Structural and Molecular Dynamic Simulation Studies of CBL-Interacting Protein Kinase CIPK and its Complexes Related to Plant Salinity Stress

doi:10.21203/rs.3.rs-3760168/v1

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Structural and Molecular Dynamic Simulation Studies of CBL-Interacting Protein Kinase CIPK and its Complexes Related to Plant Salinity Stress

https://doi.org/10.21203/rs.3.rs-3760168/v1

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Calcium-dependent signaling in plants is responsible for several major cellular events, including the activation of the salinity-responsive pathways. Calcium binds to calcineurin B-like protein (CBL), and the CBL-Ca²⁺ binds to CBL-interacting protein kinase (CIPK). The CBL-CIPK complex enhances the CIPK interaction with an upstream kinase. The upstream kinase phosphorylates CIPK that, in turn, phosphorylates membrane transporters. Targeted membrane transporter phosphorylation influences its activity and kick-starts many downstream functions, such as balancing the cytosolic Na⁺-to-K⁺ ratio. The CBL-CIPK interaction is pivotal for Ca²⁺-dependent salinity stress signaling. The plant contains multiple CBL and CIPK genes coded in their genomes. Hence, different yet specific combinations of CBL and CIPK are responsible for targeting particular ion transporters. Here, we present the computationally predicted structures of autoinhibited CIPK24 and CIPK24-CBL4 complex. The models are supported by the available structural and functional data. Models are energy-minimized and subjected to molecular dynamics (MD) simulations. MD simulations enabled us to predict the importance of conserved residues of the proteins. Finally, the work is extended to predict the CIPK24-CBL4 complex with the upstream kinase GIRK2. MD simulation on the ternary complex structure enabled us to identify the critical CIPK24-GIRK2 interactions. Together, these data could be used to engineer the CBL-CIPK interaction network for developing salt tolerance in crops.

Calcineurin B-like protein

CBL-interacting protein kinase

Protein kinase

protein-protein interactions

Plant salt tolerance

Calcium-dependent signaling in plants is responsible for several major cellular events, including the activation of salinity-responsive pathways [32, 56]. Calcium and calcineurin B-like protein (CBLs) complex binds to CBL-interacting protein kinases (CIPKs) to propagate signals in plants. CBL-CIPK complex targets membrane ion transporters that belong to the ion homeostasis membrane protein group [48, 52]. Although the targeted membrane transporters control the Na⁺-to-K⁺ ratio in the cytoplasm, some recent data suggests that the CBL-CIPK complex also regulates Ca²⁺ transporting membrane proteins [33, 48]. Interaction between CBL and CIPK is the pivotal phenomenon in Ca²⁺-dependent salinity stress signaling in plants. Different yet specific combinations of CBL-CIPK are possible that target a particular group of membrane ion transporters. For example, Arabidopsis thaliana CBL4 (SOS3) interacts with CIPK24 (SOS2) to regulate plasma membrane Na⁺/H⁺ transporter SOS1 [16]. In the same plant, CBL8 also interacts with CIPK24 to activate SOS1, but the interaction is apparent during higher salt stress [51]. CBL10, which was first reported to combine with CIPK24 for targeting a yet unknown vacuolar transporter, can also complex with CIPK8 to activate plasma membrane SOS1 transporter [24, 60]. Homologous interactions between CIPK24 and its CBL partners are variable among plants. The yeast two-hybrid method shows that physical interactions between CIPK24 and CBL4 are possible in cassava and rapeseed plants, whereas the same interaction is not valid in eggplants [28, 31, 36]. CIPK24 and CBL10 interactions are also not universal in plants [31].

CIPK consists of two distinct domains joined by a flexible loop [Figure 1A]. The N-terminal domain is a kinase domain (KD), and the C-terminal domain is a protein-protein interaction or regulatory domain (PPI/RD). Between the domains, a flexible loop is present that binds to the CBL-Ca²⁺ complex. It has been predicted that the PPI domain interacts with KD when CIPK is not phosphorylated, resulting in inactivation [17]. Upon binding to the CBL-Ca²⁺ complex, the CIPK obtains an open conformation allowing the complex to bind a variety of regulatory molecules. For example, CBL4-CIPK24 complex interact with nucleoside diphosphate kinase 2 and catalase for redox homeostasis in plants [54]. Others have proposed a glutamine-based modulation of CBL4-CIPK24 complex for the regulation of SOS1 [35]. But most importantly, CBL4-CIPK24 needs activation by upstream kinase and deactivation by the protein phosphatases. CIPK is phosphorylated by the upstream kinases like Geminivirus Rep-Interacting Kinases (GRIK1/2) primarily at its phosphate binding loop [4]. Phosphorylated CIPK is deactivated by the abscisic acid-insensitive 1/2 (ABI 1/2), which is a protein phosphatease 2C group of phosphatase [39].

CBLs are predominantly membrane localized by N-terminal myristoylation or by possessing N-terminal transmembrane helices [21, 24]. A CBL-CIPK complex is directed to the membrane for phosphorylating the target membrane transporters. Some reports suggest that CIPK can phosphorylate CBL at its C-terminal region to further enhance its activity [18, 29].

CIPK belongs to a conserved protein kinase group. All plant genomes known to date contain more than one CIPK coded in their genomes [33]. In Arabidopsis thaliana (At), there are 26 CIPK proteins encoded in its genome [25]. These proteins share 39–77% sequence identities; hence, the overall protein structure of CIPK must be the same (Suppl. Figure 1.). Only a few structures of individual domains of plant CIPK proteins are determined at atomic resolution [Figure 1B-C]. These include the crystal structures of AtCIPK14-PPI domain complex with AtCBL2 [1], AtCIPK24-PPI domain complex with AtCBL4 [47], and the KD structures of AtCIPK24 and AtCIPK23 [8]. Due to the lack of detailed CIPK structural information, there are several unanswered questions. First, what does the auto-inhibited CIPK24 structure look like? Second, what is the most probable conformation of the CBL4-CIPK24 complex? Third, how does the CBL4-CIPK24 complex enable CIPK24 interaction with the upstream kinases?

In this work, the attempt is made to computationally build CIPK models in their autoinhibited and uninhibited forms, followed by computational prediction of the CBL-CIPK complex and CBL-CIPK24-GIRK2 complex. The predicted structures are supported by the known data. Together, these data could be used to develop salt tolerance in plants.

Bioinformatics

Arabidopsis thaliana CIPK14 (Q9LZW) and CBL4 (O81223) sequences are retrieved from the UNIPORT database. The templates used in the study are summarized in suppl. table 1. Multiple sequence alignment is done using MAFFT [22] and presented using EsPript [45]. As required, structural editing of PDBs is done using PyMOL [12], COOT [15], or Chimera [43]. All data plotting figures are generated using Matlab (https://in.mathworks.com).

Computational Modeling

The templates for homology modeling were identified from the I-TASSER server [59]. Required atomic coordinates were obtained from PDB, manually edited, and used for homology modeling in MODELLER software [55]. For making the CBL-CIPK complex, the CIPK open conformation was first modeled using MODELLER software. The best-modeled structure is selected using the DOPE score.

CBL4 docking onto CIPK24 open conformation was done using multiple protein-protein docking servers, including ClusPro [26], PatchDock [49], Haddock [11], and ZDock [44]. Multiple docking servers were used to find a consensus docking position. For making the CIPK-GRIK2 complex, the GRIK2 structure was obtained from the AlphaFold model available in the Uniport Server (https://www.uniprot.org/). Protein-protein docking was done as mentioned above. The available structural and biochemical data supported the docking positions in complex structures. The modeled structures were refined and energy minimized using Rosetta Relax Protocol [] and validated using Procheck [27]. The validated structure was used for MD analysis.

Rosetta structure refinement:

Rosetta Relax protocol was employed for initial structure refinement [5, 23, 38, 53]. The protocol optimizes protein structures through a systematic multi-step process. The protocol is integrated within the Rosetta force field framework. It executes multiple iterations of amino acid side-chain reorganization and energy minimization procedures to explore and identify the most energetically favorable conformation for a given protein structure, effectively navigating local energy barriers. The relaxation process first introduces small perturbations to the protein structure via adjustments in backbone torsion angles followed by side-chain repacking and energy minimization calculations and optimizing the structure's torsional angles (Φ, Ψ, and χ_j). Notably, each perturbation step operates under a predefined weight for the van der Waals repulsive component of the all-atom scoring function (fa_rep) [2]. The orchestrated process enables the structure to traverse the local energy landscape, facilitating transitions toward new energy minima. Concurrently, clashes arising during each cycle are systematically resolved. The resultant structure with the lowest energy was selected as the input structure for subsequent Molecular Dynamics (MD) analyses, ensuring a refined starting point for further computational investigations.

Molecular Dynamic Simulations

The molecular dynamics (MD) simulation was performed using AMBER20 software with the ff19SB force field [6, 7, 41, 46]. The system was solvated with TIP3P water molecules in a cubic box and was neutralized by adding counter ions using the 'tleap' module of AMBER. Simulation involving calcium ions was done using the frcmod.ions234lm_1264_tip3p parameter file. Initially, the system's energy was minimized using a dual-step algorithm, employing the steepest descent method followed by the conjugate gradient method. The energy minimization stabilizes the system and eliminates unfavorable contacts that could lead to unrealistically high energies or aberrant trajectories. Identifying and removing these detrimental contacts was imperative to ensure the reliability and accuracy of subsequent simulations. The key parameters governing this optimization involved the system undergoing minimization with 50,000 steps of steepest descent followed by 50,000 steps of conjugate gradient methods. During minimization, positional restraints ('ntr = 1') with a restraint weight of 100.0 were utilized. The heating process was carried out under constant volume (ntb = 1) conditions without any pressure control (ntp = 0). The heating simulation was run for 50000000 steps (nstlim) using a time step of 0.002 picoseconds (dt). The SHAKE algorithm was employed to constrain bonds involving hydrogen atoms. A Langevin thermostat with a collision frequency of 1.0 ps-1 (gamma_ln) was applied. Additionally, positional restraints were applied to prevent large conformational changes during the heating process. Additional positional restraints with a weight of 100 (restraint_wt) were applied to atoms. During the first 40 million steps of the heating process, the temperature was gradually increased from 100K to 300K, and for the remaining 10 million steps, the temperature was held constant at 300K.

During equilibration, the simulation utilized periodic boundary conditions (iwrap = 1) to confine positions within the primary unit cell. Under these conditions, constant pressure was applied (ntb = 2) alongside pressure control (ntp = 1). A total of 500,000 steps were executed (nstlim) with a time step size of 0.002 picoseconds. The treatment of bond lengths and angles employed the SHAKE algorithm (ntc = 2) for hydrogen atoms and utilized the Amber force field for interactions (ntf = 2). For temperature control, Langevin dynamics were employed (ntt = 3) with a collision frequency of 1.0 ps-1 (gamma_ln). The relaxation time for pressure coupling was set to 0.1 ps (taup). Positional restraints of 100 were applied during equilibration to maintain specific atom positions within the system. The simulation was maintained at a target temperature of 300 K. The trajectory was analyzed to ensure that the system had reached a stable equilibrium state.

During the production phase of the simulation, various parameters were applied to ensure accurate modeling of the system. Constant pressure was maintained (ntb = 2) along with isotropic position scaling (ntp = 1), facilitating a stable environment for the simulation. A considerable duration of 50,000,000 steps was executed with a time step size of 0.002 picoseconds. The treatment of bond lengths and angles utilized the SHAKE algorithm (ntc = 2) for hydrogen atoms and applied the Amber force field for interactions (ntf = 2). Temperature control employed Langevin dynamics with a collision frequency of 1.0 ps-1 and a relaxation time of 0.1 picoseconds. The simulation commenced at an initial temperature of 300.0 Kelvin. These settings ensured a prolonged and stable simulation for the production phase, allowing detailed observations and analyses of the system's molecular dynamics behavior. For all calculations, a cutoff distance value of 10 was used for non-bonded interactions, beyond which no interactions are calculated. Finally, the trajectory was analyzed using CPPTRAJ and chimera software to extract relevant data, such as RMSD, RMSF, and specific analysis. All variables were explained in Amber Reference Manual (https://ambermd.org/Manuals.php)

Structure of autoinhibited CIPK

The autoinhibited conformation of CIPK24 is modeled based on the human Ca²⁺/calmodulin-dependent protein kinase, MARK1 (MAP/Microtubule affinity-regulating kinase) (6c9d), which shows a tight association between kinase domain (KD) and regulatory domain (KA1) [14]. The structural similarity between CIPK and MARK has been observed before [47]. CIPK-KD (1-300) and the MARK-KD show 45% sequence identity, whereas CIPK24-PPI (341–426) and MARK-KA1 share 29%. Superposition of CIPK24-KD (1-300) and CIPK24-PPI (340–426) crystal structures on corresponding domains of MARK1 structure (6c9d) show 1.14 Å and 1.28 Å RMSD, respectively indicating significant structural homologies between two proteins. Building the KD and PPI domain-connecting loop (301–339) is done based on the orientations of the C-terminal of KD and the N-terminal of PPI domains. The secondary structures of the domain-connecting loop are built based on the known crystal structure information and the predicted secondary structures using PSIPRED [34]. The edited model is used as a template in MODELLER, and the best-predicted structures are chosen for subsequent MD simulation. The final CIPK24 structure after simulation is shown in Fig. 2A. Procheck validation shows that the structure has 99.7% residues in the most favored and allowed regions. The overall structures of the two domains are essentially the same as in the crystal structure, with some local rearrangements of the loops. The PPI domain sits at the interface of the N- and C-lobes of the KD. The NAF/FISL loop runs parallel and in opposite directions, where it becomes close to the N-terminal end of the protein.

A total 500 ns MD simulation run shows the structure gradually reaches stability (Fig. 2B). However, at 128 ns and 243 ns, drifts are observed in the RMSD vs time graph. RMSF calculations of the autoinhibited CIPK24 reveal that many PPI domain regions undergo more fluctuations than the kinase domain (Fig. 2B inset). Analysis of the intermediate structures reveals significant structural variation around the active site loop residue Val¹⁶³, the NAF/FISL loop, and the PPI domain residues Asn³⁶⁹ and Ala³⁸². Comparison between the initial and the final CIPK structure (after simulation) reveals reorientation of the active site loop spanning the residues 160–172. Measurement shows that after simulation, the loop shifts closer to the N-terminal ²⁰GTFA²³ motif and the PPI domain (Fig. 3A). The GTFA motif is conserved in CIPK proteins and can contribute to reorienting the active site loop (160–172). During simulation, another major readjustment occurs to the domain-connecting NAF/FISL loop (301–339). RMSF calculation demonstrates that readjustment of the loop occurs during the first 100 ns cycle and shows lesser structural fluctuations during the rest of the simulation cycles. Two regions of the loop ³¹³FEMITL³¹⁸ and ³²⁴LSALF³²⁸ are initially modeled as helices based on the predicted secondary structure and the x-ray crystallography structure available (2ehb). After 500 ns simulations, the helical structure of only ³²⁴LSALF³²⁸ segment is retained. The result indicates that during the autoinhibited state, ³¹³FEMITL³¹⁸ exists as a loop structure that attains helical conformation upon CBL binding (Fig. 3B.).

The simulation data revealed that the interaction between KD and the PPI domain is weak yet stable. The average centroid distance between the domains is maintained at 25 Å. Structural comparison before and after simulation manifests subtle reorientations of the N-lobe and the PPI domains. Interdomain interactions are mediated by hydrogen bonding, electrostatic, and hydrophobic interactions. αD helix of the kinase domain is primarily involved in interdomain interactions. Glu⁹⁵, ⁹⁸Asp-Arg⁹⁹, and His¹⁰² belong to the αD helix and interact with the C-terminal residues of the domain-connecting NAF/FISL loop (Arg³³⁷ and Glu³³⁸) (Fig. 4A). Although Arg³³⁷ and Glu³³⁸ fall within the range of NAF/FISL loop, this region is also proposed to be a part of regulatory domain [54]. Transient interactions are also observed between Lys⁴⁰⁵ and His⁴¹⁴ with the αD helix residues. The corresponding αD helix of MARK1 also participates in intra-domain interaction [14]. The N-terminal of αD helix in AtCIPK24 comprises the most conserved residues ⁹³GGELFDRIVH¹⁰² in AtCIPKs and, therefore, can have a role in interdomain interactions (Suppl. Figure 2.). The 500 ns simulation further shows a steady shift of the active site loop toward the PPI domain. The active site loop movement towards the PPI domain is monitored using the distances between Cys¹⁷⁰ and Glu⁴⁰⁹. Cys¹⁷⁰ is in the active site loop, closest to the PPI domain, and Glu⁴⁰⁹ in the PPI domain shows relatively less atomic fluctuation (Fig. 2b, inset). The result shows that the Cys¹⁷⁰ to Glu⁴⁰⁹ distance is decreased (Fig. 4B-C). Cys¹⁷⁰ is a conserved residue, whereas glutamates or aspartates are found in the Glu⁴⁰⁹ position in AtCIPKs (Suppl. Figure 2.). Interestingly, Glu⁴⁰⁹ is located in the β5-α2 loop in the PPI domain, yet it undergoes less flexibility. We propose that Glu⁴⁰⁹ have an active-site loop-engaging role in the CIPK autoinhibited state.

The MARK1/KA1 domain mediates phospholipid interactions [37]. The cluster of charged residues in MARK1 is suggested to be Glu⁷⁵⁶, Arg⁷⁷¹, Lys⁷⁷³, and Arg⁷⁷⁴. The MARK1/KA1 and CIPK-PPI superimposition identified the corresponding amino acids Glu³⁹⁰, Asp⁴⁰², Arg⁴⁰⁴, and Lys⁴⁰⁵ (Suppl Figure). Although the interactions between the CIPK24/PPI domain and the membrane are not known, it is possible that CBL-mediated CIPK targeting to the membrane opens up the PPI domain and interacts with the membrane.

Structure of CIPK-CBL binary complex

The CIPK24-CBL4 complex is modeled in two steps, first by modeling the CIPK24 open conformation and then by predicting the CBL4 docking onto the CIPK24 open conformation. CIPK24 open conformation is modeled based on the human adenosine monophosphate-activated protein kinase α subunit (AMPKα; 7myj and 4rer) [30, 40, 58]. Sequence alignment between the CIPK24-KD and the AMPKα-KD shows 45% sequence identity, and the same between CIPK24-PPI and AMPKα-CTD is 20%. Superposition of CIPK24-KD (1-300) and CIPK24-PPI (340–426) crystal structures on corresponding domains of AMPKα structure (7myj) show 1.31 Å and 1.33 Å RMSD, respectively indicating significant structural homology. In the CIPK24 open conformation model, the relative positions of the NAF/FISL loop and the PPI domain are somewhat similar to that of the CBL-complex structure (2ehb) (Suppl. Figure 3.). The structure is validated using Procheck. After obtaining the CIPK24 open conformation, the CBL4-CIPK24 complex is predicted by protein-protein docking servers. Results from different protein-protein docking servers show a consensus CBL docking mode. In the final CBL4-CIPK24 complex, the PPI domain is located on the opposite side of the KD-active site, keeping the phosphorylation loop accessible to other proteins [Figure 5].

The CBL4-CIPK24 complex model is energy-minimized and validated using Procheck. Procheck validation shows that the structure has 99% residues in the most favored and allowed regions. A 300 ns MD simulation run is performed, showing the complex reaches stability at the end of the run (Fig. 6A). CBL-CIPK complex makes a transient complex, which becomes weakened during the following simulation runs. RMSF calculations show that two particular regions of the CIPK encompassing the residues Asp²⁸³-Ile²⁹¹ and Asp³³³ undergo significant atomic fluctuations (Fig. 6B-C). Asp²⁸³-Ile²⁹¹ harbors a short helical segment that flanks out and opens the NAF/FISL loop for CBL binding. Asp³³³ is located in the C-terminal of the CBL4-binding segment of CIPK24.

Interactions between the CBL4-CIPK24 are predominantly driven by hydrophobic interactions (Fig. 7). For example, the Phe³¹³ remains within hydrophobic interacting distance with CBL4/Ala¹⁶⁰, CBL4/Leu¹³³, and CBL4/Met¹⁵⁶. Phe³¹³ is part of the conserved ³¹¹NAFEMI³¹⁶ motif of the NAF/FISL loop. Phe³²⁸ hydrophobic interaction with Tyr¹⁹⁵ and Leu¹⁹³ does not change more than 0.5 Å during the 300 ns simulations. Phe³²⁸ is also a conserved residue in the CIPK24 NAF/FISL loop. The conserved Leu³²⁴ and CBL4/Phe⁸³ hydrophobic interaction also do not change more than 0.2 Å during the simulation timeframe. A few hydrogen bond interactions are observed and remain consistent during the simulation, including the interaction between the conserved Glu³¹⁴ with CBL4/Ser¹⁸⁶ and Ser³¹⁹ with CBL4/Glu¹⁴³. In the CBL4-CIPK24 complex structures, the CIPK24 residues Phe³¹³ and Ser³¹⁹ are found to be interacting with CBL4. Other CBL4-CIPK24 interactions, such as Arg³³¹ with CBL4/Asp¹⁹⁸, Lys³⁷¹ with CBL4/Glu¹⁴⁵, Glu³⁹³ with CBL4/Arg⁷¹, and Arg⁴⁰⁴ with CBL4/Glu¹⁴⁵ are mostly confined within the PPI domain of CIPK24.

CBL4 shows lesser atomic fluctuations owing to its compact structure. However, the C-terminal region around the residue Leu¹⁴⁸ shows the highest fluctuation [Figure 6C].

Structure of CBL-CIPK-GIRK ternary complex

Computational prediction of GIRK2 binding to CIPK24 is done on the CBL4-CIPK24 complex. The active site of the CBL4-CIPK24 complex is open and can be accessed by the upstream kinases. Arabidopsis GIRK2 structure was predicted using ITASSAR and compared with the alphafold structure and used for docking. The result shows that GIRK2 docks on the CIPK24-KD head-to-tale fashion, comparable to the CIPK24-KD dimer in the crystal structure. GIRK2 shows a typical Ser/Thr protein kinase fold like CIPK. Hence, it is assumed that the GIRK2-CIPK24 heterodimer mimics the CIPK-CIPK homodimer (Fig. 8).

A total 300 ns simulation shows that the structure is stable throughout the simulation (Fig. 9A). RMS fluctuation (Fig. 9B-D) shows that the significant structural rearrangement of CIPK24 is limited to the vicinity of the CBL4-binding region. On the other hand, only a few atomic fluctuations are observed in its kinase domain. GIRK2, a protein kinase, structure was compared with the closest sequence homologue murine NFκβ-inducing kinase (NIK) structure (4g3f) to identify the important amino acids [10]. The GIRK2 consists of N- and C-lobes. C-lobe comprises α helices and undergoes relatively higher structural fluctuations than the N-lobe. Functional amino acid segments in GIRK2 are identified using multiple sequence alignment and are clustered at the interface of its N- and C lobes (Suppl. Figure 4.). GIRK2 functional segments, such as the phosphate binding P-loop ¹¹³IGSGSYGKV¹²¹ and the catalytic residues ²³⁶HGDIK²⁴⁰ are located at the N- and C-lobe interface. CIPK24 active site loop residues Thr¹⁶⁸ and Thr¹⁶⁹ are close to the GIRK2 P-loop Gly¹¹⁶ and Ser¹¹⁷ residues (Fig. 10A). Simulation data reveals that the two regions have close interactions and shows the reorientation of the CIPK24 active site loop and the ordering of the GIRK2 P-loop (Fig. 10B-C). The reorientation and the ordering of the loop are not artifacts, as these regions are involved in phosphate exchange. A few other interactions surrounding the active site loop may be involved in holding the active site loop in position. For example, Glu⁹⁵ and Glu¹³⁸ of CIPK24 are observed interacting with the P-loop residue Lys¹²⁰. Glu⁹⁵ and Glu¹³⁸ positions in CIPKs are occupied with the negatively charged residue (Seq alignment). In GIRK2, Lys¹²⁰ is located at the C-terminal of the P-loop. Multiple sequence alignment of GIRK2 with other homologous protein kinases suggests that the charged or polar residue occupies the Lys¹²⁰ position. These interactions, therefore, have a role in stabilizing the active site loop.

Transient interaction between CBL and CIPK proteins regulate a multitude of protein activation pathways in plants. CIPK remain in autoinhibited state but structural alteration occurs to make complex with a specific CBL protein partner. The transient CBL-CIPK complex is phosphorylated by upstream kinases and the phosphorylated CIPK can regulate membrane transporters. Current understandings of the CIPK autoinhibited and complex structures are limited. Because, the partial CBL and CIPK structures are known and the structures of the CIPK homologous are known, attempt is taken to predict the autoinhibited and active CIPK structures.

The autoinhibited CIPK structure is predicted based on the human Ca²⁺/calmodulin-dependent protein kinase; MARK1 due to its significant structural homology with CIPK. The relative orientations of the KD and PPI domains are not confirmed experimentally, but the outcomes from the structural analysis and the simulation study affirms the predicted structure. An independent computational docking further confirms the PPI domain position on the KD domain. CIPK autoinhibited structure remains stable and converges during the simulations. Plotting the radius of gyration value during the simulation reveal that the structure remains compact. Analysis shows that the interaction between the KD and the PPI domain is weak but PPI domain does not move away form the KD structure. Interdomain interaction is maintained due to the interactions between the αD helix and the C-terminal residues from the NAF/FISL loop. Simulation data suggesting a steady shift of the active site loop towards the PPI domain and the ²⁰GTFA²³ manifests the autoinhibition mechanism. Membrane transporters and protein-lipid interaction in membrane is suggested to be one of the pivotal phenomena during salinity stress in plants [3, 13]. CIPKs activating ions transporting membrane proteins must be membrane proximal phenomena. It is not known if CIPK has any affinity towards the phospholipid membranes, which is otherwise driven by CBL proteins. Most CBL binds to the membrane via N-myristoylation or acylation whereas some CBL has integral membrane helix. Since, the CBL-CIPK mediated phosphorylation is a membrane-proximal phenomena, it is possible that CIPK might have affinity for the phospholipid membrane. Interestingly, the template MARK1, used for modeling CIPK24, has intrinsic affinity towards phospholipids through its KA1 domain. Positively charged residues are found in CIPK24-PPI domain when structural comparison is done with MARK1-KA1 domain, but in AtCIPKs, the positions of the positively charged residues are sparsely conserved. However, further study is needed to confirm if CIPKs have any interaction with the membrane.

The CIPK24 open conformation is built based on the activated conformation of the AMP-activated protein kinase (AMPK). AMPK autoinhibitory domain (AID) and the regulatory-subunit-interacting motif (RIM) control the phosphorylation-dependent activation of the KD [9]. During inactive conformation, AID remains in folded conformation whereas upon activation, it obtains an open conformation freeing the KD active site for phosphorylation [42]. The structural modeling assumes that in CIPK24 open conformation, the NAF/FISL loop brings the PPI domain to the opposite face of the CIPK that of the autoinhibited face. This conformational change is facilitated by the CBL4 binding to the NAF/FISL loop. MD simulation endorses the stability of the CBL-CIPK complex. It is observed that the interaction is mostly due to the hydrophobic interactions, and a few conserved hydrogen-bonding interactions are observed such as Glu³¹⁴-CBL4/Ser¹⁸⁶ and Ser³¹⁹-CBL4/Glu¹⁴³. CBL4/Glu¹⁴³ is located in a loop, showing the highest atomic fluctuations (Fig. 6C). The region is juxtaposition of the PPI domain and another negatively charged residue in that loop Glu¹⁴⁵ is involved in interactions with two positively charged residues of PPI domain; Lys³⁷¹ and Arg⁴⁰⁴. It is not known if PPI has any role in CBL4 binding and its interaction with PPI domain is not evident from the crystal structures. PPI is involved in protein-protein interaction; hence CBL-CIPK interaction might occur through PPI domain. Crystal structures of CBL2 and CBL4 complexes with their cognate CIPK partners reveal a role of backbone interactions between Gln³²⁰-CBL4/Glu¹⁴² [47]. A close backbone interactions between Gln³²⁰ and CBL4/Glu¹⁴³ are also observed in some intermediate simulation structures endorsing the predicted complex structure. Another observation is the CBL4-CIPK24 complex exposes the ³⁴⁰RF³⁴¹ motif allowing it to interact with the other regulatory proteins such as ABA-INSENSITIVE2 Ser/Thr protein phosphatases [39]. In case of autoinhibitory state, CIPK24 αD helix maintains a close contact with the region Gln³³⁸, hence keeping ³⁴⁰RF³⁴¹ not accessible to other molecules.

AMPK in mammals are phosphorylated by the upstream kinases LKB1 and CaMKKβ kinases [19, 20, 57]. BLAST results identified mammalian LKB1 and CaMKKβ share highest homology with the Arabidopsis thaliana GIRK1 and GIRK2. It has been shown that both GIRK1 and GIRK2 kinases of Arabidopsis thaliana can phosphorylate sucrose non-fermenting 1 (SNF1) related protein kinases such as SnRK1 [4, 50]. It is also evidenced that the SnRK kinase domain is sufficient to bind to GIRK proteins. SOS2 is a class 3 SnRK which was shown to be phosphorylated by GIRK2 [4]. For this study, GIRK2 is used because GIRK2 alpha fold model (Q5HZ38) shows least RMSD value when compared to the ITASSR GIRK2 model. The CBL4-CIPK24-GIRK2 ternary complex essentially uses the model of CBL4-CIPK24 complex. GIRK2 and CIPK24 both are protein kinases therefore share a similar fold. GIRK2 docking on CIPK shows a head-to-tail association similar to the CIPK dimer association in crystal structure. Simulation data shows that the C-lobe of CIPK has stronger interaction with the N-lobe of GIRK2 that shows lesser atomic fluctuations. Analyses of the GIRK2 protein sequences have identified critical motifs of the protein including the phosphate-binding P-loop and the catalytic bases. In GIRK2 these motifs are ¹¹³IGSGSYGKV¹²¹ and ²³⁶HGDIK²⁴⁰. The ¹¹³IGSGSYGKV¹²¹ motif is regarded as P-loop, which is a phosphate binding loop located close proximity to the CIPK active site loop. Analyzing the ternary structures before and after the simulations, revealed the CIPK active site loop reorientation and the ordering of the GIRK2 P-loop. Another part of the active site, the catalytic residues ²³⁶HGDIK²⁴⁰ also substantially moved towards the CIPK24 active site loop (Fig. 8c). Therefore it is possible that the coordinated movements of the loops are required for the phosphate transfer. Interactions between the Glu⁹⁵ and Glu¹³⁸ with Lys¹²⁰ are important for holding the active site loop in position.

Based on the structural data a series of protein-protein interaction events can be hypothesized involving CIPK activation [Figure 9]. During CIPK autoinhibition, the PPI domain binds close to the kinase domain active site. The active site loop has some inherent affinity towards the PPI domain and become closure to KD active site loop during the simulation time frame. It is also registered that, PPI and kinase domain interaction is weak, therefore, the CIPK could show basal level activity. Interaction between the PPI and KD is broken as the NAF/FISL loop is bound to CBL protein. CBL binding to the NAF/FISL loop brings the PPI domain to the opposite side of the active site face of CIPK. CIPK open conformation can bind to a variety of interacting proteins including the upstream protein kinase GIRK2. CIPK-GIRK2 binding shows a head-to-tail association. CIPK active site loop is in close contact with the GIRK2 phosphate binding loop and the catalytic loop.

CIPK is the major Ser/Thr protein kinase contributing to the activations of many critical membrane transporters essential for controlling plant biotic and abiotic stresses. CIPK is an intermediate between the Ca²⁺ sensors and their targets while acting as a hub of many protein-protein interactions. Besides interacting with the Ca²⁺ sensors, CIPK activation and deactivation are controlled by the upstream kinases and the phosphatases, respectively. Simulation data of computationally predicted CIPK24 autoinhibited structure reveals the interactions of the PPI domain with both the NAF/FISL loop and the active site loop. The two interactions might have a role in CIPK24 autoinhibition. Simulation data of the modeled CBL4-CIPK24 complex shows a stable complex primarily driven by the hydrophobic interaction. The complex shows that the PPI domain may be involved in CBL4 interaction. Molecular dynamics simulation further endorses the CIPK24-CBL4-GIRK2 ternary complex, identifying critical amino acids in GIRK2 responsible for phosphate transfer. The results could be used to design CBL, CIPK, and GIRK constructs for further structural and functional studies of the individual proteins or their complexes.

Author Contribution

T.B., S.B., and S.M. performed the sequence alignment, initial model building, protein-protein docking and interaction analysis. P.K.D. performed the simulation experiments. D.D. conceptualize the study, finalize the models, analyze the simulation data. P.K.D and D.D. wrote the manuscript

Acknowledgement

The work is supported by the Science and Engineering Research Board (SERB) Government of India Start up Research Grant no. SRG/2022/000035 dt. 26/10/2022 to Debajyoti Dutta.

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Structural and Molecular Dynamic Simulation Studies of CBL-Interacting Protein Kinase CIPK and its Complexes Related to Plant Salinity Stress

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Abstract

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Introduction

Methodology

Bioinformatics

Computational Modeling

Rosetta structure refinement:

Molecular Dynamic Simulations

Result

Structure of autoinhibited CIPK

Structure of CIPK-CBL binary complex

Structure of CBL-CIPK-GIRK ternary complex

Discussions

Conclusions and Future Direction

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Author Contribution

Acknowledgement

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