Afanasyeva Arina, Hirtreiter Angela, Schreiber Anne, Grohmann Dina, Pobegalov Georgii, McKay Adam R, Tsaneva Irina, Petukhov Michael, Käs Emmanuel, Grigoriev Mikhail, Werner Finn
Department of Biophysics, Saint Petersburg State Polytechnical University, Saint Petersburg 195251, Russia; Division of Molecular and Radiation Biophysics, Petersburg Nuclear Physics Institute, Gatchina 188300, Russia.
Division of Biosciences, Institute for Structural and Molecular Biology, University College London, Darwin Building, Gower Street, London WC1E 6BT, UK.
Structure. 2014 Apr 8;22(4):549-59. doi: 10.1016/j.str.2014.02.002. Epub 2014 Mar 6.
Eukaryotic TIP49a (Pontin) and TIP49b (Reptin) AAA+ ATPases play essential roles in key cellular processes. How their weak ATPase activity contributes to their important functions remains largely unknown and difficult to analyze because of the divergent properties of TIP49a and TIP49b proteins and of their homo- and hetero-oligomeric assemblies. To circumvent these complexities, we have analyzed the single ancient TIP49 ortholog found in the archaeon Methanopyrus kandleri (mkTIP49). All-atom homology modeling and molecular dynamics simulations validated by biochemical assays reveal highly conserved organizational principles and identify key residues for ATP hydrolysis. An unanticipated crosstalk between Walker B and Sensor I motifs impacts the dynamics of water molecules and highlights a critical role of trans-acting aspartates in the lytic water activation step that is essential for the associative mechanism of ATP hydrolysis.
真核生物的TIP49a（Pontin）和TIP49b（Reptin）AAA + ATP酶在关键细胞过程中发挥着重要作用。由于TIP49a和TIP49b蛋白及其同源和异源寡聚体组装体的性质不同，它们微弱的ATP酶活性如何促成其重要功能在很大程度上仍然未知且难以分析。为了规避这些复杂性，我们分析了古菌坎氏甲烷嗜热菌（Methanopyrus kandleri, mkTIP49）中发现的单一古老TIP49直系同源物。通过生化分析验证的全原子同源建模和分子动力学模拟揭示了高度保守的组织原则，并确定了ATP水解的关键残基。沃克B基序（Walker B motif）和传感器I基序（Sensor I motif）之间意外的串扰影响了水分子的动力学，并突出了反式作用天冬氨酸在裂解水激活步骤中的关键作用，这对于ATP水解的缔合机制至关重要。
