Antimalarial activity of cupredoxins: the interaction of Plasmodium Merozoite Surface Protein 119 (MSP119) and Rusticyanin

Antimalarial Activity of Cupredoxins THE INTERACTION OF PLASMODIUM MEROZOITE SURFACE PROTEIN 1 19 (MSP1 19 )AND RUSTICYANIN * Received for publication, February 14, 2013, and in revised form, June 7, 2013 Published, JBC Papers in Press, June 7, 2013, DOI 10.1074/jbc.M113.460162 Isabel Cruz-Gallardo ‡1 , Irene Díaz-Moreno ‡ , Antonio Díaz-Quintana ‡ , Antonio Donaire §2 , Adrián Velázquez-Campoy ¶3 , Rachel D. Curd 储4 , Kaveri Rangachari 储 , Berry Birdsall**, Andres Ramos** 5 , Anthony A. Holder 储6 , and Miguel A. De la Rosa ‡7 From the ‡ Instituto de Bioquímica Vegetal y Fotosíntesis (IBVF), cicCartuja, Universidad de Sevilla-CSIC, Avenida Américo Vespucio 49, Sevilla 41092, Spain, the § Departamento de Química Inorgánica, Facultad de Química, Universidad de Murcia, Campus Universitario de Espinardo, Murcia 30100, Spain, the ¶ Instituto de Biocomputación y Física de Sistemas complejos (BIFI), Universidad de Zaragoza, c/Mariano Esquillor, Zaragoza 50018, Spain, the 储 Parasitology Division and **Molecular Structure Division, Medical Research Council (MRC) National Institute for Medical Research, The Ridgeway, Mill Hill, London W7 1AA, United Kingdom Background: The interaction of MSP1 19 with the cupredoxin azurin inhibits the growth of Plasmodium falciparum in red blood cells. Results: Rusticyanin forms a well defined complex with MSP1 19 upon binding at the same surface area than inhibitory antibodies. Conclusion: Rusticyanin becomes an excellent therapeutic agent for malaria. Significance: Knowing the rusticyanin-MSP1 19 interface will allow the design of novel antimalarial drugs. The discovery of effective new antimalarial agents is urgently needed. One of the most frequently studied molecules anchored to the parasite surface is the merozoite surface protein-1 (MSP1). At red blood cell invasion MSP1 is proteolytically processed, and the 19-kDa C-terminal fragment (MSP1 19 ) remains on the surface and is taken into the red blood cell, where it is transferred to the food vacuole and persists until the end of the intracellular cycle. Because a number of specific antibodies inhibit erythrocyte invasion and parasite growth, MSP1 19 is therefore a promising target against malaria. Given the structural homology of cupredoxins with the Fab domain of monoclonal antibodies, an approach combining NMR and isothermal titration calorimetry (ITC) measurements with docking calculations based on BiGGER is employed on MSP1 19 -cupredoxin complexes. Among the cupredoxins tested, rusticyanin forms a well defined complex with MSP1 19 at a site that overlaps with the surface recognized by the inhibitory antibodies. The addition of holo-rusticyanin to infected cells results in parasitemia inhibition, but negligible effects on parasite growth can be observed for apo-rusticyanin and other proteins of the cupredoxin family. These findings point to rusticyanin as an excellent therapeutic tool for malaria treatment and provide valuable information for drug design. Malaria is a widely spread disease causing morbidity and mortality throughout a large part of the world. The increasing resistance of Plasmodium falciparum, the causative agent of the most deadly form of the disease, to current drugs has only increased the urgency for finding new antimalarial agents (1, 2), including an effective vaccine and new drug therapies (3, 4). There are five Plasmodium species that infect humans, whereas others infect other primates or rodents. Among this latter group, rodent parasites such as Plasmodium yoelii provide useful laboratory models for the study of malaria. The disease is caused by the replication and multiplication of the asexual blood stages in red blood cells. The merozoite form of the parasite invades the host cell, where it develops and replicates to form several new merozoites that then burst out of the cell to continue the cycle of invasion and multiplication. The invasion of red blood cells requires an initial recognition and binding mediated by parasite surface ligands, followed by reorientation and the formation of a moving junction between the erythrocyte and merozoite surfaces as the parasite enters the cell. Merozoite surface protein-1 (MSP1) 8 has been implicated in this initial binding between parasite and host cell. Located on the surface of the asexual blood-stage schizont and merozoite, MSP1 is one of the most frequently studied molecules of the parasite (5). It is synthesized as a ⬃200-kDa *This work was supported in part by Junta de Andalucía Grants P08-CVI-3876 and BIO198 (Spain) and European Social Fund-ERDF 2007–2013. 1 Supported by Junta de Andalucía Ph.D. Grant P08-CVI-3876 and GERMN bursaries for short term stays (Spain). 2 Supported by Ministerio de Economía y Competitividad Grant SAF201126611 and Fundación Séneca de la Región de Murcia Grant 15354/PI/10 (Spain). 3 Supported by Ministerio de Ciencia e Innovación Grant BFU2010-19451 (Spain). 4 Recipient of a Medical Research Council studentship. 5 Supported by Medical Research Council Grant U117574558. 6 Laboratory work supported by Medical Research Council Grant U117532067 and the European Union FP7 Network of Excellence EviMalar. 7 To whom correspondence should be addressed. Tel.: 34-954489506; Fax: 34-954460065; E-mail: [email protected]. 8 The abbreviations used are: MSP1, merozoite surface protein-1; Az, azurin; BiGGER, bimolecular complex generation with global evaluation and ranking; HSQC, heteronuclear single-quantum coherence; ITC, isothermal titration calorimetry; mAb, monoclonal antibody; MSP1 19 , MSP1 19-kDa C-terminal fragment; Pc, plastocyanin; PDB, Protein Data Bank; Rc, rusticyanin. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 29, pp. 20896–20907, July 19, 2013 © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. 20896 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288•NUMBER 29•JULY 19, 2013 at FAC BIOLOGIA/BIBLIOTECA on February 12, 2018http://www.jbc.org/Downloaded from precursor attached to the surface of the parasite via a glycosylphosphatidylinositol anchor, which undergoes a two-step proteolytic process: first, at merozoite release and then at erythrocyte invasion (6). As a result of this processing, the MSP1 is cleaved into several polypeptides that are shed from the surface in the final processing step, save a 19-kDa C-terminal fragment (MSP1 19 ). MSP1 19 is retained on the parasite surface by the glycosylphosphatidylinositol anchor and taken into the red blood cell at invasion (7–9). The role of MSP1 19 in the subsequent intracellular development of the parasite is poorly understood, although it is transferred to the developing food vacuole, where it remains until the end of the intracellular cycle and is discarded in the residual body together with products of hemoglobin digestion such as hemozoin (10). MSP1 19 is considered a promising malaria vaccine candidate due to the abundant evidence of specific antibodies inhibiting erythrocyte invasion and parasite growth, for instance, via the disruption of MSP1 proteolytic processing and intracellular parasite development (11). At the structural and functional levels, MSP1 19 is particularly well conserved among Plasmodium species (Fig. 1) (12–17), and its three-dimensional structure has been shown to consist of two epidermal growth factor (EGF)-like domains in close contact. A characteristic disulfide-bridge pattern (Fig. 1) makes MSP1 19 highly resistant to proteases (19) and may explain why MSP1 19 remains intact in the digestive food vacuole up to the end of the intracellular cycle (10). MSP1-specific immunoglobulins react with conformational epitopes of MSP1 19 . Some of these antibodies inhibit parasite invasion of erythrocytes, whereas others do not. Fine structure epitope mapping of different monoclonal antibodies (mAbs) and the use of NMR methods indicates the binding of two inhibitory antibodies to epitopes on one side of the molecule near the interface between the two EGF domains, including residues from both domains (20, 21). By contrast, non-inhibitory neutral mAbs bind elsewhere on the molecule (15, 20). Here, we have used MSP1 19 from P. yoelii, a rodent malaria parasite used as a laboratory model for vaccine studies (22) and for which both inhibitory and neutral antibodies have been partially mapped on the structure (23, 24). 9 Independently of the immunoglobulin class, complexes involving MSP1 19 are kinetically rather stable with dissociation constants in the micromolar to subnanomolar range (15, 25). 10 Because the binding affinity is similar in all cases, it has been assumed that the inhibitory effect depends mainly on steric factors, namely, epitope location, rather than the nature of the antibody. The present study has been based on the structural homology of cupredoxins with the Fab fragment of an antibody, as well as on reports of a protein from this family interacting directly with MSP1 19 and blocking the increase of parasitemia in human red blood cells infected by P. falciparum, suggesting a promising treatment (26, 27). We first used the DaliLite pairwise comparison program to identify structural similarities between cupredoxins and the Fab fragment (28). A screening was then performed combining NMR and ITC measurements with docking calculations using BiGGER which indicated that, among the cupredoxins tested, rusticyanin (Rc) provided the most effective binding to MSP1 19 . The two proteins form a well defined complex where Rc interacts at the interface between the two MSP1 19 subdomains, at a site that overlaps with the surface recognized by the inhibitory antibodies. Further, P. falciparum growth is inhibited by the presence of Rc in red blood cell cul9 R. D. Curd and A. A. Holder, unpublished data. 10 M. J. Lock and A. A. Holder, unpublished data. FIGURE 1. MSP1 19 protein. A, multiple sequence alignment of MSP1 19 with ClustalW2 (18) from a number of Plasmodium species. Sequences are colored by percent identity: black (100%), dark gray (80%), light gray (60%), and white (⬍50%). Secondary structure elements of P. yoelii MSP1 19 are shown. Orange boxes highlight cysteine residues that are conserved across different Plasmodium species, whereas red boxes highlight those present only in P. falciparum.Gold arrows stand for disulfide bonds formed by cysteine residues. The main difference between P. falciparum MSP1 19 and homologs from other Plasmodium species characterized to date lies in the disulfide bond pattern of the protein from the other species: one of the cysteine pairs in the first EGF domain is substituted by a tryptophan and a nonpolar or aliphatic residue. B, ribbon representation of the lowest energy NMR structure of P. yoelii MSP1 19 (BMRB accession number: 19233). Side chains of cysteine residues forming disulfide bridges are represented in gold. MSP1 19 -Rusticyanin Complex JULY 19, 2013•VOLUME 288•NUMBER 29 JOURNAL OF BIOLOGICAL CHEMISTRY 20897 at FAC BIOLOGIA/BIBLIOTECA on February 12, 2018http://www.jbc.org/Downloaded from tures. Interestingly, the copper site plays a key role in complex formation, because apo-Rc is not only unable to interact with MSP1 19 , but also to inhibit parasite invasion and development in infected red blood cells. EXPERIMENTAL PROCEDURES Expression and Purification of Proteins—P. yoelii MSP1 19 ( 15 N-labeled or unlabeled) was produced (essentially as described previously (29)) from a synthetic gene optimized for Pichia pastoris expression using as nitrogen source either 15 NH 4 Cl or (NH 4 ) 2 SO 4 for labeled and unlabeled protein, respectively. The 99-amino acid sequence corresponds to residues 1656–1754 of the UniProtKB entry P13828 containing the N-terminal tag HHHHHHIEGR that has little effect on the NMR spectrum (20). Secreted His 6 -tagged MSP1 19 was purified from the culture medium by nickel affinity chromatography (Ni-Sepharose 6 Fast Flow; GE Healthcare) and according to a previously elaborated protocol (12). All recombinant metalloproteins were expresed in Escherichia coli cultures in LB medium and purified according to previously elaborated procedures, namely Acidithiobacillus ferrooxidans Rc (30, 31), Nostoc sp. PCC 7119, Phormidium laminosum and poplar plastocyanins (Pc) (32–34) and Pseudomonas aeruginosa azurin (Az) (35). NMR Spectroscopy—All protein samples were concentrated in 10 mMpotassium phosphate (pH 6.5) using Millipore 3000 NMWL centricons and microcons. MSP1 19 samples ranged in concentration from 0.5 to 2 mM, whereas cupredoxins were used in the range of 2–5 mM. All NMR samples contained 10% D 2 O to adjust the lock signal. Reduction of the metal center in samples of copper(I) cupredoxins was achieved by adding sodium ascorbate, whereas oxidation of the metal center in samples of copper(II) proteins was achieved using sodium ferricyanide for Pc and Az, and sodium hexachloroiridate(IV) for Rc. In all cases, the proteins were washed extensively to remove the excess of the reducing/oxidizing agent. NMR experiments were performed in a Bruker Avance 600 MHz spectrometer at 25 °C. The sequence-specific assignment of the backbone amide groups of 15 N MSP1 19 (BMRB accession number: 19233) was achieved using standard backbone experiments (HNCACB, HNCA, etc.) and was confirmed using three-dimensional 1 H15 N NOESY-HSQC and three-dimensional 1 H15 N total correlation spectroscopy-HSQC spectra. The interaction of MSP1 19 with cupredoxins was followed by acquiring two-dimensional 1 H15 N HSQC spectra during the titration of 0.5 mM 15 N-MSP1 19 solutions with an increasing amount of oxidized or reduced cupredoxins up to a final cupredoxin:MSP1 19 molar ratio of 4:1. The pH value of the sample was verified after each titration step. Prolines, which are invisible resonances in 15 N HSQC spectra, are located at the positions 4, 15, 50, 61, 84, and 86, whereas Gly-1, Val-2, Glu-69, and Asn-73 are unassigned residues. All data processing was performed with Bruker TopSpin 2.0, and NMR analysis of line broadening perturbations of the cupredoxin-bound MSP1 19 with respect to free malarial protein was performed in the SPARKY program (36). NMR Line Width Analysis—To estimate line widths, the peaks were fitted to a Gaussian function for the 15 N and 1 H dimensions using the program SPARKY with a 10,000 steps minimization and a 0.05% tolerance. In the analysis of the line widths (⌬ ␯ 1/2 ), the overall broadening (⌬⌬ ␯ 1/2 ) obtained from signals displaying only minor line broadening was first subtracted from the line width of the corresponding signal. Then, for each residue, the differences of line widths between free and interacting MSP1 19 were calculated in every titration series (⌬⌬ ␯ 1/2 Binding ). The threshold value, used to identify a specifically broadened residue when data from the titration series were analyzed together, was defined as the average ⌬⌬ ␯ 1/2 Binding for the system plus 2 standard deviations (2S n⫺1 ). The average ⌬⌬ ␯ 1/2 Binding and standard deviation were calculated for all amides with values ⱕ10 Hz on the basis that data ⬎10 Hz clearly indicated a specifically broadened residue, and their inclusion would bias the average to a higher value. Some assigned signals of the free MSP1 19 HSQC spectrum overlap (Val-9, Asn-87, and Cys-95) or exhibit very low intensity (Gly-41 and Asn-42), so they could not be properly integrated to include them in the line width analysis. ITC—All ITC experiments were performed using VP-ITC and Auto-ITC200 instruments (Microcal; GE Healthcare) at 25 °C titrating Rc with MSP1 19 . The reference cell was filled with distilled water. The experiments consisted of 10- ␮ lor2- ␮ l injections of 0.3 mMMSP1 19 solution in 10 mMpotassium phosphate buffer (pH 6.5) into the sample cell, initially containing 6.67 ␮ MRc solution (reduced, oxidized, and apo forms) in the same buffer. All of the solutions were degassed before the titrations were performed. Titrant was injected at appropriate time intervals to ensure the thermal power signal returned to the base line prior to the next injection. To achieve homogeneous mixing in the cell, the stirring speed was kept constant at 1000 rpm in the Auto-ITC200 and at 450 rpm in the VP-ITC. The data, specifically the heat per injection normalized per mol of injectant versus molar ratio, were analyzed with Origin 7 (Microcal) using a single-site binding model. Calibration and performance tests of the calorimeter were carried out conducting CaCl 2 -EDTA titrations with solutions provided by the manufacturer. Molecular Docking Simulations—A soft docking algorithm implemented in the BiGGER software package (37) was used to determine in silico a model of the complexes MSP1 19 -Az, MSP1 19 -Pc, and MSP1 19 -Rc. The PDB coordinates files of cupredoxins were 1JZG for Az (38), 1NIN for Pc (39), and 1A3Z for Rc (40). For each run, 5000 docking geometric solutions were generated based on the complementarity of the protein surfaces. These solutions were evaluated and ranked according to their “global score” and different interaction criteria including electrostatic energy of interaction, relative solvation energy, and the relative propensity of side chains to interact. For the MSP1 19 -Rc adduct, NMR restraints were introduced in the docking calculations. All complexes graphic images were generated using the UCSF Chimera package (41). P. falciparum Cultures—Synchronized P. falciparum 3D7 late stage trophozoites at 33–36 h were used. The final parasitemia and hematocrit were between 0.1–0.2 and 2%, respectively. Red blood cells used for the assay were centrifuged to remove the buffy coat and washed twice in RPMI 1640 medium so that no white blood cells were present. The culMSP1 19 -Rusticyanin Complex 20898 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288•NUMBER 29•JULY 19, 2013 at FAC BIOLOGIA/BIBLIOTECA on February 12, 2018http://www.jbc.org/Downloaded from ture medium contained RPMI 1640 medium supplemented with 5 g/liter albumax, 0.025 g/liter gentamycin, and 0.292 g/liter L-glutamine. P. falciparum Growth Inhibition Assay—Sterile 96-well black tissue culture plates (Costar) were used routinely for every assay. The holoor apo-species of Rc and Nostoc Pc were diluted in culture medium and used in duplicate wells for each dilution (200, 100, 50, and 25 ␮ M, respectively) in a final volume of 100 ␮ l/well. Two control sets were used in duplicate wells, one set with no added cupredoxin (positive control) and one with uninfected red blood cells (negative control). The plates were incubated at 37 °C for 48 h in a gas chamber flushed with 5% CO 2 ,5%O 2 , and 90% N 2. After 48 h, supernatants were removed from each well, replaced with fresh medium containing protein, and incubated for a further 48 h in the same manner. At the end of the 96-h incubation, 25 ␮ l of SYBR Green I dye (SYBR Green I nucleic acid gel stain 10,000⫻, in dimethyl sulfoxide from Invitrogen) in lysis buffer (1 ␮ l dye to 1 ml lysis buffer), was added to each well and stored overnight at ⫺20 °C. The lysis buffer contained Tris-HCl (20 mM, pH 8.0), EDTA (2 mM), Saponin (0.16% w/v) and Triton X-100 (1.6% v/v). Plates were thawed at room temperature, and fluorescence intensity was measured with a FLUO Star Omega microplate fluorescence reader (BMG Labtech). Values were expressed in relative fluorescence units. Binding of SYBR Green is specific for parasite DNA as mature erythrocytes lack DNA and RNA. Fluorescence intensity unit was converted to percentage (%) of growth as follows: % growth ⫽(culture under Rc or Pc) ⫺(uninfected RBC)/(culture with no Rc or Pc) ⫺(uninfected RBC) ⫻100, where RBC are red blood cells. FIGURE 2. Conserved structural motifs. A, ribbon representation of the Fab fragment of mAb G17.12 (PDB ID code 1OB1). Domain A is depicted in light gray and domain B in gold.B, structural alignment of Az (upper, PDB ID code 1JZG) and A1 fragment of Fab (lower) built by DaliLite pairwise comparison server. Matching regions, with a Dali Z-score of 2.9, appear in orange.C, ribbon representation of Rc (upper, PDB ID code 1A3Z) and Pc (lower, PDB ID code 1NIN). Dali Z-scores for Rc and Pc are 2.6 and 3.1, respectively. Copper atoms are colored in blue. FIGURE 3. Ab initio docking of cupredoxins with MSP1 19 performed by BiGGER. Left, Az. Center, Pc. Right, Rc. Light yellow spheres represent the geometry centers of MSP1 19 in the 100 best solutions generated for each complex. All cupredoxins are oriented with respect to their copper center. Robertson diagrams of the cupredoxins are colored according to secondary structures: ␣ -helices in red and ␤ -strands in blue. Copper atoms are represented as blue spheres and the residues bonding to them are in gray sticks. MSP1 19 -Rusticyanin Complex JULY 19, 2013•VOLUME 288•NUMBER 29 JOURNAL OF BIOLOGICAL CHEMISTRY 20899 at FAC BIOLOGIA/BIBLIOTECA on February 12, 2018http://www.jbc.org/Downloaded from RESULTS Structural Similarities between Cupredoxins and the Fab Fragment of a mAb—The Fab fragment crystal structure of mAb G17.12 (15), shown to bind MSP1 19 , is composed of two domains, A and B, containing two regions, variable (symbolized by 1) and constant (symbolized by 2), with the canonical ␤ -sandwich fold of the immunoglobulin superfamily (Fig. 2A). Consistent with previous reports, the DaliLite pairwise comparison program (28) identified significant structural similarities between the A1 fragment of the mAb and copper-containing redox proteins with immunoglobulin fold, such as Az, Pc, and Rc. In particular, the structural alignment between the A1 fragment and Az reveals a matching region, which is localized mainly at the two antiparallel ␤ -sheets, as described previously (26; Fig. 2B), and with a Dali Z-score of 2.9. For the two other cupredoxins, Pc and Rc, this structural match is extended to the loops connecting ␤ -strands yielding Z-scores of 3.1 and 2.6, respectively (Fig. 2C). Given the structural similarity between various cupredoxins and the Fab fragment, ab initio docking approaches were performed with no experimental restraints (Fig. 3) to explore the capability of the metalloproteins to interact with MSP1 19 from P. yoelii. MSP1 19 is well conserved among the species with ⬎50% sequence identity (Fig. 1A), with conserved three-dimensional structure and common functional features (17). We used the BiGGER rigid docking algorithm to generate sets of possible orientations for the different cupredoxin probes around MSP1 19 (the target). Fig. 3 shows the distribution of MSP1 19 mass centers resulting from the 100 best solutions from each computation around the corresponding copper protein. For Az and Pc, we observe a remarkably broad dispersion of the MSP1 19 geometry centers, suggesting the lack of specific surface complementarity. By contrast, molecular docking for the Rc-MSP1 19 interaction indicates that MSP1 19 explores a well defined area of Rc surrounding its copper center. MSP1 19 Interactions with Cupredoxins by NMR—The MSP1 19 -cupredoxin interaction was monitored by recording two-dimensional 15 N HSQC NMR spectra on 15 N-MSP1 19 , both free and following the addition of Az, Pc, or Rc. The absence of changes in MSP1 19 resonances, either chemical shift perturbations or line broadening, indicates no detectable binding to Az and Pc in any of their oxidation states (Fig. 4, top and middle panels). By contrast, line width changes of certain MSP1 19 amide signals upon addition of Rc(Cu ⫹ ) suggest a specific MSP1 19 -Rc interaction (Fig. 4, bottom panel). Such observations coincide with ab initio docking simulations performed (Fig. 3) which suggested a well defined complex only between Rc and MSP1 19 . To probe in greater detail the interaction of MSP1 19 with either oxidized or reduced Rc, we analyzed the line widths of MSP1 19 resonances from 15 N HSQC spectra across several titrations. Binding to Rc results in general signal broadening due to the increase in the rotational correlation time of MSP1 19 when interacting with Rc. In addition, several MSP1 19 backbone amides clustered in one area of the structure undergo larger changes in line widths (⌬⌬ ␯ 1/2 Binding ) upon Rc binding (Figs. 5 and 6). These resonances are expected to be at or in the proximity of the area of MSP1 19 interacting with Rc. To selectively define resonances most likely to be part of the interface, threshold values (specifically, ⌬⌬ ␯ 1/2 Binding ⱖ5Hz for 15 N and ⱖ11Hz for 1 H dimension) were set (see “Experimental Procedures”). As expected, the distribution of line width changes (⌬ ␯ 1/2 ) becomes broader as the Rc:MSP1 19 ratio increases (data not shown). FIGURE4. NMRtitrationsof 15 NMSP1 19 withreduced cupredoxins.Top,Az from P. aeruginosa.Middle, Pc from Nostoc sp. PCC 7119. Bottom, Rc from A. ferrooxidans. Superimposition of 15 N HSQC spectra of free MSP1 19 (magenta) and after adding one of three cupredoxins (blue) at a cupredoxin:MSP1 19 ratio of 4:1 is shown. Arrows point out those residues that experience substantial broadening. Negligible binding to MSP1 19 was observed upon adding aliquots of Pc and Az. MSP1 19 -Rusticyanin Complex 20900 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288•NUMBER 29•JULY 19, 2013 at FAC BIOLOGIA/BIBLIOTECA on February 12, 2018http://www.jbc.org/Downloaded from At the Rc(Cu 1⫹ ):MSP1 19 ratio of 1.5:1, 14 amino acids show considerable 15 N line width changes in MSP1 19 HSQC spectra (Fig. 5, Aand B). These residues are distributed mainly along two regions of MSP1 19 , namely, at the beginning of N-terminal EGF domain involving Asp-3, Lys-5, His-6, Val-7, Asp-10, and at the end of C-terminal domain comprising Thr-85, Ala-88, Tyr-89, Phe-94, and Ser-97. In addition, Asp-24, Asp-25, and Arg-31 at the two first antiparallel ␤ -strands, along with Cys59, are also altered. Fig. 5Cshows the map of MSP1 19 residues affected by Rc addition, with colors corresponding to line broadening in the 15 N dimension. The sequential stretches of residues detailed above form the main cluster on the MSP1 19 surface, which surrounds the two EGF domain interface. Interestingly, some of these affected residues (Asp-24, Asp-25, Arg-31 and Cys-59) are located at the rear of the protein (Fig. 5C). Whereas Asp-24 and Asp-25 are close to amino acids at the N terminus, Arg-31 and Cys-59 lie near Phe-94. Because these four residues are adjacent to others placed at the EGF domain interface and involved in direct contact with Rc, these line width perturbations are probably a secondary effect of binding. Similar conclusions may be inferred from 1 H line width analysis, although protons are more sensitive to broadening (data not shown). NMR titration of oxidized Rc on MSP1 19 results in significant MSP1 19 line width perturbations at a Rc(Cu 2⫹ ):MSP1 19 ratio of 4:1, at which some signals broaden beyond the detection limit (Fig. 6A). The MSP1 19 resonances affected by Rc(Cu 2⫹ ) binding involve both N-terminal (Asp-3, Lys-5, Asp10, and Asp-13) and C-terminal (Cys-79, Thr-85, Tyr-89, Gly92, Phe-94, Ser-97, and Ser-98) regions, as described for the reduced system at a Rc(Cu 1⫹ ):MSP1 19 ratio of 1.5:1. An additional stretch (Phe-21 to Asp-24, Gly-26, Glu-29, Arg-31, and Cys-59) is also perturbed whereas His-6, Val-7, Asp-25, and Thr-27 were residues over the detection limit (Fig. 6A). Notably, the strength of the broadening observed for a 4:1 Rc(Cu 2⫹ ): MSP1 19 ratio is comparable with the one observed at a 1.5:1 Rc(Cu 1⫹ ):MSP1 19 ratio, indicating that MSP1 19 binds more weakly to the oxidized Rc. However, the MSP1 19 interacting surface involved in Rc recognition at a Rc:MSP1 19 ratio of 4:1 is independent of the cupredoxin redox state (Fig. 6). Binding of MSP1 19 to Holo-Rc by ITC—ITC measurements reveal that MSP1 19 binds to Rc either in its reduced or oxidized state with a 1:1 stoichiometry at 25 °C (Fig. 7). Notably, the interaction of MSP1 19 with Rc(Cu 1⫹ ) is exothermic with a dissociation affinity constant (K d )of2 ␮ M, whereas that with Rc(Cu 2⫹ ) is an endothermic process with lower binding affinity FIGURE 5. NMR titration of 15 N MSP1 19 with reduced Rc. A, 15 N line width differences ( 15 N⌬⌬ ␯ 1/2 Binding ) between free and Rc-bound MSP1 19 . The Rc:MSP1 19 ratios are 0.5:1 (red), 1:1 (gray), and 1.5:1 (blue). B, superposition of 15 N HSQC spectra of free MSP1 19 (magenta) and bound to Rc (blue) in a Rc:MSP1 19 ratio of 1.5:1. A subset of three representative resonances is labeled in black.C, map of MSP1 19 interface upon binding to Rc. MSP1 19 surface is rotated 90° around the vertical axes in each view. Residues are colored according to their 15 N⌬⌬ ␯ 1/2 Binding (Hz): the resonances that undergo the largest broadening (ⱖ5 Hz) are orange, and the signals with a significant line width over the detection limit ⬍5Hzareyellow. The limit of 5 Hz corresponds to a threshold value relative to the average plus 2-fold the S.D. (⌬⌬ ␯ 1/2 Binding ⱖ⬍⌬⌬ ␯ 1/2 Binding ⬎⫹2S n⫺1 ). Residues with no line width perturbation are marked in blue, whereas prolines are in gray. MSP1 19 -Rusticyanin Complex JULY 19, 2013•VOLUME 288•NUMBER 29 JOURNAL OF BIOLOGICAL CHEMISTRY 20901 at FAC BIOLOGIA/BIBLIOTECA on February 12, 2018http://www.jbc.org/Downloaded from (K d ⫽25 ␮ M) (Table 1). Such differences in K d values are in agreement with NMR titrations and suggest that MSP1 19 binds to reduced Rc with a greater affinity than to oxidized Rc. Further experiments need to be performed to explain the opposite sign in the enthalpy of both processes as it could be related to (i) changes in the protonation/deprotonation equilibrium of ionizable groups of the interacting proteins; (ii) variations in the hydrogen-bonding networks; (iii) differences in water arrangement in the vicinity of oxidized or reduced cofactors; and (iv) slight conformational modifications altering the number of solvent molecules excluded from the protein interface. FIGURE 6. Comparison between NMR titrations of 15 N MSP1 19 with either oxidized or reduced Rc at Rc:MSP1 19 ratio of 4:1. Most of the MSP1 19 signals in binding to Rc(Cu 1⫹ ) are broadening over the detection limit at the Rc:MSP1 19 ratio of 4:1, suggesting that MSP1 19 binds reduced Rc with a higher affinity. A, upper, overlap between 15 N HSQC spectra of free MSP1 19 (magenta) and oxidized Rc-bound MSP1 19 (blue). A,lower, map of MSP1 19 in the presence of oxidized Rc. B, the same as Awith reduced Rc. The 90°-rotated surface representations of MSP1 19 show residues colored according their 15 N⌬⌬ ␯ 1/2 Binding following the same color code as in Fig. 5C. Those residues broadened beyond the detection limit are highlighted in red. MSP1 19 -Rusticyanin Complex 20902 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288•NUMBER 29•JULY 19, 2013 at FAC BIOLOGIA/BIBLIOTECA on February 12, 2018http://www.jbc.org/Downloaded from The Case of Apo-Rc—To determine the role of the copper center, NMR and ITC titrations were carried out using MSP1 19 and apo-Rc. Surprisingly, NMR titrations showed no substantial line width changes even at an apo-Rc:MSP1 19 ratio of 4:1 (Fig. 8). The finding was further corroborated by ITC measurements, as the weak calorimetric profile suggests a lack of interaction (Fig. 8). Altogether, these data indicate the relevance of the copper center in the binding to MSP1 19 . Despite the small structural differences in cupredoxins reported previously in solid state, there exists a high degree of mobility of the metalbinding loops in solution in the apo form, as recently demonstrated by NMR (42, 43). The different pattern between apoand holo-Rc versus MSP1 19 could be related with the found differences in the dynamics of this site in the two forms. Docking Simulations with BiGGER—Along with the ab initio docking calculations run on the MSP1 19 -Rc(Cu ⫹ ) complex, revealing how the MSP1 19 mass center docks on the Rc metal crevice (Fig. 3), an NMR-restrained docking with BiGGER was also performed. Line width data for those MSP1 19 residues in contact with Rc at a Rc:MSP1 19 ratio of 1.5:1 were included in the run. The output is a set of docked solutions that can be ranked according to the BiGGER global score or individual scores, such as hydrophobic criteria, electrostatics, and geometrical parameters. Fig. 9 shows the best 100 solutions, as represented by Rc geometry centers, according to the global and hydrophobic scores from the program (Fig. 9A). The best scoring models predicted by restrained docking reveal how MSP1 19 leans its EGF domain interface to approach Rc (Fig. 9A). Indeed, the proximity of the surfaces on both proteins in the complex is shown in the space-filling representation (Fig. 9A). In addition to the copper center, loops connecting Rc ␤ -strands are in close contact with MSP1 19 . A deep analysis of the complex interface predicted by docking points to the N-terminal and C-terminal regions of the MSP1 19 as driving complex formation as some NMR restraints are satisfied (Fig. 9B). In the model reported, some electrostatic interactions occur (Fig. 9C) because Rc residues Lys-81 and Lys-116 are close to Glu-91 and Glu-83 in MSP1 19 . Furthermore, Lys-5 of MSP1 19 can interact with Glu-9 of Rc, although the Glu is partially buried. The Rc interaction surface is mainly hydrophobic with the exception of a single positive spot, Lys-81 and Lys-116, surrounded by Phe83, Gly-82, and Trp-7 and the copper center, which, in turn, is enclosed by Met-99, Val-98, and Pro-141. This interaction mode resembles those of the copper protein Pc and cytochrome c 6 with their photosynthetic partners, cytochrome fand photosystem I. In such complexes, Pc and cytochrome c 6 use the hydrophobic site surrounding the copper center and the heme group (site 1) as well as their charged patch (site 2; 44, 45). Inhibition of P. falciparum Growth by Holo-Rc—To assess the physiological relevance of the MSP1 19 binding to Rc, the growth of P. falciparum within red blood cells was followed in FIGURE 7. ITC titrations of MSP1 19 with both redox states of Rc. Binding assays of the complexes between MSP1 19 and reduced (left) or oxidized Rc (right) are shown. Thermograms are shown at the top and binding isotherms at the bottom, along with the dissociation constant (K d expressed in ␮ M), enthalpy (⌬H in kcal/mol). The stoichiometry value (n) was fixed to avoid degeneracy in the nonlinear regression data analysis. Typical relative errors are 20–25% for the dissociation constant and 5–10% for the stoichiometry. TABLE 1 Thermodynamic values inferred from ITC experiments The affinity of a protein-protein interaction is defined by the Gibbs energy of the binding: ⌬G⫽⫺RT ln K a .⌬Ghas two different contributions, ⌬Hand ⌬S, according to the equation: ⌬G⫽⌬H⫺T⌬S, thus several combinations of those values could yield similar binding affinities. The complex formation is entropically driven in both cases, being the enthalpic contribution to the binding unfavorable (15 kcal/ mol with oxidized Rc) or only slightly favorable (⫺1.7 kcal/mol with reduced Rc). MSP1 19 complex ⌬G⌬Hⴚ⌻⌬SK d kcal/mol kcal/mol kcal/mol ␮ M Oxidized Rc ⫺6.3 15.0 ⫺21.3 25.0 Reduced Rc ⫺7.8 ⫺1.7 ⫺6.1 2.0 MSP1 19 -Rusticyanin Complex JULY 19, 2013•VOLUME 288•NUMBER 29 JOURNAL OF BIOLOGICAL CHEMISTRY 20903 at FAC BIOLOGIA/BIBLIOTECA on February 12, 2018http://www.jbc.org/Downloaded from the presence of either holoor apo-Rc (see “Experimental Procedures”). The addition of holo-Rc to infected cells resulted in parasitemia inhibition, as culture growth decreased significantly (Fig. 10) at a concentration of 100 ␮ Muntil the point of complete inhibition at 200 ␮ Mholo-Rc. In contrast, the apo form even at high concentrations did not have a significant effect on P. falciparum growth. These data fully corroborate previous observations using NMR or ITC as much as apo-Rc is unable to bind to MSP1 19 . To discard the theory that such a parasitemia inhibition could be ascribed to the well known apoptotic role of several cupredoxins (46, 47), control experiments were run with the holoor apo-form of Pc. Holo-Pc was chosen as it does not bind to MSP1 19 , as inferred from the herein presented NMR screening (see above), but it is structurally very similar to Rc: the two proteins belong to the type I blue copper-protein family, with almost identical folding and tetrahedral copper center. Upon addition of either holoor apo-Pc (25 ␮ M) under the same culture conditions, the Plasmodium growth first slightly decays to further reach a constant value until the end of the experiment. So the Plasmodium growth does not depend on protein concentration (the percentage of growth is maintained at approximately 70% even at 200 ␮ MPc) and is practically the same with either holoor apo-Pc. Altogether these results indicate that holo-Rc inhibits parasitemia upon specific binding to MSP1 19 . FIGURE 8. NMR and ITC titrations of MSP1 19 with apo-Rc. A, 15 N line width differences ( 15 N⌬⌬ ␯ 1/2Binding ) between free MSP1 19 and in the presence of apo-Rc. The apo-Rc:MSP1 19 ratios were 1:1 (red), 1.5:1 (gray), and 4:1 (blue). B, overlap between 15 N HSQC spectra of free MSP1 19 (magenta) and in the presence of apo-Rc (blue) at an apo-Rc:MSP1 19 ratio of 4:1. C, ITC thermogram obtained from the apo-Rc-MSP1 19 titration, revealing the lack of binding between both proteins because of the flat calorimetric profile. FIGURE 9. BIGGER molecular docking of the MSP1 19 -Rc complex. A,left, best 100 models with the lowest energy values after alignment of MSP1 19 molecules with Rc geometry centers represented by spheres. Ribbon (center) and space-filling (right) representations for the best model are shown in the same orientation as on the left. MSP1 19 is represented in dark gray whereas Rc is in light blue.B, interface residues of the MSP1 19 -Rc complex. MSP1 19 and Rc are independently rotated 90° to the top and to the bottom, respectively, with regard to their orientation in A. Contacting residues are depicted in light yellow for the MSP1 19 and dark blue for Rc. C, electrostatic potential surfaces of MSP1 19 (upper) and Rc (lower) with the same orientations as in B. The electrostatic potential surfaces were created with a color ramp for positive (blue) and negative (red) potentials at 300 mMionic strength. The potentials were calculated in Chimera software (41). MSP1 19 -Rusticyanin Complex 20904 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288•NUMBER 29•JULY 19, 2013 at FAC BIOLOGIA/BIBLIOTECA on February 12, 2018http://www.jbc.org/Downloaded from