Sharma Neeru, Sonavane Uddhavesh, Joshi Rajendra
HPC - Medical and Bioinformatics Applications Group, Centre for Development of Advanced Computing, Pune University Campus, Pune - 411 007, India.
HPC - Medical and Bioinformatics Applications Group, Centre for Development of Advanced Computing, Pune University Campus, Pune - 411 007, India.
Comput Biol Chem. 2017 Aug;69:96-109. doi: 10.1016/j.compbiolchem.2017.05.008. Epub 2017 Jun 1.
The most representative member of the Ras subfamily is its HRas isoform. Ras proteins being GTPases, possess an intrinsic activity to hydrolyze the GTP molecule to GDP. During the transition phases, between active and inactive states, P-loop and switch regions show maximum variations. Various hot-spot Ras mutants (G12V, A59G, Q61L etc) have been reported, that limit the protein's conformation in the permanent active state. In the present study, we aim to explore the structural dynamics of one such crucial mutant of Ras namely A59G which belongs to the conserved Switch II region of the protein. Approximately ∼15μs of Classical Molecular Dynamics (CMD) simulations have been carried out on the mutant and wild-type complexes. Further, a metadynamics simulation of 500ns was also carried out, which suggests an energy barrier of ∼9.56kcal/mol between wild-type and mutant conformation. We demonstrate the role of water molecule in maintaining the required interaction networks in the pre-hydrolysis state, its impact on A59G mutation, distinct orientation of the Gln61 residue in two conformations, disruption of crucial Gly60 and γ phosphate and the change in the Switch II region. The outcome of our study captures the pre-hydrolysis state of the HRas protein. It also establishes the fact that this mutation makes the movement of Switch II region and the conserved DXXGQ motif highly constrained, which is known to be an important requirement for hydrolysis. This suggests that the A59G mutation may decrease the rate of intrinsic hydrolysis as well as GAP-mediated hydrolysis.
Ras亚家族最具代表性的成员是其HRas亚型。Ras蛋白作为GTP酶，具有将GTP分子水解为GDP的内在活性。在活性状态和非活性状态之间的转变阶段，P环和开关区域表现出最大的变化。已经报道了各种热点Ras突变体（G12V、A59G、Q61L等），这些突变体将蛋白质的构象限制在永久活性状态。在本研究中，我们旨在探索Ras的一种关键突变体A59G的结构动力学，该突变体属于该蛋白质保守的开关II区域。对突变体和野生型复合物进行了约15微秒的经典分子动力学（CMD）模拟。此外，还进行了500纳秒的元动力学模拟，结果表明野生型和突变体构象之间的能量屏障约为9.56千卡/摩尔。我们证明了水分子在维持水解前状态所需的相互作用网络中的作用、其对A59G突变的影响、Gln61残基在两种构象中的不同取向、关键的Gly60和γ磷酸的破坏以及开关II区域的变化。我们的研究结果捕捉到了HRas蛋白的水解前状态。这也证实了这样一个事实，即这种突变使开关II区域和保守的DXXGQ基序的运动受到高度限制，而这是水解的一个重要条件。这表明A59G突变可能会降低内在水解以及GAP介导的水解速率。
