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Culture Documents
Department of Zoology, Naihati Rishi Bankim Chandra College, West Bengal State University, Naihati, North 24 Parganas,
West Bengal, India; bDepartment of Microbiology, University of Kalyani, Kalyani, West Bengal, India; cDepartment of Chemistry,
University of Miami, 1301 Memorial Drive, Coral Gables, FL 33146, USA; dDepartment of Biochemistry, Albert Einstein
College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
Communicated by Ramaswamy H. Sarma
(Received 6 November 2012; nal version received 22 April 2013)
Comparative molecular dynamics simulations of chemotaxis protein CheY from thermophilic origin Thermotoga
maritima and its mesophilic counterpart Salmonella enterica have been performed for 10 ns each at 300 and 350 K, and
20 ns each at 400 and 450 K. The trajectories were analyzed in terms of different factors like root-mean-square deviation,
root-mean-square uctuation, radius of gyration, solvent accessible surface area, H-bonds, salt bridge content, and proteinsolvent interactions which indicate distinct differences between the two of them. The two proteins also follow dissimilar unfolding pathways. The overall exibility calculated by the trace of the diagonalized covariance matrix displays
similar exibility of both the proteins near their optimum growth temperatures. However, at higher temperatures mesophilic protein shows increased overall exibility than its thermophilic counterpart. Principal component analysis also
indicates that the essential subspaces explored by the simulations of two proteins at different temperatures are nonoverlapping and they show signicantly different directions of motion. However, there are signicant overlaps within the
trajectories and similar direction of motions are observed for both proteins at 300 K. Overall, the mesophilic protein
leads to increased conformational sampling of the phase space than its thermophilic counterpart. This is the rst ever
study of thermostability of CheY protein homologs by using protein dynamism as a main impact. Our study might be
used as a model for studying the molecular basis of thermostability of two homologous proteins from two organisms
living at different temperatures with less visible differences.
Keywords: CheY; signal transduction; MD simulation; GROMACS; thermostability; salt bridge; PCA; protein
engineering
Introduction
Chemotaxis is a biochemical signaling process in which
bacterial agellar rotation is controlled by several environmental cues such as pH, chemicals, temperature, etc.
Motile bacteria are able to change the direction of their
migration through solution in response to the concentration gradients of attractants and repellents (Spohn &
Scarlato, 2001). Chemotactic behavior requires detection
of the chemical signal, signal transduction, and subsequent motor response. Chemotaxis helps bacteria to
search for food and also escape hostile environments
(Eisenbach & Caplan, 1998). There are a number of
proteins involved in the bacterial chemotactic signal
cascade. Through several studies using molecular biology, genetics, biochemical, and biophysical methods it
*Corresponding author. Email: saugata.hazra@einstein.yu.edu
Manish Paul and Mousumi Hazra contributed equally to this work
2013 Taylor & Francis
Thermostability of CheY
downstream proteins to change their conformations
critical to some biological processes (Hoch, 2000).
The two component system formed by Che proteins
is relatively complicated than the models, involving more
than two proteins. During chemotaxis, the sensor CheA
receives signals from transmembrane chemoreceptors with
the help of CheW (Bourret, Davagnino, & Simon, 1993;
Gegner, Graham, Roth, & Dahlquist, 1992; Hazelbauer,
Berg, & Matsumura, 1993) and then transfers the
signal to the response regulator, CheY, by the process of
phosphorylation. Phosphorylated CheY (CheYP) is
dephosphorylated by its autophosphatase activity, a reaction enhanced by CheZ (Kuo & Koshland, 1989). A series
of conformational changes and proteinprotein interactions occur in between those phosphotransfer events
involving several other proteins (e.g. FliM, FliN, and
FliG), which help switch motor to reverse the direction of
agellar rotation from counterclockwise to clockwise.
The main focus of our current study is the protein
CheY. The length of this protein ranges from 120 to
130 residues (Stock, Koshland, & Stock, 1985). CheY is
composed of central patches of sheets surrounded by a
number of helices. The number of helix and
strands varies in different bacterial species. CheY protein
contains different exible loop regions in its structure.
Each CheY protein contains one receiver domain and
one response regulatory domain, respectively. It plays a
key role in the control of the bacterial movements in
response to environmental chemotactic stimuli (Volz &
Matsumura, 1991). The phosphoryl group is received by
CheY from a conserved histidine residue of histidine
kinase CheA (Djordjevic & Stock, 1998). This leads to a
conformational change in the regulatory domain and
helps it to bind with the protein FliM. Binding to FliM
leads to a complex series of interactions involving motor
proteins and in this way, CheY plays a major role in
transmitting chemical stimuli to the bacterial agella via
a signal transduction cascade (Matsumura, Rydel, Linameir, & Vacante, 1984; McEvoy, Hausrath, Randolph,
Remington, & Dahlquist, 1998).
The primary target of our study is to understand the
factors contributing towards the thermostability of CheY.
We have selected two representatives of the CheY protein, one is thermophilic and the other is mesophilic
(Dasgupta & Dattagupta, 2008; Knaggs, Salsbury,
Edgell, & Fetrow, 2007; Liang et al., 2009). Thermostability is a property of a protein which could be utilized
to measure the potential of the protein to retain its secondary structure content at its maximum tolerable
temperature. A thermophilic protein can retain its secondary structure more compactly at higher temperatures
compared to its mesophilic counterpart. Proteins that are
stable at high temperatures have attracted much interest
because they have potential industrial applications
(Bruins, Janssen, & Boom, 2001; Sagi, Khan, &
929
930
M. Paul et al.
Figure 1.
Figure 2.
Crystal structures of (A) SeCheY (PDB ID 2CHF) and (B) TmCheY (PDB ID 1TMY).
Thermostability of CheY
931
Z
2 Z
cosiptpi tdt
0
1
p2i tdt
where T = upper limit of cosine content, pi = the amplitude of the motion along the eigenvector i, t = time, and
dt = change of time. The cosine content could be taken
values between 0 (no cosine) in corroborative motion
and 1 (a perfect cosine) in noncorroborative motion.
932
M. Paul et al.
Figure 3. RMSD of the proteins as functions of time calculated for SeCheY (black) and TmCheY (red) each at 300 K (A), 350 K
(B), 400 K (C), and 450 K (D).
Thermostability of CheY
whereas SeCheY takes longer time to attain the equilibrium (Table 1). Thus, consistent with the observed thermophilicity of TmCheY, changing temperature shows
little effect on the stability of its native structure according to the MD simulation. For SeCheY, more signicant
deviations from the native structures have been observed
at higher temperatures compared to the lower ones.
Root-mean-square uctuation
A more detailed picture of differences in residue mobility within and between simulations can be obtained from
graphs of the RMSF of C atoms relative to the average
structure. Such uctuations have been calculated for its
simulations at different temperatures. RMSF value
increases with the loss of secondary structure at high
temperatures. Analysis of RMSF values clearly shows
the structural exibility of the loop region in comparison
with the rest of the protein structure. Both the TmCheY
and SeCheY have 11 loops in their structure. When the
loop regions of SeCheY and TmCheY are compared,
comparatively large exibility is observed for the earlier.
Signicant exibility has been shown in the loop
regions L1 (26), L3 (2731), L7 (7581), and L8
(8891) of SeCheY at 300 K (Figure 4(A)). At 350 K,
again a sharp increase in the exibility of those loop
regions of SeCheY has been observed. In contrast, the
loops of TmCheY remain quite stable under such conditions (Figure 4(B)). At 400 K, uctuation of the loop
regions L5 (4752) and L9 (100105) of SeCheY have
been initiated whereas the exibility of L1 and L3
increase (Figure 4(C)). At 450 K, all the 11 loops of
SeCheY become extremely exible (Figure 4(D)). In
contrary, at 300 K some loop regions of TmCheY: L4
(34, 35), L6 (5557), L9 (8386), and L11 (118, 119)
show some degree of exibility (Figure 4(A)). But at
higher temperatures these loop regions remain stable.
The RMSF values of SeCheY ranges from .06 to .97 nm
during the temperature increase of 300450 K, in contrast
933
Table 1. Average values of RMSD, Rg, and SASA and average number of proteinprotein (intramolecular) and Proteinsolvent Hbonds in SeCheY and TmCheY at different temperature simulations.
Factors
RMSD
Rg
SASA
Proteinprotein Hbond
Proteinprotein H-bond
Source
300 K
350 K
400 K
450 K
SeCheY
TmCheY
SeCheY
TmCheY
SeCheY
TmCheY
SeCheY
TmCheY
SeCheY
TmCheY
.13 .02
.09 .01
1.37 .01
1.30 .01
49.05 1.00
45.34 1.03
90.40
90.30
278.40
233.40
.12 .02
.10 .01
1.38 .01
1.31 .01
50.53 1.46
47.07 2.17
87.90
84.50
259.60
226.70
.25 .05
.15 .02
1.38 .01
1.31 .01
52.35 1.45
47.96 1.21
80.05
82.15
243.40
206.20
.42 .11
.19 .02
1.43 .13
1.32 .01
55.89 2.03
48.62 1.51
74.10
82.55
224.85
177.60
RMSD, Rg, SASA, and H-bond denote root mean square deviation, radius of gyration, solvent accessible surface area, respectively. The values of
RMSD, Rg, and SASA are given in nm.
934
M. Paul et al.
Figure 4. RMSF of the proteins according to residue numbers calculated for SeCheY (black) and TmCheY (red) each at 300 K (A),
350 K (B), 400 K (C), and 450 K (D).
Thermostability of CheY
935
Figure 5. Rg of the proteins as functions of time calculated for SeCheY (black) and TmCheY (red) each at 300 K (A), 350 K (B),
400 K (C), and 450 K (D).
936
M. Paul et al.
Figure 6. SASA of the proteins according to residues numbers calculated for SeCheY (black) and TmCheY (red) each at 300 K (A),
350 K (B), 400 K (C), and 450 K (D).
Salt bridges
Salt Bridges most often play very crucial part in the stability of proteins. A salt bridge is actually a combination
of two noncovalent interactions: hydrogen bonding and
electrostatic interactions. This is quite common in proteins and contributes towards the stability of the entropically unfavorable folded conformation of proteins.
Although noncovalent interactions are known to be relatively weak interactions, small stabilizing interactions
can add up to make an important contribution to the
overall stability of a conformer. Especially, it signicantly contributes to the thermal stability of a protein. It
is often observed while comparing between protein
homologs with different stability, salt bridges become
one of the most signicant factors to be responsible. In
our present study, we have tried to nd out the differences in salt bridges between these two structures. We
have analyzed the variation in the length of the important
salt bridges in 10 ns at 300 and 350 K temperature, and
in 20 ns at 400 and 450 K for the thermophilic TmCheY
(Table 3) and mesophilic SeCheY proteins (Table 2).
Thermostability of CheY
937
Figure 7. SASA of the proteins according to time calculated for SeCheY (black) and TmCheY (red) each at 300 K (A), 350 K (B),
400 K (C), and 450 K (D).
shortened. The shortened parts of these secondary contents unfold to become a loop structure (Figure 9(C)).
Asp12, Asp57, and Lys109 become more exible due to
this loop formation. This is the main reason for the
stretching of the corresponding salt bridges.
The only exception is Lys45NZ-Asp41OD1, which
maintains a steady length even at high temperature and
there is a decrease in length at 450 K (Figure 9(D)).
Therefore, by comparing the salt bridge contents
between the two homologues of CheY, it can be easily
inferred that thermophilic TmCheY has a greater
number of potent salt bridges which are signicant
contributors towards its structural stability at higher
temperatures.
938
M. Paul et al.
Figure 8. SASA of the proteins according to atoms numbers calculated for SeCheY (black) and TmCheY (red) each at 300 K (A),
350 K (B), 400 K (C), and 450 K (D).
Glu35
NH2OE1
NZ - Asp12OD2
Lys109
NZ - Asp57OD1
Lys45
NZ - Asp41OD1
Lys45
NZ - Asp41OD2
Lys109
300 K
350 K
400 K
450 K
.35
.36
.24
.44
.43
.31
.25
.25
.39
.25
.26
.35
.25
.28
.24
.37
.27
.36
.30
.28
300 K
350 K
400 K
450 K
.38
.29
.30
.54
.33
.23
.16
.36
.22
.22
.26
.31
.37
.28
.48
.48
All the values presented in the above two tables are in nm.
Glu32
NH2OE1
NZ-Asp9OD2
Lys104
NZ-Asp54OD1
Lys42
NZ - Glue38OE1
Lys104
All the values presented in the above two tables are in nm.
Thermostability of CheY
Figure 9.
939
Salt bridge length (in nm) comparison at starting structure (A, B) and 450 K (C, D) of SeCheY and TmCheY.
Secondary structure
Additional information on the structural exibility of
SeCheY and TmCheY has been obtained by the analysis
of time-dependent secondary structure uctuations. We
have analyzed the number of residue contents in different
secondary structures for both SeCheY and TmCheY at
four temperatures (300, 350, 400, and 450 K). The secondary structure contents in the native structures are
80.45 and 83.03% for SeCheY and TmCheY, respectively (Table 4). When the average secondary structure
contents over time and temperature is considered, differences between SeCheY and TmCheY are evident from
300 K onwards simulations. The average secondary
structure contents of SeCheY and TmCheY are 82.2 and
82.91%, respectively (300 K simulations), 81.08 and
82.56%, respectively (350 K simulations) (Table 4). At
300 K, SeCheY exhibits lower contents of -sheets, 310
helices, and greater number of residues in coil-like conformation compared to TmCheY. The same pattern is
maintained at 350 K. In TmCheY, -helix is stably maintained at high temperatures compared to SeCheY. In case
of SeCheY, original -strand content decreases with
increase in temperature. While, in TmCheY the original
-strand is maintained its stability throughout the simulation. At 400 K, the average secondary structure content
for SeCheY and TmCheY are 78.9 and 84.26%, respectively (Table 4).
At 450 K, the 4 and 5 helices are totally absent in
SeCheY, while in TmCheY; all -helix content is stabily
maintained. At 450 K simulation, average secondary
structure contents for SeCheY and TmCheY are 73.1 and
82.56%, respectively (Table 4). So, it is very clear from
the above result that with increasing temperatures, the
average SeCheY secondary structure content reduces
constantly, while TmCheY maintains almost same average secondary structure content. The above result clearly
implies that at higher temperature the structure becomes
relatively disorganized with signicant loss of the
13 (10.2%)
12 3.0 (9.5%)
10 2.8 (7.9%)
15.3 4.4 (12.1%)
15.8 5.7 (12.4%)
8 (6.8%)
13.7 3.6 (11.7%)
15.4 3.8 (13.2%)
15.1 3.2 (12.9%)
13.3 3.7 (11.5%)
0 (0%)
0 0(0%)
0 0(0%)
.25 1.1(.2%)
.5 1.7 (.4%)
0 (0%)
0 0 (0%)
0 0 (0%)
0 0 (0%)
0 0 (0%)
22 (17.2%)
23.1 1.7 (18.2%)
21.3 2.5 (16.8%)
21.4 2.6 (16.8%)
18.8 4.9 (14.8%)
25 (21.2%)
26.5 1.3 (22.7%)
26.2 1.6 (22.4%)
26.2 1.8 (22.4%)
24.7 2.7 (21.1%)
TmCheY
SeCheY
Starting
300 K
350 K
400 K
450 K
Starting
300 K
350 K
400 K
450 K
56 (43.8%)
50.1 3.1 (39.5%)
53.6 4.1 (42.2%)
41.1 6.2 (32.4%)
25.6 7.3 (20.2%)
55 (46.6%)
49.3 3.6 (42.1%)
44.6 3.3 (38.1%)
42.2 4.2 (36.1%)
4.75 4.1 (34.8%)
0 (0%)
3.3 1.7 (2.6%)
3.3 1.7 (2.6%)
2.6 2.3 (2.0%)
6.1 2.9 (4.8%)
3 (2.5%)
0 0 (0%)
.9 2.0 (.8%)
1.5 2.3 (1.3%)
2.6 2.3 (2.2%)
0 (0%)
0 0(0%)
0 0(0%)
1.1 1.4 (.9%)
.7 1.4 (.5%)
0 (0%)
0 0 (0%)
0 0 (0%)
.4 .9 (.3%)
.6 1.0 (.5%)
Turn
5-helix
-bridge
-strand
310helix
-helix
Table 4. Average secondary structure contents in SeCheY and TmCheY trajectories obtained at different temperatures and in the corresponding starting structures.
12 (9.4%)
15.9 2.2 (12.5%)
14.8 1.5 (11.7%)
18.4 2.3 (14.5%)
25.3 6.0 (19.9%)
7 (5.9%)
7.5 1.2 (6.4%)
9.5 2.6 (8.1%)
13.1 2.5 (11.2%)
14.4 2.7 (12.3%)
M. Paul et al.
Bend
940
Thermostability of CheY
Table 5. Cosine contents of SeCheY and TmCheY at different
temperature simulation.
SeCheY
TmCheY
PC1
PC2
PC1
PC2
300 K
350 K
400 K
450 K
.050
.590
.140
.030
.070
.120
.140
.160
.002
.080
.002
.090
.050
.080
.030
.270
respectively (Table 1). It has been shown that at all temperatures the average Proteinsolvent hydrogen bond
number is higher for SeCheY. This is because SeCheY
has a higher number of surface hydrophilic residues than
its thermophilic counterpart, which enables SeCheY to
make more solvent specic hydrogen bonds. On the
other hand, due to a greater number of intramolecular
941
Figure 10. Intramolecular hydrogen bonds number of proteins according to time calculated for SeCheY (black) and TmCheY (red)
each at 300 K (A), 350 K (B), 400 K (C), and 450 K (D).
942
M. Paul et al.
Figure 11. Proteinsolvent hydrogen bonds number of proteins according to time calculated for SeCheY (black) and TmCheY (red)
each at 300 K (A), 350 K (B), 400 K (C), and 450 K (D).
Thermostability of CheY
943
Figure 12. PCA for SeCheY and TmCheY. (A) and (C) represent the PCA of SeCheY at 300, and 350 K respectively. (B) and (D)
represent the PCA of TmCheY at 300, and 350 K respectively.
944
M. Paul et al.
Figure 13. PCA for SeCheY and TmCheY. (A) and (C) represent the PCA of SeCheY at 400, and 450 K respectively. (B) and (D)
represent the PCA of TmCheY at 400, and 450 K respectively.
Thermostability of CheY
945
Figure 14. Superimposition of 10 congurations obtained by projecting the C motion onto the rst and second eigenvector at
300 K (A, B) and 350 K (C, D) of SeCheY and TmCheY, respectively. Arrows are used as qualitative indicators of the motion
direction.
946
M. Paul et al.
Figure 15. Superimposition of 10 congurations obtained by projecting the C motion onto the rst and second eigenvector at
400 K (A, B) and 450 K (C, D) of SeCheY and TmCheY respectively. Arrows are used as qualitative indicators of the motion
direction.
Table 6. RMSIP between the rst 10 eigenvectors obtained for different simulation groups.
Se300 K
Se300 K
Se350 K
Se400 K
Se450 K
Tm300 K
Tm350 K
Tm400 K
Tm450 K
.973
Se350 K
Se400 K
Se450 K
Tm300 K
Tm350 K
Tm400 K
Tm450 K
.986
.988
1.000
.887
.868
1.000
.815
1.000
.773
.815
.820
.818
.742
.990
.877
.865
.887
.775
.920
.930
1.000
.879
1.000
1.000
.917
.948
.952
1.000
.889
1.000
1.000
.868
.910
1.000
.942
These values refer to the RMSIP obtained by partitioning simulations in the same group into two halves and calculate their inner products. Se and
Tm stands for SeCheY and TmCheY respectively.
Thermostability of CheY
Figure 16.
947
its thermophilic homolog TmCheY can retain its secondary structure content for a longer time-scale with increasing temperature. At 450 K, TmCheY is able to retain
83% of its secondary structure content; whereas
SeCheY can only retain 73%. The loop regions: L1
(26), L3 (2731), L7 (7581), and L8 (8891) of
SeCheY showed signicant exibility at all four temperature simulations. The residues which render maximum
exibility in those loops with increasing temperature are
Ala2, Lys6, Asp12, Met16, Lys23, Val32, Lys45, Phe52,
Thr70, Ile71, Ser75, Ala76, Met77, Ala89, Asn93,
Lys125, Leu126, Gly127, and Met128.
Our comparative analysis also nds out differences
in strength of salt bridges (Lys104NZ-Asp9OD2,
Arg15
NH2-Glu32OE1, and Lys42NZ -Glu38OE1 of TmCheY
and SeCheY). In case of SeCheY, the salt bridges are
abolished with increasing temperature which further
explains its lower thermostability. Additional residues
Arg4, Asp10, Met14, Ile21, Ile50, Ile78, Lys95, Val103,
Arg110, and Ala114 are also important in maintaining
high thermal tolerance of TmCheY.
Intramolecular hydrogen bonds also play an important role in protein stability. Our results show that there
is a loss of considerable numbers of intramolecular
hydrogen bonds in case of SeCheY at higher temperature
in comparison to TmCheY. Also, TmCheY has a comparatively more packed hydrophobic core than SeCheY.
From the PCA analysis, greater exibility in the loop
regions of SeCheY was visualized which further supports
our results.
Previous studies have shown that unlike other thermophilic proteins, it is not possible to understand the reason
behind higher thermostability of T. maritima CheY solely
by comparing the structures of the thermophilic and mesophilic counterparts at room temperature (Usher et al.,
1998). Our study clearly revealed the reason behind this
enigma. In the case of proteins like CheY and many
other, higher thermo stability is not the result of one or
two predominant factors, rather it is due to small cumula-
948
M. Paul et al.
Supplementary material
The supplementary material for this paper is available
online at http://dx.doi.10.1080/07391102.2013.799438.
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