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The molecular mechanisms underlying the hijack of host proteins by the 1918 Spanish influenza virus Qingliang Shen, Danyun Zeng, Baoyu Zhao, Veer S. Bhatt, Pingwei Li, and Jae-Hyun Cho ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00168 • Publication Date (Web): 03 Apr 2017 Downloaded from http://pubs.acs.org on April 4, 2017

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ACS Chemical Biology

The molecular mechanisms underlying the hijack of host proteins by the 1918 Spanish influenza virus Qingliang Shen, Danyun Zeng, Baoyu Zhao, Veer S. Bhatt, Pingwei Li, Jae-Hyun Cho* Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843 Corresponding Author *Phone: (979) 458-5928. Email: [email protected]

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Abstract: The 1918 Spanish influenza A virus (IAV) caused one of the most serious pandemics in history. The nonstructural protein 1 (NS1) of the 1918 IAV hijacks the interaction between human CrkII and JNK1. Little is, however, known about its molecular mechanism. Here, we performed X-ray crystallography, NMR relaxation dispersion experiment, and fluorescence spectroscopy to determine the structural, kinetic, and thermodynamic mechanisms underlying the hijacking of CrkII by 1918 IAV NS1. We observed that the interaction between a proline-rich motif in NS1 and the N-terminal SH3 domain of CrkII displays strikingly rapid kinetics and exceptionally high affinity with 100-fold faster kon and 3,300-fold lower Kd compared to those for the CrkII-JNK1 interaction. These results provide molecular insight into the mechanism by which 1918 IAV NS1 hijacks CrkII and disrupts its interactions with critical cellular signaling proteins.


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Influenza A virus (IAV) infects a wide range of avian and mammalian species.1 While influenza hemagglutinin (HA) protein determines virus-host specificity,2 nonstructural protein 1 (NS1) plays important roles in IAV replication and the evasion of antiviral immunity by hijacking and rewiring host protein-protein interactions.3, 4 Therefore, NS1 has been shown to be a potential antiviral target.5 The C-terminal tail region of NS1 shows the largest amino acid sequence variation among different virus strains.6, 7 Recent studies have shown that the C-terminal tail of the 1918 Spanish IAV NS1 contains a proline-rich motif (PRM) that binds to the N-terminal SH3 (nSH3) domain of human Crk-family proteins (CrkI, CrkII, and Crk-L) with exceptionally high selectivity.8, 9 Interestingly, common seasonal IAV NS1 proteins do not bind to the nSH3 domain of Crk because of a single amino acid (P215T) replacement in this PRM.8-10 The nSH3 domain of Crk proteins binds to PRMs in a large number of signaling proteins, including c-Jun-N-terminal kinase 1 (JNK1) and Abl kinase.11-15 Therefore, NS1 binding to Crk inhibits Crk-JNK and Crk-Abl interactions,9, 10 suppressing the host antiviral immune response (Figure 1a).8, 9 Despite its critical importance, the molecular basis by which IAV NS1 hijacks Crk remains to be elucidated. In this study, we report that the PRM of the 1918 IAV NS1 binds to the nSH3 domain of CrkII with an extremely high affinity. Furthermore, we determined the structural and kinetic mechanisms underlying the inhibition of CrkII-JNK1 and CrkII-Abl interactions by NS1. We synthesized a peptide corresponding to the C-terminal PRM of IAV-NS1 (PRMNS1) and measured its binding affinity to the nSH3 domain of CrkII (nSH3CrkII) by monitoring fluorescence intensity change upon complexation. The Kd of the nSH3CrkII-PRMNS1 complex was measured to be 6 nM (Figure 1b and Table 1), which, to our knowledge, is the strongest binding observed for any known SH3-ligand interactions. In general, the Kd for SH3-PRM complexes are in the range of 1 to 10 µM.16 Of note, the nSH3CrkII domain in isolation and in the context of full-length CrkII have the similar binding affinities to target proteins.17 IAV NS1 has been shown to interfere with CrkII-JNK1 interaction, resulting in the suppression of JNK mediated antiviral activity.9,

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Strikingly, we found that the affinity of nSH3CrkII-PRMNS1

complex is 3,300-fold higher than that of nSH3CrkII-PRMJNK complex (Figure 1c). This result supports that IAV NS1 inhibits Crk-mediated JNK1 activation resulting in impaired interferon (IFNβ) expression.9,

18

. It has also been reported that the 1918 IAV-NS1 hijacks CrkII-Abl

interaction, which is mediated by the interactions between the nSH3CrkII and PRMs of Abl (PRMAbl) (Figure 1a).10 Previously, we observed that the Kd of nSH3CrkII-PRMcAbl is ~3 µM.19 3 ACS Paragon Plus Environment


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Hence, the binding affinity between nSH3CrkII and PRMNS1 is 460-fold higher than that between nSH3CrkII and PRMAbl. This surprisingly high affinity will enable NS1 to hijack CrkII and interfere with critical protein-protein interactions mediated by CrkII in human cells. It was shown that PRMNS1 binds to nSH3CrkII with high selectivity,8 while PRMs in general have low SH3 domain selectivity.20 To elucidate the structural basis of the interaction between the nSH3CrkII domain and PRMNS1, we determined the crystal structure of nSH3CrkII-PRMNS1 complex at 1.45 Å resolution (Figure 2a and Supporting Table 1). The structure shows that PRMNS1 binds to the nSH3 domain in class-II binding mode: i.e., PRMNS1 contains core PxxPx+ motif (where + is positively charged residue and x is any amino acid).21 NMR chemical shift perturbation (CSP) analysis revealed that similar binding surface of the nSH3CrkII is involved in the interactions with PRMNS1 in solution (Supporting Figure 1). The nSH3CrkII-PRMNS1 complex structure reveals a bipartite interface (Figure 2a). The Nterminal region of PRMNS1 interacts with nSH3CrkII via hydrophobic interactions mediated by the PPLP motif. These hydrophobic interactions are commonly observed in other SH3-PRM complexes.20 In contrast, the C-terminal region forms electrostatic interactions with a negatively charged surface of nSH3CrkII domain (Figure 2a). In this region, PRMNS1 forms two sets of shortrange electrostatic interactions with the nSH3CrkII domain. One set is a salt-bridge network formed between the ε-amino group of K217 of PRMNS1 and three acidic residues (D147, E149, and D150) of the nSH3 domain (Figure 2b). Although this salt-bridge network has been observed in the complexes of nSH3CrkII and the PRMs of Abl19 and C3G,22 it is rarely observed in other SH3-PRM complexes (see Supporting Information and Supporting Table 2). The arrangement of these three acidic residues of the nSH3 domain is specifically optimized to accommodate the ε-amino group of K of a PRM. The replacement of this K with R was shown to reduce the binding affinity to the nSH3 domain by 10-fold.19, 22 In contrast, most of SH3 domains that recognize PxxPx+ (+ represents either K or R) motifs prefer to bind to PRMs containing an R at this position.23 Another important intermolecular contact is a bidentate hydrogen bond between E166 of the nSH3 and the backbone amides of Q218 and K219 of PRMNS1 (Figure 2c). To examine whether these intermolecular interactions are observed in other SH3-PRM complexes, we investigated all human SH3-PRM complex structures available in the PDB (see Supporting Information). Interestingly, none of the SH3 domains, except the nSH3CrkII, form both sets of intermolecular interactions with their cognate ligands (Supporting Table 2 and Supporting Figure 2). Therefore, these results explain why the 1918 IAV NS1 is highly specific for the nSH3CrkII domain.8 4 ACS Paragon Plus Environment

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Despite the importance in binding selectivity, these short-range electrostatic interactions have also been observed in nSH3CrkII-PRMAbl and nSH3CrkII-PRMC3G complexes, of which binding affinities are significantly lower than that of the nSH3CrkII-PRMNS1 complex.19 Therefore, other factors likely make contribution to the binding energetics between nSH3CrkII and PRMNS1. While the binding surface of the nSH3 domain is negatively charged (Figure 2a), the PRMNS1 and PRMJNK1 contain net 5 and 0 positive charges, respectively. Therefore, long-range electrostatic interaction is likely to contribute to this large difference in Kd.24, 25 To test this hypothesis, we examined the correlation between the net positive charges of PRMs and Kd. We indeed found that binding affinities correlate with the increasing net charge of PRMs (Figure 3a). Here, we included our previous results for binding of PRMAbl to the nSH3CrkII domain.19 It has been known that long-range electrostatics play an important role in accelerating association kinetics of protein-protein interactions.24, 26 Therefore, we examined the effect of net charge of PRMs on binding kinetics to the nSH3CrkII domain. We measured kon and koff of the nSH3CrkII domain with PRMNS1 and PRMJNK1 using NMR

15

N Carr-Purcell-Meiboom-Gill

relaxation dispersion (CPMG-RD) experiment (Figure 3b and 3c).27 A total of 11 and 6 backbone resonances of the nSH3 domain showed analyzable dispersion behavior upon binding of the PRMNS1 and PRMJNK1, respectively (Supporting Table 3, Supporting Figures 3 and 4). These residues are located in the interface between the nSH3CrkII and the PRMs (Supporting Figures 3 and 4). We observed flat dispersion profiles for the free nSH3 domain.28 Hence, the dispersion curve observed for the nSH3 partially saturated with PRMs is the result of the association and dissociation processes. Figure 3d shows that kon values correlate with the increasing net charge of PRMs. In this analysis, we included our previous results for binding kinetics between the nSH3CrkII and PRMsAbl.28 The kon of the nSH3 and PRMNS1 was measured to be 1.1 x 1010 M-1s-1, which is 100fold faster compared to that of nSH3CrkII and PRMJNK1 (Table 1). Furthermore, the kon of PRMJNK1, whose net charge is 0, is similar to the basal level kon (i.e. in the absence of electrostatic enhancement) between the nSH3CrkII and a PRM which is approximately 107 – 108 M-1s-1.28 Therefore, this large difference in kon is most likely due to long-range electrostatic acceleration.24, 26

Interestingly, the aforementioned short-range electrostatic interactions are conserved in all the nSH3CrkII-PRM complexes included in this analysis. Nevertheless, positively charged residues, except K217, in PRMNS1 do not form short-range interactions with negatively charged residues 5 ACS Paragon Plus Environment


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of the nSH3CrkII. Moreover, the side chain electron densities of the C-terminal positively charged residues (K219, R220 and K221) in PRMNS1 are noticeably weaker than other residues (Figure 2d), indicating conformational heterogeneity due to lack of specific interactions. Taken together, these results indicate that long-range electrostatics play an important role in binding thermodynamics and accelerating the association between CrkII and IAV NS1. These contributions of long-range electrostatics have been demonstrated in other protein-protein (or ligand) interactions.25, 29, 30 Our results suggest that this rapid association provides IAV NS1 with a temporal advantage to compete with host defense signaling processes. Rapid hijacking of CrkII by IAV NS1 would interfere with CrkII-JNK1 and CrkII-Abl interactions before host antiviral immunity pathways are activated. Moreover, the koff of nSH3CrkII-PRMNS1 is 35-fold and 15-fold slower than those of nSH3CrkII-PRMJNK and nSH3CrkII-PRMAbl, respectively. It was indicated that IAV NS1 can translocate CrkII into the nucleus.31 Therefore, the longer lifetime (i.e. 1/ koff) of the complex seems to help IAV NS1 maintain contact with CrkII during the nuclear translocation process. These results suggest that the association and dissociation kinetics of IAV NS1 and CrkII are optimized for hijacking host protein-protein interactions. Despite its importance, very little is known about the molecular basis of the 1918 Spanish IAV pathogenicity. In this study, we have provided the structural, thermodynamic, and kinetic basis of the interaction between the PRM of 1918 IAV NS1 and the nSH3 of CrkII. These results provide mechanistic insights into how IAV NS1 hijacks CrkII from its cellular binding partners, JNK1 and Abl kinase. Therefore, understanding the molecular mechanisms underlying viral hijacking of host protein-protein interactions will provide insights into the rational development of antiviral agents. The SH3 domain is one of the most prevalent protein binding modules in the human proteome and it plays important roles in diverse signaling processes32. Therefore, small-molecule inhibitors targeting SH3 domains can be a powerful chemical biology tool for modulating proteinprotein interactions33. However, the development of SH3 domain inhibitors has proven difficult because most of the SH3-ligand interactions are of low binding affinity and selectivity34. This low affinity is mainly due to the small and shallow binding interface, which are common features of undruggable targets. In light of this, understanding the molecular mechanism underlying exceptionally high affinity and selectivity of PRMNS1 to the nSH3 domain will provide insights into the design principles for potent SH3 domain inhibitors. 6 ACS Paragon Plus Environment

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Methods Protein and peptides: All protein samples for crystallization, fluorescence and NMR experiments, were prepared as described elsewhere.19 Synthetic peptides were purchased in a crude form, and further purified using reverse-phase high performance liquid chromatography in our laboratory. The N- and C-termini of peptides were acetylated and amidated, respectively. The peptide concentration was determined by measuring the UV absorption at 280 nm of a single tyrosine at the N- or C-terminal ends of the peptides. Crystallization and structure determination: 4 mM nSH3 was mixed with 5 mM PRMNS1 for the crystallization trials. The sample was crystallized by hanging drop vapour diffusion method in 0.1 M sodium acetate (pH 4.6), 30% PEG 2000 and 0.2 M ammonium sulfate. An 1.45 Å resolution dataset was collected at 120 K using an R-AXIS IV++ image plate detector mounted on a Rigaku MicroMax 007HF X-ray generator. The data were processed using iMosflm in the CCP4 package.35 The structure was determined using the nSH3 domain model (PDB ID: 5UL6) as search model using Phenix.36 The NS1 PRM peptide was modeled into the difference map manually in COOT37 and refined with Phenix. The electrostatic potential surface of nSH3 domain was calculated using APBS and PDB2PQR.38-40 NMR spectroscopy: All NMR experiments were conducted using protein samples in 20 mM sodium phosphate (pH 6.1), 80 mM NaCl, 0.02% sodium azide, 1 mM EDTA, 10 µM DSS (4,4dimethyl-4-silapentane-sulfonate), and 10% D2O at 25 °C. NMR experiments were performed on Bruker Avance 600 MHz and 800 MHz spectrometers equipped with a cryogenic probe. NMR spectra were processed with NMRPipe41 and analyzed with NMRViewJ (One Moon Scientific, Inc.) and CARA42. The temperatures of NMR sample were calibrated using the deuterated methanol-d4.43 The experimental details for binding kinetics measurements using NMR relaxation dispersion experiments are provided in the Supporting Information. Binding assay: The dissociation constants (Kd) of nSH3-PRM complexes were measured by monitoring the change of tryptophan fluorescence signal. Excitation wavelength was 295 nm. All binding assays were performed in a stirred 1-cm path length cuvette using a PTI QM-400 fluorimeter. Protein concentration used for the fluorescence-based binding assays was 0.1 µM. The measurements were done in 20 mM sodium phosphate (pH 6.1) and 80 mM NaCl at 25 °C. The Kd was calculated by assuming a 1:1 complex, and by the global fitting of the repeatedly 7 ACS Paragon Plus Environment


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measured fluorescence intensities to the eq. 4:

 [P ] + [L ] + K ± t t d ∆F = ∆Fmax   

([ Pt ] + [ Lt ] + K d ) 2[ Pt ]

2

− 4[ Pt ][ Lt ]   

(4)

where ∆F and ∆Fmax are the change and the maximum amplitude of signal change, respectively. Pt is the total protein concentration and Lt is the total ligand concentration at each titration point. The reported Kd values are the average of 3 or 4 repeated measurements. Supporting Information The Supporting Information is available free of charge via the Internet. Supplementary methods and supporting Tables 1-3 and Figures1-4. Acknowledgment This research was supported by start-up funds from Texas A&M University.


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19. Bhatt, V. S., Zeng, D., Krieger, I., Sacchettini, J. C., and Cho, J. H. (2016) Binding mechanism of the N-terminal SH3 domain of CrkII and proline-rich motifs in cAbl Biophys. J. 110, 2630-2641. 20. Ladbury, J. E., and Arold, S. T. (2011) Energetics of Src homology domain interactions in receptor tyrosine kinase-mediated signaling, Methods Enzymol. 488, 147-183. 21. Lim, W. A., Richards, F. M., and Fox, R. O. (1994) Structural determinants of peptidebinding orientation and of sequence specificity in SH3 domains, Nature 372, 375-379. 22. Wu, X., Knudsen, B., Feller, S. M., Zheng, J., Sali, A., Cowburn, D., Hanafusa, H., and Kuriyan, J. (1995) Structural basis for the specific interaction of lysine-containing prolinerich peptides with the N-terminal SH3 domain of c-Crk, Structure 3, 215-226. 23. Saksela, K., and Permi, P. (2012) SH3 domain ligand binding: What's the concensus and where's the specificity?, FEBS Lett. 586, 2609-2614. 24. Schreiber, G., Haran, G., and Zhou, H. X. (2009) Fundamental aspects of protein-protein association kinetics, Chem. Rev. 109, 839-860. 25. Joughin, B. A., Green, D. F., and Tidor, B. (2005) Action-at-a-distance interactions enhance protein binding affinity, Protein Sci. 14, 1363-1369. 26. Schreiber, G., and Fersht, A. R. (1996) Rapid, electrostatically assisted association of proteins, Nat. Struct. Biol. 3, 427-431. 27. Loria, J. P., Rance, M., and Palmer, A. G. (1999) A relaxation-compensated Carr-PurcellMeiboom-Gill sequence for characterizing chemical exchange by NMR spectroscopy, J. Am. Chem. Soc. 121, 2331-2332. 28. Zeng, D., Bhatt, V. S., Shen, Q., and Cho, J. H. (2016) Kinetic insights into the binding between the nSH3 domain of CrkII and proline-rich motifs in cAbl, Biophys J in press. 29. Tossavainen, H., Aitio, O., Hellman, M., Saksela, K., and Permi, P. (2016) Structural Basis of the High Affinity Interaction between the Alphavirus Nonstructural Protein-3 (nsP3) and the SH3 Domain of Amphiphysin-2, J Biol Chem 291, 16307-16317. 30. Borg, M., Mittag, T., Pawson, T., Tyers, M., Forman-Kay, J. D., and Chan, H. S. (2007) Polyelectrostatic interactions of disordered ligands suggest a physical basis for ultrasensitivity, Proc Natl Acad Sci USA 104, 9650-9655. 31. Ylösmäki, L., Fagerlund, R., Kuisma, I., Julkunen, I., and Saksela, K. (2016) Nuclear Translocation of Crk Adaptor Proteins by the Influenza A Virus NS1 Protein, Viruses 8, 101. 32. Kaneko, T., Li, L., and Li, S. S. (2008) The SH3 domain--a family of versatile peptide- and protein-recognition module, Front Biosci. 13, 4938-4952. 33. Nguyen, J. T., Porter, M., Amoui, M., Miller, W. T., Zuckermann, R. N., and Lim, W. A. (2000) Improving SH3 domain ligand selectivity using a non-natural scaffold, Chem. Biol. 7, 463-473. 34. Agrawal, V., and Kishan, K. V. (2002) Promiscuous binding nature of SH3 domains to their target proteins, Protein Pept. Lett. 9, 185-193. 35. (1994) The CCP4 suite: programs for protein crystallography, Acta Crystallogr D Biol Crystallogr 50, 760-763. 36. Adams, P. D., Afonine, P. V., Bunkóczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J., Hung, L.-W., Kapral, G. J., Grosse-Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner, R., Read, R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C., and Zwart, P. H. (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution, Acta Crystallogr D Biol Crystallogr 66, 213-221. 37. Emsley, P., and Cowtan, K. (2004) Coot: model-building tools for molecular graphics, Acta Crystallogr D Biol Crystallogr 60, 2126-2132. 38. Dolinsky, T. J., Czodrowski, P., Li, H., Nielsen, J. E., Jensen, J. H., Klebe, G., and Baker, N. A. (2007) PDB2PQR: Expanding and upgrading automated preparation of biomolecular structures for molecular simulations, Nucleic Acids Res 35, W522-W525. 10 ACS Paragon Plus Environment

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Table 1. Thermodynamic and kinetic parameters for the binding between the nSH3CrkII and PRMs of NS1 or JNK1.

a

Kd (µM)

kon (108 M-1 s-1)a

koff (s-1)a

PRMNS1

0.006 ± 0.002

110 ± 20

66.5 ± 10.1

PRMJNK1

22.0 ± 1.5

1.06 ± 0.06

2339.5 ± 123.5

These are the results of a global fit of CPMG-RD data


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Figure Legends Figure 1. a) Schematic diagram showing the hijacking of CrkII-JNK1 and CrkII-Abl interactions by IAV NS1. Fluorescence-monitored binding isotherms of b) PRMNS1 and c) PRMJNK1 to the nSH3 domain of CrkII. The sequences of PRMs are shown in each panel. The N- or C-terminal Y was incorporated for the quantitation of peptides. Figure 2. a) Crystal structure of nSH3CrkII-PRMNS1 complex (PDB ID: 5UL6). The protein surface is colored according to electrostatic potential at neutral pH from -5 kT (red) to +5kT (blue). b) Salt-bridge network between three acidic residues of the nSH3 domain and K217 of NS1. c) Hydrogen bonds between E166 of nSH3 and the backbone amides of Q218 and K219 of NS1. Side chains are not shown for clarity. d) σA weighted Fo – Fc map (contoured at 2.0σ) of the Cterminal region of PRMNS1 Figure 3. a) The plot of net positive charge of PRMs versus Kd values. The open circles represents the values for the nSH3CrkII and PRMsAbl taken from Bhatt et al.19 Representative NMR

15

N CPMG-RD profiles (E149) measured for the binding of the nSH3 domain with b)

PRMNS1 and c) PRMJNK. Data obtained at 14.1 Tesla and 18.8 Tesla are shown in black and red circles, respectively. d) The plot of net positive charge of PRMs versus kon values. The open circles represents the values for the nSH3CrkII and PRMsAbl taken from Zeng et al.28



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Figure 1


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Figure 2



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Figure 3


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