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Novel insights into the regulation of malarial calcium-dependent protein kinase 1 Anwar Ahmed,*,1 Kavita Gaadhe,*,1 Guru Prasad Sharma,* Narendra Kumar,† Mirela Neculai,‡ Raymond Hui,‡ Debasisa Mohanty,† and Pushkar Sharma*,1,2 *Eukaryotic Gene Expression Laboratory and †Bioinformatics Center, National Institute of Immunology, New Delhi, India; and ‡Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada Calcium-dependent protein kinases (CDPKs) are major effectors of calcium signaling in apicomplexan parasites like Toxoplasma and Plasmodium and control important processes of the parasite life cycle. Despite recently reported crystal structures of Toxoplasma gondii (Tg)CDPKs, several important questions about their regulation remain unanswered. Plasmodium falciparum (Pf)CDPK1 has emerged as a key player in the life cycle of the malaria parasite, as it may be involved in the invasion of the host cells. Molecular modeling and site-directed mutagenesis studies on PfCDPK1 suggested that several residues in the regulatory domain play a dual role, as they seem to contribute to the stabilization of both the active and inactive kinase. Mass spectrometry revealed that PfCDPK1 was autophosphorylated at several sites; some of these were placed at strategic locations and therefore were found to be critical for kinase activation. The N-terminal extension of PfCDPK1 was found to be important for PfCDPK1 activation. Unexpectedly, an ATP binding site in the NTE of PfCDPK1 was identified. Our studies highlight several novel features of PfCDPK1 regulation, which may be shared by other members of the CDPK family. These findings may also aid design of inhibitors against these important targets, which are absent from the host.—Ahmed, A., Gaadhe, K., Sharma, G. P., Kumar, N., Neculai, M., Hui, R., Mohanty, D., Sharma, P. Novel insights into the regulation of malarial calcium-dependent protein kinase 1. FASEB J. 26, 3212–3221 (2012). www.fasebj.org ABSTRACT
Key Words: autophosphorylation 䡠 structure-function Malaria is a major infectious disease that affects millions and causes a significant rate of mortality in the developing world. One of the problems in overcoming Abbreviations: aa, amino acid; CAD, CDPK activation domain; CaMK, calcium calmodulin-dependent protein kinase; CDPK, calcium-dependent protein kinase; CLD, calmodulinlike domain; Cp, Cryptosporidium parvum; DTT, dithiothreitol; KD, kinase domain; NTE, N-terminal extension; Pb, Plasmodium berghei; Pf, Plasmodium falciparum; Pv, Plasmodium vivax; RT-PCR, reverse transcription–polymerase chain reaction; Tg, Toxoplasma gondii; WT, wild type 3212
human malaria is an alarming increase in the rate of resistance exhibited by malaria parasites toward currently available drugs. Therefore, new lines of antimalarial drugs are urgently needed. The human malaria parasite Plasmodium falciparum has a complex life cycle during which it infects both the vector and the human host. It first propagates in the liver, followed by the invasion and subsequent development in the erythrocytes. Recent studies have highlighted that intracellular signaling is involved in almost all stages of parasite development (1–3). In-depth understanding of molecular mechanisms that underlie signaling pathways of malaria parasite may shed light on novel aspects of parasite biology as well as aid the targeting of key enzymes for drug design. Calcium has emerged as a major player in controlling several important signaling pathways in the parasite (4 – 6). These pathways control a wide-range of events in the parasite life cycle that include host cell invasion, sexual differentiation, asexual parasite life cycle, and development of hepatic stages (reviewed in refs. 1, 7). Calcium-dependent protein kinases (CDPKs) are major effectors of calcium signaling in malaria parasite and control some of these processes (1). These kinases are present in some species of plants, fungi, and protozoans but are absent from mammals (8, 9). Their importance in parasite signaling and absence in the host have made CDPKs attractive drug targets. The disruption of the CDPK3 gene in Plasmodium berghei abrogates gliding motility and invasion (10). P falciparum (Pf)CDPK5 was shown recently to regulate the egress of the blood stage malaria parasite from host erythrocyte (11). CDPK4 plays a key role in sexual differentiation of Plasmodium (12). Attempts to disrupt PfCDPK1 gene in Plasmodium have been unsuccessful, suggesting that it is essential for parasite growth during the blood stage development of the parasite (13). Due to its possible role in 1
These authors contributed equally to this work. Correspondence: Eukaryotic Gene Expression laboratory, National Institute of Immunology, New Delhi-110067, India. E-mail:
[email protected] doi: 10.1096/fj.12-203877 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information. 2
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invasion of host erythrocytes by the parasite, PfCDPK1 has emerged as a key enzyme of the parasite signaling machinery (13, 14). PfCDPK1 is located at the periphery of merozoites, and the N-terminal lipid modification signals contribute to its localization (14). It phosphorylates components of the glideosome assembly, such as PfGAP45 and PfMTIP (14), which possibly explains its involvement in host invasion. Given the importance of CDPKs in apicomplexans and plants, it is important to understand the molecular basis of their regulation. In addition to the catalytic kinase domain (KD), CDPKs are comprised of 4 helixloop-helix type EF hand motifs, which resemble a calmodulin-like domain (CLD). An autoregulatory domain previously known as the J domain (15), which connects the KD and the CLD, plays a key role in the regulation of CDPKs (16). Previous studies on plant and Plasmodium CDPKs suggested that the regulatory domain may interact with the CLD as well as the KD (9); whereas calcium binding to the CLD promotes its interaction with the J domain and results in dissociation of the autoinhibitory region of the J domain from the KD (16, 17). Recent crystal structures of CDPKs from Toxoplasma in apo and calcium-bound forms shed light on the regulation of CDPKs (18, 19). These structures revealed that the entire region downstream of the KD (i.e., the J domain and the CLD together) works in concert in CDPK activation; therefore, it was termed the CDPK activation domain (CAD). Despite these studies, several questions about CDPK regulation remain unanswered. Also, it is unclear how autophosphorylation contributes to the activation of CDPKs. Taking advantage of the high degree of sequence similarity between PfCDPK1 and Toxoplasma gondii (Tg)CDPKs, homology models of active and inactive PfCDPK1 were generated. On the basis of the structure and the model, site-directed mutagenesis of several residues of the CAD and the KD was performed. The kinase activity assays of these mutants highlighted several residues, which play a dual role in stabilizing the active and inactive structure of PfCDPK1. Several regulatory autophosphorylation sites that reside in different regions of CDPK1 were identified. The N-terminal extension (NTE) of PfCDPK1 plays a crucial role in its activation. Surprisingly, a Walker A-like motif was identified in the NTE of PfCDPK1. We demonstrate that this motif interacts with ATP, which may be important for PfCDPK1 activation.
MATERIALS AND METHODS Molecular cloning and site-directed mutagenesis of PfCDPK1 The PfCDPK1 (PFB0815w) coding sequence was amplified by reverse transcription–polymerase chain reaction (RT-PCR) using cDNA obtained from the asexual stages of the parasite and was cloned into a pET28a vector (Novagen, San Diego, CA, USA) using restriction sites for BamHI and XhoI. The REGULATION OF CDPKs
site-directed mutagenesis was performed by overlap extension PCR mutagenesis. The mutations were confirmed by automated DNA sequencing. Recombinant protein expression and purification For the production of 6xHis-PfCDPK1 and its mutants, corresponding plasmid constructs in pET28a vector were transformed into E. coli Bl21RIL DE3 cells. Induction of protein expression was done by adding 1 mM isopropyl 1- thio--dgalactopyranoside at 18°C for 16 h. The cell pellet was resuspended in ice-cold resuspension buffer A: 50 mM sodium phosphate, pH 7.4; 150 mM NaCl; 0.1% nonident-P40 detergent; 1 mM dithiothreitol (DTT); and protease inhibitors (10 g/l pepstatin, 10 g/l leupeptin, 1 mM benzimidin, and 1 mM PMSF). Following resuspension, sonication was performed for 7 cycles of 1 min each. To obtain the cell-free solution, centrifugation of the suspension was performed at 10,000 g for 30 min at 4°C. The cell lysate was incubated with Ni-NTA-agarose for 4 h at 4°C, followed by washing of the resin with a buffer containing 50 mM sodium phosphate, pH 7.4, and 500 mM sodium chloride 0.1% nonidet P-40. The protein was eluted by using 50 –300 mM imidazole (Sigma-Aldrich, St. Louis, MO, USA) in 50 mM sodium phosphate, pH 8.0, and 500 mM sodium chloride. Finally, the purified recombinant proteins were dialyzed against 50 mM sodium phosphate, pH 7.4; 10% glycerol; and 1 mM DTT. Protein concentration was determined by densitometry of the SDS-PAGE gels stained with Coomassie blue. Kinase assays The activity of recombinant PfCDPK1 and its mutants were assayed in a buffer containing 50 mM Tris, pH 7.5; 10 mM magnesium chloride; 1 mM DTT; and 100 M (␥-32P) ATP (6000 ci/mmol). Either recombinant PfGAP45 or 100 M Syntide-2 (PLARTLSVAGLPGKK) was used as phosphateacceptor substrates. Reactions were performed in the absence (2 mM EGTA) or presence of 2 mM calcium chloride for 40 min at 30°C. When PfGAP45, which was expressed as described previously (6), was used as a substrate, the reaction was stopped by boiling the mixture in the presence of SDS-PAGE sample buffer for 5 min followed by SDS-PAGE and autoradiography. For syntide assays, the reactions were stopped by spotting the assay mixture on the P81 phosphocellulose paper (Millipore, Bedford, MA, USA), followed by washing the paper strip with 1% orthophosphoric acid. Finally, phosphate incorporation was determined by scintillation counting. Identification of autophosphorylation sites on PfCDPK1 To identify autophosphorylation sites, an aliquot of 2 mg/ml of each protein sample in 20 mM HEPES, pH 7.5; 500 mM NaCl; and 2 mM tris(2-carboxyethyl)phosphine (TCEP) was mixed with ATP (final concentration 5 mM), CaCl2 (final concentration 10 mM), and MgCl2 (final concentration 5 mM). The mixture was kept at 4°C overnight and subjected to in-solution digestion with trypsin the next day. The sample was analyzed by LC/MS/MS (using a Thermo LTQ system; Thermo Scientific, Waltham, MA, USA). The MS/MS data were analyzed using the MASCOT search engine (Matrix Science, London, UK) and X! Tandem 2007.01.01.1 software (The Global Proteome Machine Organisation; http:// www.thegpm.org), both of which had a fragment ion mass tolerance of 0.40 Da and a parent ion tolerance of 20 ppm. Search results were analyzed using Scaffold Viewer (Proteome Software, Portland, OR, USA). 3213
The services of Applied Biomics (Hayward, CA, USA) were also used for the identification of phosphorylation sites by MALDI-TOF/TOF following a standard protocol. Briefly, the SDS-PAGE gel bands corresponding to autophosphorylated PfCDPK1 protein were reduced, alkylated, and subjected to trypsin digestion. The phosphopeptides were enriched by using Supel-Tips (Sigma-Aldrich) and desalted by Zip-tip C18 (Millipore). The eluted peptides were spotted on a MALDI plate, and MALDI-TOF MS was performed on an AB Sciex Proteomics Analyzer (AB Sciex, Foster City, CA, USA). MS spectra were acquired in positive ion mode and ⬃4000 laser shots/ per spectrum. A virtual digest was done by submitting protein sequences of interest to University of California–San Francisco Protein Prospector (http://prospector.ucsf.edu/ prospector/mshome.htm). The MS precursors matching the virtual digest were submitted for MS fragmentation. The resulting peptide masses were submitted to MASCOT search engine (Matrix Science) to search the database of Swiss-Prot. Candidates with either protein score C.I.% or ion C.I.% ⬎ 95 were considered significant. ATP binding assays The assay was performed using ␣-32P-ATP in a buffer containing 14 mM HEPES; 10 mM Tris, pH 7.4; 1 mM EDTA; and 60 mM KCl. The reaction mix was incubated at room temperature for 20 min, after which UV crosslinking was performed at 12,000 J for 10 min on ice. The reaction was stopped by boiling in SDS-PAGE sample buffer for 5 min, followed by SDS-PAGE and autoradiography. Homology modeling Homology modeling software Modeler 9v6 (University of California, San Francisco, CA, USA; http://salilab.org/ modeller/9v6/release/html) was used to build the 3-dimensional (3D) structural models of PfCDPK1 in active (aPfCDPK1) and inactive (i-PfCDPK1) conformations. The aPfCDPK1 was modeled based on the template structure of calcium bound active form of TgCDPK1 [Protein Data Bank (PDB) ID: 3HX4; http://www.wwpdb.org/]. The i-PfCDPK1 was built using crystal structures of TgCDPK1 (PDB: 3KU2) as well as TgCDPK3 (PDB: 3HZT) in the inactive form. The sequence similarity between PfCDPK1 and TgCDPK1 is ⬃70%, which is slightly less than the ⬃73% similarity that PfCDPK1 shares with TgCDPK3. The 3D models of a-PfCDPK1 and i-PfCDPK1 were very similar to TgCDPK structures, as reflected by C-␣ RMS deviation of 0.8 Å (PDB: 3HX4) and 0.3 Å (PDB: 3KU2 and 3HZT) with their template structures, respectively.
plemental Fig. S1). Since the crystal structure of TgCDPK1 was available in both active and inactive form, the model generated on the basis of TgCDPK1 structures was used to guide and analyze the biochemical experiments. Expectedly, the homology model for active PfCDPK1 was reasonably similar to the TgCDPK3 structures, as reflected by a RMS deviation of 0.8 and 0.3 Å. As was the case with TgCDPK1 crystal structures (19), the PfCDPK1 model indicated that the CH1 helix forms intimate contacts with the C lobe of the KD in the absence of calcium (Fig. 1). Previous studies on PfCDPK4 had suggested that the region corresponding to M347-K355 to be autoinhibitory (16), which was supported by TgCDPK crystal structures (19) and the PfCDPK1 model. The PfCDPK1 model suggested that M347 and F350 might form hydrophobic contacts with I157/158, F154 and Y233/234. In addition, K355 may form a salt bridge with E152/155 (Fig. 2Ai). To probe whether these interactions between the CH1 helix and the KD are essential for keeping the enzyme inactive, site-directed-mutagenesis studies were carried out. PfGAP45, a physiological substrate for PfCDPK1 (6, 14), and/or syntide, a classical substrate peptide for calcium calmodulin-dependent protein kinases (CaMKs), were used to assess the activity of PfCDPK1. We had expected the mutation of M347, F350 and K355 to severely impair CH1 and KD interaction and possibly cause an increase in calcium-independent activity. The F350A and M347A mutants demonstrated a significantly lower level of activity than wild-type (WT) PfCDPK1. In contrast, the K355A mutant was almost as active as
RESULTS Dual role of CH1 helix residues F350 and M347 in PfCDPK1 regulation Previously published crystal structures of TgCDPK1, TgCDPK3, and Cryptosporidium parvum(Cp)CDPK1 in apo and calcium-bound states revealed unexpected changes in the conformation as a result of calcium binding (19). Further studies were needed to identify key interactions that are responsible for CDPK activation. The structures of TgCDPK1/3 were used to generate homology models for PfCDPK1. TgCDPK3 shares slightly higher sequence homology with PfCDPK1 compared to TgCDPK1 (see Materials and Methods, Sup3214
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Figure 1. Homology modeling of PfCDPK1. A) Domain architecture of PfCDPK1. It has an N-terminal lipid modification signal and a short NTE, which precedes the KD, followed by the CAD, which comprises of the CH1 helix, previously termed the J domain, and 4 EF hand motifs, which resemble a CLD. B) Homology model of PfCDPK1 in inactive and active form was generated using the crystal structure of apoTgCDPK1 and calcium-bound TgCDPK1, respectively. KD is shown in green, CH1 helix in red, and CLD in yellow. Some of the residues in the CH1 helix (magenta) that are part of the autoinhibitory segment and that may be involved in interactions with the KD residues (white) are shown as sticks.
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Figure 2. Identification of interdomain interactions involved in PfCDPK1 regulation. A) Homology model of inactive (i) and active (ii) PfCDPK1 revealed residues that may be involved in interactions between CH1 helix (red) and the KD (blue sticks, i) or between CH1 helix and CLD (blue sticks, ii). M347, F350, and K355 may interact with the KD in the inactive PfCDPK1 (i) and with the CLD in the active kinase (ii). Note that the K355 side chain points away from the CLD in the active structure (ii). B) Model of the active PfCDPK1 suggests that some of the KD residues (white sticks) that interact with the inhibitory segment of CH1 helix (Ai) are engaged interactions with the KD (cyan sticks) residues. C–E) Equal amounts of recombinant PfCDPK1 (WT) or indicated mutants were incubated either with recombinant PfGAP45 (top panel) or syntide (bottom panel). Subsequently, phosphate incorporation in the substrates was detected by phosphorimaging of SDS-PAGE gel in the case of PfGAP45 or by scintillation counting when syntide was used as the substrate. Decrease in the activity of the mutants in syntide assays was calculated by treating the activity of WT PfCDPK1 as 100%. Average of 2 independent measurements is shown; error bars ⫽ se.
PfCDPK1 (Fig. 2C). In the absence of calcium, these mutants did not exhibit any activity (data not shown). The model of active PfCDPK1 suggested that M347 and F350A might be buried in a hydrophobic pocket that comprises of residues like F441, V428/444, and F430 (Fig. 2Aii). Therefore, it is reasonable to suggest that these interactions may be critical for stabilizing the active structure, as the mutation of M347 and F350 renders an inactive enzyme. The side chain of K355, in the active structure is away from the CLD, which explains almost unaltered activity of the K355A mutant. As mentioned above, the PfCDPK1 model indicated that several KD residues, including E152, E155, F154, I157, I158, Y233, and Y234, were engaged with the autoinhibitor portion of the CH1 helix (Fig. 2A). Alanine scanning mutagenesis was carried out to evaluate these contacts. The mutation of F154 and I157/ 158 caused a significant decrease in the kinase activity (Fig. 2D). In the active kinase, these residues are likely REGULATION OF CDPKs
to interact with the other KD residues (Fig. 2B). Therefore, it is probable that the perturbation of these interactions is deleterious for the kinase activity. Furthermore, the mutation of E152, which along with E155 forms a triad with K355 in the inactive enzyme (Fig. 2A), significantly abrogated PfCDPK1 activity. In contrast, the E155A mutation was resistant (Fig. 2E). An acidic residue complementary to E152 has been implicated in stabilizing ATP interaction in other protein kinases as well (20). It is possible that E152 plays a similar role in PfCDPK1, which makes it important for PfCDPK1 activity. PfCDPK1 is autophosphorylated at multiple sites It is known that PfCDPK1 is autophosphorylated, but only one such modified site has been reported (21). Preliminary phosphopeptide mapping experiments suggested that it may be phosphorylated at 10 or more 3215
residues (data not shown). Tandem MS/MS spectrometry was used to identify the autophosphorylation sites on PfCDPK1 as well as Plasmodium vivax (Pv)CDPK1 (Fig. 3A). Nine phosphopeptides from PfCDPK1 (Fig. 3B) were identified, and MS/MS resulted in the identification of 10 autophosphorylation sites (Fig. 3A and Supplemental Fig. S2). PfCDPK1 was autophosphorylated at several positions in the KD. Interestingly, the CH1 helix, the hinge region that separates the CH1 helix from CLD and the NTE, were also autophosphorylated (Fig. 3A). Several autophosphorylated residues detected on PfCDPK1 could not be identified in the autophosphorylated PvCDPK1 samples (Fig. 3A and Supplemental Fig. S3) and vice versa, which may be due to spurious phosphorylation or technical reasons, such as low abundance of corresponding phosphopeptides. Furthermore, several autophosphorylated S/T residues in PvCDPK1 are replaced by nonphosphorylable residues in PfCDPK1 and vice versa (Fig. 3A). Regulation of PfCDPK1 by autophosphorylation: activation loop autophosphorylation The model of PfCDPK1 indicated that several of the autophosphorylated S/T residues were present in the key regions of PfCDPK1 (Fig. 3A). These residues were mutated to test whether their autophosphorylation contributes to PfCDPK1 regulation. First, the role of the activation loop residue T231 (Fig. 4A) was determined. A T231A mutant exhibited a significant decrease in autophosphorylation as well as its ability to phosphorylate PfGAP45 and syntide (Fig. 4C). T231 is part of the
TAYY motif, which is conserved in most CDPKs (Fig. 3A). Therefore, the autophosphorylation of this motif is likely to play a key role in their regulation. Consistent with this, we have previously demonstrated autophosphorylation of T234 of PfCDPK4 to be critical for catalytic activation (16). Regulation of PfCDPK1 by autophosphorylation: CAD or hinge region autophosphorylation Since the structures of CDPKs indicate that the CH1 helix and the preceding hinge region undergoes a major conformational change on calcium binding, it was worth exploring whether phosphorylation of this region influences PfCDPK1 activation. MS data indicated that S335 of CDPK1, which resides in the hinge region (Fig. 4B), is autophosphorylated (Fig. 3). The mutation of S335A caused an almost complete loss of PfCDPK1 activity, including autophosphorylation. When S335 was mutated to a phosphate-mimicking negatively charged D, a significant amount of autophosphorylation and substrate phosphorylation was recovered. These results suggested that the autophosphorylation of S335 might be necessary for PfCDPK1 activation. Although T339 of PfCDPK1, which is in close vicinity of S335 (Fig. 3A, B), was not identified as a phosphorylation site by MS, it was mutated to probe whether the mutation of hinge region was sensitive specifically to S335. In contrast to S335A, T339A did not cause a significant change in activity (Fig. 4C). MS results indicated that T371 is autophosphorylated on PvCDPK1 (Fig. 3A and Supplemental Fig. S3);
Figure 3. Identification of PfCDPK1 autophsphorylation site by mass spectrometry. A) Autophosphorylated PfCDPK1 and PvCDPK1 were subjected to mass spectrometry for identification of phosphorylation sites. Phosphorylation sites identified by tandem MS/MS (Supplemental Figs. S2 and S3) are encircled in the ClustalW alignment. The various domains of PfCDPK1 are indicated by bars and are color coded: cyan, NTE, green, KD, pink, hinge region; red, CH1 helix; yellow, CLD. B) Amino acid (aa) sequence of PfCDPK1 phosphopeptides that were identified by MS studies (Supplemental Fig. S2). Asterisks indicate phosphorlation sites identified by MS/MS.
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Figure 4. Role of autophosphorylation of the activation loop and the hinge region in PfCDPK1 activation. A, B) Location of identified or putative phosphorylation sites (orange sticks) identified by MS (Fig. 3) in the activation loop of PfCDPK1 (A) or in the CH1 helix and the hinge region (B) is indicated using the homology model of the active PfCDPK1. K193, which is in proximity to T231, is shown as sticks. Color coding of various domains is the same as indicated in preceding figures. C) Equal amounts of PfCDPK1 or its indicated phosphorylation site mutants were incubated with PfGAP45 (top panel) or syntide (bottom panel) in kinase assays as described earlier. The autophosphorylation of PfCDPK1 and phosphorylation levels of PfGAP45 were detected by phosphorimaging (top panel) or by scintillation counting (bottom panel). Bottom panel: change in activity of mutants was calculated by treating the activity of WT PfCDPK1 as 100%.
however, a phosphopeptide spanning the corresponding T369 in PfCDPK1 could not be identified. In addition to T369, two other possible phosphorylation sites exist in this region of the CH1 helix of PfCDPK1. We evaluated the importance of autophosphorylation of CH1 residues S366, T369/T370 for PfCDPK1 regulation. While the mutation of S366A caused only a marginal change in PfCDPK1 activity, the T369/370A double mutant exhibited both significantly reduced autophosphorylation and phosphorylation of PfGAP45 (Fig. 5B). The model for PfCDPK1 suggested that T369 may be in the vicinity of R456 in the CLD (Fig. 5A). Therefore, its phosphorylation may result in a salt bridge to stabilize the active conformation. To test this, a R456A mutant was generated. The kinase assays revealed that the R456A mutant was significantly less active than the WT PfCDPK1, which suggests that R456
Figure 5. Autophosphorylation of CH1 helix residues is important for PfCDPK1 activity. A) Putative autophosphorylation sites in the CH1 region, T369 and T370, are indicated in orange; R456, which is in close vicinity of T369, is shown in cyan. B) Activity and autophosphorylation of equal amounts of PfCDPK1 or its mutants T366A, T369/T370A was tested using PfGAP45 as substrate. C) Kinase assays were performed using equal amounts of PfCDPK1 or R456A mutant using PfGAP45. Subsequent to SDS-PAGE of the reaction mix (bottom panel), autoradiography was performed (top panel). REGULATION OF CDPKs
may be involved in interactions crucial for PfCDPK1 activation (Fig. 5C). Based on these observations, it is reasonable to conclude that phosphorylation of T369 may stabilize the active conformation by interacting with the CLD. NTE is critical for PfCDPK1 regulation Although the available crystal structures of apicomplexan CDPKs were crystallized with only a short segment of NTE, the structures of TgCDPK1 and CpCDPK3 in their active conformations clearly showed interaction of this region with the N lobe of the CAD (19). To study the role of the NTE in PfCDPK1 activation, deletion mutants of the NTE were generated (Fig. 6A). Mutants with either the first 50 residues or the first 30 residues of the NTE truncated were inactive (Fig. 6B). In contrast, deletion of the first 20 aa of PfCDPK1 was not found to be deleterious to its activity (Fig. 6B). Therefore, residues 20–30 are possibly important for PfCDPK1 activation. While a low sequence similarity was found between the NTE of PfCDPK1 and TgCDPK1, a few residues may be conserved in the NTE of the two kinases. For instance, corresponding to L32 of TgCDPK1, PfCDPK1 has L37. TgCDPK1-NTE latches onto a cleft in the CAD as the backbone oxygen of L32 forms a hydrogen bond with Lys338, which may contribute to stabilize the active conformation of the protein. The observation that the T30 mutant (which contains L37 that corresponds to L32 of TgCDPK1) remained inactive suggested that the region upstream of L37 may also be important for PfCDPK1 activation. NTE of PfCDPK1 binds ATP On close inspection of the sequence of the regulatory segment 20 –30 in NTE, a GxxxGKS motif was noted, which is consistent with the known motifs of the class of 3217
Figure 6. Regulation of PfCDPK1 by its NTE. A) ClustalW comparison of NTEs of PfCDPK1 and TgCDPK1. B) Various truncation mutants of PfCDPK1 lacking the entire NTE (⌬N) or the first 40 aa (T40), 30 aa (T30), or 20 aa (T20) were expressed and used for activity assays with syntide as the substrate. Percentage activity of various mutants in comparison to the WT PfCDPK1 (WT) was determined; data are means of 3 experiments. Error bars ⫽ se.
TgCDPK1 PfCDPK1
nucleotide-binding domain known as Walker A (ref. 22 and Fig. 7A). This observation prompted us to test whether NTE interacted with ATP. PfCDPK1_NTE exhibited ATP binding, and mutation of K27 in the GKS motif resulted in a significant loss in this binding (Fig. 7B), confirming that NTE may interact with ATP via this motif. Interestingly, CDPK1s of other Plasmodium spp. lack an obvious ATP-binding motif in their NTE (Fig. 7A), which was supported by negligible ATP binding of their NTEs (Fig. 7B). Next, we tested the influence of the interaction between ATP and NTE on the kinase activity of PfCDPK1. The kinase activity assays revealed that K27 to A mutation causes a marked reduction in PfCDPK1 activity (Fig. 7C). Even though these data establish that the additional ATP-binding site in the NTE of PfCDPK1 may be important for its activation, further studies need to be performed to gain insights into the underlying mechanism.
Figure 7. NTE of PfCDPK1 interacts with ATP, which may be important for its activation. A) ClustalW alignment of the NTEs of PfCDPK1, Plasmodium berghei (Pb)CDPK1, and PvCDPK1. GxxxGKS, a Walker A-like motif present in PfCDPK1, is boxed. B) NTEs of PfCDPK1, PbCDPK1, and PvCDPK1 or K27A mutant (NTE_K27A) of PfCDPK1-NTE were expressed as 6xHis- tagged recombinant proteins and were used for UV cross-linking with radiolabeled ATP. Reaction mix was separated by SDS-PAGE (bottom) followed by phosphorimaging (top). C) Activity of PfCDPK1 and its K27A mutant was assessed by using syntide as substrate. Percentage decrease in activity on mutation was calculated by treating the activity of PfCDPK1 as 100%. Data are means of 3 independent experiments; error bars ⫽ se. 3218
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DISCUSSION Previous studies suggested that the junctional domain may perform dual function. It was proposed that the N-terminal residues may be engaged in autoinhibitory interactions with the KD and the C-terminal portion interacts with the CLD on calcium binding (16). The crystal structures of T. gondii and C. parvum CDPKs revealed major domain rearrangement brought about by calcium binding. These crystal structures revealed that the CH1 helix, which is a component of the CAD along with CLD, undergoes major rearrangement on calcium binding (19, 23): The CLD wraps around the CH1 helix, which is distorted into 3 shorter helices. As a result, the entire CAD moves away from the front face of the KD, resulting in severing of interdomain interactions that stabilize the inactive structure. Given the high degree of homology between PfCDPK1 and TgCDPK1/3, it is not surprising that homology modeling suggests a similar rearrangement for PfCDPK1. Several residues involved in key interactions are conserved in the two kinases. The N terminus of the CH1 helix (L344-K355) may act as an autoinhibitor by making intimate contacts with the KD. The mutation of M347 and F350 rendered an inactive kinase, and inspection of the model provided an answer to this observation. In the active PfCDPK1, M347 and F350 make key hydrophobic contacts with V428, V444, F430, and F441 (Fig. 2A), which are likely to be crucial for the active enzyme. Since these residues are conserved in most CDPKs (Supplemental Fig. S1), similar interactions may also be critical for the active conformation of other CDPKs. In contrast, K355, which forms a triad with E152 and E155 in the inactive enzyme and stabilizes the autoinhibitory interactions, was dispensable for the active PfCDPK1, as it does not seem to interact intimately with the CLD. Several of the KD residues involved with CH1 helix reside in helix D of the KD, and the mutation of F154, I157, and I158 caused a significant loss of activity. The model for the active kinase suggests that these residues may form hydrophobic interactions with the other KD residues (Fig. 2B), which in the light of these results may be important. An acidic residue complementary to E152 is also found in other protein kinases that interact with the ribose ring of ATP (20, 24). In the case of CaMKII, complementary
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E96 has been implicated in substrate and ATP binding (20). The mutation of E152, and not E155, significantly decreased the kinase activity of PfCDPK1 (Fig. 2E). Since it is in the vicinity of the ATP binding pocket, it is indeed possible that it stabilizes the interaction with ATP, as is the case with other kinases. It was unexpected to find PfCDPK1 autophosphorylated at a minimum 9 residues. As observed in the case of PfCDPK4 (16), the autophosphorylation of the T loop at T231 also seems to be necessary for PfCDPK1 activation. The published TgCDPKs in reported crystal structures were in the unphosphorylated form (18, 19); therefore, site-directed mutagenesis and enzyme activity studies were needed to understand the role of phosphorylation of various sites. The PfCDPK1 model helped explain the results obtained with phospho-site mutants. In the inactive structure, the T loop seems to obstruct the catalytic cleft, and calcium-induced conformational changes push it out of the cleft. The model suggested that K193 side chain may be in the proximity of the T231 side chain (Fig. 4A). Therefore, it is possible that the phosphorylation may strengthen the interaction of two residues and help anchor the loop in the active orientation. The autophosphorylation of T286 in CaMKII, which is at the start of the autoinhibitory helix, leads to CaMindependent constitutive activation of the enzyme (25). While a complementary S/T is absent in PfCDPK1, S335 and T339 are part of the small hinge region that separates the KD from the CH1 helix (Fig. 4B). The negative charge acquired as a result of the autophosphorylation of S335 seems to be indispensable for PfCDPK1 activity. Interestingly, PvCDPK1 does not have a phosphorylable S/T at this position (Fig. 3). T371, which is present on the CH1 helix of PvCDPK1, was autophosphorylated. Therefore, we probed the role of T369, which is the corresponding residue in PfCDPK1, and the adjacent T370. As a result of distortion of the CH1 helix on calcium binding to the CLD, T369/370 reside on a small loop that separates the two shorter helices. The double mutant T369A/T370A exhibited a significant decrease in the kinase activity. T369 is in the close vicinity of R456 (⬃4.0 A), which in turn may be involved in a salt bridge with E373 (Fig. 5A). Therefore, it is likely that the phosphorylation of T369 causes a rearrangement in the bonding network, which may be nucleated by an interaction with R456. The loss in the activity of T369A or R456A mutants supports this possibility. While this report was in preparation, a phosphoproteomic study of Plasmodium proteins (26) reported that PfCDPK1 may be phosphorylated at multiple sites (S34, S64, S98, S217, S220, T231, S345, S406). S64 phosphorylation was also identified in another recent study (27). We identified some of these sites to be autophosphorylated in PfCDPK1 (S64, S217, S220, T231) and in addition found other sites (S17, S28, T100, S117, S204, S335), which were not previously identified in the published study. Notably, we confirmed the autophosphorylation of several of these by site-directed mutagenesis and assessed their effect on PfCDPK1 regulation, as discussed above. It is indeed possible that some of the sites identiREGULATION OF CDPKs
fied by Treeck et al. (26) on PfCDPK1 in the parasite, which we were unable to identify by MS, are a result of phosphorylation of PfCDPK1 by other kinases. It will therefore be worth investigating their role in PfCDPK1 regulation. Although the results are not shown here, it is important to indicate that none of the phosphorylation site mutants exhibited significant activity in the absence of calcium. Several phospho-S/T-mimicking D/E mutants were created, which also failed to exhibit any calcium independent activation. From these data, it appears unlikely that a phosphate group at any of the described sites makes PfCDPK1 constitutively active as observed in the case with T286 phosphorylation of CaMKII (25). Despite sharing significant sequence homology in the KD and CAD, CDPKs have a variable NTE. The similarity among the NTEs of Plasmodium CDPK1 is also extremely low (Fig. 7A and Supplemental Fig. S1). However, our studies indicate that the NTE is critical for PfCDPK1 activity. TgCDPK1 was crystallized with a short portion of its NTE, its structure demonstrates that the short NTE may interact with the CAD and L32 may serve as a “latch” (19). While this leucine is conserved on PfCDPK1 at position 37, deletion studies suggest that a larger region of the NTE, starting at position 20, is essential for PfCDPK1 activation (Fig. 6A). Due to the lack of structural evidence, it is difficult to speculate how NTE may interact with CAD or KD. The identification of a secondary functional ATP-binding motif is highly unusual in protein kinases and was therefore unexpected. In prokaryotic protein kinase Hpr, which is very different in structure from eukaryotic protein kinases, a Walker A motif is present which is used for phosphotransfer to its substrate (28). It is apparent that the disruption of ATP binding by mutation of K27 significantly abrogates the kinase activity (Fig. 6A). Strikingly, this motif is not conserved in Plasmodium berghei (Pb)CDPK1 and PvCDPK1 (Fig. 7A) and their NTE does not seem to interact with ATP. Clearly, it will be interesting to understand how ATP binding to this motif contributes to PfCDPK1 activation and the functional implications of this binding. Our results demonstrate that the activation of PfCDPK1 is a multistep process (Fig. 8B). Calcium binding to the EF hand motifs causes major domain rearrangement leading to key interactions between the CH1 helix/J domain and the CLD. Some of the residues (M347, F350) that nucleate the active structure are part of the autoinhibitory region and are responsible for engaging key KD residues in the active enzyme. The autophosphorylation of the enzyme in various domains is necessary for it to achieve maximal catalytic activation; the T-loop phosphorylation at T231 may be important for stabilizing its orientation; the phosphorylation of the flexible hinge region at S335 seems to be necessary for kinase activity; and the phosphorylation of the CH1 T369 may stabilize the active conformation via interactions with the CLD. Our studies also indicate that the NTE of PfCDPK1 may be critical for its activation; although the precise mechanism needs to be worked out. Since a reasonable sequence similarity occurs 3219
Figure 8. Model for PfCDPK1 regulation. A) Residues that were implicated in forming key interactions in PfCDPK1 activation are indicated. B) a) In the absence of calcium, the CAD (CH1⫹CLD) masks and interacts with the KD (circled). b) Calcium binding to the CLD (yellow) causes a major conformational change and domain rearrangement. CH1 helix (red) is distorted into 3 smaller helices, and the active conformation is stabilized as a result of interaction between the CLD and the CH1. The residues, which are involved in interaction with the KD in the inactive kinase, are now critical for CH1-CLD interaction in the active CDPK (circled). c, d) As a result of these events, the KD and its catalytic cleft are free, and autophosphorylation of several residues may cause local conformational changes that might further stabilize the active kinase and make the kinase conducive for catalysis facilitating substrate entry into the catalytic cleft (arrow). It may be possible to target PfCDPK1 by disrupting interactions in pockets I and II of the CLD-CH1 interface (purple circles).
between PfCDPK1 and other CDPKs, the proposed model may help explain the regulation of the CDPK family. However, variations in regions like the NTE or in putative phosphorylation sites suggest that subtle differences may exist in their cellular regulation. Typically, protein kinase inhibitors are generated by targeting the ATP binding pocket or the substrate binding cleft. Our studies have highlighted other pockets in the active CAD, which may be targeted to develop CDPK inhibitors. The J-domain/CH1-helix region of PfCDPK4 are well conserved (16). Previously, we have used peptides corresponding to regions in the J domain/CH1 helix of PfCDPK4 that not only inhibit PfCDPK4 but also inhibit PfCDPK1. Three peptides used in this study either corresponded to part of the autoinhibitory (peptide II: NIRQFQSTQ) and downstream (peptide III: TQKLAQAALLYM) regions of the CH1 helix or encompassing both these regions. The present studies shed more light on the mechanism via which these peptides may operate; while peptide II may target pocket I and/or the KD, peptide III is likely to interact with pocket II. Preliminary studies on the basis of the structural information have suggested that better peptides can be designed to target these sites more effectively and specifically (unpublished results).
REFERENCES
P.S. received a Swarnajayanti Fellowship grant from the Department of Science and Technology, India, and his laboratory is supported by the Signalling in Life Cycle Stages of Malaria Parasites (MALSIG) project funded by the European Union under FP7. This work was also supported in part by U.S. National Institutes of Health grant R01AI075459 from the National Institute of Allergy and Infectious Diseases. The assistance rendered by Shivang Vachharajani, Ashutosh Upadhyay and Sudhir Kumar in generating constructs, protein expression and kinase assays is acknowledged.
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