Deciphering the structural basis of the broad substrate specificity of myo-inositol monophosphatase (IMP) from Cicer arietinum

Journal Pre-proof Deciphering the structural basis of the broad substrate specificity of myo-inositol monophosphatase (IMP) from Cicer arietinum

Prakarsh K. Yadav, Prafull Salvi, Nitin Uttam Kamble, Bhanu Prakash Petla, Manoj Majee, Saurabh C. Saxena PII:

S0141-8130(19)36380-9

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.11.098

Reference:

BIOMAC 13879

To appear in:

International Journal of Biological Macromolecules

Received date:

12 August 2019

Revised date:

7 November 2019

Accepted date:

11 November 2019

Please cite this article as: P.K. Yadav, P. Salvi, N.U. Kamble, et al., Deciphering the structural basis of the broad substrate specificity of myo-inositol monophosphatase (IMP) from Cicer arietinum, International Journal of Biological Macromolecules(2019), https://doi.org/10.1016/j.ijbiomac.2019.11.098

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© 2019 Published by Elsevier.

Journal Pre-proof Deciphering the Structural Basis of the Broad Substrate Specificity of myo-Inositol Monophosphatase (IMP) from Cicer arietinum Author names and affiliations: Prakarsh K Yadav1, Prafull Salvi2, Nitin Uttam Kamble3, Bhanu Prakash Petla3, Manoj Majee3, Saurabh C. Saxena1*

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1. Department of Biotechnology, Delhi Technological University, Shahbad Daulatpur, Bawana road, Delhi 110042, India 2. Agriculture Biotechnology Department, National Agri-Food Biotechnology Institute, Mohali, Punjab 140308, India 3. National Institute of Plant Genome Research (NIPGR), Aruna Asaf Ali Marg, New Delhi 110067, India

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*Corresponding author

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Dr. Saurabh C. Saxena, Assistant Professor Department of Biotechnology, Delhi Technological University, Shahbad Daulatpur, Bawana road, Delhi 110042, India E-mail address: [email protected] Tel.: +91-11- 27294668

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Journal Pre-proof Abstract Myo-inositol monophosphatase (IMP) is a crucial enzyme in the inositol biosynthetic pathway that dephosphorylates myo-inositol 1-phosphate and other inositol phosphate derivative compounds to maintain the homeostasis of cellular inositol pool. In our previous research, we have biochemically and functionally characterized IMP enzyme from chickpea (CaIMP), which was able to catalyze diverse substrates. We cloned, overexpressed recombinant CaIMP protein and purified it and further characterized the CaIMP with its three main substrates viz. galactose

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1-P, inositol 6-P and fructose 1, 6-bisP. Homology model of CaIMP was generated to elucidate the factors contributing to the broad

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substrate specificity of the protein. The active site of the CaIMP protein was analysed with

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respect to its interactions with the proposed substrates. Structural features such as, high B-factor and flexible loop regions in the active site, inspired further investigation into the static and

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dynamic behaviour of the active site of CaIMP protein. The electrostatic biding of each of the key substrates was assessed through molecular docking. Furthermore, molecular dynamics

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simulations showed that these interactions indeed were stable for extended periods of time under physiological conditions. These experiments conclusively allowed us to establish the primary

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factors contributing to the promiscuity in substrate binding by CaIMP protein.

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Keywords: Myo-inositol monophosphatase; Broad substrate catalysis; Structural analysis; Molecular simulation; Molecular docking

Introduction Inositol, a member of the cyclitols family, is an essential factor for the precise cellular metabolic functioning in all organisms. Myo-inositol is served as a precursor for the synthesis of inositol phospholipids and inositol phosphates. Biological activities of inositol are also associated with numerous biological process including gene regulation, hormone signalling, membrane tethering, stress tolerance, and raffinose family oligosaccharide synthesis (Loewus and Loewus, 1983; Loewus and Murthy, 2000; Majumder et al., 1997; Kaur et al., 2013; Salvi et al., 2016; 2018). 2

Journal Pre-proof Myo-inositol monophosphatase (IMP) is functionally a hydrolase, is a crucial enzyme in the inositol biosynthetic pathway that dephosphorylates myo-inositol 1-phosphate and other inositol phosphate derivative compounds to maintain the homeostasis of cellular inositol pool (Gillaspy et al., 1995). Therefore, the functional significance of IMPase attributes to its requirement for both pathways i.e. the de novo biosynthesis of inositol and the recycling of inositol through the PI pathway. Thus, IMPase may have function in regulatory pathways that utilize free inositol (Berridge and Irvine, 1989; Bone et al., 1992; Rajjou and Debeaujon, 2008). IMP is a broad substrate enzyme; belong to a metal dependent phosphatase family (Berridge and

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Irvine, 1989). After the first report of IMPase gene cloned from bovine brain tissue, there have been several studies on the IMP enzyme from several prokaryote and eukaryotes (Diehl et al.,

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1990). In humans, IMPase is uncompetitively inhibited by therapeutic concentrations of Li +

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results in depletion of inositol, and phosphoinositides that leads to decreased synthesis of the second messenger IP3 and is, therefore, exploited as a biological target for the treatment of

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manic depression (Atack et al., 1995; Bone et al., 1992; Fauroux and Freeman, 1999; Hallcher

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and Sherman, 1980; Kennedy et al., 1990; Saiardi and Mudge., 2018). The biochemical analysis of mammalian IMP (bovine, human, rat) also revealed the bi-functional nature of IMP enzyme catalysis (Parthasarathy et al., 1994, 1997). It was shown that, kiwifruit (Actinidia deliciosa)

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encode a plant homologue of the mammalian IMPase that also harbour galactose 1-P phosphatase activity (Laing et al., 2004). Likewise, (Conklin et al., 2006) reported that

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Arabidopsis, VTC4, encodes an L-Gal-1Pase/IMPase. Thus, similar to mammalian IMPase, plant

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IMPase also encodes a bifunctional enzyme, and shows coordinate regulation of inositol and galactose metabolism. Interestingly, soyabean IMPase showed multiple substrate specificity and catalyzes three different substrate including myo-inositol 1-phosphate, phytate and the sodium pyrophosphate (Islas-Flores and Villanueva, 2007). Unlike animals, plants IMPase are relatively less studied and their biochemical properties are examined in relatively limited species such as arabidopsis, chickpea, tomato, barely etc. (Fu et al., 2008; Gillaspy et al., 1995; Saxena et al., 2013; Styer et al., 2004). We previously had analysed a differentially expressed IMP gene from Cicer arietinum which positively contribute towards seed germination and abiotic stress tolerance (Saxena et al., 2013). The biochemical analysis of CaIMP indicated that it catalyses the removal of phosphate from galactose 1-phosphate and myo-inositol 1-phosphate and participate in myo-inositol and ascorbate biosynthesis (Saxena et al., 2013). Moreover, the enzyme also 3

Journal Pre-proof showed considerable activity with fructose 1, 6-bisphosphate. However, the physiological significance as well as the structural attribute of the broad substrate specificities of IMP is not well understood. Here, the three-dimensional structure and its detailed analysis revealed the presence of important amino acid residues that participate in different substrate binding. However, despite the presence of F-6-P motif in IMP the biochemical activity with fructose 1, 6bisP is obscure. The purified protein fractions were used to study the detailed biochemical characterization and illustrate the kinetic comparisons of the recombinant CaIMP enzyme with three substrates, i.e., galactose 1-P, inositol 6-P, fructose 1, 6-bisP. The possible causes of the

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kinetic differences associated with structural conformation of CaIMP for their substrates are

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discussed, based on the docking analysis, active site residues interaction, and the conformational changes at structure level of the IMPase. Overall, the structural and catalytic activity analysis of

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CaIMP enzyme aid a foundation for a better understanding of the regulation of IMPase in

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different cellular/metabolic pathways and thus inositol accumulation in plants. Material methods:

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Cloning, overexpression and protein purification of CaIMP

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CaIMP cloned into expression vector pET-23b (Novagen) were used from our previous study (Saxena et al., 2013). The clone was freshly expressed and the extracts were analyzed by 12%

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SDS-PAGE (Laemmli, 1970). Solubilization and purification of the recombinant CaIMP protein was done according to the procedure described by (Majee et al., 2004; Petla et al., 2016).

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Dialyzed expressed protein sample was purified using nickel charged affinity columns (GE Healthcare) following the manufacture’s protocol and used for further biochemical analysis (Saxena et al., 2013; Rabbani et al., 2011). IMP Assay IMP was assayed by colorimetric estimation of released inorganic phosphate (Pi) after enzymatic hydrolysis of suitable substrate with malachite green as described by Baykov et al. (1988). A typical 100 µl reaction mixture contained 30 mM various substrates like galactose 1-P, inositol 6-P, fructose 1, 6-bisP, 3 mM MgCl2, in 50 mM tris-HCl (pH 8) buffer. The reaction was carried out at 37°C for 1hour using ~10 µg of purified enzyme. After the incubation, 700 µl of deionized

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Journal Pre-proof water and 200 µl of malachite green solution were added to develop color and subsequently determine the released inorganic phosphate spectrophotometrically at A630. Three-Dimensional Computational Modelling The homology modelling of CaIMP, (UniProt ID: A0A1S2XI20), includes following stages: (a) The target protein sequence was obtained from NCBI-Genpept database and subsequently submitted to BLASTp (Altschul et al., 1990). The database chosen for BLASTp was PDB (Berman et al., 2000), which resulted in the identification of 1IMA|A| PDB, human inositol

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monophosphatase with bound myo-inositol, as suitable template for creating full atom three-

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dimensional model of CaIMP. This template was chosen because it was bound to myo-inositol and hence would provide the near-native conformation of protein-substrate complex. (b) The

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three-dimensional structure of target protein was generated by uploading the peptide sequence to the I-TASSER webserver (Yang and Zhang, 2015; Zhang, 2008). The active site residues of the

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generated model were predicted using the active site prediction tool hosted by Indian Institute of

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Technology-Delhi (Singh et al., 2011). The magnesium ion was added by overlapping with 1IMA, this protein ion complex was used for further structural analysis. The built model was energy minimized to remove any steric clashes, by utilizing the Steepest Descent Algorithm with

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convergence criteria of r.m.s gradient of 0.1 kcal/mol/Å in GROMACS-5.0.7. The minimized model was evaluated for Ramachandran Plot and stereo-chemical validation using PROCHECK

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Molecular Docking

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(Laskowski et al., 1993, 1996; Rabbani et al., 2014).

The substrates identified through the IMP assay, galactose 1-P, inositol 6-P, fructose 1, 6-bisP, were docked into the protein active site by using the molecular docking program AutoDock 4.2 (Morris et al., 2009; Trott and Olson, 2009; Rabbani et al., 2017a; b; 2018). Here the gird box was redefined and torsional flexibility was allowed for the same residues, Lys 38, Asp 94 and Cys 196. Docking was carried out by Lamarckian Genetic Algorithm using default software parameters. The docking poses were assessed with respect to the crystal structure PDB ID: 1IMA. The conformers with lowest binding energy in the majority cluster were selected. This allowed docking of putative substrate in a conformation like the natural substrate, myo-inositol 1phosphate. The selected poses were then energy minimized before subsequent MD simulations 5

Journal Pre-proof for relaxing the protein ligand-complex. This minimized structure was then used for analysing the protein-substrate interactions. Molecular Dynamics The molecular dynamics simulation of the apo protein and substrate conjugated structure were carried out by using GROMACS-5.1.4 (Abraham et al., 2015; Berendsen et al., 1995), with GROMOS 54a7 force field. Topologies for ligand hetero-atoms were generated by PRODRG server, while considering complete charges and chirality of atoms. Each structure was placed in a

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dodecahedron box extending 10Å from each extremity of protein, to compensate for

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compressibility during dynamics. The box was solvated by using the SPC216 solvent system which is pre-equilibrated at 300K and the net charge of system was neutralized by adding

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appropriate amount Na+ ions, by random replacement of solvent molecules. This solvent-protein system was then energy minimized with backbone restraints to remove hydrogen clashes and

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optimize solvent molecules. The cut-off criteria were forces less than 100.0 kJ/mol/nm or

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50,000. This optimized structure was equilibrated in two steps at 300 K. First, a 100ps simulation of NVT ensemble followed by another 100 ps of simulation in NPT ensemble by using the Leapforg Dynamics integrator and 1fs time step. Verlet scheme was employed for neighbour

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search, updating after every 10 steps, under Periodic Boundary Conditions and harmonic constraints were applied by LINCS algorithm. Temperature coupling was done by Berendsen

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coupling algorithm and pressure coupling was done by Parrinello-Rahman algorithm, long range

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electrostatic interactions were computed by Particle Mesh Ewald (PME) algorithm. After these equilibration steps, production run was carried out under NPT ensemble and no constraints for 50 ns using identical parameter. Results and Discussion Purification and kinetic characterization of CaIMP with galactose 1-P, inositol 6-P, fructose 1, 6-bisP IMP has been known to harbour broad substrate specificities in few organisms including Arabidopsis, Glycine max, Methanococcus and Actinidia deliciosa to name a few (Gillaspy et al., 1995; Maesen, 1987; Nardozza et al., 2013; Nourbakhsh et al., 2015). Previously, we have performed the biochemical analysis of the IMP from chickpea which revealed that CaIMP 6

Journal Pre-proof utilizes galactose 1-P and inositol 6-P as preferred substrate (Saxena et al., 2013). Though, CaIMP also exert considerable activity with fructose 1, 6-bisP, its biochemical analysis has not been performed yet. We have therefore sought out to measure the kinetic parameter of CaIMP with fructose 1, 6-bisP substrate utilization. To address this, we again purified the CaIMP protein and used purified protein fraction to measure IMP activity with galactose 1-P, inositol 6-P, fructose-1, 6-bisP as substrates. Further, Km and Vmax along with optimum temperature and pH were measured as described by Rabbani et al (2012; 2015). The kinetic parameters are presented in Figure 1E. With the fructose 1,6-bisP substrate, the Km value was least out of three substrates

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which showed its less affinity with CaIMP in comparison with galactose 1-P and inositol 6-P.

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The biochemical results clearly indicated that CaIMP is much sensitive for galactose 1-P and inositol 6-P as compared to fructose 1, 6-bisP. As the later substrate is known to be involved in

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gluconeogenesis, probably CaIMP action is attributed to ensure the coordinated regulation of glycolysis/gluconeogenesis pathway by controlling the unusual rise in fructose 1, 6-bisP level.

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Also, we cannot discount the possible role of broad substrate catalysis of CaIMP to regulate

environment.

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Sequence analysis of IMP protein

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inositol level for cellular signalling as well as in stress tolerance during unfavourable

BLAST search of IMP sequence (Uniport ID A0A1S2XI20), on RCSB-PDB server showed

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greatest similarity with human inositol monophosphatase protein. We chose the substrate bound

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structures, PDB ID: 1IMA for modelling the CaIMP protein, among the multiple structural variants available from human IMP. This structure depicted D-Myo inositol bound to the IMP protein, which is the natural substrate for this enzyme. This allowed us to generate a model which had conformation similar to that of substrate bound enzyme and thus, mimicking the “active state” of CaIMP enzyme. Sequence alignment showed that the key residues in active site were well conserved, including the Mg2+ ion binding residues, Glu71, Asp91, Asp94 and Asp221. There was considerable similarity in the side chains involved in recognition of phosphate moiety (Figure 2).

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Journal Pre-proof Three-Dimensional Computational Modelling The three-dimensional structure of target protein (CaIMP) was generated through I-TASSER (Yang and Zhang, 2015) and the best model based on the quality was selected for future analysis. The Ramachandran plot of the CaIMP model had 94.03% residues in the favoured region while 5.97% residues were present in outlier region. This plot is shown in the Supplementary Figure S1. The generated model of CaIMP had an r.m.s. deviation of 0.6828 Å from the template model of

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human inositol monophosphatase (PDB ID: 1IMA). The overall topology of the model protein

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was identical to the x-ray diffraction structure of human IMP. The region involved in recognition of sugar moiety is largely appeared in a flexible loop with much flexibility than other region of

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the proteins. This flexibility of loop region endows the protein to accommodate different sugar moiety as a substrate. The region involved in recognition of sugar moiety was largely in loop

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region, thereby allowing the protein to accommodate variation in the sugar moiety of the

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substrate. This loop region had greater flexibility when compared to other regions of the protein. The modelled CaIMP protein has a pI value of 5.05 and a high density of positively charged residues at the phosphate binding region of the protein active site. This region is responsible for

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interacting with and stabilizing the Mg2+ ion in the protein. The amino acids contributing to this are Glu71, Asp91, Asp94 and Asp221. Together these residues form a pocket for the Mg 2+ ion to sit

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and interact stably with the phosphate moiety of the ligand. There are three loops in particular

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which interact with the sugar moiety of the inositol-1-phosphate molecule, which are Glu163Thr166, Gly194-Cys196 and Phe216-Asp221. These loop regions for Van der Waals interactions as well as electrostatic interactions with the sugar moiety. The modelled CaIMP protein carries a high density of positively charged residues at the phosphate binding region of the active site. The amino acids (Glu71, Asp91, Asp94 and Asp221) of this region of CaIMP are involved in the stable interaction of the Mg2+ ion with the phosphate moiety of the ligand. There is a highly flexible region between Gln31 and Glu46 which has also been reported in both the crystal structures 5EQ9 and 1IMA. This region consists of a loop of amino acids that are not interacting with the active site, which has been proposed to act as a flap to the active site of SaIMP, (Bhattacharyya et al., 2012). This flap can act as a shutter to control the entry and exit of substrate and product from the protein active site. This high flexibility region between Gln31 and 8

Journal Pre-proof Glu46 for CaIMP was also verified by B-factor analysis of the generated model, where high Bfactor showed greater flexibility (Supplementary Figure S2). Similarly, the active site residues loop regions were found to have comparatively high B-factor, this motivated further investigation into the dynamics of these loop regions to determine how this flexibility contributes to the substrate promiscuity for CaIMP protein. Overall Structure of CaIMP The CaIMP protein is a single domain phosphatase consisting of 6 major α helices, namely Asn4-

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Tyr30, Glu46- Leu62, Cys196-Cys205, Lys170-Lys186 and finally Ala223-Ala232. These six helices form the bulk of the Secondary structure observed in the CaIMP protein. In addition to this, there are

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three minor α helices, Glu72- Ala75, Thr96- His101 and Pro219- Asp221. These are the primary

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helical components of the CaIMP protein. The β strands in CaIMP are present as two antiparallel β sheet elements. Together, they from the key structural features of the CaIMP protein.

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These secondary structure elements are conserved in all the available crystal structures, on RCSB

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server, of the IMP protein. However, the key component of the IMP class of proteins are the loop regions. Apart from connecting the secondary structure elements, these loop regions are the

Active site of Ca-IMP

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primary component of the CaIMP active site (Figure 3).

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The active site for CaIMP protein consists exclusively of loop region. Primarily, there are 6 loop

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regions that form the active site, Gln31- Glu46, Ile69- Glu72, Asp91- Thr96, Ser157- Thr172, Lys186Ser195 and Thr214- Gly218. These six loop regions are responsible for stabilizing the Mg 2+ ion which is the cofactor for CaIMP. Additionally, they form the cavity on which the substrate binds. Since, CaIMP is a phosphatase, the presence of Mg2+ ion is essential for the catalytic activity of the protein. The absence of metal ion or even a substitution of Mg2+ to Li+ makes the protein catalytically inactive. It can be reasoned from the analysis of CaIMP active site, that in absence of Mg2+ it is not possible to stabilize the highly negative phosphate moiety of the substrate. Thus, the position of the metal ion is essential for the catalytic activity of the CaIMP protein. This specificity is observed in case of the metal ion only, as the protein is reported to be promiscuous in terms of the substrates it can bind to. The many phosphorylated substrates are reported to interact with and bind to the IMP protein. This promiscuity can be largely attributed 9

Journal Pre-proof to the prevalence of loop region in the active site of the IMP protein. In case of CaIMP protein, the previously mentioned loop regions cumulatively stabilize the Mg2+ ion and the substrate to which the protein interacts. From an electrochemical perspective, the substrates to CaIMP are highly polar and have multiple hydroxyl groups on the sugar moiety; additionally the active site lacks any hydrophobic patches (Figure 5). This complementarity ensures that the substrates and protein form strong interactions prior to dephosphorylation. The flexibility of loop regions ensures that the protein is capable of binding to a variety of substrates. This allows the CaIMP protein to form transient interactions which modulate themselves according to the substrate

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bound to the protein. These unique features in the CaIMP protein inspired further investigations

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into the static and dynamic behaviour of the CaIMP active site.

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Molecular Docking

The orientation of inositol 1-phosphate was determined from the PDB structure of human IMP,

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PDB ID 1IMA. Since the crystal structure had natural substrate bound to it, the orientation of

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surrounding loop region was assumed to be accurate. As a result, when the CaIMP model was generated from same crystal structure, the conformation of these loops was conserved. In the crystal structure PDB ID: 1IMA, the metal ion Mg 2+ is replaced with Gd2+ to ensure that the

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enzyme is in inhibited state and the substrate protein interactions could be captured in the crystal structure. However, it leads to the argument that since the enzyme is in inhibited state it is no

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longer the native conformation of the protein-substrate interactions. This is partly true,

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nonetheless our concern was to map the interactions between inositol-1-phosphate’s sugar moiety and the protein loop region which is on opposite from the metal ion. Thus, it could be reasonably concluded that the interactions between the sugar moiety and the protein loop region are accurately representative and can be used to study the interactions in our model protein of CaIMP. Using the docking methodology described previously (Figure 4), interaction energy for CaIMP and inositol-1-phosphate in best pose was found to be -10.32 kcal/mol. Here conformational flexibility was allowed for the side-chain of amino acids, Glu71, Lys83, Asp94 and Cys196 (Figure 4A). Similarly, docking was carried out for fructose-1, 6-bisphosphate, with conformational flexibility for Lys38, Asp42, Glu71, Asp91, Cys196, Glu213 and Asp221. This allowed us to determine the biding energy of fructose-1, 6-bisphosphate as -9.78 kcal/mol (Figure 4B). Another substrate 10

Journal Pre-proof reported to be catalysed by IMP protein is galactose-6-phosphate. Similar methodology was used to carry out the docking of galactose-6-phosphate and CaIMP protein. The docking energy of the best pose in this case was -11.01kcal/mol (Figure 4C). These docking values suggest that from the three compound whose Km values with CaIMP were determined, galactose 1-P (Km=0.017) forms the strongest interactions, hence it exhibits highest specific activity with CaIMP (Figure 1E). The specific activity of inositol 1-P is closely followed with a Km value of 0.020, corresponding to it inositol 1-P has docking energy of -10.32 kcal/mol.

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Finally, lowest specific activity within these three compounds is shown by fructose 1, 6-bisP (Km=0.028), and its corresponding docking energy was determined to be -9.78 kcal/mol. These

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findings exhibited the strong correlation between the biochemical and computational findings of

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the relation between CaIMP protein and different substrates.

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Static and Dynamic analysis of CaIMP

The docked conformation of substrate-protein interaction was validated by observing the

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stability of protein ligand interactions in a computational simulation environment. The protein ligand complexes were studied for 100 nanoseconds in a virtual simulation box. Here the protein

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acclimatizes to the new environment and forms interactions with ligands by altering the conformation of its main chain. Prior to interpreting the simulation results of protein-ligand

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complexes, the apo protein was studied for 100 ns, here it was observed that the protein is largely stable. There is translation motion in certain loop regions of the protein but this motion is

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profound in region of active site (Figure 6A), thereby providing evidence that this region has higher flexibility as compared to the other regions in the protein. Next simulation, aimed to study the stabilizing effect of Mg 2+ ion on the metal ion interacting region of the protein, in particular the region surrounding residues Glu 71, Asp91, Asp94 and Asp 221

. As expected, the region had conformational rigidity upon binding of metal ion. Over the

course of dynamics of 100ns, this region had comparatively lower Root Mean Square Fluctuation (RMSF) as compared to apo structure dynamics. In absence of Mg2+ the residues in loop region from Ile69 to Glu81 are highly flexible, because interactions with Mg2+ are not present. However, in presence of Mg2+ this region gets anchored by interactions between the Mg 2+ ion and carboxylate head of Glu71 and translation motion previously observed is not present. Over the 11

Journal Pre-proof course of 100ns simulation it was also observed that interaction occurs between Cys 196 and Asp 221

. This interaction was made possible as Asp221 is freed from Mg2+ interaction thereby allowing

it to interact with Cys196. This gives rigidity to loop and helix consisting of amino acids from Glu213 to Ala223, restricting its conformational flexibility. These interactions cumulatively stabilize the protein in presence of Mg2+ ion. The flexibility in active site loop regions was also corroborated by the RMSF plot of simulation data. High flexibility in the region between Gln31 and Glu46, and the loop regions of the active site was evident from the RMSF plot

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(Supplementary Figure S3). These simulations established the baseline reference for the motion of protein under different

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conditions. Utilizing this information, the dynamics of protein-substrate complexes were carried out. For CaIMP and inositol-1-phosphate, the equilibrated complex of protein and ligand showed

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that the primary interactions that stabilize inositol-1-phosphate are electrostatic in nature. The

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phosphate moiety is stabilized by interactions with Mg2+ ion and hydroxyl group of Thr96, carboxylate head of Glu71 amine group of Lys38 and backbone of Leu43. Cumulatively these

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interactions anchor the phosphate moiety and keep the inositol-1-phosphate molecule attached to the active site. The sugar moiety of inositol-1-phosphate is stabilized by interactions with

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hydroxyl group of Thr166, backbone of Cys196 and Phe216, carboxylate head of Asp 94 and Asp221. The above-mentioned interactions ensured that the inositol-1-phosphate molecule stays anchored

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to the active site and retains its original conformation for the entirety of molecular dynamics

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simulation. Since the region surrounding the inositol-1-phosphate molecule is predominantly loop region, the interactions initially observed are transient in nature. The molecule forms new interactions over the course of simulation. However, the most significant of these interactions was with Arg168. This interaction is not possible earlier due to conformational constraints shown by the protein. Thus, the binding of inositol-1-phosphate validated the assumption, that binding of the IMP protein to substrates would lower the conformational deviations in the loop region surrounding the active site (Figure 6B). Fructose-1, 6-bisphosphate has a phosphate group on either of its ends and each of these phosphate tails is uniquely anchored by the CaIMP protein. The first phosphate group is stabilized in a manner similar to that of inositol-1-phosphate. The primary stabilizing interaction is with Mg2+, additionally carboxylate head of Glu71 and Asp91, and backbone interactions with Leu93 and Asp94 contribute to anchoring the first phosphate 12

Journal Pre-proof group. The second phosphate group however forms unique interactions, due to the high flexibility of the sugar chain. There are multiple rotamers of the linear fructose sugar chain. The most stable of these rotamers has the second phosphate group interacting with the highly flexible loop region between Gln31 and Glu46. The second phosphate group is stabilized by electrostatic interactions with amine head of Lys28 and Lys167, carboxylate head of Asp42 and hydroxyl group of Thr166. The sugar chain of fructose-1, 6-bisphosphate is stabilized by interactions with carboxylate head of Asp94 and Asp221, along with backbone interactions with Asp94 and Phe216. These interactions are highly overlapping with those of inositol-1-phosphate, thus, validating the

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energy minimized conformation of fructose-1, 6-bisphosphate (Figure 6C). However, after the Molecular Dynamics simulation of 100ns, the interactions between the second phosphate group

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are lost. The flexibility in between Gln31 and Glu46 allows this loop region to move freely. This

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makes the second phosphate group to lose its interactions and become free. The first phosphate group is held tightly in its place over the entire duration of simulation. In this conformation the

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fructose-1, 6-bisphospahte molecule is anchored at one end by interaction which is similar to that

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of the inositol-1-phosphate after 100ns of simulation.

Galactose-1-phosphate molecule is structurally similar to inositol-1-phosphate; hence the docked

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pose of galactose-1-phosphate should be nearly identical to inositol-1-phosphate docked pose. The phosphate group of galacotse-1-phosphate molecule forms very strong interactions with the

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Mg2+ ion, along with side chain interaction occurring with the carboxylate head of Glu71, hydroxyl group of Thr96 and the amine head of Lys96. These interactions together anchor the

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phosphate group of galactose-1-phosphate to the protein active site. Similar to inositol-1phosphate, the galactose-1-phosphate molecule also interacts with carboxylate group of Asp221 and the backbone of Cys196. This orientation of CaIMP and galactose-1-phosphate complex was held for the entire duration of the molecular dynamics simulation which further substantiates the molecular docking pose (Figure 6D). During the simulation it was observed that the modelled protein and the complexes with various ligands, held their conformation and there were no structural rearrangements. There were only minor oscillations in the loop region comprising of the active site. However, the flexible region between Gln31 and Glu46 did undergo fluctuations, but it had little influence on the protein ligand interactions. These claims are substantiated by the simulation parameters. The RMSF graph 13

Journal Pre-proof shows that the region of flexibility is localized to the loop regions comprising of the active site. Additionally, the Radius of Gyration plot shows that the protein is sufficiently compact during the course of simulation. The Root Mean Square Deviation (RMSD) shows that the proteinligand complexes were stable for the entire duration of simulation and the duration of production run, 100 nanoseconds, was sufficient for reaching convergence (Supplementary Figure S3). Further MD statistics of Potential Energy, Pressure and Temperature for the entire duration of simulation (100ns) is available in Supplementary Figure S4.

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Conclusion

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Through this study, we were able to elucidate the factors responsible for the interaction between CaIMP and inositol-1-phosphate, its substrate. Additionally, the ability of CaIMP for the

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dephosphorylation of fructose-1, 6-bisphosphate and galactose-1-phosphate was identified. Further computational analysis of the static and dynamic interactions between the Ca-IMP

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protein and its substrates led to the identification of the key structural features in the protein

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which lead to its substrate promiscuity. Acknowledgments

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This work was supported by the Science and Engineering Research Board (SERB), Government of India under the scheme of “Start Up Research Grant (Young Scientist)- Elucidating the

from

drought

tolerant

legume

Chickpea

(Cicer

arietinum)”

(Grant

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IMPL2)

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Functional and Regulatory Aspects of inositol Monophosphatase like Proteins (IMPL1 and

no:YSS/2014/001012/LS).

Compliance with ethical standards Conflict of interest: The authors declare that they have no conflict of interest.

Figure Legends Figure 1: Biochemical analysis of CaIMP with galactose 1-P, inositol 6-P and fructose 1, 6-bisP A) Effect of pH and temperature on inositol mono phosphatase activity with galactose 1-P, inositol 6-P and fructose 1,6-bisP. Specific activity of purified CaIMP was 14

Journal Pre-proof measured at different pH (pH-5 to pH-10) and temperature (0°C to 60°C) profile. C) CaIMP activity with different divalent cations and D) with different concentration of Mg+2.

For each analysis 10 µg of purified recombinant CaIMP was used. Error bar

represent ± SD of three replicate.

Figure 2: The multiple sequence alignment of CaIMP sequence with the sequence of Ca-IMPL1 and Ca-IMPL2. Additionally, the AtIMP (Arabidopsis thaliana), IMPL1 and IMPL2

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have been included. 1IMA_IMLP1_str sequence refers to the FASTA sequence of the PDB ID: 1IMA, similarly, 5EQ9_IMPL2_str refers to the FASTA sequence of the

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PDB ID: 5EQ9. The Orange boxes represent the α helices in the protein, the grey

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arrow represents the β strands and blue line represents the loop region of the protein. Figure 3: This is the overall structure of the model of the CaIMP protein. The secondary

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structure elements are visible as cyan cartoons. The pink region represents the highly flexible region, between Gln 31 and Glu 46, the yellow region represents the

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remaining 5 loops which cumulatively from the active site of the protein. The Mg 2+

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ion is represented as the green sphere.

Figure 4: The docking interactions of the CaIMP protein (cyan) with; A. inositol-1-phosphate

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(green), B. fructose-1, 6-bisphosphate (pink) and C. galactose-1-phosphate (brown). The black dashes represent the electrostatic interactions between the protein side

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chains and the ligand.

Figure 5: The electrostatic surface potential of the CaIMP protein, highlighting the active site cavity of the protein. The red colour represents the positively charged amino acids, blue colour represents negatively charged amino acids and white represents the electrically neutral amino acids. In the highly positive active site cavity of the protein, the docked ligands can be seen. A. inositol-1-phosphate (green), B. fructose-1, 6bisphosphate (yellow) and C. galactose-1-phosphate (brown). Figure 6: The different MD states of the CaIMP protein and its ligands. The cyan colour structure represents the initial state and the green colour structure represents the state after 100ns. A. the apo protein showing minimal structural changes. B. the inositol-115

Journal Pre-proof phospahte molecule retains its orientation in the CaIMP active site. C. One phosphate end of fructose-1, 6-bisphosphate molecule remains anchored at the active site. D. the galactose-1-phosphate molecule remains at the active site over the 100ns of MD simulation. References: Abraham, M.J., Murtola, T., Schulz, R., Páll, S., Smith, J.C., Hess, B., and Lindahl, E. (2015). GROMACS: High performance molecular simulations through multi-level parallelism from

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