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ABSTRACT
In the case of Rubisco Like Protein (RLP) Chromohalobacter salexigen BKL 5 (RLP CS), which is
halophilic, the presence of salt bridges (SB) is not too numerous but efficient enough to maintain stability in high
salt stress. This study was to determine the effect of mutations SB at position 185 (D185/WT) on the stability and
activity of RLP CS through an in-silico approach by replacing aspartic acid with glutamic acid and alanine (D185E
and D185A). The methods used were Molecular Dynamics Simulations (MDS) and Molecular Docking (MD).
The MDS was used to study molecular characteristics at the atomic level, while MD was for the interaction patterns
of proteins and ligands. The results of the MDS analysis carried out at 10ns showed that the mutation at position
185 with alanine and glutamic acid changed the size/dimensional of RLP CS and affected its overall geometric
structure. Interestingly, even though the structure has changed, the activity of the protein remains relatively
constant, which is indicated by the results of MD, and has a relatively similar binding energy value of around -6.3
Kcal/mol.
Keywords: Chromohalobacter salexigen BKL 5, molecular docking, molecular dynamic simulation, mutation,
saltbridge
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JURNAL KIMIA (JOURNAL OF CHEMISTRY) 18 (1), JANUARI 2024: 1 - 7
valuable in deciphering the functional previous study which showed that RLP CS has
mechanisms of proteins and other biomolecules significant differences in glutamic acid and
in uncovering the structural basis of disease and alanine compared to its non-halophilic
in the design and optimization of small homologs (unpublished). Sequences containing
molecules, peptides and proteins mutant residues were homologated with
(Hollingsworth & Dror, 2018). AlphaFold to obtain a 3D structure in the form
The results of previous observations of PDB files (Miyazono & Tanokura, 2022).
showed that the saltbridge in halophilic RLP The selected structures were validated with
only has half of its homologous RLP saltbridge ERRAT and SAVE (Messaoudi et al., 2013).
which is mesophilic (unpublished). A more The results of the homologation were
detailed observation of the RLP CS saltbridge analyzed for energy in the ground state with the
shows that the saltbridge at position D185 is SPDV Tool (Guex & Peitsch, 1997) then
unique because it has the most interactions and superimposed with RLP WT to find out the
is closest to the active site of the protein which difference in geometry in the form of RMSD
is thought to have a major contribution in using Chimera (Pettersen et al., 2004).
maintaining the stability (conformation) of RLP
CS to function optimally. This study was Molecular Dynamic Simulation
conducted to determine how much influence the Molecular dynamics simulations can be
saltbridge has on the function and stability of used as a procedure to study molecular
RLP cs. Tests were carried out through the characteristics at the atomic level, such as
process of mutation of aspartic acid with other protein conformation changes that occur. In this
residues (glutamic acid and alanine) and study protein molecular dynamics (MDS)
analyzed using a computational approach, simulations were carried out using the
namely Molecular dynamic simulation and Gromacs-based method performed on the
molecular docking methods. It is hoped that the WebGro server (Oostenbrink et al., 2004;
results of this study can provide an overview of Abraham et al., 2015).
how the saltbridge influences the stability and Proteins were prepared using the
activity of RLP CS. GROMOS96 43a1 force field. SPC was chosen
as a model solvent (a triclinic water box with a
MATERIAL AND METHODS size of 50 × 75 × 70 Å) for protein complexes.
This system is neutralized by adding sodium or
Material and Tool chlorine ions based on the total charge. To
The 3D structure of RLP CS in the form minimize the system before MD, the steepest
of PDB files, called as Wild Type (WT). Protein descent (5000 steps) algorithm was applied.
sequences in FASTA format containing MD simulations were carried out in the
mutants at position 185, hereinafter referred to presence of 0.15 M NaCl using constant
as D185E (asp mutation to glutamic acid) and temperature (300 K) and pressure (1.0 bar). The
D185A (aspartic acid mutation to alanine). The approximate number of frames per simulation is
computational analysis was operated on ACER 1000. The simulation time is set to 10 ns. The
Nitro with i7 processor, 32 GB RAM, parameters analyzed were RMSD, RMSF, Rg,
Windows 10, GPU GTX 1060 6 GB and 64-bit SASA and the number of hydrogen bonds
operating system formed. Graph preparation and visualization
using Originlab 2019 and GraPhadPrism 8.
Method
The first step is identification and Molecular Docking
visualization of the areas/residues of amino To predict the interaction patterns of
acids to be mutated using the Chimera tool, proteins and ligands, a molecular docking
followed by the following steps. process was carried out. In this study the
method used was Autodock Vina and the MGL
Homologation of mutational 3D structures Tool. The ligand used was RUBP whose
Mutations were carried out on the structure was obtained from a web-based portal
aspartic acid amino acid residue at position 185 (Pubchem) in SDF format. The protein used is
(D185/WT) by replacing it with glutamic acid the PDB structure of the RLP WT file and the
(D185E) and alanine (D185A). The selection of mutants prepared using the MGL Tool (Morris
these two types of residues was based on et al., 2009). All polar hydrogens were removed
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Effect of The D185 Mutation on the Stability and Functionality of Rubisco Like Protein (Rlp) from
Chromohalobacter Salexigen BKL 5
(I. Sudarmanto, M. S. Rohman, W. T. Artama)
from the ligand and a Gasteiger type charge was at position 185 is shown in Figure 1. In the
added. Thereafter the receptor files were figure it can be seen that aspartic acid at position
prepared by adding hydrogen and a Kollman 185 binds to arginine (R) at 146,221 and 394
charge. Then all files are converted to pdbqt which forms a salt bridge which is important to
format for docking. The grid is arranged maintain conformation, especially the active
according to the structure in such a way as to site area.
cover the active site area of the RLP protein. The results of the homologation with
After preparation of the ligand and receptor, AlphaFold which have been validated (data not
docking was carried out using Autodock Vina shown) are then analyzed for energy and
(Walker & Causgrove, 2009). The best docking compared with WT which shows that the two
position (the most negative binding energy mutants are geometrically different (Table 1).
value) was chosen for visualization. This is consistent with initial predictions that
Visualization of docking results using LigPlot. changing or even removing the saltbridge will
affect the stability of the RLP CS (WT) protein,
considering that the saltbridge is very important
RESULT AND DISCUSSION to maintain the structure of a protein. The small
RMSD value of the superimposed results on the
Visualization of SB at position 185 and two mutants indicates that the two mutants are
Homologation of Mutant Structures geometrically smaller than the WT structure of
Previous studies revealed that the RLP CS and this is reinforced by the energy
aspartic acid (asp/D) residue at position 185 is value which also changes. This indicates that
unique because it has interactions with several mutations in the saltbridge area will reduce
other residues to produce a relatively large stability.
amount of saltbridge (SB) compared to other
residues that make up SB. Visualization of SB
Figure 1. Position of Aspartic Acid at Number 185 (A) and the Salt-bridge Interaction Formed (B)
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JURNAL KIMIA (JOURNAL OF CHEMISTRY) 18 (1), JANUARI 2024: 1 - 7
4
Effect of The D185 Mutation on the Stability and Functionality of Rubisco Like Protein (Rlp) from
Chromohalobacter Salexigen BKL 5
(I. Sudarmanto, M. S. Rohman, W. T. Artama)
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JURNAL KIMIA (JOURNAL OF CHEMISTRY) 18 (1), JANUARI 2024: 1 - 7
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Effect of The D185 Mutation on the Stability and Functionality of Rubisco Like Protein (Rlp) from
Chromohalobacter Salexigen BKL 5
(I. Sudarmanto, M. S. Rohman, W. T. Artama)
Inhibitory Activity of ACEIs Against Force Field Based on The Free Enthalpy
SARS-CoV-2 Targeting the hACE2 of Hydration and Solvation: The
Receptor. Frontiers in Chemistry. 9 GROMOS Force-Field Parameter Sets
Bosshard, H. R., Marti, D. N., Jelesarov, I. 2004. 53A5 and 53A6. Journal of
Protein Stabilization by Salt Bridges: Computational Chemistry. 25(13):
Concepts, Experimental Approaches 1656–1676
and Clarification of Some Pettersen, E.F., Goddard, T.D., Huang, C.C.,
Misunderstandings. Journal of Couch, G. S., Greenblatt, D.M., Meng,
Molecular Recognition. 17(1): 1–16 E.C., Ferrin, T.E. 2004. UCSF Chimera:
Guex, N., Peitsch, M.C. 1997. SWISS-MODEL A Visualization System for Exploratory
and the Swiss-Pdb Viewer: An Research and Analysis. Journal of
Environment for Comparative Protein Computational Chemistry. 25(13):
Modeling. Electrophoresis. 18(15): 1605–1612
2714–2723 Rácz, A., Mihalovits, L.M., Bajusz, D.,
Hollingsworth, S.A., Dror, R.O. 2018. Héberger, K., & Miranda-Quintana,
Molecular Dynamics Simulation for R.A. 2022. Molecular Dynamics
All. Neuron. 99(6): 1129–1143 Simulations and Diversity Selection by
Karplus, M., McCammon, J.A. 2002. Molecular Extended Continuous Similarity
Dynamics Simulations of Indices. Journal of Chemical
Biomolecules. Nature Structural Information and Modeling. 62(14):
Biology. 9(9): 646–652 3415–3425
Kumar, S., Nussinov, R. 2002. Close-Range Shukla, R., Shukla, H., Tripathi, T. 2018.
Electrostatic Interactions in Proteins. Activity loss by H46A Mutation in
ChemBioChem. 3(7): 604-610 Mycobacterium Tuberculosis Isocitrate
Meng, X.Y., Zhang, H.X., Mezei, M., Cui, M. Lyase is Due To Decrease in Structural
2011. Molecular Docking: A Powerful Plasticity and Collective Motions of
Approach for Structure-Based Drug The Active Site. Tuberculosis.
Discovery. Current Computer Aided- 108:143–150
Drug Design. 7(2): 146–157 Takano, K., Ogasahara, K., Kaneda, H.,
Messaoudi, A., Belguith, H., Ben Hamida, J. Yamagata, Y., Fujii, S., Kanaya, E.,
2013. Homology Modeling and Virtual Kikuchi, M., Oobatake, M., Yutani, K.
Screening Approaches to Identify 1995. Contribution of Hydrophobic
Potent Inhibitors of VEB-1 β- Residues to the Stability of Human
Lactamase. Theoretical Biology and Lysozyme: Calorimetric Studies and X-
Medical Modelling. 10(1) ray Structural Analysis of the Five
Miyazono, K., Tanokura, M. 2022. New Era in Isoleucine to Valine Mutants. Journal of
Structural Biology with The Alphafold Molecular Biology. 254(1): 62–76
Program. Translational and Regulatory Takano, K., Tsuchimori, K., Yamagata, Y.,
Sciences. 4(2): 48–52 Yutani, K. 2000. Contribution of Salt
Morris, G.M., Huey, R., Lindstrom, W., Sanner, Bridges Near The Surface of A Protein
M.F., Belew, R. K., Goodsell, D. S., to The Conformational Stability,.
Olson, A.J. 2009. AutoDock4 and Biochemistry. 39(40): 12375–12381
AutoDockTools4: Automated Docking Tian, W., Chen, C., Lei, X., Zhao, J., Liang, J.
with Selective Receptor Flexibility. 2018. CASTp 3.0: Computed Atlas of
Journal of Computational Chemistry. Surface Topography of Proteins.
30(16): 2785–2791 Nucleic Acids Research. 46(W1):
Musafia, B., Buchner, V., Arad, D. 1995. W363–W367
Complex Salt Bridges in Proteins: Walker, K. D., Causgrove, T. P. 2009.
Statistical Analysis of Structure and Contribution of Arginine-Glutamic
Function. Journal of Molecular Acid Salt Bridges to Helix Stability.
Biology. 254(4): 761–770 Journal of Molecular Modeling. 15(10):
Oostenbrink, C., Villa, A., Mark, A.E., Van 1213–1219
Gunsteren, W.F. 2004. A Biomolecular