Article pubs.acs.org/JPCB
Cite This: J. Phys. Chem. B 2019, 123, 4858−4866
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 590000, Vietnam
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‡
S Supporting Information *
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 PRLR. Dimerization of the receptor through its transmembrane (TM) domain is a key step for understanding its signaling and related issues. Our multiscale simulation results revealed that its TM domain can form dimers in a membrane environment with distinct states stabilized by different residue motifs. On the basis of the simulated data, an activation mechanism of PRL 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 PRLR 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 PRL-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), PRLR, erythropoietin receptor, and thrombopoietin receptor.3 In addition to the role in lactation and mammary gland development, 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 PRLR. The human PRLR (hPRLR) is a single-pass transmembrane (TM) receptor containing 598 amino acids. It consists of an Nterminal extracellular domain (ECD) for ligand binding, a TM domain (TMD), 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. © 2019 American Chemical Society
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 CG one. The bottom line is the sequence of hPRLR-TMD.
Structurally, the ECD 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,8 and more recently, hPRLR-ICD has been characterized by biophysical characterization and NMR data.9 Since then, the TMD structure and Received: March 1, 2019 Revised: May 16, 2019 Published: May 17, 2019 4858
DOI: 10.1021/acs.jpcb.9b01986 J. Phys. Chem. B 2019, 123, 4858−4866
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The Journal of Physical Chemistry B
Subsequent to the identification of predominant conformational states via CG simulations, representative structures were then 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 hPRLR.
full structural model of hPRLR have been described by combining the NMR data and computational modeling.10 More importantly, the structure of the hPRLR-TMD monomer was determined in micelles, mimicking a membrane environment in this previous study.10 In addition, 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, hPRLR can form dimerization independently on the ligand binding.11,12 Nevertheless, the structures and dynamics of the homodimer of hPRLR-TMD that are the key for understanding signal-transfer mechanisms have not been well characterized yet. 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 (LH) structures. Because 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 results in a relatively poor understanding. Moreover, hPRLR does not possess any conventional motifs, that is, GxxxG or GxxG, that is largely believed to support the homodimer formation as in GHR or other single-pass 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 the hPRLR-TMD dimer may differ from that of the GHR dimer. Coarse-grained (CG) MD simulations and enhanced sampling methods have been developed to predict the dimer structure and 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 improve computational approaches.19−25 Molecular simulations in both CG dynamics and atomistic modeling have recently been successfully applied to investigate the homodimer and heretodimer formations of several single-pass TM peptides by counting a single GxxxG motif 20,26,27 or double GxxxG motif 28 and particular motifs.29−32 A number of receptors such as EphA229 and EGFR33 have the ability of switching between different dimeric conformations because 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 CG 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 two-dimensional density landscape of dimer conformations of hPRLR-TM in an explicit phosphatidylcholine (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.
2. COMPUTATIONAL DETAILS 2.1. CG Simulations. The initial structure of the hPRLRTM domain (Figure 1) was obtained from the 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 CG representations compatible with the MARTINI 2.2 force field35,36 (using the martinize.py script). The initial systems of protein with membrane bilayer were generated by performing 200 ns simulations, wherein the POPC molecules were selfassembled around the two position-restrained TM domains. The simulation using a self-assembly protocol was previously described.37,38 Each hPRLR-TM/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 ensured 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 μs of length time. Each of the performed replicas was different in the starting configuration of POPC molecules around the two TM domains because they were independently self-assembled to enhance the sampling. 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 LennardJones 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 by making use of the same way as the wild-type system. All the performed simulations are summarized in Table 1. 2.2. All-Atom Simulations. The representative structures relevant to the three main conformational states of hPRLR dimer obtained from CG simulations were converted to allatom 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 allatom 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 Parrinello−Rahman barostat.42,43 The temperature was maintained at 300 K by means of the Nosé−Hoover thermostat.44,45 Long-range Coulomb interactions were treated by making use of the smooth particle mesh Ewald method,46,47 with a real-space cutoff of 1.2 nm. The van der Waals interaction was shifted between 1.0 and 1.2 nm. All bond 4859
DOI: 10.1021/acs.jpcb.9b01986 J. Phys. Chem. B 2019, 123, 4858−4866
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The Journal of Physical Chemistry B Table 1. Summary of Performed Simulationsa TM domain
force field
simulation time
hPRLR-WT hPRLR-A218L hPRLR-A222L hPRLR-A218L + A222L hPRLR-W214V hPRLR-W230V hPRLR-W214V + W230V hPRLR-S1 hPRLR-S2 hPRLR-S3 EphA2
CG, MARTINI CG, MARTINI CG, MARTINI CG, MARTINI CG, MARTINI CG, MARTINI CG, MARTINI AA, CHARMM36 AA, CHARMM36 AA, CHARMM36 CG, MARTINI
99 × 3 μs, 1 × 10 μs 100 × 3 μs 100 × 3 μs 100 × 3 μs 100 × 3 μs 100 × 3 μs 100 × 3 μs 3 × 200 ns 3 × 200 ns 3 × 200 ns 100 × 3 μs
over the all simulation frames belonging to each dimer state. All visualizations were done with the VMD software.51
3. RESULTS 3.1. Predominant Right-Handed Conformation of hPRLR-TMD Dimer in a POPC Bilayer. Although the hPRLR 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 hPRLRTMD 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 hPRLRTMDs 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 terms of a crossing angle (Ω) and an interhelical distance, dAA. The distribution of conformation of hPRLRTMD 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 right-handed (RH) conformation and positive for a LH conformation. As can be clearly seen from Figure 2A, the three main states (i.e., S1, S2, and S3) of the dimer conformation at different interhelical distances are observed. The S1 and S2 states are RH structures sharing the same crossing angle (Ω) (roughly −24°) but have dissimilar distances (dAA) at ∼5.0 and ∼7.2 Å, respectively. State S3 adopts a LH structure with Ω ≈ 10° and dAA ≈ 11 Å. This LH conformation is able to switch to RH packing through a long time scale (10 μs) simulation (Figure 2C). Importantly, the simulated distributions indicate that the RH helical structure of hPRLR-TMD dimer is predominant and state S1 is the most favored state (cf. Figure 2A,B).
a
hPRLR-S1, hPRLR-S2, and hPRLR-S3 are the systems corresponding to three main states S1, S2, and S3 of hPRLR (see more details in the Results section).
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 2.3. Trajectory Visualization and Analysis. The characteristics of the hPRLR-TM conformational homodimer such as crossing angles and interhelix distances along the simulated trajectories were analyzed on the basis of tools implemented within GROMACS and our local scripts conducted on a large number of resulting trajectories. A crossing angle (Ω) 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
Figure 2. Homodimer formation of hPRLR-TM. (A) Averaged distribution of hPRLR-TM homodimers in POPC membrane projected on a crossing angle (Ω) and interhelical distance (dAA) between A217 and A217 of the two chains. (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 10 μs. (D) Representative snapshots showing the RH and LH conformations. The membrane head groups are represented as a brown cloud, and water molecules are not shown for clarity. 4860
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The Journal of Physical Chemistry B Our simulated homodimer structures of hPRLR-TMD are consistent with the experimental observation that confirms the ligand-independent dimerization of hPRLR in carcinoma cells through a significant role of its TM domain.11 Although the dimer structure of hPRLR has not been determined yet by NMR or X-ray techniques, lipid bilayers with different components were suggested to mediate to the hPRLR-TMD dimer formation.10 From our simulations, different conformational states of hPRLR-TMD dimer in the POPC bilayer are displayed and reflect a distinct interhelical interaction via several potential motifs in its TMD. We would note that our dimer structures of hPRLR-TMD were 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 RTKs,57 and others.58,59 3.2. TM 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 analyzed (Figure 3). In the most predominant conformation (i.e., state S1 shown 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 RH packing and a smaller interhelical 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 RH dimer 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. Although the crossing angle of S2, with its lower propensity, is the same as that in 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 toward 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 RH conformations of S1 and S2, neither the A218xxxA222 nor the polar S221xxxC225 motifs show their role in the LH structure of state S3. Instead, residues T212, S216, and particularly W230 adopt high probabilities of interhelix 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 LH packing. Overall, various residues stabilize each dimer state of hPRLR-TMD wherein A218xxxA222 is for S1, S221 and C225 for S2, and 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 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
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 cultured scale on the right describes the relative populations. The protein sequence at the bottom highlights the key residues involved in interhelical contacts.
and A222L was performed in the POPC bilayer. These simulations show that substitution of residue A218 and/or A222 strongly impacts the conformation of the dimer. Specifically, S3 corresponding to the LH conformation turns out to be predominant in either single- or double-point mutations (see Figure 4). Interestingly, RH structures (in S1 and S2) almost disappear in the case of mutation on both A218L and A222L, and state S3 with LH packing becomes the most preferred one. A conformational change also occurs when mutating residue Trp214 to Val, wherein the LH structure becomes predominant (Figure S1, Supporting Information). This suggests that the residue W214 slightly contributes to the stability of the RH one but not as strongly as the AxxxA motif. Residue Trp230 shows a significantly high propensity of mutual interaction in state S3 (Figure 3C), which suggests its central role in forming the LH 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 4861
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Figure 4. Simulated distributions for three mutant systems A218L, A222L, and A128 + A222L, derived from 100 replicated simulations of each.
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 RH and LH 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 CG 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, and S3) was back-mapped to the 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 slightly changed because of the different resolutions. The RH angle shifted from ∼−25° to ∼−40°, whereas the LH structure changes from ∼10° to ∼7° of the crossing angle (Figure S2, Supporting Information). These shifts are acceptable because of 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 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, Supporting Information).
depicted in Figure 5. It is apparent that the probability of LH angle (Ω > 0) significantly goes down as compared to the WT
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 gray shade. (B) Representative CG structures of S1 and S3 highlighting the interaction interface of key residues.
and A218L + A222L mutation systems. Consequently, the RH conformation becomes completely dominant in the mutant W230V system (Figure 5A). Interestingly, as can be seen in Figure 5B, two distinguishable configurations of W230 and W214 appear in the LH and RH dimer structures. Upon formation of the RH 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 LH structure of S3. The interinteraction of W230 contributes to a stabilization of the LH 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. 3.4. Comparison with Structural Dimerization of EphA2. It is instructive to compare our predicted dimeric structures of hPRLR-TMD with other extensively studied TM 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 LH (∼+15°) dimer is stabilized by a heptad repeat motif while the RH conformation with the crossing angle of −45° is
4. DISCUSSION Our multiscale simulations reveal three distinct states of the hPRLR TM dimer with two-handed dimer packing conformations. The RH 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 S221xxxC225 for the latter. Residue W230 tends to give rise to formation of the LH state (S3). Simulated distribution landscape demonstrates that the state with RH crossing angle and smaller interhelix distance (S1) is the most predominant one. Within the cytokine family, 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 RH state (S1). Substitution of two residues 4862
DOI: 10.1021/acs.jpcb.9b01986 J. Phys. Chem. B 2019, 123, 4858−4866
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Figure 6. (A) Simulated distribution landscape of homodimer EphA2 derived from 100 independent CG simulations. The cultured 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 the key residues involved in interhelical contacts.
Ala to Leu strongly impacts the conformational state of homodimer of hPRLR-TMD where the RH 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 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 hPRLR-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 because the RH crossover conformation increases the distance between the 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 C-terminal distance of two monomers was previously probed to be an active state involved in the JAK2 activation of other member GHR of
Figure 7. Suggested mechanism of hPRLR activation. Helical structures highlight the key residues W124 and W230 in purple bond representation, as well as A218 and A222 in orange bond representation.
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 longtime dynamics (Figure 2B). Our present study suggests that the 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 4863
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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.
predominant state (state S2 shown 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. It should be noted that the RH 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 CG metadynamics 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 the highlight of two symmetrical tryptophans. From the LH (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 RH state (S2) stabilized by S221xxxC225 residues, and intercontact does not exist between the tryptophan residues. The other helix, in turn, rotates with respect to its helix axis, ultimately leading to the RH 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 PRL ligand to the ECD of predimerized PRLR. Such a binding of PRL causes a rotation of the helix TM domain, consequently altering the conformations of TM dimers from the near-parallel (LH) conformation to the RH crossover conformation. The distant C-termini of TM domains adapt to the interactions between associated JAK2s and ultimately activate a signal (see Figure S3, Supporting Information).63 Our findings also indicate that mutations of A218L and A222L can prevent the activation since the only LH state provide the greatest distribution, and the RH conformation becomes rarely found. In contrast, the mutation W230V can promote PRLR activation.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.9b01986.
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Distribution landscape of the mutant system W214V, EphA2 distribution landscape, atomistic refinement of PRLR-TMD, and inactive and active models of hPRLR (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone: +842837755037. ORCID
Huynh Minh Hung: 0000-0002-4853-5106 Tran Dieu Hang: 0000-0002-1487-0686 Minh Tho Nguyen: 0000-0002-3803-0569 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors are indebted to KU Leuven (IRO scholarships and PDM fellowships). The authors thank Ton Duc Thang University (Demasted) for support.
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REFERENCES
(1) Chen, W. Y.; Ramamoorthy, P.; Chen, N. Y.; Sticca, R.; Wagner, T. E. A Human Prolactin Antagonist, HPRL-G129R, Inhibits Breast Cancer Cell Proliferation through Induction of Apoptosis. Clin. Cancer Res. 1999, 5, 3583−3593. (2) Clevenger, C. V.; Furth, P. A.; Hankinson, S. E.; Schuler, L. A. The Role of Prolactin in Mammary Carcinoma. Endocr. Rev. 2003, 24, 1−27. (3) Liongue, C.; Ward, A. C. Evolution of Class I Cytokine Receptors. BMC Evol. Biol. 2007, 7, 120−130. (4) Bogorad, R. L.; Courtillot, C.; Mestayer, C.; Bernichtein, S.; Harutyunyan, L.; Jomain, J.-B.; Bachelot, A.; Kuttenn, F.; Kelly, P. A.; Goffin, V.; et al. Identification of a Gain-of-Function Mutation of the Prolactin Receptor in Women with Benign Breast Tumors. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 14533−14538. (5) Tworoger, S. S.; Eliassen, A. H.; Zhang, X.; Qian, J.; Sluss, P. M.; Rosner, B. A.; Hankinson, S. E. A 20-Year Prospective Study of Plasma Prolactin as a Risk Marker of Breast Cancer Development. Cancer Res. 2013, 73, 4810−4819. (6) Wennbo, H.; Gebre-Medhin, M.; Gritli-Linde, A.; Ohlsson, C.; Isaksson, O. G.; Törnell, J. Activation of the Prolactin Receptor but Not the Growth Hormone Receptor Is Important for Induction of Mammary Tumors in Transgenic Mice. J. Clin. Invest. 1997, 100, 2744−2751. (7) Broutin, I.; Jomain, J.-B.; Tallet, E.; van Agthoven, J.; Raynal, B.; Hoos, S.; Kragelund, B. B.; Kelly, P. A.; Ducruix, A.; England, P.; et al. Crystal Structure of an Affinity-Matured Prolactin Complexed to Its Dimerized Receptor Reveals the Topology of Hormone Binding Site 2. J. Biol. Chem. 2010, 285, 8422−8433. (8) Dagil, R.; Knudsen, M. J.; Olsen, J. G.; O’Shea, C.; Franzmann, M.; Goffin, V.; Teilum, K.; Breinholt, J.; Kragelund, B. B. The WSXWS Motif in Cytokine Receptors Is a Molecular Switch Involved
5. CONCLUSIONS Dimerization of hPRLR through its TM domain emerges as a key step for an understanding of its signaling mechanism and related issues. Although the dimeric structures of hPRLR were not solved experimentally by NMR or X-ray techniques, our multiscale simulation results revealed that its TM domain can undergo dimerization in a membrane bilayer in three different states, namely, two RH conformations and one LH state. This observation on the dimerization of hPRLR is consistent with previous experimental studies that confirmed a ligandindependent dimerization of hPRLR in cells through an important role of its TM domain.11 Our simulated distribution landscape demonstrated that the state with RH 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 PRLR. From the simulated results, an activation mechanism of PRL was proposed in some details to understand the conformational change of its receptor, which is essential in emitting a signal. Our better knowledge of PRLR structure and its protein−protein interaction considerably contributes to a further understanding of the PRLR signaling action and help to develop new PRLR signaling-based strategies for PRLrelating disease such as breast cancer and prostate cancer. Our 4864
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