Article pubs.acs.org/jmc
Ligand-Induced Conformational Change of Plasmodium falciparum AMA1 Detected Using 19F NMR Xiaopeng Ge,†,§ Christopher A. MacRaild,‡,§ Shane M. Devine,‡ Cael O. Debono,‡ Geqing Wang,‡ Peter J. Scammells,‡ Martin J. Scanlon,‡ Robin F. Anders,† Michael Foley,*,† and Raymond S. Norton*,‡ †
Department of Biochemistry, La Trobe University, Melbourne 3086, Victoria, Australia Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville 3052, Victoria, Australia
‡
S Supporting Information *
ABSTRACT: We established an efficient means of probing ligand-induced conformational change in the malaria drug target AMA1 using 19F NMR. AMA1 was labeled with 5fluorotryptophan (5F-Trp), and the resulting 5F-Trp resonances were assigned by mutagenesis of the native Trp residues. By introducing additional Trp residues at strategic sites within a ligand-responsive loop, we detected distinct conformational consequences when various peptide and small-molecule ligands bound AMA1. Our results demonstrate an increase in flexibility in this loop caused by the native ligand, as inferred from, but not directly observed in, crystal structures. In addition, we found evidence for long-range allosteric changes in AMA1 that are not observed crystallographically. This method will be valuable in ongoing efforts to identify and characterize therapeutically relevant inhibitors of protein−protein interactions involving AMA1 and is generalizable to the study of ligand-induced conformational change in a wide range of other drug targets.
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INTRODUCTION There are approximately 200 million cases of malaria each year, resulting in over 600000 deaths, mostly caused by Plasmodium falciparum infections.1 The RTS,S malaria vaccine is showing some efficacy in an ongoing phase III trial,2−4 but antimalarial drugs will continue to play a critical role in controlling and treating the disease for the foreseeable future. However, the repeated emergence of drug-resistant parasites has made it essential that the development of new antimalarial drugs continues.5,6 A range of new potential drug targets has been identified as a result of sequencing the parasite’s genome and from a more detailed understanding of the parasites’ life cycle at the molecular level.7−9 To cause disease, parasite merozoites must invade host erythrocytes, a process that involves initial attachment, reorientation, tight-junction formation, and engagement of the parasite actin−myosin motor to drive the parasite into the erythrocyte.10,11 The mechanisms underlying these processes are unique to Plasmodium and related parasites, making them potential targets for the development of new antimalarial drugs.12,13 Apical membrane antigen 1 (AMA1) is a well-characterized protein involved in parasite invasion that is being studied as a potential target of new antimalarial drugs.13 After release from the merozoite micronemes, PfAMA1 forms a complex with another parasite antigen, Pf RON2, which is located at the moving junction.14,15 Recent conditional knockdown studies have generated some controversy regarding the precise role of this complex,16,17 but vaccine studies have nonetheless shown that targeting AMA1 can markedly inhibit development of asexual blood-stages of P. falciparum,18−20 and antibodies and © 2014 American Chemical Society
peptides targeting AMA1 have been shown to inhibit erythrocyte invasion by blocking the AMA1−RON2 interaction.21−23 Structures of the AMA1 ectodomain revealed the presence of a hydrophobic cleft that is conserved across apicomplexan parasites24 and has been suggested to be the principal ligand-binding site on AMA1.21,25 The recent cocrystal structure of AMA1 with RON2L, a peptide corresponding to the extracellular region of RON2, confirmed that the hydrophobic cleft is the site of RON2 binding.26,27 This structure also revealed a conformational change in which RON2L displaces the AMA1 domain II (DII) loop to expose a positively charged binding site that interacts with a complementary charged region at the N-terminus of peptide RON2L.26,27 R1, an AMA1-binding peptide selected from a peptide library displayed on phage,25,28 competes with RON2L for binding to this site and induces a similar conformational change of the AMA1 DII loop.27 A diverse range of ligands, including the peptides RON2L and R1, monoclonal antibodies and shark antibodies (IgNARs), bind to the hydrophobic cleft and block the interaction between AMA1 and RON2.25,27,29−31 This has stimulated research to identify inhibitors of this interaction that may be valuable drug leads. Two approaches, high-throughput screening and fragment-based ligand discovery, have been used to identify small molecules that bind to AMA1 and compete with peptide RON2L or R1.32,33 To understand the mechanisms by which these small molecules bind to AMA1, and to better Received: March 13, 2014 Published: July 28, 2014 6419
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evaluate their potential as inhibitors of the AMA1−RON2 interaction, we have explored methods that would allow detection of conformational changes of the DII loop induced by ligand binding. 19 F NMR spectroscopy has proved sensitive to ligandinduced conformational changes in a range of other proteins of biological and therapeutic interest.34−37 In this study, we have applied 19F NMR spectroscopy to the detection of ligandinduced movements of the DII loop of PfAMA1. Tryptophan residues were inserted into specific sites within the DII loop by site-directed mutagenesis, then all Trp in the protein were substituted with 5-fluorotryptophan (5F-Trp) by expression in the presence of glyphosate. 19F NMR was then used to identify small AMA1-binding molecules among our current fragment hits32 that induced a conformational change in AMA1 similar to that of known inhibitors. This approach will be used to characterize other AMA1-binding molecules that are being assessed as drug leads. More generally, it highlights the power of 19F NMR as an efficient probe of protein−ligand binding and associated conformational change.
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RESULTS AND DISCUSSION Construction of AMA1 Mutants. Because none of the four Trp residues in AMA1 is in the flexible DII loop, mutant forms of 3D7 PfAMA1108−434 (corresponding to domains I and II of the AMA1 ectodomain) were created, either by replacing individual aromatic residues in the loop with Trp or by inserting an additional Trp in the middle of the loop. The insertion site was chosen at a highly exposed site, between two small residues (A372 and S373), where insertion was deemed least likely to perturb the structure of the DII loop (Figure 1A). Each of the mutant proteins was expressed on the surface of phage38 so that they could readily be assessed for their ability to bind the conformationally dependent monoclonal antibodies (mAb) 1F9 and 4G231 and peptide RON2L (Table 1). All three reagents bound the replacement mutant F367W and the insertion mutant IS373W equivalently to wild-type AMA1 (Figure 1). In contrast, the binding of the mAbs and peptide to the other replacement mutants was reduced (F360W, F379W, and F385W) or abolished (Y353W and Y390W). Consequently, mutants F367W and IS373W were selected for the 19F NMR studies described below. These mutants, together with wild-type AMA1 DI+II, were expressed in Escherichia coli as 5FTrp-labeled recombinant proteins by growing the bacteria in minimal media supplemented with glyphosate to inhibit aromatic amino acid metabolism,39 and with 5F-Trp, Phe, and Tyr. Expression, Purification, and Characterization of FTrp-AMA1 and Its Mutants. The 5F-Trp-labeled recombinant proteins were isolated and refolded following the previously published procedure,40 modified by the addition of 0.6 M L-arginine to the refolding buffer. This resulted in a high yield of each form of AMA1 DI+II, all of which were judged to be >95% pure by nonreducing SDS-PAGE. Mass spectrometry confirmed the identity of the expressed constructs and was consistent with 5F-Trp incorporation to the level of approximately 85% (data not shown). The conformationally dependent mAb 1F9 reacted well on Western blots with all three forms of AMA1, indicating the presence of correctly refolded protein (Figure 2A). The binding affinities of 5F-TrpAMA1 for the peptides RON2L and R1 were found to be 5 and 23 nM, respectively, using SPR measurements. The replacement mutant F367W bound both peptides with affinities nearly
Figure 1. (A) Location of tryptophan residues and the domain II loop in AMA1. The structure of AMA1 DI+II (PDB ID 2Z8V) showing the locations of the four tryptophan residues, W110, W298, W315, and W399 (yellow). None of the four Trp residues is located in the domain II loop (blue) or hydrophobic cleft (green). Also shown are the sites of Trp mutation, F367 (red), and insertion, S373. (B) mAb reactivity of AMA1 DII loop mutants displayed on the surface of phage. Phage displaying wild-type and mutant forms of AMA1 were allowed to bind to immobilized mAb 1F9, mAb 4G2, and RON2L peptide. The average of duplicated wells is shown, and error bars indicate the range.
Table 1. Comparison of the Reactivity of Phage-Displayed AMA1 DII Loop Mutants and Wild-Type AMA1a reactivity with AMA1-binding ligands phage-displayed AMA1
mAb 1F9
mAb 4G2
peptide RON2L
wild-type AMA1 replacement mutant Y353W replacement mutant Y360W replacement mutant F367W* replacement mutant F379W replacement mutant F385W replacement mutant Y390W insertion mutant IS373W*
+++ − + +++ + ++ − +++
+++ − ++ +++ − + − +++
+++ − ++ +++ + + − +++
* indicates the two mutant forms of AMA1 DI+II selected for expression in E. coli as 5F-Trp recombinant proteins. +++ indicates an OD >2.5 for mAbs and >1.5 for peptide, ++ indicates an OD >1.5 for mAbs and >1 for peptide, + indicates an OD >0.5 for mAbs and >0.5 for peptide, − indicates an OD 90% purity (GL Biochem, China). Both peptides with two cysteine residues were cyclized with correctly formed intramolecular disulfide bonds. AMA1 binding fragments 5-(1H-benzo[d]imidazo-2-yl)benzene-1,3diamine (1),43 2-(piperidin-1-yl)-1H-benzo[d]imidazol-6-amine (2),44 and 5-(4-aminophenyl)-4-methylthiazol-2-amine (3)45 were synthesized by established procedures. Phage-Displayed AMA1 Mutants. All AMA1 mutant genes in the pHENH6 vector were transformed into Escherichia coli TG1 strain, and the cells were grown at 37 °C in 2YT medium with shaking until the culture reached log phase. The culture was subsequently left stationary at room temperature for 15 min to allow the regeneration of the F-pilus. Phages were then rescued from the E. coli cells by adding 108 helper phage, followed by incubation at 37 °C stationary for 1 h. Finally, 70 μg/mL of kanamycin was added to the culture, which was then incubated at 37 °C overnight with shaking. The overnight culture was centrifuged at 7700g for 10 min, and phage in the supernatant was precipitated using 5× PEG solution (30% (w/v) PEG8000, 2.6 M NaCl). Following incubation at 4 °C for 2 h, the precipitated phage were centrifuged at 15000g for 30 min, and the phage pellet was resuspended in 1 mL of PBS and stored at −80 °C for subsequent analysis. Phage ELISA. Microtiter plates were coated with 0.2 μg/well of mAbs 1F9 or 4G2 overnight at 4 °C. For peptide RON2L, a neutravidin plate was coated overnight with 0.2 μg/well of biotinylated RON2L peptide in PBS/0.1% Tween/0.05% BSA. Phage expressing the desired proteins were diluted 1 in 10 in PBS−Tween and allowed to interact with the immobilized antigen for 1 h with shaking. After washing, the phage were subsequently detected using a horseradish peroxidase (HRP)-conjugated anti-M13 monoclonal antibody. Binding was visualized using 100 μL/well of 3,3′,5,5′-tetramethylbenzidine solution, and the reaction was stopped by adding 100 μL/well of 1 M HCl. The absorbance was then read at 450 nm. Recombinant 5F-Trp-AMA1 and Its Mutants Proteins. The genes for wild-type 3D7 PfAMA1108−434 and its mutants W110F,
Figure 5. 19F NMR spectra of 5-5F-Trp-F367W AMA1 bound to small molecule ligands 1 (A), 2 (B), and 3 (C). Spectra are shown for free FTrp-F367W AMA1 (black) and in the presence of 1 mM (red) and 3 mM (blue) ligand.
bind to the hydrophobic cleft, more than 30 Å from W110. Although we cannot rule out the possibility of a secondary binding site for each of these ligands close to W110 on DII, the generality of this effect across peptide and small molecule ligands suggests a long-range conformational coupling between the two sites, such that the binding of these ligands at the hydrophobic cleft induces conformational change at this distant site. The detection of allosteric structural change of this nature by 19F NMR is well established.34,42 As noted above, any such conformational coupling is not evident from the available crystal structures, suggesting that it may be dynamic in nature. It is, however, consistent with the extensive changes seen in the 1 H−15N HSQC spectrum of AMA1 DI+II on binding these ligands (our unpublished data). 6424
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W298F, W315F, F367W, and IS373W were cloned into the expression vector pET26b(+) and transformed into E. coli BL21 (DE3) strain. Bacteria were grown overnight at 37 °C in LB medium, containing 100 μg/mL of ampicillin, and subsequently diluted 1 in 10 in M9 medium supplemented with 2% glucose (w/v), 1× BME vitamins, 1× trace elements, 2 mM MgSO4, and 0.1 mM CaCl2. Then 1 g/L of glyphosate, 50 mg/L of Phe, 50 mg/L of Tyr, and 36 mg/L of 5F-Trp were added to the medium when the cell density reached ∼0.4. After shaking for 1 h, the expression of 5F-Trp-labeled proteins was induced by addition of 1 mM (final) IPTG for 3 h, then cells were harvested by centrifugation at 5000g for 15 min. Protein Refolding, Purification, and Characterization. 5FTrp-labeled AMA1 and its mutants were expressed, solubilized, and isolated as described.20 The recombinant proteins were subsequently refolded overnight in the conventional refolding buffer with the addition of 0.6 M L-Arg.46 To correctly form the five intramolecular disulfide bonds in AMA1 DI+II,47 reduced glutathione and oxidized glutathione were then added to the refolding buffer at final concentrations of 0.1 and 0.025 M, respectively. After refolding, the proteins were purified through a HiTRAP QFF (GE Healthcare) ionexchange column and the sample buffer was changed to 20 mM phosphate buffer, pH 7.4. The binding of conformationally sensitive mAbs to F-Trp-AMA1 and its mutants was assessed using Western blot. All protein samples were run on SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was subsequently probed with 1 μg/mL of monoclonal antibody 1F9, and proteins were detected using HRP-conjugated antimouse antibody (1 in 2000 dilution, Millipore). Surface Plasmon Resonance. All SPR analyses were performed using ProteOn XPR 36 platform (BioRad) and carried out in PBS− Tween (0.1%) at 25 °C using a constant flow rate of 30 μL/min. AMA1 was immobilized on a GLC sensor chip following the manufacturer’s recommended protocol. Peptides RON2L and R1 were flowed across the surface of the chip at a constant flow rate for 8 min, then dissociated from the AMA1 surface for 800 s by flowing PBS−Tween across the surface of the chip. Peptides were tested at a range of concentrations (50−3.125 nM) and a lane without AMA1 was included as a negative control. Signals generated by buffer-only or DMSO-only control experiments were subtracted from the resulting sensorgrams to indicate the binding of the peptides to AMA1. All peptide samples were diluted in PBS−Tween, and the surface of the chip was regenerated by injecting 30 μL of 0.85% phosphoric acid (flow rate = 100 μL/min) in all channels in the vertical orientation of the ProteOn XPR36 fluidics. NMR Spectroscopy. 19F NMR spectra were acquired on a Bruker Avance III spectrometer operating at 564 MHz (600 MHz 1H frequency) and at 25 °C using a TCI triple-resonance cryoprobe, with the 1H channel tuned for 19F. Samples contained ∼100 μM 5F-Trp AMA1 in 20 mM sodium phosphate, pH 7.4, with 0.005% trifluoroethanol as internal reference for 19F chemical shift (−1.375 ppm relative to trace TFA in water) and signal intensity and 7% 2H2O for the spectrometer lock signal. R1 and RON2L were added from 1 mM stocks in 20 mM sodium phosphate, pH 7.4, to a final peptide:AMA1 ratio of 1.2:1, while trRON2 was used at a ratio of 5:1 to account for its lower affinity. Small molecule ligands of AMA1 were added from 100 mM stocks in H2O (2), ethanol (1), or DMSO (3). Spectra were typically acquired as 2K scans of 8K complex points over a 150 ppm sweep width, for a total experimental time of less than 40 min. Spectra were processed using 20 Hz exponential line-broadening prior to Fourier transformation. Line widths were estimated by leastsquares fits to a sum of Lorentzians.
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Article
AUTHOR INFORMATION
Corresponding Authors
*For R.S.N.: phone, +61 3 9903 9167; fax, +61 3 9903 9582; Email, [email protected]. *For M.F.: phone, +61 3 9479 2158; E-mail, m.foley@latrobe. edu.au. Author Contributions §
X.G. and C.A.M. contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Krishnarjuna Bankala for assistance with mass spectrometry. This work was supported in part by an Australian National Health and Medical Research Council (NHMRC) project grant (1025150). R.S.N. acknowledges fellowship support from the NHMRC.
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ABBREVIATIONS USED 5F-Trp, 5-fluorotryptophan; AMA1, apical membrane antigen 1; DII, domain II; DI+II, domains I and II; mAb, monoclonal antibody
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REFERENCES
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ASSOCIATED CONTENT
S Supporting Information *
Table showing 19F line widths of 5F-Trp resonances of F367W AMA1 in the presence of RON2L and R1; figure showing the location of the native Trp residues in AMA1, and figures showing 1H NMR spectra of AMA1 mutants. This material is available free of charge via the Internet at http://pubs.acs.org. 6425
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G.; Anders, R. F.; Miura, K.; Haynes, J. D.; Batchelor, A. H. Overcoming antigenic diversity by enhancing the immunogenicity of conserved epitopes on the malaria vaccine candidate apical membrane antigen-1. PLoS Pathog. 2013, 9, e1003840. (23) Srinivasan, P.; Beatty, W. L.; Diouf, A.; Herrera, R.; Ambroggio, X.; Moch, J. K.; Tyler, J. S.; Narum, D. L.; Pierce, S. K.; Boothroyd, J. C.; Haynes, J. D.; Miller, L. H. Binding of Plasmodium merozoite proteins RON2 and AMA1 triggers commitment to invasion. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 13275−13280. (24) Bai, T.; Becker, M.; Gupta, A.; Strike, P.; Murphy, V. J.; Anders, R. F.; Batchelor, A. H. Structure of AMA1 from Plasmodium falciparum reveals a clustering of polymorphisms that surround a conserved hydrophobic pocket. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 12736− 12741. (25) Harris, K. S.; Casey, J. L.; Coley, A. M.; Masciantonio, R.; Sabo, J. K.; Keizer, D. W.; Lee, E. F.; McMahon, A.; Norton, R. S.; Anders, R. F.; Foley, M. Binding hot spot for invasion inhibitory molecules on Plasmodium falciparum apical membrane antigen 1. Infect. Immun. 2005, 73, 6981−6989. (26) Tonkin, M. L.; Roques, M.; Lamarque, M. H.; Pugniere, M.; Douguet, D.; Crawford, J.; Lebrun, M.; Boulanger, M. J. Host cell invasion by apicomplexan parasites: insights from the co-structure of AMA1 with a RON2 peptide. Science 2011, 333, 463−467. (27) Vulliez-Le Normand, B.; Tonkin, M. L.; Lamarque, M. H.; Langer, S.; Hoos, S.; Roques, M.; Saul, F. A.; Faber, B. W.; Bentley, G. A.; Boulanger, M. J.; Lebrun, M. Structural and functional insights into the malaria parasite moving junction complex. PLoS Pathog. 2012, 8, e1002755. (28) Harris, K. S.; Casey, J. L.; Coley, A. M.; Karas, J. A.; Sabo, J. K.; Tan, Y.; Dolezal, O.; Norton, R. S.; Hughes, A. B.; Scanlon, D.; Foley, M. Rapid optimisation of a peptide inhibitor of malaria parasite invasion by comprehensive N-methyl scanning. J. Biol. Chem. 2009, 284, 9361−9371. (29) Coley, A. M.; Gupta, A.; Murphy, V. J.; Bai, T.; Kim, H.; Foley, M.; Anders, R. F.; Batchelor, A. H. Structure of the malaria antigen AMA1 in complex with a growth-inhibitory antibody. PLoS Pathog. 2007, 3, 1308−1319. (30) Henderson, K. A.; Streltsov, V. A.; Coley, A. M.; Dolezal, O.; Hudson, P. J.; Batchelor, A. H.; Gupta, A.; Bai, T.; Murphy, V. J.; Anders, R. F.; Foley, M.; Nuttall, S. D. Structure of an IgNAR−AMA1 complex: targeting a conserved hydrophobic cleft broadens malarial strain recognition. Structure 2007, 15, 1452−1466. (31) Collins, C. R.; Withers-Martinez, C.; Bentley, G. a.; Batchelor, A. H.; Thomas, A. W.; Blackman, M. J. Fine mapping of an epitope recognized by an invasion-inhibitory monoclonal antibody on the malaria vaccine candidate apical membrane antigen 1. J. Biol. Chem. 2007, 282, 7431−7441. (32) Lim, S. S.; Debono, C. O.; MacRaild, C. A.; Chandrashekaran, I. R.; Dolezal, O.; Anders, R. F.; Simpson, J. S.; Scanlon, M. J.; Devine, S. M.; Scammells, P. J.; Norton, R. S. Development of inhibitors of Plasmodium falciparum apical membrane antigen 1 based on fragment screening. Aust. J. Chem. 2013, 66, 1530−1536. (33) Srinivasan, P.; Yasgar, A.; Luci, D. K.; Beatty, W. L.; Hu, X.; Andersen, J.; Narum, D. L.; Moch, J. K.; Sun, H.; Haynes, J. D.; Maloney, D. J.; Jadhav, A.; Simeonov, A.; Miller, L. H. Disrupting malaria parasite AMA1−RON2 interaction with a small molecule prevents erythrocyte invasion. Nature Commun. 2013, 4, 2261. (34) Liu, J. J.; Horst, R.; Katritch, V.; Stevens, R. C.; Wüthrich, K. Biased signaling pathways in β2-adrenergic receptor characterized by 19 F-NMR. Science 2012, 335, 1106−1110. (35) Pomerantz, W. C.; Wang, N.; Lipinski, A. K.; Wang, R.; Cierpicki, T.; Mapp, A. K. Profiling the dynamic interfaces of fluorinated transcription complexes for ligand discovery and characterization. ACS Chem. Biol. 2012, 7, 1345−1350. (36) Luck, L. A.; Barse, J. L.; Luck, A. M.; Peck, C. H. Conformational changes in the human estrogen receptor observed by 19F NMR. Biochem. Biophys. Res. Commun. 2000, 270, 988−991. (37) Evanics, F.; Kitevski, J. L.; Bezsonova, I.; Forman-Kay, J.; Prosser, R. S. 19F NMR studies of solvent exposure and peptide 6426
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binding to an SH3 domain. Biochim. Biophys. Acta 2007, 1770, 221− 230. (38) Coley, A. M.; Campanale, N. V.; Casey, J. L.; Hodder, A. N.; Crewther, P. E.; Anders, R. F.; Tilley, L. M.; Foley, M. Rapid and precise epitope mapping of monoclonal antibodies against Plasmodium falciparum AMA1 by combined phage display of fragments and random peptides. Protein Eng. 2001, 14, 691−698. (39) Kim, H. W.; Perez, J. A.; Ferguson, S. J.; Campbell, I. D. The specific incorporation of labelled aromatic amino acids into proteins through growth of bacteria in the presence of glyphosate. Application to fluorotryptophan labelling to the H(+)-ATPase of Escherichia coli and NMR studies. FEBS Lett. 1990, 272, 34−36. (40) Gupta, A.; Bai, T.; Murphy, V.; Strike, P.; Anders, R. F.; Batchelor, A. H. Refolding, purification, and crystallization of apical membrane antigen 1 from Plasmodium falciparum. Protein Expression Purif. 2005, 41, 186−198. (41) Tonkin, M. L.; Crawford, J.; Lebrun, M. L.; Boulanger, M. J. Babesia divergens and Neospora caninum apical membrane antigen 1 (AMA1) structures reveal selectivity and plasticity in apicomplexan parasite host cell invasion. Protein Sci. 2012, 22, 114−127. (42) Song, L.; Teng, Q.; Phillips, R. S.; Brewer, J. M.; Summers, A. O. 19 F-NMR reveals metal and operator-induced allostery in MerR. J. Mol. Biol. 2007, 371, 79−92. (43) Mikroyannidis, J. A. Aromatic polyamides and polyimides with benzimidazole or benzoxazinone pendent groups prepared from 5-(2benzimidazole)- or 5-(2-benzoxazinone)-1,3-phenylenediamine. Polymer 1996, 37, 2715−2721. (44) Carpenter, A. J.; Al-Barazanji, K. A.; Barvian, K. K.; Bishop, M. J.; Britt, C. S.; Cooper, J. P.; Goetz, A. S.; Grizzle, M. K.; Hertzog, D. L.; Ignar, D. M.; Morgan, R. O.; Peckham, G. E.; Speake, J. D.; Swain, W. R. Novel benzimidazole-based MCH R1 antagonists. Bioorg. Med. Chem. Lett. 2006, 16, 4994−5000. (45) Lee, B. W.; Lee, S. D. [5,5] Sigmatropic shift of N-phenyl-N′-(2thiazolyl)hydrazines and N,N′-bis(2-thiazolyl)hydrazines into 2amino-5-(p-aminophenyl)thiazoles and 5,5′-bis(2-aminothiazole) derivatives. Tetrahedron Lett. 2000, 41, 3883−3886. (46) Anders, R. F.; Crewther, P. E.; Edwards, S.; Margetts, M.; Matthew, M. L.; Pollock, B.; Pye, D. Immunisation with recombinant AMA-1 protects mice against infection with Plasmodium chabaudi. Vaccine 1998, 16, 240−247. (47) Hodder, A. N.; Crewther, P. E.; Matthew, M. L.; Reid, G. E.; Moritz, R. L.; Simpson, R. J.; Anders, R. F. The disulfide bond structure of Plasmodium apical membrane antigen-1. J. Biol. Chem. 1996, 271, 29446−29452.
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Ligand-Induced Conformational Change of Plasmodium falciparum AMA1 Detected Using 19F NMR Xiaopeng Ge,†,§ Christopher A. MacRaild,‡,§ Shane M. Devine,‡ Cael O. Debono,‡ Geqing Wang,‡ Peter J. Scammells,‡ Martin J. Scanlon,‡ Robin F. Anders,† Michael Foley,*,† and Raymond S. Norton*,‡ †
Department of Biochemistry, La Trobe University, Melbourne 3086, Victoria, Australia Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville 3052, Victoria, Australia
‡
S Supporting Information *
ABSTRACT: We established an efficient means of probing ligand-induced conformational change in the malaria drug target AMA1 using 19F NMR. AMA1 was labeled with 5fluorotryptophan (5F-Trp), and the resulting 5F-Trp resonances were assigned by mutagenesis of the native Trp residues. By introducing additional Trp residues at strategic sites within a ligand-responsive loop, we detected distinct conformational consequences when various peptide and small-molecule ligands bound AMA1. Our results demonstrate an increase in flexibility in this loop caused by the native ligand, as inferred from, but not directly observed in, crystal structures. In addition, we found evidence for long-range allosteric changes in AMA1 that are not observed crystallographically. This method will be valuable in ongoing efforts to identify and characterize therapeutically relevant inhibitors of protein−protein interactions involving AMA1 and is generalizable to the study of ligand-induced conformational change in a wide range of other drug targets.
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INTRODUCTION There are approximately 200 million cases of malaria each year, resulting in over 600000 deaths, mostly caused by Plasmodium falciparum infections.1 The RTS,S malaria vaccine is showing some efficacy in an ongoing phase III trial,2−4 but antimalarial drugs will continue to play a critical role in controlling and treating the disease for the foreseeable future. However, the repeated emergence of drug-resistant parasites has made it essential that the development of new antimalarial drugs continues.5,6 A range of new potential drug targets has been identified as a result of sequencing the parasite’s genome and from a more detailed understanding of the parasites’ life cycle at the molecular level.7−9 To cause disease, parasite merozoites must invade host erythrocytes, a process that involves initial attachment, reorientation, tight-junction formation, and engagement of the parasite actin−myosin motor to drive the parasite into the erythrocyte.10,11 The mechanisms underlying these processes are unique to Plasmodium and related parasites, making them potential targets for the development of new antimalarial drugs.12,13 Apical membrane antigen 1 (AMA1) is a well-characterized protein involved in parasite invasion that is being studied as a potential target of new antimalarial drugs.13 After release from the merozoite micronemes, PfAMA1 forms a complex with another parasite antigen, Pf RON2, which is located at the moving junction.14,15 Recent conditional knockdown studies have generated some controversy regarding the precise role of this complex,16,17 but vaccine studies have nonetheless shown that targeting AMA1 can markedly inhibit development of asexual blood-stages of P. falciparum,18−20 and antibodies and © 2014 American Chemical Society
peptides targeting AMA1 have been shown to inhibit erythrocyte invasion by blocking the AMA1−RON2 interaction.21−23 Structures of the AMA1 ectodomain revealed the presence of a hydrophobic cleft that is conserved across apicomplexan parasites24 and has been suggested to be the principal ligand-binding site on AMA1.21,25 The recent cocrystal structure of AMA1 with RON2L, a peptide corresponding to the extracellular region of RON2, confirmed that the hydrophobic cleft is the site of RON2 binding.26,27 This structure also revealed a conformational change in which RON2L displaces the AMA1 domain II (DII) loop to expose a positively charged binding site that interacts with a complementary charged region at the N-terminus of peptide RON2L.26,27 R1, an AMA1-binding peptide selected from a peptide library displayed on phage,25,28 competes with RON2L for binding to this site and induces a similar conformational change of the AMA1 DII loop.27 A diverse range of ligands, including the peptides RON2L and R1, monoclonal antibodies and shark antibodies (IgNARs), bind to the hydrophobic cleft and block the interaction between AMA1 and RON2.25,27,29−31 This has stimulated research to identify inhibitors of this interaction that may be valuable drug leads. Two approaches, high-throughput screening and fragment-based ligand discovery, have been used to identify small molecules that bind to AMA1 and compete with peptide RON2L or R1.32,33 To understand the mechanisms by which these small molecules bind to AMA1, and to better Received: March 13, 2014 Published: July 28, 2014 6419
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evaluate their potential as inhibitors of the AMA1−RON2 interaction, we have explored methods that would allow detection of conformational changes of the DII loop induced by ligand binding. 19 F NMR spectroscopy has proved sensitive to ligandinduced conformational changes in a range of other proteins of biological and therapeutic interest.34−37 In this study, we have applied 19F NMR spectroscopy to the detection of ligandinduced movements of the DII loop of PfAMA1. Tryptophan residues were inserted into specific sites within the DII loop by site-directed mutagenesis, then all Trp in the protein were substituted with 5-fluorotryptophan (5F-Trp) by expression in the presence of glyphosate. 19F NMR was then used to identify small AMA1-binding molecules among our current fragment hits32 that induced a conformational change in AMA1 similar to that of known inhibitors. This approach will be used to characterize other AMA1-binding molecules that are being assessed as drug leads. More generally, it highlights the power of 19F NMR as an efficient probe of protein−ligand binding and associated conformational change.
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RESULTS AND DISCUSSION Construction of AMA1 Mutants. Because none of the four Trp residues in AMA1 is in the flexible DII loop, mutant forms of 3D7 PfAMA1108−434 (corresponding to domains I and II of the AMA1 ectodomain) were created, either by replacing individual aromatic residues in the loop with Trp or by inserting an additional Trp in the middle of the loop. The insertion site was chosen at a highly exposed site, between two small residues (A372 and S373), where insertion was deemed least likely to perturb the structure of the DII loop (Figure 1A). Each of the mutant proteins was expressed on the surface of phage38 so that they could readily be assessed for their ability to bind the conformationally dependent monoclonal antibodies (mAb) 1F9 and 4G231 and peptide RON2L (Table 1). All three reagents bound the replacement mutant F367W and the insertion mutant IS373W equivalently to wild-type AMA1 (Figure 1). In contrast, the binding of the mAbs and peptide to the other replacement mutants was reduced (F360W, F379W, and F385W) or abolished (Y353W and Y390W). Consequently, mutants F367W and IS373W were selected for the 19F NMR studies described below. These mutants, together with wild-type AMA1 DI+II, were expressed in Escherichia coli as 5FTrp-labeled recombinant proteins by growing the bacteria in minimal media supplemented with glyphosate to inhibit aromatic amino acid metabolism,39 and with 5F-Trp, Phe, and Tyr. Expression, Purification, and Characterization of FTrp-AMA1 and Its Mutants. The 5F-Trp-labeled recombinant proteins were isolated and refolded following the previously published procedure,40 modified by the addition of 0.6 M L-arginine to the refolding buffer. This resulted in a high yield of each form of AMA1 DI+II, all of which were judged to be >95% pure by nonreducing SDS-PAGE. Mass spectrometry confirmed the identity of the expressed constructs and was consistent with 5F-Trp incorporation to the level of approximately 85% (data not shown). The conformationally dependent mAb 1F9 reacted well on Western blots with all three forms of AMA1, indicating the presence of correctly refolded protein (Figure 2A). The binding affinities of 5F-TrpAMA1 for the peptides RON2L and R1 were found to be 5 and 23 nM, respectively, using SPR measurements. The replacement mutant F367W bound both peptides with affinities nearly
Figure 1. (A) Location of tryptophan residues and the domain II loop in AMA1. The structure of AMA1 DI+II (PDB ID 2Z8V) showing the locations of the four tryptophan residues, W110, W298, W315, and W399 (yellow). None of the four Trp residues is located in the domain II loop (blue) or hydrophobic cleft (green). Also shown are the sites of Trp mutation, F367 (red), and insertion, S373. (B) mAb reactivity of AMA1 DII loop mutants displayed on the surface of phage. Phage displaying wild-type and mutant forms of AMA1 were allowed to bind to immobilized mAb 1F9, mAb 4G2, and RON2L peptide. The average of duplicated wells is shown, and error bars indicate the range.
Table 1. Comparison of the Reactivity of Phage-Displayed AMA1 DII Loop Mutants and Wild-Type AMA1a reactivity with AMA1-binding ligands phage-displayed AMA1
mAb 1F9
mAb 4G2
peptide RON2L
wild-type AMA1 replacement mutant Y353W replacement mutant Y360W replacement mutant F367W* replacement mutant F379W replacement mutant F385W replacement mutant Y390W insertion mutant IS373W*
+++ − + +++ + ++ − +++
+++ − ++ +++ − + − +++
+++ − ++ +++ + + − +++
* indicates the two mutant forms of AMA1 DI+II selected for expression in E. coli as 5F-Trp recombinant proteins. +++ indicates an OD >2.5 for mAbs and >1.5 for peptide, ++ indicates an OD >1.5 for mAbs and >1 for peptide, + indicates an OD >0.5 for mAbs and >0.5 for peptide, − indicates an OD 90% purity (GL Biochem, China). Both peptides with two cysteine residues were cyclized with correctly formed intramolecular disulfide bonds. AMA1 binding fragments 5-(1H-benzo[d]imidazo-2-yl)benzene-1,3diamine (1),43 2-(piperidin-1-yl)-1H-benzo[d]imidazol-6-amine (2),44 and 5-(4-aminophenyl)-4-methylthiazol-2-amine (3)45 were synthesized by established procedures. Phage-Displayed AMA1 Mutants. All AMA1 mutant genes in the pHENH6 vector were transformed into Escherichia coli TG1 strain, and the cells were grown at 37 °C in 2YT medium with shaking until the culture reached log phase. The culture was subsequently left stationary at room temperature for 15 min to allow the regeneration of the F-pilus. Phages were then rescued from the E. coli cells by adding 108 helper phage, followed by incubation at 37 °C stationary for 1 h. Finally, 70 μg/mL of kanamycin was added to the culture, which was then incubated at 37 °C overnight with shaking. The overnight culture was centrifuged at 7700g for 10 min, and phage in the supernatant was precipitated using 5× PEG solution (30% (w/v) PEG8000, 2.6 M NaCl). Following incubation at 4 °C for 2 h, the precipitated phage were centrifuged at 15000g for 30 min, and the phage pellet was resuspended in 1 mL of PBS and stored at −80 °C for subsequent analysis. Phage ELISA. Microtiter plates were coated with 0.2 μg/well of mAbs 1F9 or 4G2 overnight at 4 °C. For peptide RON2L, a neutravidin plate was coated overnight with 0.2 μg/well of biotinylated RON2L peptide in PBS/0.1% Tween/0.05% BSA. Phage expressing the desired proteins were diluted 1 in 10 in PBS−Tween and allowed to interact with the immobilized antigen for 1 h with shaking. After washing, the phage were subsequently detected using a horseradish peroxidase (HRP)-conjugated anti-M13 monoclonal antibody. Binding was visualized using 100 μL/well of 3,3′,5,5′-tetramethylbenzidine solution, and the reaction was stopped by adding 100 μL/well of 1 M HCl. The absorbance was then read at 450 nm. Recombinant 5F-Trp-AMA1 and Its Mutants Proteins. The genes for wild-type 3D7 PfAMA1108−434 and its mutants W110F,
Figure 5. 19F NMR spectra of 5-5F-Trp-F367W AMA1 bound to small molecule ligands 1 (A), 2 (B), and 3 (C). Spectra are shown for free FTrp-F367W AMA1 (black) and in the presence of 1 mM (red) and 3 mM (blue) ligand.
bind to the hydrophobic cleft, more than 30 Å from W110. Although we cannot rule out the possibility of a secondary binding site for each of these ligands close to W110 on DII, the generality of this effect across peptide and small molecule ligands suggests a long-range conformational coupling between the two sites, such that the binding of these ligands at the hydrophobic cleft induces conformational change at this distant site. The detection of allosteric structural change of this nature by 19F NMR is well established.34,42 As noted above, any such conformational coupling is not evident from the available crystal structures, suggesting that it may be dynamic in nature. It is, however, consistent with the extensive changes seen in the 1 H−15N HSQC spectrum of AMA1 DI+II on binding these ligands (our unpublished data). 6424
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W298F, W315F, F367W, and IS373W were cloned into the expression vector pET26b(+) and transformed into E. coli BL21 (DE3) strain. Bacteria were grown overnight at 37 °C in LB medium, containing 100 μg/mL of ampicillin, and subsequently diluted 1 in 10 in M9 medium supplemented with 2% glucose (w/v), 1× BME vitamins, 1× trace elements, 2 mM MgSO4, and 0.1 mM CaCl2. Then 1 g/L of glyphosate, 50 mg/L of Phe, 50 mg/L of Tyr, and 36 mg/L of 5F-Trp were added to the medium when the cell density reached ∼0.4. After shaking for 1 h, the expression of 5F-Trp-labeled proteins was induced by addition of 1 mM (final) IPTG for 3 h, then cells were harvested by centrifugation at 5000g for 15 min. Protein Refolding, Purification, and Characterization. 5FTrp-labeled AMA1 and its mutants were expressed, solubilized, and isolated as described.20 The recombinant proteins were subsequently refolded overnight in the conventional refolding buffer with the addition of 0.6 M L-Arg.46 To correctly form the five intramolecular disulfide bonds in AMA1 DI+II,47 reduced glutathione and oxidized glutathione were then added to the refolding buffer at final concentrations of 0.1 and 0.025 M, respectively. After refolding, the proteins were purified through a HiTRAP QFF (GE Healthcare) ionexchange column and the sample buffer was changed to 20 mM phosphate buffer, pH 7.4. The binding of conformationally sensitive mAbs to F-Trp-AMA1 and its mutants was assessed using Western blot. All protein samples were run on SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was subsequently probed with 1 μg/mL of monoclonal antibody 1F9, and proteins were detected using HRP-conjugated antimouse antibody (1 in 2000 dilution, Millipore). Surface Plasmon Resonance. All SPR analyses were performed using ProteOn XPR 36 platform (BioRad) and carried out in PBS− Tween (0.1%) at 25 °C using a constant flow rate of 30 μL/min. AMA1 was immobilized on a GLC sensor chip following the manufacturer’s recommended protocol. Peptides RON2L and R1 were flowed across the surface of the chip at a constant flow rate for 8 min, then dissociated from the AMA1 surface for 800 s by flowing PBS−Tween across the surface of the chip. Peptides were tested at a range of concentrations (50−3.125 nM) and a lane without AMA1 was included as a negative control. Signals generated by buffer-only or DMSO-only control experiments were subtracted from the resulting sensorgrams to indicate the binding of the peptides to AMA1. All peptide samples were diluted in PBS−Tween, and the surface of the chip was regenerated by injecting 30 μL of 0.85% phosphoric acid (flow rate = 100 μL/min) in all channels in the vertical orientation of the ProteOn XPR36 fluidics. NMR Spectroscopy. 19F NMR spectra were acquired on a Bruker Avance III spectrometer operating at 564 MHz (600 MHz 1H frequency) and at 25 °C using a TCI triple-resonance cryoprobe, with the 1H channel tuned for 19F. Samples contained ∼100 μM 5F-Trp AMA1 in 20 mM sodium phosphate, pH 7.4, with 0.005% trifluoroethanol as internal reference for 19F chemical shift (−1.375 ppm relative to trace TFA in water) and signal intensity and 7% 2H2O for the spectrometer lock signal. R1 and RON2L were added from 1 mM stocks in 20 mM sodium phosphate, pH 7.4, to a final peptide:AMA1 ratio of 1.2:1, while trRON2 was used at a ratio of 5:1 to account for its lower affinity. Small molecule ligands of AMA1 were added from 100 mM stocks in H2O (2), ethanol (1), or DMSO (3). Spectra were typically acquired as 2K scans of 8K complex points over a 150 ppm sweep width, for a total experimental time of less than 40 min. Spectra were processed using 20 Hz exponential line-broadening prior to Fourier transformation. Line widths were estimated by leastsquares fits to a sum of Lorentzians.
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AUTHOR INFORMATION
Corresponding Authors
*For R.S.N.: phone, +61 3 9903 9167; fax, +61 3 9903 9582; Email, [email protected]. *For M.F.: phone, +61 3 9479 2158; E-mail, m.foley@latrobe. edu.au. Author Contributions §
X.G. and C.A.M. contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Krishnarjuna Bankala for assistance with mass spectrometry. This work was supported in part by an Australian National Health and Medical Research Council (NHMRC) project grant (1025150). R.S.N. acknowledges fellowship support from the NHMRC.
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ABBREVIATIONS USED 5F-Trp, 5-fluorotryptophan; AMA1, apical membrane antigen 1; DII, domain II; DI+II, domains I and II; mAb, monoclonal antibody
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REFERENCES
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ASSOCIATED CONTENT
S Supporting Information *
Table showing 19F line widths of 5F-Trp resonances of F367W AMA1 in the presence of RON2L and R1; figure showing the location of the native Trp residues in AMA1, and figures showing 1H NMR spectra of AMA1 mutants. This material is available free of charge via the Internet at http://pubs.acs.org. 6425
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