Cell Biochemistry and Biophysics

, Volume 67, Issue 2, pp 623–633

Investigation of Binding Phenomenon of NSP3 and p130Cas Mutants and Their Effect on Cell Signalling

Authors

  • Balu K.
    • Bioinformatics Division, School of Bio Sciences and TechnologyVellore Institute of Technology University
  • Vidya Rajendran
    • Bioinformatics Division, School of Bio Sciences and TechnologyVellore Institute of Technology University
  • Rao Sethumadhavan
    • Bioinformatics Division, School of Bio Sciences and TechnologyVellore Institute of Technology University
    • Bioinformatics Division, School of Bio Sciences and TechnologyVellore Institute of Technology University
    • Human Genetics Foundation
Original Paper

DOI: 10.1007/s12013-013-9551-6

Cite this article as:
Balu K., Rajendran, V., Sethumadhavan, R. et al. Cell Biochem Biophys (2013) 67: 623. doi:10.1007/s12013-013-9551-6

Abstract

Members of the novel SH2-containing protein (NSP3) and Crk-associated substrate (p130Cas) protein families form a multi-domain signalling platforms that mediate cell signalling process. We analysed the damaging consequences of three mutations, each from NSP3 (NSP3L469R, NSP3L623E, NSP3R627E) and p130Cas (p130CasF794R, p130CasL787E, p130CasD797R) protein with respect to their native biological partners. Mutations depicted notable loss in interaction affinity towards their corresponding biological partners. NSP3L469R and p130CasD797R mutations were predicted as most prominent in docking analysis. Molecular dynamics (MD) studies were conducted to evaluate structural consequences of most prominent mutation in NSP3 and p130Cas obtained from the docking analysis. MD analysis confirmed that mutation in NSP3L469R and p130CasD797R showed significant structural deviation, changes in conformations and increased flexibility, which in turn affected the binding affinity with their biological partners. Moreover, the root mean square fluctuation has indicated a rise in fluctuation of residues involved in moderate interaction acquired between the NSP3 and p130Cas. It has significantly affected the binding interaction in mutant complexes. The results obtained in this work present a detailed overview of molecular mechanisms involved in the loss of cell signalling associated with NSP3 and p130Cas protein.

Keywords

Cell signallingFlexibilityBinding affinityInteractionsHydrogen bondsMolecular dynamics

Abbreviations

BCAR1

Breast cancer anti-oestrogen resistance protein 1

BSA

Buried surface area

Crk

Cysteine-rich receptor like kinases

FAT

Focal adhesion target

Rg

Radius of gyration

SASA

Solvent accessible surface area

Introduction

Cell signalling is an essential characteristic of multi-cellular organism that governs basic cellular activities and coordinates cell actions which help in development, tissue repair, immunity and normal tissue homeostasis. Its importance lies in the fact that any kind of discrepancy in cell signalling may lead to spectrum of diseases such as cancer, autoimmunity and diabetes [1]. Hence, by understanding the mechanism of cell signalling, diseases may be treated effectively. Members of the novel SH2-containing protein (NSP) and Crk-associated substrate (Cas) protein families form multi-domain signalling platforms that mediate cell migration and invasion through a collection of distinct signalling motifs [2]. The NSP family protein contains three members namely NSP1, NSP2 and NSP3, all of which have a similar architecture, with an N-terminal SH2 domain [3]. The adapter protein NSP3 is encoded by a gene named SH2D3C and it is a member of cytoplasmic protein family which involved in cell migration [4]. P130Cas (Crk-associated substrate) is a member of Cas family of protein and also known as BCAR1 which is involved in various cellular events like migration, adhesion, survival, transformation and invasion [57]. The Cas family proteins are a family of adhesion docking molecules that mediate protein–protein interactions and contribute to a number of signal transduction pathways [3]. The NSP structure tightly binds with the FAT domain of Cas protein using an extended interface. The NSP and Cas effectively interact with each other to form various combinations of signalling modules that regulate cellular processes [2]. When Cas and NSP proteins get together, they help in cell migration. Furthermore, the NSP3–p130Cas complex is essential in neuronal cells for proper olfactory development, and its absence causes a human developmental disorder called Kallmann syndrome [8, 9]. Moreover, the Cas proteins act as scaffolds to regulate protein complexes controlling migration and chemotaxis, apoptosis, cell cycle, and differentiation and have more recently been linked to a role in progenitor cell function [10]. The NSP3 (SHEP1)–Cas-L signalling node is crucial for B-cell migration and maturation [11, 12]. The experimental studies revealed that the mutations in NSP3 (NSP3L469R, NSP3L623E, NSP3R627E) and p130Cas (p130CasF794R, p130CasL787E, p130CasD797R) were shown to affect the binding affinity with their corresponding biological partners and alters cell migration, adhesion and invasion processes [2, 1113].

In this work, we have highlighted the interaction behaviour of NSP3 and p130Cas protein, and mechanism behind the loss of binding interactions between their biological partners (Fig. 1). Study of protein conformational changes occurring at their protein–protein interaction sites has enabled us to investigate the root causes behind several pathological cases of cell signalling cascades [1423]. The main objective of this work is to gain in-depth molecular insights into NSP3 and p130Cas interaction pattern and to elucidate the root cause behind mutation-induced NSP3–p130Cas-mediated cell signalling loss. Binding behaviour of NSP3 and p130Cas proteins was studied with the aid of docking and molecular dynamic simulation approaches. Results showed that mutations in NSP3 and p130Cas protein caused structural damage in complex by increasing the flexibility which ultimately led to disturbance of the cell signalling.
https://static-content.springer.com/image/art%3A10.1007%2Fs12013-013-9551-6/MediaObjects/12013_2013_9551_Fig1_HTML.gif
Fig. 1

The interactional behaviour of NSP3 and p130Cas protein, how the mutations affect binding interactions between the biological partners which leads to disturbance of the cell signalling process. NSP3 protein functional domain (in maroon colour) residues range from 386 to 698 and p130Cas protein functional domain (in green colour) residues range from 739 to 872. Mutation in NSP3 protein showed in black colour, whereas mutation in p130Cas protein showed in purple colour. Formation of NSP3–p130Cas complex is vital for cell signalling process (Color figure online)

Materials and Methods

Dataset Collection

Crystal structure of NSP3–p130Cas complex (PDB ID: 3T6G) [2] was obtained from protein data bank (PDB) [24]. We considered monomer of NSP3 (chain A) and monomer of p130Cas (chain B) for our studies. In order to build the mutant structures, we induced the point mutations in monomer of NSP3 (NSP3L469R, NSP3L623E and NSP3R627E) and monomer of p130Cas (p130CasF794R, p130CasL787E and p130CasD797R) proteins by using SPDB viewer package [25]. These structures were energetically optimized by applying the all-atom OPLS force field available in GROMACS package 4.5.3 [26].

Protein–Protein Interactions

The HADDOCK protocol [27, 28] was used to dock the optimized structures of native and mutant of NSP3 and p130Cas. HADDOCK is a series of scripts that run in combination with ARIA [29, 30] and CNS [31]. The docking process in HADDOCK is driven by ambiguous interaction restraints (AIRs), which are derived from the available experimental information on the residues involved in the intermolecular interaction. The  predicted interface surface residues in NSP3 (position number 386, 387, 388, 454, 461, 465, 468, 469, 470, 471, 595, 596, 598, 613, 614, 615, 616, 617, 620,623, 624, 627, 630 and 631) together with interface residues on the partner proteins (residues between 742 and 831 on p130Cas), which used as active residues and residues neighbouring the active residues used as passive residues. At the first stage of the docking protocol (see also the HADDOCK website at http://www.nmr.chem.uu.nl/haddock), consisting of randomization of orientations and rigid body energy minimization, we have calculated 1,000 complex structures. The 200 solutions with the lowest intermolecular energies have been selected for semi-flexible simulated annealing in torsion angle space. The resulting structures have been then refined in explicit water. Finally, the solutions have been clustered using a threshold value of 1.5 Å for the pairwise backbone root mean square deviation (RMSD) at the interface, and the resulting clusters have been ranked according to their average interaction energy (defined as the sum of van der Waals, electrostatic and AIRs energy terms) and buried surface area. One lowest energy structure of the lowest intermolecular energy cluster was selected for analysis. This lowest energy structure displayed no AIR restraint violations (within a threshold of 0.3 Å) and was accepted as the final docked structure for the complex.

HADDOCK scoring is performed according to the weighted sum (HADDOCK score) of different energy terms which includes van der Waals energy, electrostatic energy, distance restraints energy, direct RDC restraint energy, inter-vector projection angle restraints energy, diffusion anisotropy energy, dihedral angle restraints energy, symmetry restraints energy, binding energy, desolvation energy and buried surface area. Along with HADDOCK score and total interaction energy, we reported van der Waals energy, electrostatic energy, restraints violation energy, desolvation energy and buried surface area. Intermolecular contacts (hydrogen bonds and non-bonded contacts) were analysed with DIMPLOT, which is part of the LIGPLOT software [32]. The default settings were used (3.9 Å heavy atoms distance cut-off for non-bonded contacts; 2.7 and 3.3.5 Å proton–acceptor and donor–acceptor distance cut-offs, respectively, with minimum 90° angles (D–H–A, H–A–AA, D–A–AA) for hydrogen bonds [33].

Molecular Dynamics Simulation

Molecular dynamics simulation was performed by using Gromacs 4.5.3 package [26] running on a single 2.8 GHz Pentium IV IBM machine with 3 GB RAM and running Ubuntu 11.10 Linux package. Complex structure of native and mutant of NSP3–p130Cas was used as starting point for MD simulations. Systems were solvated in a rectangular box with TIP3P water molecules at 10 Å marginal radius. At physiological pH, the complex was found to be positively charged; thus, in order to make the simulation system electrically neutral, we added four chloride ions (CL) to the simulation box using the genion tool that accompanies with Gromacs package. Initially, the solvent molecules were relaxed while all the solute atoms were harmonically restrained to their original positions with a force constants 100 kcal/mol for 5,000 steps. After this, whole molecular system was subjected to energy minimization for 5,000 iterations by steepest descent algorithm implementing GROMOS96 43a1 force field. Berendsen temperature coupling method [34] was used to regulate the temperature inside the box. Electrostatic interactions were computed using the Particle Mesh Ewald method [35]. The ionization states of the residues were set appropriate to pH 7 with all histidines assumed neutral. The pressure was maintained at 1 atm with the allowed compressibility range of 4.5 × 10−5 atm. SHAKE algorithm was used to constrain bond lengths involving hydrogens, permitting a time step of 2 fs. Van der Waals and coulomb interactions were truncated at 1.0 nm. The non-bonded pair list was updated every 10 steps and conformations were stored every 0.5 ps. Position restraint simulation for 500 ps was implemented to allow solvent molecules to enter the cavity region of structure. Finally, systems were subjected to MD simulation for 10 ns. We then computed the comparative analysis of structural deviations in native and mutant structure. RMSD, RMSF, SAS and Rg analysis were carried out by using g_rms, g_rmsf, g_sas and g_gyrate tool, respectively. Numbers of distinct hydrogen bonds are formed by specific residues to other amino acids within the protein during the simulation (NH bond) were calculated using g_hbond. NH bond was determined on the basis of donor–acceptor distance smaller than 0.35 nm and of donor-hydrogen-acceptor. We used g_analyze tool to calculate the average values and standard deviations of simulation output data. All the graphs were plotted using XMGRACE [36] program.

Principal Component Analysis

The calculation of the eigenvectors and eigenvalues, and their projection along the first two principal components, was carried out using essential dynamics (ED) method according to the protocol [37] within the Gromacs software package. The principle component analysis or ED is a technique that reduces the complexity of the data and extracts the concerted motion in simulations that are essentially correlated and presumably meaningful for biological function [37]. In the ED analysis, a variance/covariance matrix was constructed from the trajectories after the removal of the rotational and translational movements. A set of eigenvectors and eigenvalues was identified by diagonalizing the matrix. The eigenvalues represent the amplitude of the eigenvector along the multidimensional space, and the displacement of atoms along each eigenvector shows the concerted motions of protein along each direction. The movements of structures in the essential subspace were identified by projecting the Cartesian trajectory coordinates along the most important eigenvectors from the analysis. Backbone C-alpha bonds trajectories were obtained using g_covar and g_anaeig of Gromacs utilities.

Result and Discussion

Typical in silico docking approaches depend on defining the interface of complexes based on the surface geometry complementarity and amino acid pairwise affinities of the 3D structure of the unbound molecules without a prior inclusion of any experimental information [38, 39]. We selected crystal structure of 3T6G protein for our analysis and considered monomer of NSP3 (chain A) and monomer of p130Cas (chain B). Novel SH2 containing protein (NSP) and Crk-associated substrate (Cas) protein families form multi-domain signalling platforms that mediate cell migration and invasion through a collection of diverse signalling motifs. Hence, we docked monomer of NSP3 with monomer of P130Cas by HADDOCK server. In our analysis, we called native NSP3 and p130Cas protein as native complex, NSP3L469R–p130Cas complex as group Ia, NSP3L623E–p130Cas complex as group Ib and NSP3R627E–p130Cas complex as group Ic, respectively; similarly, NSP3–p130CasF794R complex as group IIa, NSP3–p130CasL787E complex as group IIb and-NSP3–p130CasD797R complex as group IIc, respectively.

Detailed analysis of docked complex exposed notable features. Calculation of HADDOCK score is very important to understand the affinity level between the biological partners. A HADDOCK score is defined to rank the structures after each docking stage. It is a weighted sum of intermolecular electrostatic (Elec), van der Waals (vdw), desolvation (Dsolv), AIR energies and a buried surface area (BSA) [38, 40]. Overall, HADDOCK score of native complex showed a score of −197.4 ± 9.8, whereas Group I (a–c) showed scores −168.7 ± 12.5, −192.0 ± 2.5 and −178.0 ± 1.2, respectively, and Group II (a–c) showed scores −182.4 ± 5.3, −189.2 ± 1.0 and −163.5 ± 1.1, respectively. Higher negative values of HADDOCK score for native complex indicated high binding affinity towards their corresponding biological partners (NSP3 and P130Cas) when compared to Group I and II complexes.

Buried surface area (BSA) is used to quantify protein surface which is not exposed to water. A higher BSA value enables close proximity between the biological complexes. In Table 1, native complex showed maximum BSA of 2,778.6 ± 63.2 Å2. Group Ia and IIc showed the minimum BSA value of 2,696.7 ± 85 Å2 and 2,639.0 ± 93.9 Å2, respectively. Other group I and II complexes showed intermediate value of BSA. Deliberation of desolvation component (the loss of interactions with the water phase) is important because it overcompensates the HADDOCK score and results in an opposite effect [41, 42]. Desolvation energy, restraints violation energy and BSA have good correlation with docking score and interaction energy of complex during docking simulation. The combined value of native complex is significantly higher than Group I and II complexes, thus indicating the effect of gain of function. Native complex enables more close interaction between NSP3 and P130Cas than Group I and II complexes (Table 1). Docking analysis between NSP3 and p130Cas proteins was performed to evaluate the interaction behaviour. The result clearly indicated that the mutation had caused severe structural and functional consequences on the protein and had affected its binding interactions.
Table 1

Statistical analysis of protein–protein docking result obtained by Haddock

Protein type

HADDOCK score

Total interaction energy (Kcal mol−1)

Van der waal energy (Kcal mol−1)

Electrostatic energy (Kcal mol−1)

Desolvation energy (Kcal mol−1)

Restraints violation energy (Kcal mol−1)

Buried surface area (Å2)

Native complex

−197.4 ± 9.8

−562.401

−94.9 ± 4.5

−447.3 ± 30.2

−6.5 ± 3.9

15.8 ± 4.54

2,778.6 ± 63.2

Group Ia

−168.7 ± 12.5

−506.026

−87.6 ± 6.0

−394.1 ± 46.3

−3.4 ± 12.5

11.0 ± 4.52

2,696.7 ± 85.3

Group Ib

−192.0 ± 2.5

−515.516

−94.3 ± 14.5

−448.1 ± 79.0

−10.0 ± 2.4

19.0 ± 6.45

2,747.5 ± 107.1

Group Ic

−178.0 ± 1.2

−490.275

−90.6 ± 2.3

−384.2 ± 19.4

−11.6 ± 3.5

11.4 ± 4.51

2,706.8 ± −80.9

Group IIa

−182.4 ± 5.3

−520.853

−96.3 ± 6.9

−457.9 ± 33.7

−4.2 ± 5.4

13.0 ± 2.91

2,711.1 ± 71.9

Group IIb

−189.2 ± 1.0

−530.409

−104.9 ± 4.8

−440.0 ± 29.3

−6.5 ± 8.2

19.7 ± 4.96

2,753.2 ± 42.6

Group IIc

−163.5 ± 1.1

−450.683

−78.8 ± 6.0

−435.4 ± 61.4

−0.5 ± 14.7

18.9 ± 2.96

2,639.0 ± 93.9

RMSD = RMSD from overall lowest energy structure

Hydrogen bonds are by far the most important specific interactions in biological recognition processes and particularly essential in determining the binding specificity [14, 43, 44]. The intermolecular hydrogen bonds can provide favourable free energy to the binding [4446]. The number of intermolecular hydrogen bonds was calculated for native, group I and II complexes and were listed in Table 2. The number of intermolecular hydrogen bonds had shown for all complexes in Fig. 2 and Fig. S1. Native complex (NSP3–p130Cas) showed a total of 18 hydrogen bond formations at their interaction locus while NSP3L469R–p130Cas complex (Group Ia) and NSP3–p130CasD797R complex (Group IIc) showed 14 hydrogen bonds and it is depicted in Fig. 2a–c. Other mutant complexes showed intermediate number of hydrogen bonds and it is showed in supplementary material (Fig. S1). Protein–protein docking analysis and intermolecular hydrogen bonding patterns specified that the native complex showed tight interaction between NSP3 and P130Cas proteins, whereas the mutation had caused severe interaction losses. The three factors namely, score difference in docking process, variation in interaction energy and differences in buried surface of complex might correspond to conformational changes at protein-binding surface due to mutation. NSP3L469R and p130CasD797R mutations were predicted as most prominent in docking analysis.
Table 2

Number of hydrogen bonds of native and mutant NSP3–P130Cas complex

Protein type

Number of H-bonds

Native complex

18

Group Ia

14

Group Ib

17

Group Ic

14

Group IIa

15

Group IIb

15

Group IIc

14

H-bonds Hydrogen bonds

https://static-content.springer.com/image/art%3A10.1007%2Fs12013-013-9551-6/MediaObjects/12013_2013_9551_Fig2_HTML.gif
Fig. 2

Residue interaction at protein–protein interface in NSP3–p130Cas complex. a NSP3–p130Cas complex b NSP3L469R–p130Cas complex and c NSP3–p130CasD797R complex. The colour coding represents NSP3 in brown colour, p130Cas in pink colour. Hydrogen bonding interactions are denoted by dashed lines. Residues involved in the hydrophobic interactions are shown as starbursts. This illustration was prepared by Ligplot (Color figure online)

Further, we conducted molecular dynamics (MD) simulations to study the conformational fluctuations and dynamic behaviour of native (NSP3–P130Cas) complex, and most prominent of mutant I (NSP3L469R–p130Cas) and mutant II (NSP3–p130CasD797R) complex which were observed in docking analysis. We highlighted RMSF of C-alpha carbon by trajectory analysis obtained through MD simulation. Distinct NH bond analyses were performed to understand the flexible behaviour of residues. In order to verify the convergence criterion during the MD simulations, we examined the RMSD and energy fluctuations throughout the simulations. We calculated RMSD for all the Cα atoms from the starting structure, which were considered as a vital condition to measure the convergence of the protein system concerned (Fig. 3). In Fig. 3, native and both mutant complex proteins showed similar fashion of deviation till 250 ps from their starting structure, resulting in a backbone RMSD of ~0.1 to 0.20 nm during the simulations. This deviation in both mutant complexes persists till 250 ps from starting structure but after this, both mutant complexes showed more deviation. Mutant I and II attained approximately ~0.22 to 0.31 nm and ~0.15 to 0.26 of backbone RMSD till 1,800 ps while native structure maintained RMSD value of ~0.12 to 0.21 nm. Between periods of 1,800 and 5,700 ps, native structure maintained deviation between 0.15 and 0.25 nm and mutant I showed deviation between ~0.22 and 0.31 nm, while mutant II showed ~0.15 to 0.26 nm. Mutant II retained the maximum deviation till the end, and near to 8,720 ps, it attained RMSD value of ~0.34 nm. From their initial structure, it was specified that native complex structure exhibited minimum deviation but both the mutant complexes showed maximum deviation (Fig 3). The average value of RMSD during the simulation time period in native and mutant complex is signified in Table 3. We observed a maximum energy value in both mutant complexes as compared to native complex and it was depicted in Fig. 4. Moreover, energy plot clearly indicated that both mutant complexes were less energetically stable as compared to native complex.
https://static-content.springer.com/image/art%3A10.1007%2Fs12013-013-9551-6/MediaObjects/12013_2013_9551_Fig3_HTML.gif
Fig. 3

Time evolution of backbone is shown as a function of time of the native and mutant NSP3–p130Cas complex structures at 300 K. The symbol coding scheme is as follows: native complex (in black colour), mutant I complex (in red colour) and mutant II complex (in green colour) (Color figure online)

Table 3

Average values of RMSD, Rg, SASA and average number of protein–protein H-bonds in native and mutant NSP3–P130Cas complex

 

Native complex

Mutant I complex

Mutant II complex

RMSD

0.23

0.27

0.28

Rg

2.35

2.39

2.40

SASA

110.54

118.52

117.33

Protein solvent H-bonds

363.36

354.67

349.59

The values of RMSD, Rg and SASA are given in nm

RMSD root mean square deviation, Rg radius of gyration, SASA solvent accessible surface area, H-bonds hydrogen bonds

https://static-content.springer.com/image/art%3A10.1007%2Fs12013-013-9551-6/MediaObjects/12013_2013_9551_Fig4_HTML.gif
Fig. 4

Plot of total energy as a function of time for the MD simulations. The symbol coding scheme is as follows: native complex (in black colour), mutant I complex (in red colour) and mutant II complex (in green colour) (Color figure online)

A more detailed picture of differences in residue movements within and between simulations could be obtained from graphs of the RMSF of Cα atoms relative to the average structure (Fig. 5). In Fig. 5, both mutant complexes exhibited more flexibility when compared to native complex. The increased flexibility of both mutant complexes affected its binding behaviour towards their biological partners and it was the reason for showing less docking scores. We conducted radius of gyration (Rg) and solvent accessible surface area (SASA) analysis to further evaluate the conformational changes in native and mutant complexes. The native complex showed Rg value of ~2.34 nm at 0 ps, ~2.37 nm at 350 ps, ~2.36 nm at 1,550 ps, ~2.35 nm at 4,500 ps, ~2.35 nm at 7,450 ps and ~2.34 nm at 10,000 ps. Mutant I complex showed Rg value of ~2.37 nm at 0 ps, ~2.40 nm at 350 ps, ~2.40 nm at 1,550 ps, ~2.38 nm at 4,500 ps, ~2.39 nm at 7,450 ps and ~2.37 at 10,000 ps. Mutant II complex showed Rg value of ~2.38 nm at 0 ps, ~2.41 nm at 350 ps, ~2.39 nm at 1,550 ps, ~2.40 nm at 4,500 ps, ~2.40 nm at 7,450 ps and ~2.40 nm at 10,000 ps, respectively. Native curve did not differ greatly and maintained Rg values of ~2.32 to 2.36 nm, indicating that the native conformation was mostly conserved throughout the simulation time. Both mutant structures showed large fluctuation in Rg and that is between ~2.37 to 2.40 nm (mutant I) and ~2.38 to 2.42 nm (mutant II), respectively. The average value of Rg during simulation time period in native and mutant complexes is specified in Table 3 (Fig. 6).
https://static-content.springer.com/image/art%3A10.1007%2Fs12013-013-9551-6/MediaObjects/12013_2013_9551_Fig5_HTML.gif
Fig. 5

RMSF of the backbone C-alpha atoms of native and mutant NSP3–p130Cas complex structure at 300 K is shown. The symbol coding scheme is as follows: native complex (in black colour), mutant I complex (in red colour) and mutant II complex (in green colour) (Color figure online)

https://static-content.springer.com/image/art%3A10.1007%2Fs12013-013-9551-6/MediaObjects/12013_2013_9551_Fig6_HTML.gif
Fig. 6

Radius of gyration of Cα atoms of native and mutant of NSP3–p130Cas versus time at 300 K. The symbol coding scheme is as follows: native complex (in black colour), mutant I complex (in red colour) and mutant II complex (in green colour) (Color figure online)

The variation of SASA of the native and mutant structures with time was shown in Fig. 7. Both mutant complexes showed higher values of SASA with time, while native complex showed smaller values. The average value of SASA during the simulation time period in native and mutant complex is signified in Table 3. The large fluctuation in the radius of gyration in both mutant complex indicated that the protein might be undergoing a significant structural transition. This was also supported by the fluctuations in SASA (Fig. 7). In Rg and SASA plot, the major structural transition was occurred between 1,300 and 9,400 ps, which showed the variations in native and mutant protein complexes.
https://static-content.springer.com/image/art%3A10.1007%2Fs12013-013-9551-6/MediaObjects/12013_2013_9551_Fig7_HTML.gif
Fig. 7

Solvent accessible surface area (SASA) of native and mutant of NSP3–p130Cas versus time at 300 K. The symbol coding scheme is as follows: native complex (in black colour), mutant I complex (in red colour) and mutant II complex (in green colour) (Color figure online)

Protein flexibility is directly proportional to intramolecular NH bonds between amino acid residues [47, 48]. Intermolecular NH bonds were calculated for both mutant and native complex during the simulation time and it was shown in Fig. 8. More intermolecular NH bonds help to maintain rigidity in native complex while less affinity of the mutant complex in NH bonds formation with neighbouring residues made it more flexible. The presence of more hydrogen bonds in native complex made it rigid and showed close interaction between the NSP3 and p130Cas while both mutant complex exhibited more flexibility behaviour and also showed not as much participation in NH bond formation (Table 3). From the RMSF and NH bond analysis, it is confirmed that due to less number of hydrogen bond, the mutant structures attained more flexible conformation.
https://static-content.springer.com/image/art%3A10.1007%2Fs12013-013-9551-6/MediaObjects/12013_2013_9551_Fig8_HTML.gif
Fig. 8

Average number of intermolecular hydrogen bonds in native and mutant NSP3–p130Cas versus time at 300 K. The symbol coding scheme is as follows: native complex (in black colour), mutant I complex (in red colour) and mutant II complex (in green colour) (Color figure online)

Essential dynamics (ED) analysis was used to obtain the better view of dynamical mechanical property of the investigated structures. To further support our MD simulation result, the large-scale collective motions of the native, mutant I and II complex using ED analysis were conducted. The dynamics of two proteins were best achieved via characterization of its phase space behaviour. The eigenvectors of the covariance matrix were called its principle components. The change of particular trajectory along each eigenvector was obtained by this projection. The spectrum of the corresponding eigenvalues (Fig. 9a–b) indicated that the fluctuation of the system was basically restricted within the first two eigenvectors. The projection of trajectories obtained at 300 K onto the first two principal components (PC1, PC2) showed the motion of native and two mutant protein complexes in phase space. On these projections, we saw clusters of stable states. Two features were very apparent from these plots. Initially, the clusters were well defined in native than mutant complexes. Finally, both mutant complexes covered a larger region of phase space particularly along PC1 plane than native and it is showed in Fig. 9a–b. Our observation consequently supported with the idea of higher flexibility in both mutant complex than native at 300 K. The overall flexibility of native, mutant I and II complexes was calculated by the trace of the diagonalized covariance matrix of the Cα atomic positional fluctuations. We obtained the following values for native and mutant (I and II) complexes as 19.34, 38.15 and 74.14 nm2, respectively, and again it confirmed the overall increased flexibility in both mutant complexes than native at 300 K. The whole concept was cleared after analysing the snapshots of complex conformation during simulation (Fig. 10). Both mutant complex started to form an open conformation after 0 ns timescale and continued till the end of 10 ns, whereas no major unfolding and structural perturbation were observed in native complex. After observing docking and molecular dynamics simulation results, it was confirmed that mutation in NSP3 and P130Cas structure got expanded and flexible conformation affected the binding interaction between its biological partners which might led to disturbance of cell signalling.
https://static-content.springer.com/image/art%3A10.1007%2Fs12013-013-9551-6/MediaObjects/12013_2013_9551_Fig9_HTML.gif
Fig. 9

Projection of the motion of the protein in phase space along the first two principal eigenvectors at 300 K. a. Native complex (black) versus mutant I complex (red). Native (black) versus mutant II complex (green) is shown in b (Color figure online)

https://static-content.springer.com/image/art%3A10.1007%2Fs12013-013-9551-6/MediaObjects/12013_2013_9551_Fig10_HTML.gif
Fig. 10

Surface representation of conformational states of native and mutant NSP3–p130Cas complex at different time intervals during simulations. NSP3 (native/mutant) protein is shown in brick red colour and p130Cas (native/mutant) protein is shown in dark green colour (Color figure online)

Conclusion

The molecular mechanisms involved in the loss of signalling process associated with NSP3 and p130Cas mutant proteins were poorly understood. In this analysis, we highlighted the damaging consequences of mutations in NSP3 (NSP3L469R, NSP3L623E, NSP3R627E) and p130Cas (p130CasF794R, p130CasL787E, p130CasD797R) and their role in inducing interaction affinity loss with their corresponding biological partners. NSP3L469R and p130CasD797R mutations were predicted as most prominent in docking analysis. Intermolecular NH bond analysis of native complex showed good interaction between NSP3 and p130Cas, whereas the mutant complexes showed less proximity between the biological partners. Conformational analysis of NSP3 and p130Cas was studied using classical MD simulation. Both mutant complexes showed significant structural deviation and alteration in conformation as compared to native complex. The rise in flexibility level in mutant complexes had affected their binding affinity and bond stability. Mutant complexes showed large fluctuation as observed in Rg and SASA analysis. Essential dynamic studies clearly proved that mutation in NSP3 and p130Cas protein attained more degree of flexibility in phase space. Our in silico analysis provided a detailed molecular insight into the mutation that induces the loss of cell signalling and binding interaction between NSP3 and p130Cas.

Acknowledgments

Authors gratefully acknowledge the management of Vellore Institute of Technology University for providing the facilities to carry out this work. We thank the anonymous reviewers for their helpful comments and critical reading of the manuscript.

Conflict of interest

  Authors have no potential conflict of interest to disclose.

Supplementary material

12013_2013_9551_MOESM1_ESM.tif (313 kb)
Supplementary material 1 (TIFF 313 kb)

Copyright information

© Springer Science+Business Media New York 2013