Docking of Smaller Ligands in the Heme Pocket of Hell’s Gate Globin IV

Farrukh Jamil*, Samra Raheel and Haroon Rasheed

Department of Biosciences, COMSATS Institute of Information Technology (CIIT), Sahiwal, Punjab

ABSTRACT

HGbIV is unique truncated hemoglobin (tHb) that differs from other bacterial tHbs due to its large size, polar heme pocket residues and large cavity for exogenous ligand in the heme pocket. The crystal structures of HGbIV showed that its heme pocket can accommodate and interact with bulky PO4-3 ion. Here, we have docked different smaller molecules/ion like O2, NO, CO, CO2, NO3-1, CH3COO-1 and CN-1 in the heme pocket of HGbIV. Analysis of the structure showed that the smaller ligands docked far away from most of the heme pocket residues namely His70(B9), His71(B10), Ser97(E11) and Trp137(G8), and only PO4-3 docked at a position where it can interact with all these residues. It appears that HGbIV is not designed for transport of smaller gaseous molecule like the other heme containing proteins and it may be a phosphate sensor.

Article Information

Received 19 January 2018

Revised 23 February 2018

Accepted 17 March 2018

Available online 24 May 2018

Authors’ Contribution

FJ designed the study. SR and HR performed the experiments, collected and analyzed the data. FJ gathered the data, supervised and prepared the manuscript.

Key words

Truncated hemoglobins, Phosphate sensor, Oxygen carrier, Enzymes.

DOI: http://dx.doi.org/10.17582/journal.pjz/2018.50.4.sc9

* Corresponding author: [email protected];

[email protected]

0030-9923/2018/0004-1565 $ 9.00/0

Copyright 2018 Zoological Society of Pakistan

Truncated hemoglobins (tHbs) are heme containing proteins (Hbs) that are ubiquitous to bacteria, protozoa, nemertea, cyanobacteria and plants (Ascenzi and Pesce, 2017; Wittenberg et al., 2002). These proteins are almost 20 to 40 amino acid short than novertebrate Hbs and constitute a separate lineage in the globin superfamily (Bustamante et al., 2016; Wittenberg et al., 2002; Vinogradov et al., 2013). Structurally, tHbs are based on 2/2 α-helical fold, showing an editing in classical 3/3 α-helical globin fold (Pesce et al., 2000). Although, real physiological roles of different bacterial tHbs have not yet been established, though different studies have proposed different roles for tHbs such as oxygen and nitrogen transport, as sensor, oxygen carrier, ligand storage and in nitric oxide detoxification (Dikshit et al., 1992; Couture et al., 1999; Pesce et al., 2000; Ouellet et al., 2002, 2003; Igarashi et al., 2011; Jamil et al., 2014; Minaeva et al., 2017; Ascenzi et al., 2017).

tHGbs are further classified into four phylogentic groups: group I (tHbN), group II (tHbO), group III (tHbP) and group IV (tHbQ) (Vinogradov et al., 2013; Bustamante et al., 2016). These groups separate from each other due to different heme pocket residues located at topological positions (B9, B10, E7, E11, E14 and G8). The most highly conserved residues in group II tHgbs are Phe, Tyr and Trp at B9, B10 and G8 positions, respectively (Wittenberg et al., 2002). These residues play a vital role in stabilization of heme-bound ligand as reported in crystal structures of different bacterial tHbs (Pesce et al., 2000; Bonamore et al., 2005; Ilari et al., 2007; Igarashi et al., 2011; Jamil et al., 2014; Tariq et al., 2016; Rana et al., 2017).

A truncated hemoglobin, designated Hell’s Gate Globin IV (HGbIV), has been reported from a thermophilic, acidophilic obligate methotroph bacterium Methylacidiphilim infernorum (Hou et al., 2008). HGbIV differs from all the other structurally characterized group II tHbs due to large size (>197) compared to the typical size of other group II tHbs (120-140), and by their polar heme pocket residues: His(B9), His(B10), Ser(E11) and Trp(G8). The crystal structure of HGbIV (PDB: 4NK1) showed a bulky heme-bound phosphate ligand in the heme pocket (Jamil et al., 2014). It is observed that the two consecutive His residues at B9 and B10 positions provide more space for phosphate ion, suggesting that HGbIV heme pocket is designed for bulky ligand. This finding was reinforced by another crystal structure of HGbIV (4NK2) showing a very large electron density for an unknown heme bound ligand (Jamil et al., 2014).

This study is designed to determine the possible interaction of HGbIV with different already reported smaller ligands, such as O2, NO, CO, CO2, CH3COO-1, CN-1 for other bacterial tHbs (Dikshit et al., 1992; Couture et al., 1999; Pesce et al., 2000; Ouellet et al., 2002, 2003; Igarashi et al., 2011). It will be interesting to understand how smaller ligands will be stabilise in the bulky heme pocket of HGbIV.

Methods

The PDB files of HGbIV (4NK1) and different ligands (SO4-2, NO3-1, CH3COO-1 and CN-1, O2, NO, CO and CO2) were retrieved from RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/home.do) (Berman et al., 2000). Chimera was used for the energy minimization of HGbIV and ligands (Petterson et al., 2004). PDBQT files were generated by using the autodock 4 and autodock vina (Trott and Olson, 2010), and Gasteiger charges were added to convert protein and all ligands into PDBQT files. By using Autodock Vina, targeted docking was performed at the heme pocket of HGbIV with the above mentioned ligands and structure was visualized by using Chimera (Petterson et al., 2004).

Results

A targeted docking strategy was used for docking a PO4-3 ion in the heme pocket of HGbIV (4NK1) where it docked with the binding affinity of -17.16 KJ/mol (Fig. 1A; Table I). Superimposition of the docked PO4-3 ion in HGbIV to the real 3D structure of HGbIV showed that the ion docked at same orientation as observed in crystal structure of HGbIV. Moreover, it shows similar interactions with heme pocket residues namely His70(B9), His71(B10), Ser97(E11) and Trp137(G8) as observed in its real 3D crystal structure of HGbIV (Jamil et al., 2014). SO4-2 ion is a structural analogue of PO4-3 ion, it docked in the heme pocket of HGbIV relatively far (2.67 Ǻ) from heme-iron compared to the position of PO4-3 ion with binding affinity of -17.16 KJ/mol (Fig. 1B; Table I). Analyses of the structure showed that most of the heme pocket residues can stabilize SO4-2 ion except Ser97(E11) which is relatively far (3.34 Ǻ) away (Fig. 1B).

Table I.- Ligands and their binding affinity.

CH3COO-1, NO3-1 and CN-1 ions docked in the heme pocket of HGbIV at a distance of 3.89, 3.11 and 3.53 Ǻ away from the heme iron with binding affinities of -12.13,-11.30 and -5.44 KJ/mol, respectively (Fig. 1C-E). Analyses of the structures showed that the docked CH3COO-1 ion is stabilized by Ser97(E11) and Trp137(G8), whereas the His70(B9) and His71(B10) are far away (> 3.2 Ǻ) from the ion. Previously, CH3COO-1 ion has been reported in a crystal structure of a tHb from Thermobifida fusca (tf-tHgb). A notable difference between the two structures is long distance of His(B10) from the docked CH3COO-1 ion in HGbIV compared to that of Tyr(B10) in tf-tHgb. On the other hand, the Trp(G8) plays a vital role in interacting and stabilizing the acetate ion in tf-tHgb as well as in HGbIV. Analysis of the docked NO3-1 ion in HGbIV showed that only His70(B9) can interact with NO3-1while the other heme pocket residues are far away (> 3.2 Ǻ) (Fig. 1D). The CN-1 ion docked very far from all the heme pocket residues so no residue can form hydrogen bonding with it (Fig. 1E).

CO2, O2, CO and NO were docked in the heme pocket of HGbIV at distance 3.87, 5.23, 4.38, 3.33, 3.51 Ǻ away from the heme iron with binding affinity -10.04, -8.37, -6.69 and -5.85 KJ/mol, respectively (Table I; Fig. 1F-I). Analyses of these structures showed that the CO2 is stabilized by interaction of Ser97(E11) and Trp137(G8), whereas CO and O2 are stabilised only by Ser97(E11). On the other hand, NO may only interact with Trp137(G8).

Discussion

This work is an extension of our previous work in which we have determined three dimensional structure of HGbIV under two different conditions (Jamil et al., 2014). Structurally, HGbIV belongs to group II tHbs with several unique features. For instance, HGbIV is a large protein (194 amino acids) compared to other bacterial group II tHbs (120-140). Second, the heme pocket of HGbIV is more polar and host two His residues at B9 and B10 positions compared to highly conserved Phe(B9) and Tyr(B10) residues in other bacterial tHbs . Third, the crystal structure of HGbIV (4NK1) showed that the heme pocket can accommodate a bulky heme-bound phosphate ion. On the other hand, smaller ligands like CH3COO-1, NO3-1, CO2, O2, CO, NO, CN-1 and H2O have been reported in other bacterial tHbs (Pesce et al., 2000; Bonamore et al., 2005; Ilari et al., 2007; Igarashi et al., 2011; Jamil et al., 2014). In this study we docked smaller ligands in the heme pocket of HGbIV.

The PO4-3, SO4-2, CH3COO-1, NO3-1, CO2, O2, CO, NO and CN-1 were docked in the heme pocket of HGbIV with the binding affinities of -17.16,- 17.16, -12.13, -11.30, -10.04, -8.37, -6.69, -5.85 and -5.44 Kj/mol, respectively (Table I). Analysis of these values clearly showed that the heme pocket of HGbIV is designed for bulky ligands like PO4-3 as it is only ligand that can interact with all the active site residues namely His70(B9), His71(B10), Ser97(E11) and Trp137(G8) (Fig. 1). On the other hand, smaller heme-iron bound molecules are not so stabilized in this pocket due to far location of the active site residues. In other group II bacterial tHbs smaller heme-bound ligands are mostly stabilized by highly conserved Tyr(B10) which is replaced by His71(B10) in HGbIV. In conclusion, it appears that heme pocket of HGbIV is specific for bulky ligands and we forsee remarkable displacement in HGbIV for smaller ligands stabilization.

Acknowledgement

We gratefully acknowledge Higher Education Commission (HEC) of Pakistan for grants to this study.

Statement of conflict of interest

Authors have declared no conflict of interest.

References

Ascenzi, P. and Pesce, A., 2017. J. Biol. Inorg. Chem., 22: 1141-1150. https://doi.org/10.1007/s00775-017-1490-z

Ascenzi, P., Ciaccio, C., Gasperi, T., Pesce, A., Caporaso, L. and Coletta, M., 2017. J. Biol. Inorg. Chem., 22: 977-986. https://doi.org/10.1007/s00775-017-1476-x

Berman, H.M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T.N., Weissig, H., Shindyalov, I.N. and Bourne, P.E., 2000. Nucl. Acids Res., 28: 235-242. https://doi.org/10.1093/nar/28.1.235

Bonamore, A., Ilari, A., Giangiacomo, L., Bellelli, A., Morea, V. and Boffi, A., 2005. FEBS J., 272: 4189-4201. https://doi.org/10.1111/j.1742-4658.2005.04831.x

Bustamante, J.P., Radusky, L., Boechi, L., Estrin, D.A., Ten-Have, A. and Martí, M.A., 2016. PLoS Comput. Biol., 12: e1004701. https://doi.org/10.1371/journal.pcbi.1004701

Couture, M., Yeh, S.R., Wittenberg, B.A., Wittenberg, J.B., Ouellet, Y., Rousseau, D.L. and Guertin, M., 1999. Proc. natl. Acad. Sci. USA, 96: 11223-11228. https://doi.org/10.1073/pnas.96.20.11223

Dikshit, R.P., Dikshit, K.L., Liu, Y.X. and Webster, D.A., 1992. Arch. Biochem. Biophys., 293: 241-245. https://doi.org/10.1016/0003-9861(92)90391-9

Hou, S., Makarova, K.S., Saw, J.H., Senin, P., Ly, B.V., Zhou, Z., Ren, Y., Wang, J., Galperin, M.Y., Omelchenko, M.V., Wolf, Y.I., Yutin, N., Koonin, E.V., Stott, M.B., Mountain, B.W., Crowe, M.A., Smirnova, A.V., Dunfield, P.F., Feng, L., Wang, L. and Alam, M., 2008. Biol. Direct, 3: 26. https://doi.org/10.1186/1745-6150-3-26

Igarashi, J., Kobayashi, K. and Matsuoka, A., 2011. J. Biol. Inorg. Chem., 16: 599-609. https://doi.org/10.1007/s00775-011-0761-3

Ilari, A., Kjelgaard, P., Von-Wachenfeldt, C., Catacchio, B., Chiancone, E. and Boffi, A., 2007.Arch. Biochem. Biophys, 457: 85-94. https://doi.org/10.1016/j.abb.2006.09.033

Jamil, F., Teh, A.H., Schadich, E., Saito, J.A., Najimudin, N. and Alam, M., 2014. J. Biochem., 156: 97-106. https://doi.org/10.1093/jb/mvu023

Minaeva, E., Zalutskaya, Z., Filina, V. and Ermilova, E., 2017. PLoS One, 12: e0186851. https://doi.org/10.1371/journal.pone.0186851

Ouellet, H., Juszczak, L., Dantsker, D., Samuni, U., Ouellet, Y.H., Savard, P.Y., Wittenberg, J.B., Wittenberg, B.A., Friedman, J.M. and Guertin, M., 2003. Biochemistry, 42: 5764-5774. https://doi.org/10.1021/bi0270337

Ouellet, H., Ouellet, Y., Richard, C., Labarre, M., Wittenberg, B.A. and Guertin, M., 2002. Proc. natl. Acad. Sci. USA, 99: 5902-5907. https://doi.org/10.1073/pnas.092017799

Pesce, A., Couture, M., Dewilde, S., Guertin, M., Yamauchi, K., Ascenzi, P., Moens, L. and Bolognesi, M., 2000. EMBO J., 19: 2424-2434. https://doi.org/10.1093/emboj/19.11.2424

Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C. and Ferrin, T.E., 2004. J. Comput. Chem., 25: 1605-1612. https://doi.org/10.1002/jcc.20084

Rana, N., Ehsan, N., Ihsan, A. and Jamil, F., 2017. Pakistan J. Zool., 49: 1261-1265.

Tariq, F., Khalid, Q., Sehgal, S.A., Mannan, S. and Jamil, F., 2016. Pakistan J. Zool., 48: 1805-1810.

Trott, O. and Olson, A.J., 2010. J. Comput. Chem., 31: 455-461.

Vinogradov, S.N., Tinajero-Trejo, M., Poole, R.K. and Hoogewijs, D., 2013. Biochim. Biophys. Acta, 1834: 1789-1800. https://doi.org/10.1016/j.bbapap.2013.03.021

Wittenberg, J.B., Bolognesi, M., Wittenberg, B.A. and Guertin, M., 2002. J. biol. Chem., 277: 871-874. https://doi.org/10.1074/jbc.R100058200