Structural Investigation of Human Prolactin Receptor Transmembrane

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Structural Investigation of Human Prolactin Receptor Transmembrane Domain Homodimerization in a Membrane Environment Through Multiscale Simulations. Huynh Minh Hung, Tran Dieu Hang, and Minh Tho Nguyen J. Phys. Chem. B, Just Accepted Manuscript • Publication Date (Web): 17 May 2019 Downloaded from http://pubs.acs.org on May 17, 2019

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Structural Investigation of Human Prolactin Receptor Transmembrane Domain Homodimerization in a Membrane Environment through Multiscale Simulations Huynh Minh Hung,%,& Tran Dieu Hang,%,& and Minh Tho Nguyen§,#,* §

Computational Chemistry Research Group, Ton Duc Thang University, Ho Chi Minh City,

700000 Vietnam #

Faculty of Applied Sciences, Ton Duc Thang University, Ho Chi Minh City, 700000 Vietnam

%

Department of Chemistry, KU Leuven, Celestijnenlaan 200F, B-3001 Leuven, Belgium.

&

Department of Chemistry, Quy Nhon University, Quy Nhon, Vietnam


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Abstract. It is well established that prolactin (PRL) and its receptor (PRLR) are associated with hundreds of biological functions. They have been postulated to be linked to breast and prostate cancers, and PRLR signaling has attracted considerable medical and pharmaceutical interest in the development of compounds targeting the PRLR. Dimerization of the receptor through its transmembrane (TM) domain is a key step for understanding of its signaling and related issues. Our multiscale simulations results revealed that its TM domain can form dimers in a membrane environment with distinct states stabilized by different residue motifs. Based on the simulated data, an activation mechanism of prolactin with the importance of two symmetrical tryptophan residues was proposed in detail to determine the conformational change of its receptor which is essential for signal transduction. The better knowledge of prolactin receptor structure and its protein-protein interaction can considerably contribute to a further understanding of PRLR signaling action and thereby help to develop some new PRLR signaling-based strategies for prolactin-related diseases.

1. Introduction

Prolactin (PRL) is a pituitary hormone that contributes to the growth and differentiation of the mammary epithelial cells required for lactation.1,2 PRL acts through the prolactin receptor (PRLR) which belongs to the class I cytokine receptor family whose members amount to more than 40.3 These cytokine receptors are key regulators of many biological processes such as lactation, growth, myelopoiesis, erythropoiesis and metabolism. Some important cytokine receptors include the growth hormone receptor (GHR), the prolactin receptor (PRLR), the erythropoietin receptor (EPOR), and the thrombopoietin receptor.3 In addition to the role in lactation and mammary gland development, the PRLR and its primary ligand PRL have been postulated to be linked to breast and prostate cancers.4–6 PRLR signaling has thus attracted considerable medical and pharmaceutical interest in the development of chemical compounds targeting the PRLR.


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The human PRLR is a single-pass transmembrane receptor containing 598 amino acids. It consists of an N-terminal extracellular domain (ECD) for ligand binding, a transmembrane domain (TM), and a C-terminal intracellular domain (ICD) containing two membrane-proximal regions that are conserved among cytokine receptors (Figure 1). The ICD orchestrates downstream signaling which is mediated by associated kinases including Janus kinases (primarily Jak2), Stat5, phosphatidylinositol 3 phosphate kinase/Akt (PI3K/Akt), and the mitogen-activated protein kinase pathway. Structurally, the ECD domain of PRLR was initially characterized in a complex with PRL using both X-ray and solution-state NMR spectroscopies.7 Some years later, Dagil et al. solved the NMR spectrum of the unbound PRLR-ECD domain,8 and more recently the hPRLR-ICD has been characterized by biophysical characterization and NMR data.9 Since then, the transmembrane domain structure and full structural model of the hPRLR have been described by combining NMR data and computational modelling.10 More importantly, the structure of hPRLR-TMD monomer was determined in micelles mimicking a membrane environment in this previous study.10 In addition, the PRLR-TMD was suggested to play an essential role in a dimerization process and acts as signal transducers across the membrane bilayer.10,11 Similar to other cytokine receptors, the hPRLR can form dimerization independently on the ligand binding.11,12 Nevertheless, structures and dynamics of the homodimer of hPRLR-TMD that are the key for understanding signal transfer mechanisms, have not been well characterized yet.


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Figure 1. Structural model of full-length hPRLR, modified from ref.10 Close-up view is visualization of the TM domain in two resolutions, namely all-atom and coarse-grained one. The bottom line is the sequence of the hPRLR-TMD.

A recent study13 proposed a model for signal transmission of GHR, another member of the cytokine receptor family, where its homodimer TMD conformation switches between parallel and left-handed structures. Since the hPRLR belongs to the same family, it can in fact share such a mechanism. However, alanine insertion studies suggested that the activation mechanism of the two receptors GHR and PRLR14,15 may differ from each other, but this is still in a relatively poor understanding. Moreover, the hPRLR does not possess any conventional motifs, i.e. GxxxG or GxxG, that is largely believed to support the homodimer formation as in GHR or other singlepass TM peptides.16–18 In this context, alternative potential motifs need to be proposed for the formation of hPRLR dimer with various structures and consequently, packing configurations of hPRLR-TMD dimer may differ from that of GHR dimer.


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Coarse-grained MD simulations and enhanced sampling methods have been developed to predict the dimer structure and to examine the dynamics of TM peptides in membrane environment, in which the well studied Glycophorin A (GpA) with a common GxxxG motif was mostly used as a representative to develop and to improve computational approaches.19–25 Molecular simulations in both coarse-grained dynamics and atomistic modelling have recently been successfully applied to investigate the homo-dimer and hereto-dimer formations of several single-pass TM peptides by counting a single GxxxG motif20,26,27 or double GxxxG motif28 and particular motifs.29–32 A number of receptors such as the EphA229 and EGFR33 have the ability of switching between different dimeric conformations since they contain multiple motifs that stabilize the corresponding structure. Molecular dynamic simulations thus emerge as an effective computational tool to investigate the inherent conformational changes of TM receptors. In view of the importance of the dimerization process mentioned above, we set out to use the powerful multiscale simulations approach which combines a coarse-grained method with the MARTINI force field34,35 and all-atom simulations with the CHARMM force field, to investigate the conformational structure and dynamics of the hPRLR-TMD homodimer with the aim to understand its signal transmission mechanisms. Through a large replica (hundreds) of CG simulations to ensure sampling, along with proper statistical analyses, we constructed a twodimensional density landscape of dimer conformations of hPRLR-TM in an explicit POPC membrane bilayer. Key motif residues that support and stabilize each conformation found were identified by sampled contacting maps over all the independent CG simulations. Mutant systems were also performed to ensure the role of the identified key residues in the dimerization and the transformation between various configurations of hPRLR. Subsequent to the identification of predominant conformational states via CG simulations, representative structures were then


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converted to all-atom simulations to refine their atomistic structures and further characterize the detailed conformations and interactions of dimeric hPRLR-TM domain. The key finding of our present work is that the hPRLR homodimer exists in multiple structural states stabilized by appropriate motifs of residues, thus suggesting a potential activation mechanism of the hPRLR.

2. Computational details

CG simulations. The initial structure of hPRLR-TM domain (Figure 1) was obtained from NMR structure10 with the PDB ID of 2N7I. The two helix TM domains were placed separately at 5 nm of spatial distance. The two separated helices were then converted to coarse-grained (CG) representations compatible with the MARTINI 2.2 force field35,36 (using martinize.py script). The initial systems of protein with membrane bilayer were generated by performing 200 ns simulations, wherein POPC molecules were self-assembled around the two position-restrained TM domains. The simulation using a self-assembly protocol was previously described.37,38 Each hPRLRTM/POPC system contains slightly different compositions, approximately 350 POPC, 4990 waters and 99 NaCl molecules. NaCl molecules were added at a physiological concentration of 0.15 M and ensure the system charge being neutral. The MARTINI standard water model was used in combination with the MARTINI 2.2 force field for proteins and the MARTINI 2.0 for lipids. A set of one hundred generated systems were simulated independently for 3 s of length time. Each of the performed replicas was different in the starting configuration of POPC molecules around the two TM domains since they were independently self-assembled to enhance the sampling.


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Table 1. Summary of performed simulations. hPRLR-S1, hPRLR-S2, hPRLR-S3 are the systems corresponding to three main states S1, S2, S3 of hPRLR (see more details in the results section) TM domain

Force field

Simulation time

hPRLR-WT

CG, MARTINI

99 x 3µs, 1 x 10µs

hPRLR-A218L

CG, MARTINI

100 x 3µs

hPRLR-A222L

CG, MARTINI

100 x 3µs

hPRLR-A218L+A222L

CG, MARTINI

100 x 3µs

hPRLR-W214V

CG, MARTINI

100 x 3µs

hPRLR-W230V

CG, MARTINI

100 x 3µs

hPRLR- W214V +W230V

CG, MARTINI

100 x 3µs

hPRLR-S1

AA, CHARMM36

3 x 200ns

hPRLR-S2

AA, CHARMM36

3 x 200ns

hPRLR-S3

AA, CHARMM36

3 x 200ns

EphA2

CG, MARTINI

100 x 3µs

CG simulations were conducted using the GROMACS 4.6.5 program39 with semi-isotropic pressure coupling at 1 bar by Berendsen barostat with a time constant of 4.0 ps and a compressibility of 1 × 10-5 bar-1. The temperature was coupled weakly to a heat bath of 300 K, using the Berendsen thermostat with a coupling time of 1.0 ps. The electrostatic interactions were shifted from 0 to 12 Å while the Lennard-Jones potential was shifted to zero between 9 and 12 Å. An integration time step of 20 fs was chosen. Simulations of the mutant protein are carried out making use of the same way as the wild type system. All the performed simulations are summarized in Table 1.


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All-atom simulations. The representative structures relevant to the three main conformational states of hPRLR dimer obtained from CG simulations were converted to all-atom resolution using the BACKWARD algorithm.40 Parameters for the POPC lipid molecule were also built for mapping all-atom representation. Prior to product simulation, each all-atom system was energy minimized and equilibrated with position restraints on the protein backbone atoms. The CHARMM36 force field41 was used for both proteins and lipid to perform the all-atom simulations. The pressure was coupled semi-isotropically to a pressure bath at 1 bar through the ParrinelloRahman barostat.42,43 The temperature was maintained at 300 K by means of the Nose-Hoover thermostat.44,45 Long-range Coulomb interactions were treated making use of the smooth particle mesh Ewald (PME) method,46,47 with the real-space cut off of 1.2 nm. The van der Waals interaction was shifted between 1.0 and 1.2 nm. All bond lengths were constrained by the linear constraint solver LINCS algorithm48 with an integration time step of 2 fs. Each all-atom simulation was carried out for 200 ns using GROMACS, version 5.1.49 Trajectory visualization and analysis. The characteristics of hPRLR-TM conformational homodimer such as crossing angles and inter-helix distances along the simulated trajectories were analysed on the basis of tools implemented within GROMACS and our local scripts conducted on a large number of resulting trajectories. A crossing angle () is defined as an angle formed between two vectors along the helix segment of the TM domain. The simulated distribution landscape was performed by normalizing the two-dimensional histogram employed in MATLAB 2015.50 The intramolecular contacts between residues from CG simulations were calculated from the minimum distance truncated at 0.55 nm. The contact numbers were normalized over the all simulation frames belonging to each dimer state. All visualizations were done with the VMD software.51


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3. Results 3.1. Predominant Right-Handed Conformation of hPR-TMD Dimer in POPC Bilayer.

Although the human prolactin receptor TM domain contains no classical dimerization GxxxG zipper, the SmallxxxSmall (SmxxxSm) motif is present in this receptor TMD. An AxxxA motif or polar residues in the hPRLR-TMD can be a potential factor for homodimerization. Multiscale molecular dynamics were used in this study to investigate the dimerization of hPRLR-TMD. A set of 100 CG simulations of systems where two separated wild type hPRLR-TMDs were placed in a surrounding POPC bilayer, was performed for 3 µs per each simulation. Consequently, a selfhomodimerization was observed in all simulations after at most 1 µs. The structure of the hPRLR-TMD homodimer is characterized in term of a crossing angle (Ω) and an inter-helical distance, dAA. The distribution of conformation of hPR-TMD dimer averaged over all performed CG simulations is illustrated in Figure 2. The angle Ω parameter distinguishes the handedness of the dimer structure wherein this angle is negative for a righthanded conformation and positive for a left-handed conformation. As clearly seen from Figure 2A, the three main states (i.e. S1, S2, S3) of the dimer conformation at different interhelical distances are observed. The S1 and S2 states are right-handed structures sharing the same crossing angle (Ω) (roughly -24o) but have dissimilar distances (dAA) at ~5.0 Å and ~7.2 Å, respectively. The state S3 adopts a left-handed structure with Ω ~ 10o and dAA ~ 11 Å. This left-handed conformation is able to switch to right-handed packing through a long-time scale (10 µs) simulation (Figure 2C). Importantly, the simulated distributions indicate that the right-handed helical structure of hPRLRTMD dimer is predominant and the state S1 is the most favored state (cf. Figures 2A and 2B).


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Figure 2. Homodimer formation of hPRLR-TM. A. Averaged distribution of hPRLR-TM homodimers in POPC membrane projected on crossing angle () and interhelical distance (dAA) between A217 and A217 of the two chain. B. Crossing angle distribution of the homodimer derived from one hundred CG simulations. C. Time evolution of the crossing angle analyzed from a typical extended long-time scale of ten s, and D. Representative snapshots showing the right-handed (RH) and left-handed (LH) conformations. The membrane headgroups are represented as a brown cloud, and water molecules are not shown for clarity.

Our simulated homodimer structures of hPRLR-TMD are consistent with the experimental observation that confirms the ligand-independent dimerization of the hPRLR in carcinoma cells through a significant role of its TM domain.11 Although the dimer structure of hPRLR has not been


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determined yet by NMR or X-ray techniques, lipid bilayers with different components were suggested to mediate to the hPR-TM dimer formation.10 From our simulations, different conformational states of hPRLR-TMD dimer in POPC bilayer are displayed and reflect a distinct interhelical interaction via several potential motifs in its transmembrane domain. We would note that our dimer structures of hPRLR-TMD was discovered in a single POPC lipid bilayer which is the most dominant in the plasma membrane. However, lipid complexity in the biological membrane that contains hundreds of different lipids52 may impact on the dimer conformation, as well as the membrane protein stability and function as explored in the case of amyloid C99 protein,53,54 EGF receptor,55,56 RTKs57 and others.58,59

3.2. Transmembrane interaction interface

To identify the residues that actually respond to the structure of each state found in the conformational distribution landscape, contacting maps between each residue were analysed (Figure 3). In the most predominant conformation (i.e. state S1 in Figure 2A), three residues W124, A218 and A222 in each chain are found to adopt the highest propensity to interact with each other. Importantly, motif A218xxxA222 presents an important role in this conformation. This motif tends to stabilize the dimer structure S1 with a right-handed packing and a smaller inter-helical distance at dAA ~ 0.5 nm. This is not the well-known GxxxG zipper, but it turns out to be a more general SmxxxSm motif. The alternative motif SmxxxSm was also found to be involved in a number of other right-handed dimers receptors.33,60,61 Residue Trp124 also plays a role in stabilizing this conformation when showing a high propensity of intercontact between tryptophans in the two helix TM domains.


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Figure 3. hPRLR-TM contact maps of each pair of residues calculated for three distribution states, namely A: S1, B: S2 and C: S3. The coloured scale on the right describes the relative populations. The protein sequence at the bottom highlights key residues involved in interhelical contacts. Although the S2’s crossing angle, with its lower propensity, is the same as that in the state S1, the distance dAA is dissimilar. This implies another motif in stabilizing this dimer structure. Indeed, the contacting map (Figure 3B) shows that two polar residues S221 and C225 (S221xxxC225) contribute towards the formation of the S2 structure. A218xxxA222 motif interaction becomes less preferred in this case with a longer dAA (see Figures 1A and 2B). Polar contacts related to serine (SxxS motif) have recently been found to stabilize toll-like receptor TL4 dimer.30 In contrast to the right-handed conformations of S1 and S2, neither the A218xxxA222 nor the polar S221xxxC225 motifs show their role in the left-handed structure of the state S3. Instead, residues T212, S216 and particularly W230 adopt high probabilities of inter-helix interaction (Figure 3C) in the dimerization of this conformation. The aromatic residue Trp230 shows the highest contacting propensity, suggesting its importance in this case of left-handed packing. Overall, various residues stabilize each dimer state of hPRLR-TMD wherein the A218xxxA222 is for S1, the S221 and C225 for S2 and the W230 for S3. Mutation of these residues may change the helical packing structure of the dimer.

3.3. Effects of mutation of key residues on homodimer conformation of hPRLR-TMD.

To further confirm the role of the AxxxA motif in the stabilization of the most dominant confirmation of the hPRLR-TM dimer, a set of simulations of the single and double alanine mutation A218L and A222L was performed in POPC bilayer. These simulations show that a substitution of residue A218 and/or A222 strongly impacts the conformation of the dimer. Specifically, the S3 corresponding to the left-handed conformation turns out to be predominant in


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either single- or double-point mutations (see Figure 4). Interestingly, right-handed structures (in S1 and S2) almost disappear in case of mutation on both A218L and A222L, and the state S3 with left-handed packing becomes the most preferred one.

Figure 4. Simulated distributions for three mutant systems A218L, A222L and A128+A222L, derived from 100 replicated simulations of each.

A conformational change also occurs when mutating residue Trp214 to Val, wherein the lefthanded structure becomes predominant (Figure S1, SI file). This suggests that residue W214 somewhat contributes to the stability of the right-handed one but not as strongly as the AxxxA motif. Residue Trp230 shows a significantly high propensity of mutual interaction in the state S3 (Figure 3C), which suggests its central role in forming the left-handed structure of the receptor TM domain. In order to ensure this result, we performed another set of 100 simulations of mutation W230V in the POPC bilayer, and the resulting distribution of the crossing angle is depicted in Figure 5. It is apparent that the probability of left-handed angle (Ω > 0) significantly goes down as compared to the WT and Α218L+ A222L mutation systems. Consequently, the right-handed conformation becomes completely dominant in the mutant W230V system (Figure 5A).


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Interestingly, as seen in Figure 5B, two distinguishable configurations of W230 and W214 appear in the left-handed and right-handed dimer structures. Upon formation of the right-handed structure in the most predominant state (S1), residues W230 in the two helix chains are exposed in two opposite directions without any interaction while those of W214 contact firmly with each other. By contrast, the interacting pattern completely changes in the left-handed structure of S3. The inter-interaction of W230 contributes to a stabilisation of the left-handed dimer structure. In addition, the configuration of A218xxxA222 motif is opposite in both left- and right-handed structures wherein the AxxxA region faces inside the homodimer interface for the former and outside for the latter.


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Figure 5. A. Crossing angle distribution comparing three mutant systems with respect to the WT system where W230V is depicted in red, A218L+ A222L in green, and WT in black with grey shade. B. Representative CG structures of S1 and S3 highlighting the interaction interface of key residues.


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3.4. Comparison with structural dimerization of EphA2

Figure 6. A. Simulated distribution landscape of homodimer EphA2 derived from 100 independent CG simulations. The coloured scale on the right describes the relative population. EphA2 contact maps of each pair of residues calculated for two distribution state, namely (B) EphA2-S1 and (C) EphA2-S2. The protein sequence of EphA2 at the bottom highlights key residues involved in interhelical contacts.


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It is instructive to compare our predicted dimeric structures of hPRLR-TMD with other extensively studied transmembrane proteins. One of the most important TM helix structures is the human tyrosine kinase receptor, EphA2, which can form different dimeric conformations via various motifs. Its left-handed (~ +15o) dimer is stabilized by a heptad repeat motif while the righthanded conformation with the crossing angle of -45o is characterized by a glycine zipper motif .62 Our simulated results of EphA2 using the same multiscale simulation method employed in the study of hPRLR-TM are consistent with previous experimental62 and MD simulations29 results. The two main states with the right-handed and left-handed parallel structures are found from our present simulation study and the latter exhibits predominantly in a POPC bilayer (Figure 6). It should be noticed that the crossing angle of TMDs driven from coarse-grain MD simulations is often smaller than that observed in NMR or atomistic modelling.19 Such a consistent result of EphA2 also supports the multiscale simulation approach to determine the main conformations of dimerization and the motifs responding for each conformational state.

3.5. Structure refinement by atomic simulation

The representative structure of each conformation state (i.e. S1, S2, S3) was back-mapped to atomistic structure and simulated in a POPC bilayer for 200 ns by all-atom MD simulations (three replicas of each). In all the all-atom simulations, the hPRLR-TM dimer is stable keeping the handed packing configuration, even though the crossing angle Ω is somewhat changed due to the different resolutions. The right-handed angle shifted from ~ -25o to ~ -40o whereas the left-handed structure changes from ~10o to ~7o of crossing angle (Figure S2, SI file). These shifts are acceptable due to the low resolutions of CG simulations, and the angle derived from atomistic simulations is closer to that obtained from the experimental NMR structures.19 Additionally, as


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expected, the configurations of key residues W214 and W230, A218 and A222 corresponding to the three states remain unchanged as compared to the CG simulations (Figure S2, SI file).

4. Discussion

Our multiscale simulations reveal three distinct states of the hPRLR TM dimer with two handeddimer packing conformations. The right-handed one consists of two states (S1 and S2) with the same crossing angle but different inter distance dAA. The residue motifs that correspond to the stabilization of each state, are W214xxxA218xxxA222 for the former and SxxxC for the latter. Residue W230 tends to give rise to formation of the left-handed state (S3). Simulated distribution landscape demonstrates that the state with right-handed crossing angle and smaller interhelix distance (S1) is the most predominant one. Within the cytokine family, the PRLR is expected to exert its functions via a similar activation mechanism as that proposed for the GHR. Accordingly, two different handed dimer conformations (LH and RH) should be inactive and active states. The hPRLR-TMD dimer is able to switch between these different conformations. Let us stress that the AxxxA motif plays an essential role in formation and stabilization of the most predominant right-handed state (S1). Substitution of two residues Ala to Leu strongly impacts the conformational state of homodimer of hPR-TMD where the right-handed dimer is almost completely transferred to the counterpart. Thus, both inactive and active states should be switched to each other through the mutation of both A218L and A222L. In addition, we also find that W214 and W230 significantly impact the conformational change of the hPRLR-TMD dimeric structure. Residue W214 is involved in the interaction interface and stabilization of the dimer. Mutation W214V causes the reduction of LH structure probability as compared to the RH one. More importantly, the key amino acid W230 at the interacting interface of the hPRLR-TM dimer shows its essential role in forming the RH conformation. Substitution of


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this residue by Val causes a significant change in the crossing angle distribution where the RH becomes predominant, whereas the LH configuration is nearly dissolved. Mutation of W230V therefore also gives rise to a switch between the active and inactive states of the hPR-TMD dimer. Proposed mechanisms of activation On the basis of the simulated data and discussion presented above, we would now suggest a potential activation mechanism of hPRLR which is illustrated in Figure 7. In this mechanistic model, the RH structure S1 stabilized by motif W214xxxA218xxxA222 acts as the active state since the right-handed crossover conformation increases the distance between C-termini of helixes. PRLR does not possess an intrinsic tyrosine kinase activity but can send a signal through associated cytoplasmic proteins, for example, Janus protein kinase 2 (JAK2). The distant C-termini adapt an interplay between the associated JAK2s, ultimately activating a signal. The growth in the Cterminal distance of two monomers was previously probed to be an active state involved in the JAK2 activation of other member GHR of the class I cytokine receptor family.13 In addition, the RH conformation S1 of hPRLR is found to be the most dominant state among various possible states, and the LH state is able to switch to this conformational state through long-time dynamics (Figure 2B). Our present study suggests that TM polar residues in the core of hPRLR (i.e. S221 and C225) are involved in the dimerization to form a stable conformation but not predominant state (state S2 in Figure 1). A previous mutagenesis study on hPRLR also indicated that C225 partially contributes to the dimerization, and the intermolecular disulfide bond between these residues is not involved in the dimerization. The latter may act as an intermediate state during the transformation between active and inactive states.


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Figure 7. A suggested mechanism of hPRLR activation. Helical structures highlight the key residues W124 and W230 in purple in bond representation, as well as A218 and A222 in orange bond representation.

It should be noted that the right-handed active state of Epidermal growth factor receptor also represents a predominant conformation with the lowest free-energy minimum, as investigated in a recent study through coarse-grained meta-dynamics simulations.33 Our simulated data suggest that the activation of hPRLR can be caused by a rotation of the two helix monomers via two steps with


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the highlight of two symmetrical tryptophans. From the left-handed (near parallel) conformation, wherein residue W214 is exposed in two opposite directions while W230 face and interact with each other, one helix rotates along its axis. This brings about a conformational change to a righthanded state (S2) stabilized by S221xxxC225 residues, and inter-contact does not exist between the tryptophan residues. The other helix, in turn, rotates with respect to its helix axis, ultimately leading to the right-handed conformation or the active state, and the motif W214xxxA218xxxA222 responds to stabilize this conformation as discussed above. We note that the helix rotation may be caused by the binding of prolactin ligand to the extracellular domain of pre-dimerized prolactin receptor. Such a binding of prolactin causes a rotation of the helix TM domain, consequently altering the conformations of TM dimers from the near parallel (left-handed) conformation to the right-handed crossover conformation. The distant C-termini of TM domains adapt to the interactions between associated JAK2s, and ultimately activate a signal (see Figure S3, SI file).63 Our findings also indicate that mutations of A218L and A222L can prevent the activation since the only left-handed state provide the greatest distribution, and the right-handed conformation becomes rarely found. In contrast, the mutation W230V can promote an PRLR activation.

5. Conclusions

Dimerization of human prolactin receptor through its TM domain emerges as a key step for an understanding of its signaling mechanism and related issues. While the dimeric structures of hPRLR were not solved experimentally by NMR or X-ray techniques, our multiscale simulations results revealed that its TM domain can undergo dimerization in a membrane bilayer in three different states, namely two right-handed conformations and one left-handed state. This observation on the dimerization of hPRLR is consistent with previous experimental studies that


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confirmed a ligand-independent dimerization of the hPRLR in cells through an important role of its TM domain.11 Our simulated distribution landscape demonstrated that the state with righthanded crossing angle and smaller interhelix distance (S1) is the most predominant one among the three conformational states. Each state was identified to be stabilized by different motifs, and any mutation on such motifs largely impacts on the dimeric packing conformation of the prolactin receptor. From the simulated results, an activation mechanism of prolactin was proposed in some details to understand the conformational change of its receptor which is essential in emitting a signal. Our better knowledge of prolactin receptor structure and its protein-protein interaction considerably contributes to a further understanding of the PRLR signalling action and help to develop new PRLR signalling-based strategies for prolactin-relating disease such as breast cancer and prostate cancer. Our present approach to investigate the dimer formation and to identify key motifs that support the stabilization of homodimeric conformations, can further be utilized to extend the study of other members of the important class 1 cytokine receptor family or of other single-pass receptors.

ASSOCIATED CONTENT Supporting Information. Figures for distribution landscape of the mutant system W214V, EphA2 distribution landscape, atomistic refinement of PRLR-TMD, inactive and active models of hPRLR. ACKNOWLEDGMENTS The authors are indebted to KU Leuven (GOA program and IRO scholarship) and the Vietnam Ministry of Education and Training for doctoral scholarships (Program 911). We thank Ton Duc Thang University (Demasted) for support.


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AUTHORS INFORMATION Corresponding Author: Email: [email protected] (MTN). Phone: +84-2837755037. ORCID: Minh Tho Nguyen: 0000-0002-3803-0569 Huynh Minh Hung: 0000-0002-4853-5106 Tran Dieu Hang: 0000-0002-1487-0686


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Structural Investigation of Human Prolactin Receptor Transmembrane

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