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JURNAL KIMIA (JOURNAL OF CHEMISTRY) 18 (1), JANUARI 2024 p-ISSN 1907-9850

DOI: https://doi.org/10.24843/JCHEM.2024.v18.i01.p01 e-ISSN 2599-2740

EFFECT OF THE D185 MUTATION ON THE STABILITY AND FUNCTIONALITY OF


RUBISCO LIKE PROTEIN (RLP) FROM CHROMOHALOBACTER SALEXIGEN BKL 5

I. Sudarmanto 1,2, M. S. Rohman2,3*, W. T. Artama2,4


1
Chemistry Study Program, Department of Sciences, Institute Technology of Sumatera, Jl Terusan
Ryacudu, WayHui, Lampung Selatan, 35365, Lampung
2
Post Graduate Program of Biotechnology, Post Graduate Program, University of Gadjah Mada, Jl
Teknika Utara, Catur Tunggal, Sleman, 55281, Yogyakarta
3
Department of Agricultural Microbiology, Agriculture Faculty, University of Gadjah Mada,
Karang Malang, Catur Tunggal, Sleman, 55281, Yogyakarta
4
Veterinary Medicine Faculty, University of Gadjah Mada, Jl Fauna No 2, Karang Gayam, Catur
Tunggal, Sleman, 55281, Yogyakarta
*Email: saifur@ugm.ac.id

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

INTRODUCTION a protein or other molecular system will move


over time, based on general models of physics
Saltbridge (SB) is defined as the governing interatomic interactions (Karplus and
electrostatic interaction between two oppositely McCammon, 2002; Rácz et al., 2022). These
charged groups: anionic carboxylates from simulations can capture a wide variety of
glutamic acid (E) or aspartic acid (D), and important biomolecular processes, including
cationic ammonium from arginine (R) or lysine conformational changes, ligand binding, and
(K) (Bosshard et al., 2004; Musafia et al., 1995). protein folding, revealing the positions of all
Salt bridge interactions in proteins can provide atoms at femtosecond temporal resolution. Such
structural and functional conformation and simulations can also predict how biomolecules
contribute to protein recognition, degradation, will respond at the atomic level to perturbations
catalysis, and stability. (Albeck et al., 2000; such as mutations, phosphorylation,
Kumar & Nussinov, 2002). protonation, or the addition or removal of
Mutation analysis is a useful procedure ligands.
for estimating the contribution of several factors Simulations can also predict the behavior
to the conformational stability of a protein. The of proteins and other biomolecules in full
effects of mutations on protein stability differ atomic detail and at very fine temporal
from site to site, depending on their resolution. Substantial improvements in the
environment within the structure (Takano et al., speed, accuracy, and accessibility of
1995; Takano et al., 2000). One approach that is simulations, together with the proliferation of
often used to predict the effect of mutations on experimental structural data, have increased the
proteins is MDS. Molecular dynamics (MD) attractiveness of biomolecular simulations for
simulations can predict how individual atoms in experimentalists. Simulations have proven

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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

2
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)

Table 1. Characteristics of WT and Mutation Results

Protein RMSD ΔG Binding affinity Volume active site


Compare to WT KJ/mol KCal/mol (SA) Å3
WT 0 22.444.898 -6,3 376.486
D185E 0,403 20.364.660 -6,2 364.192
D185A 0,685 20.182.055 -6,3 372.971

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JURNAL KIMIA (JOURNAL OF CHEMISTRY) 18 (1), JANUARI 2024: 1 - 7

Molecular Dynamics hydrophobic amino acids in protein to water


RMSD is a parameter commonly used to (solvent). At D185E the SASA value will also
determine the stability of a system (protein). decrease because even though the saltbridge is
RMSD can describe the conformational and still formed, the glutamic acid side chain is
geometric changes of a protein. The greater the longer so that it will affect the distance the
RMSD value, the more unstable the protein will saltbridge is formed (longer).
be and vice versa. The MDS analysis shows that The number of hydrogen bonds in a
the RMSD value of the mutant is greater than protein describes the atomic level interactions.
that of WT which illustrates that the It also validates the compactness or shape of the
conformation of the mutant protein changes and structure. The compact structure of proteins is
becomes unstable compared to WT (Figure generally advantageous for intramolecular
2b). This happens because there is no salt bridge hydrogen bond interactions. In order to predict
that allows the protein to lose its stability. how the mutation affects the structural stability,
Although the D185E mutant still has a the intramolecular hydrogen bonds of WT and
saltbridge, its RMSD value is still greater than the mutant are shown in Figure 3. It was
that of WT, indicating that aspartic acid is the observed that the WT protein and the mutant
best residue that can maintain the stability of the protein exhibited different numbers of
RLP protein in a halophilic state. hydrogen bonds. In general, D185A has fewer
The conformational geometry of WT and hydrogen bonds than WT and D185E. This
mutant proteins can be observed by studying the strengthens the notion that the structure of
radius of gyration (Rg), SASA, and hydrogen D185A is more compact because fewer
bond analysis (Shukla et al., 2018). The Rg of a interactions involving hydrogen bonds are
protein describes the size of the protein formed in its molecule.
globally, the higher the value, the more open the The results of the comparison of Rg and
protein tends to be and vice versa. A small Rg SASA and the number of hydrogen bonds in the
value indicates that the protein is in a compact molecule against WT and mutants strengthen
state while a large Rg value indicates that the the notion that the mutation in D185 affects the
protein has a smaller folding structure. In this conformation and geometry of RLP globally.
study, both mutants showed smaller Rg values RMSF is a parameter that can describe
than their WT, especially the D185A mutant the mobility/flexibility of individual amino acid
(Figure 2d). This shows that D185A has residues. Rigid structures such as helices and
smaller dimensions than WT and tends to be sheets will have a high RMSF value while loops
more folded than WT and D185E. This fact was and turns will have a low RMSF value.
further confirmed by observing the SASA Calculation of the RMSF value in the RLP WT
parameters and the number of hydrogen bonds and mutants shows that the RMSF value of the
formed. mutant has a lower value compared to the RLP
SASA is defined as protein accessibility WT (Figure 2a). This fact strengthens the
to water (solvent). Lower SASA value notion that mutant RLP has higher cohesiveness
determines a compact structure and vice versa. than WT which causes the amino acid residues
SASA can also describe folding patterns in to be more bound. Observation of the residues
proteins, where a small SASA value indicates in the active site area (190-320) shows that the
that there is high folding. In this case, the WT residue in that area has almost no difference
and mutant proteins show different SASA between WT and its mutants which indicates
patterns, the D185A area has the smallest value that the active site area is conserved and stable.
compared to D185E and WT. (Figure 2c). This
occurs because the aspartic acid mutation with
alanine (D185A) will remove the saltbridge so
that the protein structure will fold in response to

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)

Molecular Docking does not cause changes in protein activity. This


Molecular docking is performed to fact occurs because the active site region which
predict changes in active site residues after is responsible for protein activity has not
mutation by observing the interactions between changed which is confirmed by the results of
the ligand and amino acid residues in the active analysis using CastP 3.0 (Tian et al., 2018)
site area and measuring the binding energy which indicates that the active site volume is not
formed (Meng et al., 2011; Al-Karmalawy et significantly different (Table 1) which is also
al., 2021). The ligand used was Ribulose 1.5 validated by the results of the analysis of the
Biphosphate as a substrate for the Rubsico Like RMSF value showed that there was no change
Protein from Chromohalobacter salexigen BKL in the RMSF value of WT and mutants in the
5 (RLP CS) protein analogue. amino acid residues located at positions 190-
The docking results showed that there 320 (area in the square line) (Figure 2a). The
was no change in the interaction between the interaction of the ligand and active site protein
ligand and the active site of the protein which in WT (Figure 4) used as example of docking
was characterized by similar binding energy illustration.
values (Table 1). This shows that the mutation

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JURNAL KIMIA (JOURNAL OF CHEMISTRY) 18 (1), JANUARI 2024: 1 - 7

CONCLUSION RUBP : Ribulose Biposphate


SASA : Solvent Accessibilty Surface Area
The aspartic acid (asp) mutation at SB : Saltbridge
position 185 which is responsible for the WT : Wild Type
formation of saltbridge (SB) with alanine (ala)
and glutamic acid (glu) changes the structure of
the RLP CS globally as indicated by a change ACKNOLEDGEMENT
in Rg value of mutant. Interestingly, although
the global structure of the mutation results We thank the Institute Technology of
changes, the volume of the active region tends Sumatera for awarding the scholarship and
to remain the same and the interaction of the Hibah Bersaing Pasca Sarjana UGM for
ligand-amino acid residues at the active site funding this study.
does not change as indicated by the binding
energy values of the RLP WT and RMSF values REFERENCE
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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)

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