: an α subunit and the shared βc subunit as described earlier. 5 The α receptor subunit also comprises IL-5Rα or CD125 for F I G U R E 1 IL-3 and IL-3Rα proteins. 3D structures and schematic representation of (A) human IL-3 and (B) human IL-3Rα both taken from PDB ID: 5UV8. (C) A general view of the IL-3Rα/IL-3 interaction and (D) a zoom over the predicted conserved or hot regions represented with dots. These conserved residues are R128 c , T131 c , and F132 c of IL-3, and S203 c , F232 c and N233 c of IL-3Rα (the " c " character designates a conserved residue). Disulfide bridges are represented using spheres. D2, D3, domains 2 and 3; ECD, extracellular domain; ICD, intracellular domain; NTD, N-terminal domain; SP, signal peptide; TM, transmembrane; α1, A, B, C, and D, α helices. *IL-3 mutated position 135. IL-5 and GMRα, CSF2Rα or CD116 for GMCSF. They are transmembrane proteins; their extracellular part is divided into three fibronectin type III (FnIII) domains that formed β-sandwich structures: the Nterminal domain (NTD) followed by the D2 and D3 domains (see Figure 1A). Each domain is made up of β strands (from A to G in Figure 1C). As those βc receptors share a common signal transducing receptor, their biological activities are regulated through a similar mechanism. However, IL-3Rα/IL-3 interaction, the initial step of the signaling process, displays a lower affinity ($100-200 nM) compared to other family complexes (IL-5Rα/IL-5, $1-2 nM, and GMRα/GM-CSF, $ 2-10 nM). 9 Mutagenesis studies have highlighted a superkine from literature which corresponds to the cytokine with a Lysine to Tryptophan mutation (K135W), resulting in an increased binding affinity for IL-3Rα. 10,11 Recently, the IL-3Rα/IL-3 interaction became an important target in therapeutic research since this protein-protein interaction (PPI) is associated to various human disorders. [12][13][14] Indeed, IL-3Rα is overexpressed in hematological malignancies like acute myeloid leukemia (AML) or blastic plasmacytoid dendritic cell neoplasm (BPDCN). 15   amplifies inflammation response in murine sepsis and high plasma levels of IL-3 are related to high mortality in human sepsis. 16 IL-3 promotes activation of basophils that are involved in pathogenesis of allergic diseases. 14,17,18 Given the relevance of IL-3Rα/IL-3 complex as therapeutic target, different studies have been conducted on this system. Crystal structures of those proteins, alone or in a complex form have been resolved. [19][20][21][22] The tridimensional (3D) structure of IL-3Rα with the blocking antibody CSL362, reveals that the receptor has a wrench-like conformation with two possible forms: closed and open ( Figure S1). 19 In the former, the angle between NTD and D2 is about 55 allowing for instance a van der Waals (vdW) interaction between A72 and F199. The open complex has its NTD and D2 domains more distant (angle about 95 ). The 3D structure of IL-3Rα/IL-3 complex shows that the NTD, D2 and D3 domains also have a wrench-like conformation around IL-3 (see Figure 1C). In a preliminary study, we carried out a sequence and structure analysis of IL-3 wild-type (WT) and K135W, IL-3Rα, and others family members through an evolutionary point of view. 23 We pinpointed the high conservation of the cytokine or receptor structural architectures across evolution, using multiple sequence alignments including human and other species, and within the βc family members. Moreover, the IL-3Rα/IL-3 interface has been characterized as a large interface involving the NTD domain in one side, and the D2-D3 domains in the other. Among interfacing residues, only few ones belonging to D2-D3 are evolutionary conserved and can be dispatched into two potential conserved or hot regions (see Figure 1D).
In this study, we pursued the investigation of IL-3Rα/IL-3 interaction focusing on protein dynamics and the understanding of binding affinity, by using molecular dynamics (MD) approaches to go beyond structure and sequence. To achieve our goal, classical MD simulations were performed on the human IL-3Rα/IL-3 complex as well as the unbound human IL-3Rα and IL-3. The impact of the superkine (K135W mutation on IL-3) and therefore, of the binding affinity enhancement, was explored.

| Simulation systems
In this study, we were interested in various systems surrounding IL-3 and its receptor IL-3Rα. Therefore, we evaluated the human IL-3Rα/ IL-3 complex as well as the mutated complex human IL-3Rα/IL-3 K135W. We also considered apo state of these different complexes.
Some refinements were realized for the study based on Uniprot reference sequences (Table S1). First, protein sequences in all crystal structures were renumbered to fit the numbering of the reference sequence. It is important to notice that a different sequence enumeration may be found in literature, since the mutated position 135 might be 116. Engineered mutations found in crystal structure (Table S1) were reverted, thanks to SCWRL tool 26 in PyMOL. 27 An exception was made for the K135W mutation present in the chain I of the human IL-3 Rα/IL-3 K135W complex (PDB ID: 5UWC) as this mutation enhanced receptor binding affinity. 10,11 In order to complete the structure of non-solved residues and fill the gaps in structures, unresolved residues were rebuilt with Modeler 9.17. 28 2.2 | System preparation pK values of ionizable groups were calculated at a pH of 7.4 and hydrogen atoms were added to the different structures using H++ system version 3.2 (http://biophysics.cs.vt.edu/H++). 29 Then, structures were prepared with the solution builder tool of CHARMM-GUI webserver (https://www.charmm-gui.org/). 30 During this preparation, proteins were solvated in a rectangular box of explicit TIP3P water molecules with edges at least 10 Å from the protein. Na + and Clions at 0.15 M were added to neutralize the system.

| MD simulations
Classical MD simulations were carried out on the different systems using GROMACS software version 2019.1 31 with the CHARMM36m force field. 32 Initially, the energy of solvated structures was minimized during 10 000 cycles of steepest descend algorithm that was dismissed when the overall force was lower than 1000 kJ mol À1 nm À1 .
The equilibration stage was performed in different steps. Firstly, the minimized system was heated to 300 K and then equilibrated with a timestep of 2 fs during 100 ps under NVT conditions (constant volume and temperature). Positional restraints of 400 kJ mol À1 nm À1 on backbone and 40 kJ mol À1 nm À1 on side chain atoms were applied. In the following step, the same equilibration step was applied but under NPT conditions (constant pressure and temperature). Then, in four NPT equilibration steps, positional restraints were gradually decreased until they were canceled. The Nosé-Hoover temperature coupling was used to maintain a constant temperature. The pressure was set to 1 bar using a Parrinello-Rahman barostat with a coupling constant of 5 ps and a compressibility of 4.5 e-05 bar À1 . All bonds involving hydrogen atoms were restrained using LINear Constraint Solver (LINCS) algorithm. Long range electrostatic interactions were computed using the Particle-Mesh Ewald (PME) method with a cut-off about 12 Å. At the same time, the vdW energies were smoothly switched to zero between 10 and 12 Å. Trajectories were saved during 1 μs production stage under NPT conditions with a timestep of 2 fs. Three independent production runs with random initial velocities were performed for each system.

| Analysis of MD trajectories
For all systems, trajectories were centered in the periodic box and concatenated to reach a total of 3 μs of simulation (30 000 frames), using standard GROMACS tools. Protein Blocks (PBs) were also used for analysis purpose. PBs are structural elements for the description of local structures of proteins. 34,35 There are 16 independent PBs (from a to p), each assigned to a structure type like central α-helix, terminal α-helix, or coil. The PBxplore tool 36 were used to compute a density map of PBs along protein sequences and the equivalent number of PBs (N eq ) which is a local measurement of deformability or local flexibility. 27 N eq allows a fine description of local protein dynamics as follow: rigid (N eq of 1.0), flexible (N eq of 4.0), high flexible (N eq of 6.0) and disorder (N eq of 8.0 and higher). 37 Principal components analysis (PCA) was performed using MDAnalysis library version 1.0 in Python version 3.4 and trajectory containing 3000 over the initial 30 000 snapshots were projected onto the principal component 1 and 2 (PC1 and PC2).
Contacts displayed during the simulation were analyzed using CONAN tool. 38 Contact maps were generated for all systems with a stride of 1 ns and three different cutoff values have been selected. A main cut-off distance value of 12 Å beyond which any residue pair is ignored. An "inter" cut-off value of 5 Å under which interactions are formed and a "high inter" cut-off of 10 Å that marked the disruption of interactions. k-medoid clustering of the trajectories based on the RMSD between contact maps were also carried out. K135W system representing a closed conformation. In theory, binding free energy of protein-protein interaction can be expressed as follow:

| Binding energy
where the total free energies of the complex as well as the isolated proteins are considered. Each individual entity can have a free energy determined by: and the water probe size of 1.4 Å. 41 The energy contribution of residues to the binding were also estimated including residues that are less than 6 Å between the receptor and the cytokine or superkine. We consider a residue to be important to the interaction if it contributes ≤ À2 kcalmol À1 . One must note that we decide here to not take into account the entropic term of the calculation, since our goal is to extract insights of residues of importance for the interaction. By consequent, our ΔG binding from above is in fact similar to the enthalpic term ΔH.

| Graphic generation
Visualization and graphics were made using VMD version 1.9.4 42

| The structural architecture of IL-3Rα/IL-3
IL-3 and IL-3Rα are proteins that present structural specificities. The cytokine has four principal α helices A to D with an additional one, named α1, between A and B helices. The extracellular part of IL-3Rα is mainly constituted by β-strands arranged into NTD, D2, and D3 domains (see Figure 1). These three domains are separated by hinge regions with loops and a disulfide bond is present between C76 of NTD and C195 of D2. MD simulations were performed on these two apo proteins and their complex form, wild-type (WT) and mutated (K135W) ( Figure S3, Table S1). To evaluate the protein local structure sampled during the simulation, the equivalent number of PBs (N eq ) were employed. N eq described a rigid (N eq of 1.0), flexible (N eq of 4.0), high flexible (N eq of 6.0) and disorder (N eq of 8.0 and higher) local structure. 37 In the case of IL-3 apo, flexible regions were detected in loop between C and D helices (loop C-D) and high flexible to disorder regions were found between A and α1 helices ( Figures 2D and S4).
Regarding IL-3Rα apo, except for the C-terminal, high N eq are not found, although NTD contains the largest number of flexible residues (N eq around 3 or 4-see Figure 2). These observations are also found using the root mean square fluctuation (RMSF) ( Figures 2C   and S4,S5).
The complex formed by IL-3 and IL-3Rα has been crystallized (PDB ID 5UV8). High B-factor values are observed at IL-3Rα and IL-3 protein extremities (see Figure  F I G U R E 3 NTD and D2 contacts on IL-3Rα/IL-3. (A) IL-3Rα structure oriented towards NTD and D2 domains shows the different contact patches named i  Contacts between the loop E-F of NTD (ii) and β-strands F and G of D2 (ii' and iii' respectively), including the disulfide bond between NTD C76 and D2 C195, are more persistent during all the simulation (80%-100%), while others (i-i', i-ii', and ii-i' contacts in Figure 3B) are occasional (20%-40%-see Figure 3). Thus, due to the loss of certain contacts between the two domains, NTD can be near (closed conformation) or distant to D2 (open conformation).
According to simulations, motions of IL-3Rα are not restraint to the opening and closing of the NTD regarding D2. Intriguingly, as seen in Figure Figure 4A. As presumed, the cluster 1 in Figure 4B indicates that the loop E-F of NTD (or patch ii) and β-strand G of D2 (or patch iii') are close while in cluster 0, they are distant. Indeed, the Cα distance between A72 (patch ii of NTD) and F199 (patch iii' of D2), vary along the simulation as the two residues can be at a distance less than 10 Å or higher than 12 Å ( Figure 4C). In open conformations, only the disulfide bond remained as contact between NTD and D2. One can notice that in crystal structure of the IL-3Rα/CSL362 complex, the Cα distances between A72 and F199 are 6.6 Å and 12.9 Å for closed and open forms respectively ( Figure S1). We also found cluster (see cluster 2 in Figure 4B) in which the patch ii of NTD is far away from the patch iii' of D2, except for the disulfide bond, but get closer to another contact patch, i' or the loop C-D of D2. For instance, from 2600 to 3000 ns in Figure 4C, the   (Table S3).

| Molecular basis of IL-3Rα/IL-3 binding
According to energy terms in Table 1 In addition, we have previously highlighted few conserved residues during evolution. These conserved residues, located at the complex interface, are arranged in two distinct regions in IL-3Rα or IL-3 (see Figure 1D). 23 Surprisingly, these conserved residues are not part of the strongest contribution of the binding as seen in Figure 5 as their values scarcely reach a ΔG binding of À2 kcal mol À1 . An exception may T A B L E 1 Binding energies components (in kcal mol À1 ) for the different conformations.

ΔE
The conserved IL-3 residues R128 c , T131 c , F132 c , and I39 c constituted an extended subpocket located in helices A and D. V201, K235, R237, E276, R277, V278, Y279, E280 located in the D2-D3 hinge region and the D3 domain, are potential partners of IL-3 conserved residues (see Figure 6B). IL-3Rα partner residues V201, V278, and Y279 are anchored in the IL-3 subpocket and principally form hydrophobic interactions with F132 c or I39 c . Although a relatively modest contribution to the binding energy, the three residues have a similar contribution in all states: around À2 kcal mol À1 for V201, around À5 kcal mol À1 for V278 and around À2 kcal mol À1 for Y279.
Interestingly, Y279 displays the largest non-polar contribution (vdW interaction and non-polar solvation) of IL-3Rα residues (see Figure S11). The remaining partner residues contributed to the inter- In open state, R255 switches interaction to D41. In closed state, the residue seems to lose IL-3 interaction and is mostly exposed to the solvent.
In summary, different residues could play an important role in the IL-3Rα/IL-3 binding. In all conformations, we detected residues, considered as key residues important to the binding energy of the complex. Evolutionary conserved residues are unlikely to make the greatest energetic contribution, although IL-3Rα residue N233 c appear to be relevant in all states. Per-residue contributions highlight other IL-3Rα residues, K235 and Y279 in all states. Concerning the contribution of cytokine, the importance of E138 belonging to helix D and D40 from helix A is revealed. We can observe also that newly formed salt bridges, exclusively present in the alternative-open form, can explain why the binding energy seems higher for this conformational state.

| Impact of a single mutation in IL-3Rα/IL-3
Several years ago, mutational studies resulted in the development of a "superkine": the IL-3 K135W mutant, with an increased biological and binding activities. 10,11,43 Therefore, in this study, the difference between IL-3Rα/IL-3 wild-type (WT) and IL-3Rα/IL-3 K135W were studied through MD simulations.
Firstly, we identify some conformational differences between complexes involving IL-3 WT and IL-3 K135W. In complex, the cytokine and the superkine did not present major differences in terms of RMSF and N eq and the mutated position is rigid (see Figures 2A and   S5). An exception is made in the IL-3 region between helices A and α1, where the mutation appears to cause slight local flexibilities (N eq between 3 and 4- Figure S5). However, the most noticeable effect concerns the NTD domain of IL-3Rα bound to the mutated IL-3 (noted IL-3Rα "K135W" in the study). The latter still display the most RMSF values among domains, but this mobility is attenuated in IL-3Rα "K135W" comparing to IL-3Rα "WT" (see Figure 2A). The NTD domain of IL-3Rα "K135W" is then more rigid than IL-3Rα apo or IL-3Rα "WT" in our simulations and presents a different behavior related to its D2 domain. Indeed, Cα contacts cluster analysis indicate that NTD and D2 are mainly close during the simulation (about 87% over the trajectory of 3 μs- Figure S10). As seen in Figure 4D, contact patch A72 (i) is mainly close to F199 (patch iii') over simulations. As the result of the mutation, the IL-3Rα/IL-3 K135W complex preferentially adopts a closed conformation.
Secondly, the mutation of the charged lysine into an aromatic residue has considerable impact in the binding. Binding energy computation on trajectories representing closed conformations of IL-3Rα/IL-3 K135W indicate a correlation with literature. Indeed, IL-3Rα/IL-3 K135W has the strongest binding energy (À122 kcal mol À1 ) comparing to IL-3Rα/IL-3 WT regardless of its conformations (see Table 1).  Tables 1 and S3).

An exception is
Finally, the receptor/superkine interaction presents some particularities at a residue level. In our previous study, we have suggested that W135 in the IL-3 K135W is anchored in the IL-3Rα evolutionary conserved region forming a π-stacking network with surrounding residues Y279, F281 (IL-3Rα residues), F132 c , and F56 (IL-3). 23 Indeed, the representative closed conformation of IL-3Rα/IL-3 K135W shows that W135 establishes a parallel π-stacking with Y279, which is also near the IL-3 residue F132 c . Not surprisingly, the binding energy analysis reveals that the K135 position of IL-3 WT does not contribute to the IL-3Rα/IL-3 complex formation in every state (see Figure 5B).
Moreover, W135 is the largest total non-polar contribution for IL-3 F I G U R E 7 IL-3Rα/IL-3 K135W interaction. Binding energy contribution of (A) IL-3Rα "K135W" residues and (B) IL-3 K135W residues, compared with residues of the wild-type (WT) complex in the closed form. Representative structures of the closed state oriented towards (C) IL-3Rα "K135W" conserved region and (D) IL-3 K135W conserved region. For each protein, the conserved region has two types of residues highlighted in lines: (1) Conserved residues are represented in yellow (R128 c , T131 c , F132 c , I39 c for IL-3 K135W and, S203 c , F232 c , and N233 c for IL-3Rα "K135W") and the residues, on the opposite side, interacting with these conserved regions are in stick; (2) The partners of conserved residues from the other side or protein are in green.
residues as well as its partner Y279 for IL-3Rα "K135W" residues ( Figure S12). It is also important to notice that IL-3Rα residues N233 c and E276, which interacts with K135, lost their contributions or became unfavorable to the binding in the case of W135. Comparing to IL-3Rα/IL-3 WT closed form, or others (see Figure 6), we observed a displacement of IL-3 K135W residue E138 for which the lateral chain is not completely anchored in the conserved IL-3Rα "K135W" subpocket (see Figure 7C). Thus, IL-3Rα E138 moves away from the 135 position to form salt bridge with IL-3Rα "K135W" R277 or K255, residues surrounding the subpocket. As for IL-3 WT, E138 of the superkine has a significant impact on the binding and even higher compared to the WT. Its IL-3Rα "K135W" partner residue R277 has an increased contribution while K255 has a decreased one. Hence, this high affinity complex IL-3Rα/IL-3 K135W seems governed principally by IL-3Rα residues Y279, K235 and IL-3 residues E138, D40 as for IL-3Rα/IL-3 WT, even with variable binding contributions. As IL-3 residue W135, IL-3Rα residue R277 also distinguishes the two complexes since it has the strongest IL-3Rα contribution in IL-3Rα/IL-3 K135W complex.

| DISCUSSION
As a PPI complex, the human IL-3Rα/IL-3 has a large interface that can be divided into two distinct sites commonly called site 1a and 1b.
The site 1b is composed of the IL-3 helix α1 and loop C-D on one side and the IL-3Rα NTD domain on the other. According to MD simulations, this NTD domain maintained its β-sandwich structure principally formed by β-strands. However, NTD depicts motions that induce a variation in contacts with D2 and/or IL-3. Hence, the mobility of NTD residues is related to the existence of three different conformations: (1) a closed conformation with various contacts with D2, (2)  In the site 1a, IL-3Rα D2 and D3 domains interact with helices A, D, and loop A-α1 of IL-3. This site has been referred to as the recognition site for the IL-3Rα/IL-3 binding in literature. 9,19 In a previous study, we have detected conserved regions in both receptor and cytokine that could play a role in the binding. 23 However, these conserved regions are not among the most energetical contributors to the interaction. They formed shallow binding clefts or sub-pockets in which key residues or hot spots that seem important to the binding are anchored. Considering IL-3Rα interface, the interaction is centered at the IL-3 α-helix D and taking the residue at position 132 as a starting point (i th ), the residue positions i + 3rd and i + 7th, altogether are in the same face of the α-helix and projected into the IL-3Rα conserved subpocket. Those α-helix positions are commonly involved in a specific and/or selective binding in PPIs. 45 The only hydrophobic residue corresponds to the i th position that is also conserved across evolution (F132 c ). 23 At i + 3rd and i + 7th positions are respectively located K135 and N139, two hydrophilic residues. Moreover, a supplementary position (i + 6th) has one of the most important residues in terms of binding energy contribution, E138. In IL-3, E138 side chain, in the vicinity of K135, is also projected into the subpocket. Remarkably, a hydrophobic residue is inserted at the i + 3rd position (135) of the superkine, resulting in a reorientation of E138 main chain which is no longer located at the same face than the others (i th , i + 3rd, and i + 7th). Coming from another IL-3 α-helix (A), D40 seems to have an important energetical contribution but this residue is not anchored into the conserved cleft and its contribution is reduced due to the mutation. Mutagenesis studies have revealed the importance of F132 c and D40 as essential to the binding association. 11,46 Nevertheless, the most impressive role concerns the residue K135 or W135 (in the case of low or high affinity for the receptor respectively). The K135W mutation facilitates binding association and reduces the dissociation. 46  mutation has no effect on the binding, 19 albeit the mutation into another hydrophobic residue, alanine, may explain this finding. K235, the greatest IL-3Rα polar contribution in the low affinity complex, became somewhat less relevant in the high affinity complex since the contribution is dispatched to R277 residue.
As reported in Figure 8, our results provided an interpretation for the binding mechanisms of the cytokine and the superkine. Several years ago, differences between cytokine and superkine binding mechanism have been attributed to their binding kinetics which assigned a slow association and fast dissociation rates to IL-3 WT whereas IL-3 K135W has the reverse. 46 Hence, the cytokine could rapidly leave the α subunit receptor and it is difficult to precise in which conformation at this point, though open conformation is less advantageous to the binding than the others in terms of binding free energies. Otherwise, the interaction with the superkine leads to the formation of a stable closed conformation with a suitable presentation of key residues at the interface and increased vdW interactions. In that case, the NTD domain clamps the superkine and reduced its release. Finally, both binary complexes are able to recruit the βc receptor and the signalization process. Our proposed hypothesis is of great importance regarding the therapeutic interest of this system. This biological mechanism, especially conformational changes derived from our sampling could be more explore using enhanced simulations techniques to confirm these conformational states and determine their transition pathways and characterize more deeply the energetic landscape and the molecular mechanisms associated to the complex formation. In addition, our findings could be relevant for any drug design approaches targeting the IL-3Rα/IL-3 axe since it can use multiple receptor conformations or potential hotspots for hit identification to lead optimization. highlighted different key residues that could play a role in the low or high binding affinity. Surprisingly, residues conserved across evolution are generally excluded from these key residues but constituted binding clefts in which lateral chains of key residues are anchored.

| CONCLUSION
Regarding the importance of IL-3 and IL-3Rα in therapeutic research, these structural outcomes could represent a promising starting point for the PPI modulation.

AUTHOR CONTRIBUTIONS
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
F I G U R E 8 A hypothesis for the IL-3Rα/IL-3 binding mechanism considering wild-type (yellow) and mutant (green). Dashed arrows indicate the uncertainty about the direction of changes.