Agonistic Molecules for

Oct 21, 2014 - The 16 Eph receptors constitute the largest family of receptor tyrosine kinases, and their interactions with 9 ephrin ligands initiate ...
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Dynamic Principle for Designing Antagonistic/Agonistic Molecules for EphA4 Receptor, the Only Known ALS Modifier Haina Qin,† Liang-Zhong Lim,† and Jianxing Song* Department of Biological Sciences, Faculty of Science, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore S Supporting Information *

ABSTRACT: Additional to involvement in diverse physiological and pathological processes such as axon regeneration, synaptic plasticity, and cancers, EphA4 receptor has been recently identified as the only amyotrophic lateral sclerosis (ALS) modifier. Previously, we found that two small molecules bind the same EphA4 channel at almost equivalent affinities but mysteriously trigger opposite signaling outputs: one activated but another inhibited. Here, we determined the solution structure of the 181residue EphA4 LBD, which represents the first for 16 Eph receptors. Further NMR dynamic studies deciphered that the agonistic and antagonistic effects of two small molecules are dynamically driven, which are achieved by oppositely modulating EphA4 dynamics. Consequently, in design of drugs to target EphA4, the dynamic requirement also needs to be satisfied in addition to the classic criteria. For example, to increase the survival of ALS patients by inhibiting EphA4, the drugs must enhance, or at least not suppress, the EphA4 dynamics.

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via circular dichromism (CD), NMR, and isothermal titration calorimetry (ITC).9 Very recently, C1 has been demonstrated to rescue ALS disease phenotypes in the zebrafish model by inhibiting EphA4.2,3 On the other hand, using CD, NMR, and ITC, we have revealed that doxazosin could also bind the same channel of the EphA4 LBD as C1 at almost equivalent affinities (Kd values of 20.44 ± 2.12 and 12.39 ± 0.65 μM for C1 and doxazosin, respectively) and again induced no significant change of secondary and tertiary structures.10 Unexpectedly, doxazosin acted to agonize, rather than antagonize, EphA4. So, a mechanistic mystery arises as how two small molecules achieve opposite signaling effects by binding the same channel with almost equivalent affinities. Recently, protein dynamics have been recognized to play key roles in various biological functions.11−15 Here, we decided to resolve the mystery by examining the dynamic effects of doxazosin and C1 on the EphA4 LBD using NMR spectroscopy. To achieve this, we first determined the NMR structure of the 181-residue EphA4 LBD, which represents the first solution structure for Eph receptors and thus allowed the visualization of the loop confirmations previously invisible in crystal structures. As seen in Figure 1A, the EphA4 LBD has a well-defined β-barrel core, with a backbone RMS deviation of only 0.68 Å for the 10 selected structures (Supporting Information Table S1), while the loops show dramatic variations in different structures (Figure 1B). Previously, the ligand binding domains of Eph receptors including EphA4 have been characterized to adopt two different forms: namely open

he 16 Eph receptors constitute the largest family of receptor tyrosine kinases, and their interactions with 9 ephrin ligands initiate signaling networks controlling many physiological and pathological processes.1 EphA4 receptor not only plays key roles in inhibition of the regeneration of injured axons, synaptic plasticity, platelet aggregation, and cancers, but it was recently identified to be the only disease modifier of amyotrophic lateral sclerosis (ALS) in both animal models and in humans.2,3 EphA4 thus represents a promising drug target for treating these human diseases, particularly for ALS. The Eph receptors are membrane-anchored and their ectodomains have the same modular structure, consisting of a unique N-terminal ligand-binding domain (LBD) followed by a cysteine-rich domain (CRD), composed of sushi and epidermal growth factor (EGF)-like domains, as well as two fibronectin (FN) type III repeats. The LBD has ∼180 residues adopting a jellyroll β-sandwich architecture, which has been shown to be sufficient for binding to ephrins and an antagnostic MSP domain.4−6 The formation of a complex between an Eph receptor and an ephrin is driven by the insertion of the ephrin G−H loop into the hydrophobic channel of Eph receptor and therefore, discovery and design of small molecules targeting this ephrin binding channel represents a promising strategy to manipulate Eph-mediated signaling events. The small molecules can serve as functional probes and as potential agents to treat a variety of human diseases.1−3,6−10 Indeed, previously, two small molecules 4- and 5-(2,5 dimethyl-pyrrol-1-yl)-2-hydroxybenzoic acids, called C1 and C2, were identified to antagonize the EphA4-mediated signaling,8 and we have showed that they acted by occupying the ligand-binding channel of the EphA4 LBD without significantly perturbing its secondary and tertiary structures © XXXX American Chemical Society

Received: May 26, 2014 Accepted: October 21, 2014

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Figure S1C) as well as the characteristic NOEs (Supporting Information Figure S1D and E). Furthermore, the D−E and J− K loops invisible in unbound crystal structures have been found to have close contacts in the NMR structure, as evident from the presence of several long-range NOEs between them: Met32-NH with Ile131-HD; Met32-HB with Asp133-HA; Met32-HB with Asp133-HA; and Ile131-HG with Ile31-HD. We subsequently acquired the 15N backbone relaxation data of the EphA4 LBD in the free state, in complex with doxazosin or C1 (Supporting Information Figure S2), which were further analyzed using “model-free” formalism by DYNAMICS.18,19 For the free EphA4 LBD, only data of 126 out of 175 nonproline residues were analyzed because of missing HSQC peaks or poor-quality of the relaxation data for some residues that result from the relatively large protein size, presence of many exposed loop residues, or existence of the μs−ms conformational exchanges. The squared generalized order parameters, S2, reflect the ps−ns backbone rigidity, with values ranging from zero for isotropic internal motions to unity for completely restricted motion.15,18−20 The free EphA4 residues have an average S2 of 0.86, with many β-barrel residues even having S2 > 0.95 (Figure 1C and D), indicating that the β-barrel backbones are very rigid. In contrast, residues in loops, as well as in the characteristic short β-sheet, have S2 < 0.80, suggesting that their backbones are less rigid, consistent with the solution structure (Figure 1A and B). Model-free analysis also outputs Rex, which reflects the μs−ms conformational exchanges. Remarkably, as seen in Figure 2C, 74 out of 126 fitted residues have Rex > 2 Hz and 15 residues have Rex > 6 Hz, which are distributed over the whole molecule. To confirm this, we further acquired CPMG-based relaxation dispersion data and indeed 46 residues over the whole molecule show significant responses, with ΔR2(τcp) > 1.5 Hz (Figure 1E and F), suggesting that the whole EphA4 LBD indeed undergoes significant μs−ms conformational exchanges even in the free state. In particular, the residues on the short β-sheet comprised of Phe126-Thr127Q128 and Met136-Lys137-Leu138 have large ΔR2(τcp) values, indicating that they have considerable conformational exchanges on the μs−ms time scale. This result thus provides a dynamic basis for the previous observation that even in the free state, the EphA4 LBD could adopt different conformations over the J−K loop in crystal structures.9,17 Moreover, significant μs− ms conformational exchanges are also observed on Ser101 in the H−I loop, which is responsible for the dimerization upon being activated by binding to ephrin ligands. Interestingly, the H−I loop orientation in the NMR structure has a significant difference from that in the crystal structure (Supporting Information Figure S1A). This implies that the H−I loop may undergo an exchange between two conformations in solution. The residues with the μs−ms conformational exchanges detected by the relaxation dispersion experiments are less than those indicated by Rex, which has been generally observed on other proteins.15,18−20 So why does the free EphA4 LBD undergo μs−ms conformational exchanges? Recently, we determined the crystal structure of the free EphA5 LBD.15 Intriguingly, despite having 87% sequence homology to EphA4, the free EphA5 LBD adopts an open conformation (Figure 2A). We have subsequently characterized its backbone dynamics by analyzing 15 N backbone relaxation and CPMG-based dispersion data collected under the same conditions as EphA4 here.15 Interestingly, although EphA4 and EphA5 have very similar overall rotational diffusion time τc, they show very different

Figure 1. NMR structures and dynamics of the EphA4 LBD in the free state. (A) Superimposition of 10 selected NMR structures of the EphA4 LBD in ribbon with β-strands in yellow, helices in red, and loops in green. (B) Superimposition of 10 selected NMR structures in sausage with β-strands in cyan and the rest in red. (C) Generalized squared order parameter (S2) of the EphA4 LBD in the free state. Red indicates residues with S2 < 0.8 (average value − STD). (D) NMR structure with residues having S2 < 0.8 colored in red. (E) ΔR2(τcp), difference of effective transverse relaxation rate R2(τcp) at 80 and 960 Hz, with those >3.0 Hz colored in red. (F) NMR structure with residues having ΔR2(τcp) > 1.5 Hz colored in purple and >3.0 Hz displayed as red spheres.

and closed forms mainly based on the conformations of the high-affinity ephrin-binding channel.4,9,16,17 Most free Eph LBDs assume a closed conformation with the D−E loop close to the J−K loop, which is characteristic of the presence of a short β-sheet within the J−K loop (Supporting Information Figure S1A). Upon forming the complex, in order to accommodate the ephrin-B2 G−H loop, the Eph LBDs transform into an open form, in which the D−E loop moves away from the J−K loop and the characteristic β-sheet becomes an unstructured loop (Supporting Information Figure S1B). Comparison of the current NMR structure with previous crystal structures of the EphA4 LBD in the free-state9 and in complex with ephrin-B216 reveals that in solution the EphA4 LBD assumes a closed form (Supporting Information Figure S1A and B). In the NMR structure, the presence of the characteristic short β-sheet is clearly evidenced by the negative values of the differences of Cα and Cβ secondary chemical shifts (observed − random coil) (Supporting Information B

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Figure 2. Structures and dynamics of the EphA4 and EphA5. (A) Superimposition of the EphA4 NMR (red) and EphA5 crystal (blue) structures. (B) Generalized squared order parameter (S2) of the EphA4 (red) and EphA5 (blue) LBDs in the free states. (C) Residue-specific Rex of the EphA4 (red) and EphA5 (blue) LBDs. Surface representations of the high-affinity ephrin-binding channels of EphA4 (D) and EphA5 (E). The ephrinbinding channels of EphA4 in complex with C1 (F) and doxazosin (G), we previously constructed based on NMR binding results (refs 9 and 10).

protected in the H−D exchange experiments.17 In contrast, the free EphA5 LBD has a completely open channel, which appears to be accessible to water molecules (Figure 2E), as evident from the lacking of protected amide protons in H−D exchange results with EphA5.15 Previously, it has been elegantly demonstrated that generation of a buried cavity by mutating T4 lysozyme provoked a global μs−ms conformational exchanges.19 Therefore, here, we propose that, analogous to the cavity mutant of T4 lysozyme,19 the existence of a relatively buried cavity in the EphA4 ephrin-binding channel may be largely responsible for its global μs−ms dynamics. The significant difference in the channel may also account for the

hydrodynamic properties (Supporting Information Table S2). Furthermore, model-free analysis revealed that most EphA5 residues have much smaller S2 (with an average S2 of 0.73) than those of EphA4 (Figure 2B). On the other hand, only several EphA5 residues have Rex (Figure 2C), thus implying that the EphA5 is lacking of μs−ms conformational exchanges. This result was confirmed by the absence of the CPMG-based dispersions for almost all EphA5 residues.15 Examination of the ephrin-binding channels deciphers that due to the close contact between the D−E and J−K loops, the free EphA4 LBD has a close channel with a cavity inside (Figure 2D), whose amide protons were found to be largely C

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Figure 3. μs−ms backbone dynamics of the EphA4 LBD triggered by binding C1. (A) The EphA4 LBD structure in which spheres are used to indicate residues with significant increases in relaxation dispersions upon binding C1. (B−J) Relaxation dispersion data (dots) of the C1-bound EphA4 LBD at both 500 and 800 MHz and their fitting curves (lines).

(Figures 3 and 4A). For example, the residues on G−H and H−I loops, as well as C-tail which have no direct contact with C1, also display large increases in μs−ms conformational exchanges. To gain a quantitative insight into C1-induced μs− ms conformational exchanges, we fitted the CPMG relaxation dispersion data of good quality at both 800 and 500 MHz by assuming a two-state exchanges,21 which include those of the residues on D, E, K, M β-strands; as well as J−K loop and C-tail (Figure 3 and Supporting Information Table S3). The results reveal that these residues undergo conformational exchanges from a major (>95%) to a minor conformations with Kex of ∼2000 Hz, except for the C-tail residues which exchange much faster, with Kex > 9000 Hz (Supporting Information Table S3). We also calculated the scaling factor α based on the static magnetic field dependence of Rex,22 and the results revealed that the exchanges of residues Met32 on the D-strand and Leu138 on the K-strand with α < 1 are in slow exchange regime, while the residues Val29, Ile31, Arg40, Ser101, Ile109, Gly132, N139, and Val169, with 1 < α < 1.5 are in intermediate exchange. The rest are within fast exchange regime (Supporting Information Table S3). Interestingly, residues Ser101 and Ile109 in the H−I loop within intermediate exchange, which is far away from the ligand binding channel but has very different orientations in the NMR and crystal structures (Supporting Information Figure S1A), have large differences in 15N chemical shifts between two states (Supporting Information Table S3). Furthermore, the C-tail residues Leu178 and Thr179, which are

different hydrodynamic properties for the free EphA4 and EphA5 LBDs (Supporting Information Table S2). Indeed, the binding of C1 and doxazosin to the channel significantly altered the hydrodynamic properties of the EphA4 LBD (Supporting Information Table S2). Because in the free EphA5 LBD the channel is completely open and accessible to water molecules, its backbone rigidity is lower than that of the EphA4 LBD (Figure 2B), probably due to having a larger water accessibility surface. Previously, using model-free analysis, we have demonstrated that upon binding doxazosin, a large number of residues had increased S2 but reduced Rex, which indicates an overall rigidification of the EphA4 LBD on both ps−ns and μs−ms time scales.10 Here, we performed model-free analysis of the relaxation data for the EphA4 LBD in complex with C1. Interestingly, upon binding C1, only 13 residues have increased S2, but 29 residues have reduces S2 (Supporting Information Figure S3A), implying that the EphA4 LBD might have slightly increased ps−ns backbone motions. Most strikingly, upon binding C1, most residues have significantly increased Rex (Supporting Information Figure S3B), implying a dramatic increase in μs−ms conformational exchanges over the whole EphA4 LBD. We thus collected the CPMG relaxation dispersion data for the EphA4 LBD in complex with C1 or doxazosin. Upon binding C1, residues over several regions have large increases of responses, which are not limited to the channel residues D

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conformations for the residues that have no direct contact with C1. In contrast, upon binding doxazosin, many EphA4 residues have reductions of the CPMG responses, in particular for Ser101 (Figure 4A), implying that the doxazosin binding suppressed μs−ms conformational exchanges, consistent with our previous results on the EphA4-Dox complex using modelfree analysis.10 So why does doxazosin-binding reduce but C1binding enhance μs−ms backbone dynamics of the EphA4 LBD? As protein dynamics have been recently revealed to be modulated by a global correlation network,6,11−24 the answer to this question might not be straightforward. As shown in Figure 2F, doxazosin appears to fill/cap the cavity by establishing extensive contacts with the channel-forming residues, which include not only those on the D−E and J−K loops, but also Val72-Cys73-Asn74, Thr104-Leu105-Arg106, and Ile192Ala193 on the convex β-strands, as we previously identified using NMR.10 As such, the proper filling/capping of the cavity may act equivalently to remove the cavity, thus significantly suppressing the global μs−ms conformational exchanges of the EphA4 LBD. In contrast, upon binding, C1 appears to fail to properly fill/cap the channel cavity (Figure 2G), because it could only bind a small portion of the channel residues from the D−E loop, E-strand, and J−K loop but not from the convex β-strands as we previously characterized using NMR.9 As such, the existence of the remaining cavity might provoke large conformational fluctuations as the cavity size is also critical for the global dynamics.19 On the other hand, C1 and doxazosin appear to bind the EphA4 LBD in very different modes as judged from their ITC profiles.9,10 Interestingly, the C1-binding is exothermic while the doxazosin-binding is endothermic. In other words, the doxazosin-binding is highly entropy-driven. The entropy increase upon binding doxazosin might at least partly result from releasing the water molecules on the surface or within the cavity of the EphA4 channel. However, it is extremely challenging to define how the perturbation leads to the dynamic allostery in the context of the correlation motion network. For example, using correlation analysis of the molecular dynamics simulation data, we recently deciphered that the SARS 3C-like protease has a global correlation network essential for its catalytic function. Remarkably, a mutation N214A on the extra domain is able to globally decuple the network, thus inactivating the catalysis.14,24 In contrast, another three-residue mutation enhances the catalysis by only slightly altering the correlation pattern.24 Thus, it is also possible that slight differences in binding/unbinding of C1 and doxazosin are sufficient to trigger the opposite dynamic effects. These mechanisms are not necessarily mutually exclusive and may act simultaneously. It has been well established that Eph receptors get activated through the dimerization/clustering, and the structural mechanism has been recently decoded for EphA4.4,25,26 Briefly, upon binding ephrin, two EphA4 ectodomain molecules become dimerized through two relatively small regions: one on the EphA4 LBD and another on the sushi domain. Amazingly, the LBD residues for dimerization are mostly located on H−I loop, H- and J-strands, which have no direct contact with ephrin. As such, EphA4 dimerization must be initiated by alteration of conformations and dynamics of the dimerization residues upon binding ephrin. In this context, the opposite dynamic effects onto the EphA4 LBD by C1 and doxazosin rationalize why C1 acts as an antagonist but

Figure 4. Mechanisms by which C1 and doxazosin trigger opposite signaling outputs. (A) Difference or ΔR2(τcp) (at 80 and 960 Hz) between the EphA4 LBD in complex with C1 (blue) or doxazosin (red) and the free state. (B) Schematic representation of the membrane-anchored EphA4 receptor: LBD, the ligand binding domain; CRD, cysteine-rich domain, consisting of sushi and EGFlike domains; and FN, fibronectin domains. (C) Upon binding doxazosin, μs−ms dynamics of the EphA4 LBD and sushi domain are dramatically reduced, which triggers the ectodomains dimerization mainly over regions of the LBD and sushi domain, followed by further clustering. As such, the EphA4-mediated signaling will be activated even without the ephrin-binding. (D) In contrast, upon binding C1, μs−ms dynamics of the LBD and sushi domain significantly increased, thus disfavoring the dimerization. Furthermore, as the ligand binding channel is occupied by C1, the binding of ephrin to the EphA4 LBD is physically inhibited. Together, the EphA4 receptor remains inactivated.

unstructured in NMR structure and also far away from the channel, also undergo fast exchanges. These results imply that the C1-binding is able to trigger the exchanges of very different E

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doxazosin as an agonist. Briefly, as the EphA4 dimerization is expected to also occur on μs−ms time scale, in the free state, its dimerization is mostly suppressed (Figure 4B), due to the presence of the global μs−ms conformational exchanges. However, upon binding doxazosin, the global μs−ms conformational exchanges are suppressed, thus allowing the dimerization/clustering to occur. Consequently, EphA4 is activated (Figure 4C). In contrast, upon binding C1, the global μs−ms conformational exchanges were radically provoked over the majority of residues including those having no contact with C1 but involved in dimerization, thus disfavoring the dimerization/ clustering. Furthermore, the occupancy of the channel by C1 also physically reduces the ephrin binding. Taken together, EphA4 remains inactivated (Figure 4D). Interestingly, previous NMR studies indicated that NMR resonances of the EphA4 LBD were also significantly broadened by binding to other EphA4 antagonists, including other small molecules,9,27 peptides,28 and even the 125-residue MSP domain, a wellfolded endogenous antagonist.6 This implies that these antagonists might also be able to provoke significant μs−ms dynamics of EphA4 but further NMR dynamic investigations are certainly needed. In conclusion, our present study reveals the following: (1) Protein dynamics play a key role in the EphA4 activation by modulating its dimerization/clustering. (2) Small molecules do exist to impose agonistic and antagonistic effects onto an Eph receptor by oppositely modulating its dynamics. This opens up an avenue for the “dynamically-driven” drug design. (3) It appears that only the dynamic effects can distinguish whether a small molecule is agonistic or antagonistic to EphA4. Therefore, additional to the classic criteria, the dynamic requirement needs to be absolutely satisfied in design of drugs to target EphA4. For example, to increase the survival of ALS patients by inhibiting EphA4, the drugs must enhance, or at least not suppress, the EphA4 dynamics. Furthermore, the dynamically driven signaling might also be utilized by other receptors via dimerization/clustering.

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The authors declare no competing financial interest.

ACKNOWLEDGMENTS

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REFERENCES

This study is supported by Ministry of Education of Singapore (MOE) Tier 2 Grant 2011-T2-1-096 (R154-000-525-112) and National Medical Research Council (NMRC) grant R154-000454-213 to Jianxing Song. We thank Dr. Jingsong Fan for the assistance of collecting and processing NMR data.

(1) Pasquale, E. B. (2008) Eph-ephrin bidirectional signaling in physiology and disease. Cell 133, 38−52. (2) Van Hoecke, A., Schoonaet, L., Lemmens, R., Timmers, M., Staats, K. A., Laird, A. S., Peeters, E., Philips, T., Goris, A., Dubois, B., Andersen, P. M., Al-Chalabi, A., Thijs, V., Turnley, A. M., van Vught, P. W., Veldink, J. H., Hardiman, O., Van Den Bosch, L., GonzalezPerez, P., Van Damme, P., Brown, R. H., Jr, van den Berg, L. H., and Robberecht, W. (2012) EPHA4 is a disease modifier of amyotrophic lateral sclerosis in animal models and in humans. Nat. Med. 18, 1418− 1422. (3) Faruqi, M. (2012) EPHA4 inhibition rescues neurodegeneration in ALS. Nat. Rev. Drug Discovery 11, 747. (4) Nikolov, D. B., Xu, K., and Himanen, J. P. (2013) Eph/ephrin recognition and the role of Eph/ephrin clusters in signaling initiation. Biochim. Biophys. Acta 1834, 2160−2165. (5) Tsuda, H., Han, S. M., Yang, Y., Tong, C., Lin, Y. Q., Mohan, K., Haueter, C., Zoghbi, A., Harati, Y., Kwan, J., Miller, M. A., and Bellen, H. J. (2008) The amyotrophic lateral sclerosis 8 protein VAPB is cleaved, secreted, and acts as a ligand for Eph receptors. Cell 133, 963−977. (6) Lua, S., Qin, H., Lim, L., Shi, J., Gupta, G., and Song, J. (2011) Structural, stability, dynamic, and binding properties of the ALScausing T46I mutant of the hVAPB MSP domain as revealed by NMR and MD simulations. PLoS One 6, e27072. (7) Noberini, R., Lamberto, I., and Pasquale, E. B. (2012) Targeting Eph receptors with peptides and small molecules: Progress and challenges. Semin. Cell Dev. Biol. 23, 51−57. (8) Noberini, R., Koolpe, M., Peddibhotla, S., Dahl, R., Su, Y., Cosford, N. D., Roth, G. P., and Pasquale, E. B. (2008) Small molecules can selectively inhibit ephrin binding to the EphA4 and EphA2 receptors. J. Biol. Chem. 283, 29461−29472. (9) Qin, H., Shi, J., Noberini, R., Pasquale, E. B., and Song, J. (2008) Crystal structure and NMR binding reveal that two small molecule antagonists target the high affinity ephrin-binding channel of the EphA4 receptor. J. Biol. Chem. 283, 29473−29484. (10) Petty, A., Myshkin, E., Qin, H., Guo, H., Miao, H., Tochtrop, G. P., Hsieh, J. T., Page, P., Liu, L., Lindner, D. J., Acharya, C., MacKerell, A. D., Jr, Ficker, E., Song, J., and Wang, B. (2012) A small molecule agonist of EphA2 receptor tyrosine kinase inhibits tumor cell migration in vitro and prostate cancer metastasis in vivo. PLoS One 7, e42120. (11) Smock, R. G., and Gierasch, L. M. (2009) Sending signals dynamically. Science 324, 198−203. (12) Nussinov, R., Tsai, C. J., and Ma, B. (2013) The underappreciated role of allostery in the cellular network. Annu. Rev. Biophys. 42, 169−189. (13) Boehr, D. D., Nussinov, R., and Wright, P. E. (2009) The role of dynamic conformational ensembles in biomolecular recognition. Nat. Chem. Biol. 5, 789−796. (14) Shi, J., Han, N., Lim, L., Lua, S., Sivaraman, J., Wang, L., Mu, Y., and Song, J. (2011) Dynamically-driven inactivation of the catalytic machinery of the SARS 3C-like protease by the N214A mutation on the extra domain. PLoS Comput. Biol. 7, e1001084. (15) Huan, X., Shi, J., Lim, L., Mitra, S., Zhu, W., Qin, H., Pasquale, E. B., and Song, J. (2013) Unique structure and dynamics of the EphA5 ligand binding domain mediate its binding specificity as

The details of the samples preparation, NMR experiments, structure calculation and analysis of the NMR dynamic data are provided in Supporting Information. S Supporting Information *

Details of the samples preparation, NMR experiments, structure calculation and analysis of the NMR dynamic data. This material is available free of charge via the Internet at http:// pubs.acs.org. Accession Codes

The NMR structures of the EphA4 ligand-binding domain have been deposited in PDB with ID of 2LW8.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 65 65161013. Fax: 65 6779 2486. E-mail: dbssjx@nus. edu.sg. Author Contributions †

H.N.Q. and L.Z.L. contributed equally. J.X.S. conceived and designed the experiments. H.N.Q., J.X.S., L.Z.L. performed the experiments and analyzed the data. J.X.S. and H.N.Q. wrote the paper. F

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revealed by X-ray crystallography, NMR, and MD simulations. PLoS One 8, e74040. (16) Qin, H., Noberini, R., Huan, X., Shi, J., Pasquale, E. B., and Song, J. (2010) Structural characterization of the EphA4-Ephrin-B2 complex reveals new features enabling Eph-ephrin binding promiscuity. J. Biol. Chem. 285, 644−654. (17) Qin, H., Lim, L., and Song, J. (2012) Protein dynamics at Eph receptor−ligand interfaces as revealed by crystallography, NMR, and MD simulations. BMC Biophys. 5, 2−10. (18) Fushman, D., Cahill, S., and Cowburn, D. (1997) The mainchain dynamics of the dynamin pleckstrin homology (PH) domain in solution: Analysis of 15N relaxation with monomer/dimer equilibration. J. Mol. Biol. 266, 173−194. (19) Mulder, F. A., Hon, B., Mittermaier, A., Dahlquist, F. W., and Kay, L. E. (2002) Slow internal dynamics in proteins: Application of NMR relaxation dispersion spectroscopy to methyl groups in a cavity mutant of T4 lysozyme. J. Am. Chem. Soc. 124, 1443−1451. (20) Palmer, A. G. (2004) NMR characterization of the dynamics of biomacromolecules. Chem. Rev. 104, 3623−3640. (21) Kleckner, I. R., and Foster, M. P. (2011) An introduction to NMR-based approaches for measuring protein dynamics. Biochim. Biophys. Acta 1814, 942−968. (22) Millet, O., Loria, J. P., Kroenke, C. D., Pons, M., and Palmer, A. G., III (2000) The static magnetic field dependence of chemical exchange line broadening defines the NMR chemical shift time scale. J. Am. Chem. Soc. 122, 2867−2877. (23) van den Bedem, H., Bhabha, G., Yang, K., Wright, P. E., and Fraser, J. S. (2013) Automated identification of functional dynamic contact networks from X-ray crystallography. Nat. Methods 10, 896− 902. (24) Lim, L., Shi, J., Mu, Y., and Song, J. (2014) Dynamically-driven enhancement of the catalytic machinery of the SARS 3C-like protease by the S284-T285-I286/A mutations on the extra domain. PLoS One 9, e101941. (25) Seiradake, E., Schaupp, A., Ruiz, D. T., Kaufmann, R., Mitakidis, N., Harlos, K., Aricescu, A. R., Klein, R., and Jones, E. Y. (2013) Structurally encoded intraclass differences in EphA clusters drive distinct cell responses. Nat. Struct Mol. Biol. 20, 958−964. (26) Xu, K., Tzvetkova-Robev, D., Xu, Y., Goldgur, Y., Chan, Y., Himanen, J. P., and Nikolov, D. B. (2013) Insights into Eph receptor tyrosine kinase activation from crystal structures of the EphA4 ectodomain and its complex with ephrin-A5. Proc. Natl. Acad. Sci. U.S.A. 110, 14634−14639. (27) Noberini, R., De, S. K., Zhang, Z., Wu, B., Raveendra-Panickar, D., Chen, V., Vazquez, J., Qin, H., Song, J., Cosford, N. D. P., Pellecchia, M., and Pasquale, E. B. (2011) A disalicylic acid-furanyl derivative inhibits ephrin binding to a subset of Eph receptors. Chem. Biol. Drug Des. 78, 667−678. (28) Lamberto, I., Qin, H., Noberini, R., Premkumar, L., Bourgin, C., Riedl, S. J., Song, J., and Pasquale, E. B. (2012) Distinctive binding of three antagonistic peptides to the ephrin-binding pocket of the EphA4 receptor. Biochem. J. 445, 47−56.

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