Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhan Nagar, Kolkata 700 064, India
1 To whom correspondence should be addressed. e-mail: jiban{at}cmb2.saha.ernet.in
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Abstract |
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Keywords: docking studies/in silico mutation/molecular dynamics simulation/reactive site loop/winged bean chymotrypsin inhibitor
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Introduction |
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The relationship between the protein structure and its dynamic nature, required to get the adoptability during proteinprotein interaction, is a key question in biology, and is yet to be answered explicitly. Molecular dynamics (MD) simulation is an important tool to understand not only the effect of mutations on the structure, but more importantly, the dynamic nature of the protein molecule, as it can provide information about the structure at the atomic level on a reasonable time scale. Hence, in this work, we have replaced Asn14 with other residues in silico and MD simulations have been carried out on solvated native WCI as well as on its mutants. The residues having long/bulky side chains were excluded because we observed in N14K that the Lys side chain was not accommodated in the cavity and it folded back. Initially we started with N14G, where the side chain is fully truncated, followed by N14A, having just a methyl group as its side chain. Subsequently, mutations with hydrophobic residues like Val (N14V) and Leu (N14L) were also carried out and the results obtained from these mutants prompted us to replace the 14th residue with small, but not so polar, side chains like Thr (N14T) and Ser (N14S).
The resulting simulated structures of the mutants have been analysed in this paper, in terms of hydrogen-bonding interactions, dihedral angles and water orientation in the loop region. In each case, the snapshots obtained from the trajectory of the dynamics were docked at the active site of chymotrypsin to quantify the deviations in the canonical conformation in terms of the interaction energy and occurrence of conserved hydrogen bonds, as seen in the serine proteaseprotease inhibitor complexes.
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Materials and methods |
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The computation described in this paper utilized our X-ray coordinates of WCI (PDB code: 1EYL) as the starting models. The water molecules and sulphate ions were deleted from the X-ray structure. The MD simulations were performed on a SiliconGraphics O2 Workstation, using the DISCOVER-III module of Insight-II from Molecular Simulation Inc. (San Diego, CA, USA). Asn14 of WCI was mutated in silico to Gly, Ala, Ser, Thr, Val and Leu using the Insight-II Biopolymer module and the lowest energy rotamer for the mutated residues was chosen, whenever necessary. To start with, hydrogen atoms were generated at pH 7.0 and minimized, keeping all the heavy atoms fixed. Potentials were assigned using the CVFF force-field with a distance-independent dielectric constant of 4.0. No cross-terms were used in the energy expressions. For the treatment of non-bonded interactions, an atom-based method was used where non-bonded interactions were evaluated with a cut-off distance of 9.5 Å. Simulations in vacuo are known to suffer from serious artifacts, such as an excessive deviation from the native conformation. Moreover, in our present study, the understanding of the structural and dynamic properties of the solventprotein interaction is an important task for the comprehension of protein functionality. Hence, the simulations were performed on the solvated protein molecules. Initially, a subset was defined in each case that included the following residues of the reactive site loop and neighbouring scaffold: Asp1Gly16, Arg59Val75. Then, the solvation of the defined subset was done, by creating a water shell of 10 Å capping using the SOAK utility of the DISCOVER-III module. The water molecules thus generated were minimized (steepest descent followed by conjugate gradient) and simulated for 30 ps, keeping the protein molecules fixed. Subsequently, the energy minimization followed by MD simulations were carried out on the solvated subset, fixing the rest of the protein molecule. The minimizations were performed using the steepest descent algorithm (down to a gradient of <100 kcal mol1 Å1) followed by conjugate gradient minimization (down to <10 kcal mol1 Å1 and then 0.001 kcal mol1 Å1) successively. This minimized assembly of the subset and solvent was used as a starting point for NVT (constant volume and temperature) MD to generate possible stable conformations. The simulations were carried out using the Verlet velocity algorithm (Swope and Anderson, 1982).
The major disadvantage in the MD simulations performed here is that the present computational system does not permit simulations for long enough. In order to partially overcome this disadvantage, high temperature MD simulations were carried out for WCI and all of its mutants. The kinetic energy for MD simulations was provided initially by a thermal bath at a constant temperature of 300 K and then the temperature was elevated up to 500 K. In each case, a total simulation run of 850 ps was performed with a 1 fs time step. An initial run of 250 ps at 300 K was followed by a 400 ps run at 500 K and again a 200 ps run at 300 K. After a 200 ps thermalization at 500 K, when the time dependence of the potential energy showed a stationary behaviour, a trajectory of 200 ps at 500 K followed by another 200 ps at room temperature were performed, collecting one conformation in every 0.2 ps. The last 100 ps dynamics run at 300 K was considered as the production run for the analysis.
Docking and analysis of hydrogen-bonding interactions
Twenty snapshots, with an interval of 5 ps, were collected from the last 100 ps production run and used for the analysis of the hydrogen-bonding pattern in the loop region as well as for the docking studies with chymotrypsin. Two different serine protease inhibitor complexes, STIPPT (PBD code: 1AVW; Song and Suh, 1998) and OVO-CHY (PBD code: 1CHO; Fujinaga et al., 1987
), were used as initial templates for the docking experiments. In the case of the STIPPT template, the chymotrypsin molecule was first superposed on PPT and then the backbone of the (P3P2') region of the reactive site loop of each snapshot was superposed with the corresponding part of STI to bring it to the peptide-binding groove of the enzyme. For OVO-CHY as the template, only the reactive site loop (P3P2') of each snapshot was superposed on the corresponding part of OVO and the complexed chymotrypsin coordinate of OVO-CHY was used without any modification. The program MULTIDOCK (Jackson et al., 1998
) was used next to refine the interface of the initial docked complex by rigid body minimization for better evaluation of the possible enzymeinhibitor interactions.
Two different distance criteria were used to define the hydrogen-bonding pattern, one in the loop region of the inhibitors and the other in the docked enzymeinhibitor interface. In the loop region, two O or N atoms were considered as hydrogen bonded if the donoracceptor distance is <3.6 Å and the donorhydrogenacceptor angle is >120°. A protein O or N atom was considered to form a water-mediated interaction if a water O was <3.6 Å from each of them and if the angle subtended by these atoms at the water O atom was >90°. To analyse the hydrogen-bonding pattern, a 3.8 Å distance cut-off was used in the case of the enzymeinhibitor interface. This is because a certain amount of structural changes occur during complex formation, but the program we used for docking, MULTIDOCK, performs only a rigid body motion with different side chain rotamers, without considering the flexibility in the backbone conformation of the protein during complexation. The hydrogen bond and the van der Waals contacts in the loop region and in the enzymeinhibitor interface were monitored using the program CONTACT of the CCP4 suite (Collaborative Computational Project, 1994) and O (Jones et al., 1991
).
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Results |
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In N14G, where the side chain is truncated, and in N14A, having just a methyl group, hydrogen bonds between the side chain of the 14th residue and the loop residues are not possible. However, in both cases, the hydrogen bond involving the main chain N atom of the14th residue and the P2' carbonyl oxygen occurs with 100% frequency. Here, water molecule(s) play a role in the hydrogen-bonding interactions with the loop residues. In N14G, a water molecule Wat159 occupies a position that corresponds to the ND1 atom of Asn14. Using this bridging water molecule, the Ser OG atom of the P4 residue forms two water-mediated hydrogen bonds (Figure 5a) with the P2 carbonyl oxygen and the P1' OG, having 85 and 60% frequency of occurrence, respectively. Three water molecules are observed in the vicinity of the reactive site loop of N14A, which form a network of hydrogen bonds and connect the carbonyl oxygen atoms of P2, P1 and P1' (Figure 5b) with a frequency of occurrence ranging from 80 to 100%.
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Discussion |
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In our present MD study, the manifestation of mutations at Asn14 is explored in more detail, since we have replaced Asn14 with residues of different sizes and electrostatic nature. The reactive site loop of the mutants, when superposed on the simulated structure of WCI, shows more deviation in the P1P2' region compared with the P4P2 region, though the hydrogen-bonding interaction of the 14th residue with the P4 residue is not observed for any of these mutants. It seems that the ß-strand ending at P4, which is a part of the ß-barrel, restricts the conformational freedom immediately after it, i.e. in the P4P2 region (Figure 1). The C-terminal side of the scissile bond is far from any such secondary structural element and the change in the hydrogen-bonding pattern in this region is likely to affect the local conformation. Hence, all the hydrogen bonds observed in the reactive site loop region, may not be of equal importance, rather the hydrogen bonds that stabilize the C-terminal side of the scissile bond may play a more important role in maintaining its canonical conformation. The hydrogen-bonding pattern in the loop region of N14T and the smaller deviation of its loop supports the above fact. Here, the OG atom of Thr14 forms two hydrogen bonds with the P1' and P2' carbonyl oxygen and the Thr14 N···P2'O bond is well restored. This may be the reason for N14T maintaining the canonical conformation like that of WCI. On the other hand, in N14S, the hydroxyl group of Ser14 is oriented in such a manner that it cannot form any direct hydrogen bond with the loop residues, rather a water molecule is observed to bridge the P2 and P1' carbonyl oxygen with the Ser14 hydroxyl group. Moreover, the absence of the hydrogen bond, connecting P2'O with Ser14 N, results in a small conformational change in the C-terminal side of the scissile bond.
The loop deformation caused in N14L and N14V is a combination of the hydrogen bond disruption and/or steric repulsion. The size of the Leu side chain is comparable with that of Asn and therefore the terminal methyl groups of the Leu14 side chain come close to the carbonyl oxygen of P2 and P1'. These hydrophobic groups of Leu14 exert a repulsive force on Ser66 (P1'), resulting in a distortion of canonical conformation. The deformation at P1P1' of N14V is not due to any such steric effect, as the size of the Val side chain is smaller compared with Leu and in this case the deviation is mainly due to complete disruption of hydrogen bonds around the P1P1' region.
In both N14G and N14A, the main chain contact of the 14th residue with the P2' carbonyl oxygen is present and hydrogen bonds involving the loop residues are mediated through water molecules. The difference in the loop conformation between them, however, lies in the nature of their hydrogen-bonding pattern. Two water-mediated hydrogen bonds are present in the loop region of N14G, and among them the hydrogen bond that connects P4 and P1' with 60% frequency of occurrence is the only contributor in the loop stability. A squeezing of the N14G loop, where the C distance between P3P2' is 10.24 Å compared with 11.30 Å of WCI, may be a result of the above fact.
On the other hand, in N14A, the methyl group of Ala14, being not too big to exert any repulsive force on the loop like Leu or Val nor too small to leave a vacant space inside the loop like Gly, can partially play the role of a spacer. Moreover, the flexible C-terminal part of the scissile bond is getting stabilized through a network of hydrogen bonds involving three water molecules and the carbonyl oxygen atoms of P2, P1 and P1'. These two facts are possibly acting in tandem to stabilize the reactive site loop of N14A, whereas this is not the case in N14G, having only water molecule, Wat159, near the loop.
Analysis of the docking results of WCI and the mutants indicate that the deformations in the loop region are manifested in the canonical interactions with the cognate enzyme (Table III). All the important canonical hydrogen-bonding/hydrophobic interactions are identified in each of the snapshots of WCI, when docked with chymotrypsin. But the complete retention of the standard hydrogen-bonding pattern at the enzymeinhibitor interface is not observed for any of these mutants. The interaction energy of the P1 residue with the enzyme, which is energetically most important in chymotrypsin-like protease inhibitor and determines its specificity, is compared for different mutants. This value, obtained from MULTIDOCK, is highest for WCI (9.2 kcal mol1) and is comparable with N14T (8 kcal mol1) and N14A (8.2 kcal mol1). N14G (4.9 kcal mol1) and N14S (5.8 kcal mol1) show intermediate values and the values are a minimum for N14L (2.4 kcal mol1) and N14V (2 kcal mol1).
An examination of Figure 4 shows that the deformations in the loop region are least for N14T and N14A. N14T maintains the canonical interactions with almost 100% frequency of occurrence. N14S and N14G show the intermediate deformation, whereas the deformation is highest for N14L and N14V. In N14G, the three hydrogen bonds made by the P1 residue with the enzyme, are found with 100% frequency of occurrence and the P3···S3 interaction occurs with only 67% frequency. The absence of P2'···S2' interaction here may be an effect of the squeezing in the reactive site loop. The worst interaction with chymotrypsin has been observed for N14L and N14V. In N14L, a deviation from canonical conformation is found in the P1'P2' region and a flipping of the P3 carbonyl oxygen (1.5 Å away from that of WCI), causes a complete disruption of the P3···S3 bond (distance 4.5 Å). Though the P2'···S2' interaction occurs here with 70% frequency, the distance of this hydrogen bond is
3.8 Å. In N14V, almost all the interactions occur with lesser frequency. The P1 carbonyl oxygen of this mutant does not even point towards the oxy-anion hole and is not in a position to form bonds with Gly193 N. Flipping of the P3 carbonyl oxygen is also seen here causing only a 15% occurrence of the P3···S3 interaction. So, it seems that the presence of these two hydrophobic residues in the 14th position causes some deformations in the reactive site loop region and the cumulative effect of these deformations results in a major disruption of enzymeinhibitor interactions.
Conclusions
A number of MD simulations and docking studies are presented in this paper, and here we summarize the significant observations. Along with the role of Asn14, this study also helped us to understand the conformational variability and range of motions possible for the reactive site loop of different mutants. During MD simulation, we observed that the loop has an in-built stability, which is further supplemented by the scaffolding residues. The superpositioning data demonstrate that the N-terminal side of the scissile bond, which is close to ß-barrel, may not require hydrogen bonds for its stabilization, while the C-terminal side is far from any such secondary structural element and needs stability through hydrogen-bonding interactions. Along with Gly, the choice of hydrophobic residues like Val and Leu are ruled out at the 14th position of the inhibitor, as they can dramatically affect the binding site conformation and hence the canonical interactions. But a residue like Thr can be predicted here as it can stabilize the C-terminal part of the scissile bond through hydrogen bonds. Hence, it is clear that, despite its in-built stability, the loop needs a spacer of suitable size and charge for the best maintenance of its canonical conformation and, from that point of view, Asn is the most suitable one to complement the environment inside the loop.
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Acknowledgements |
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References |
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2 Collaborative Computational Project, Number 4 (1994) Acta Crystallogr. D, 50, 760763.[CrossRef][ISI][Medline]
Dattagupta,J.K., Podder,A., Chakrabarti,C., Sen,U., Mukhopadhyay,D., Dutta,S.K. and Singh,M. (1999) Proteins: Struct. Funct. Genet., 35, 321331.[CrossRef][ISI][Medline]
Fujinaga,M., Sielecki,A.R., Read,R.J., Ardelt,W., Laskowski,M.,Jr and James,M.N.G. (1987) J. Mol. Biol., 195, 397.[ISI][Medline]
Huber,R., Kukla,D., Bode,W., Schwager,P., Bartels,K., Deisenhofer,J. and Steingemann,W. (1974) J. Mol. Biol., 89, 73101.[ISI][Medline]
Jackson,R.M., Gabb,H.A. and Sternberg,M.J.E. (1998) J. Mol. Biol., 276, 265285.[CrossRef][ISI][Medline]
Jones,T.A., Zou,J.Y., Cowan,S.W. and Kjeldgaard,M. (1991) Acta Crystallogr. A, 47, 110119.[CrossRef][ISI][Medline]
Kortt,A.A. (1980) Biochim. Biophys Acta, 624, 237248.[ISI][Medline]
Laskowski,M.,Jr, Kato,I., Kohr,W.C., March,C.J. and Bogard,W.C. (1980) Protides Biol. Fluids, 28, 123128.
Marquart,M., Walter,J., Deisenhofer,J., Bode,W. and Huber,R. (1983) Acta Crystallogr. B, 39, 480490.[CrossRef][ISI]
McPhalen,C.A. and James,M.N.G. (1987) Biochemistry, 26, 261269.[ISI][Medline]
De Meester,P., Brick,P., Lloyd,L.F., Blow,M.D. and Onesti,S. (1998) Acta Crystallogr. D, 54, 589597.[CrossRef][ISI][Medline]
Onesti,S., Brick,P. and Blow,D.M. (1991) J. Mol. Biol., 217, 153176.[ISI][Medline]
Ravichandran,S., Dasgupta,J., Chakrabarti,C., Ghosh,S., Singh,M. and Dattagupta,J.K. (2001) Protein Eng., 14, 349357.
Robertus,J.D., Alden,R.A., Birktoft,J.J., Kraut,J., Powers,J.C. and Wileox,P.E. (1978) Biochemistry, 11, 24392449.
Song,H.K. and Suh,S.W. (1998) J. Mol. Biol., 275, 347363.[CrossRef][ISI][Medline]
Swope,W.C. and Anderson,H.C. (1982) J. Chem. Phys., 76, 637649.[CrossRef][ISI]
Wagner,G., Hyberts,S.G., Heinz,D.W. and Grütter,M.G. (1990) DNA Protein Complexes and Protein, Vol. 2. Adeninc Press, Guilderl, NY, pp. 93101.
Received October 17, 2002; revised June 6, 2003; accepted June 19, 2003.