Thermodynamic, enzymatic and structural effects of removing a salt bridge at the base of loop 4 in (pro)caspase-3.
Thermodynamic, enzymatic and structural effects of removing a salt bridge at the base of loop 4 in (pro)caspase-3.
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DOI：
10.1016/j.abb.2011.01.011
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发表时间：
2011-04-01
影响因子：
3.9
通讯作者：
Clark AC
中科院分区：
文献类型：
--
作者：
Walters J;Swartz P;Mattos C;Clark AC
环2、2'和4之间的相互作用（称为环束）稳定半胱天冬酶-3的活性位点。环4（L4）由于其位于活性位点和二聚体界面之间而特别令人感兴趣。我们已经破坏了K242和E246之间的盐桥在L4的基础上，以确定其在整体构象稳定性和维持活性位点环境中的作用。稳定性测量显示，只有K242 A单突变体降低了二聚体的稳定性，而单突变体和双突变体都表现出比野生型胱天蛋白酶-3低得多的活性。半胱天冬酶-3变体的结构研究显示K242参与稳定螺旋5的疏水相互作用，在二聚体界面附近，E246的作用似乎是中和疏水簇内K242的正电荷。总的来说，结果表明E246和K242在procaspase-3中是重要的，因为它们与相邻残基相互作用，而不是彼此相互作用。相反，半胱天冬酶-3中K242-E246盐桥的形成对于L4环的准确、稳定构象和成熟酶中适当的活性位点形成是需要的。
Interactions between loops 2, 2’ and 4, known as the loop bundle, stabilize the active site of caspase-3. Loop 4 (L4) is of particular interest due to its location between the active site and the dimer interface. We have disrupted a salt bridge between K242 and E246 at the base of L4 to determine its role in overall conformational stability and in maintaining the active site environment. Stability measurements show that only the K242A single mutant decreases stability the dimer, whereas both single mutants and the double mutant demonstrate much lower activity compared to wild-type caspase-3. Structural studies of the caspase-3 variants show the involvement of K242 in hydrophobic interactions that stabilize helix 5, near the dimer interface, and the role of E246 appears to be to neutralize the positive charge of K242 within the hydrophobic cluster. Overall, the results suggest E246 and K242 are important in procaspase-3 for their interaction with neighboring residues, not with one another. Conversely, formation of the K242–E246 salt bridge in caspase-3 is needed for an accurate, stable conformation of loop L4 and proper active site formation in the mature enzyme.