Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
PLoS One. 2013 Aug 19;8(8):e71835. doi: 10.1371/journal.pone.0071835. eCollection 2013.
Human dynamin-1-like protein (DNM1L) is a GTP-driven molecular machine that segregates mitochondria and peroxisomes. To obtain insights into its catalytic mechanism, we determined crystal structures of a construct comprising the GTPase domain and the bundle signaling element (BSE) in the nucleotide-free and GTP-analogue-bound states. The GTPase domain of DNM1L is structurally related to that of dynamin and binds the nucleotide 5'-Guanylyl-imidodiphosphate (GMP-PNP) via five highly conserved motifs, whereas the BSE folds into a pocket at the opposite side. Based on these structures, the GTPase center was systematically mapped by alanine mutagenesis and kinetic measurements. Thus, residues essential for the GTPase reaction were characterized, among them Lys38, Ser39 and Ser40 in the phosphate binding loop, Thr59 from switch I, Asp146 and Gly149 from switch II, Lys216 and Asp218 in the G4 element, as well as Asn246 in the G5 element. Also, mutated Glu81 and Glu82 in the unique 16-residue insertion of DNM1L influence the activity significantly. Mutations of Gln34, Ser35, and Asp190 in the predicted assembly interface interfered with dimerization of the GTPase domain induced by a transition state analogue and led to a loss of the lipid-stimulated GTPase activity. Our data point to related catalytic mechanisms of DNM1L and dynamin involving dimerization of their GTPase domains.
人源动力蛋白 1 样蛋白（DNM1L）是一种 GTP 驱动的分子机器，可将线粒体和过氧化物酶体进行分隔。为了深入了解其催化机制，我们测定了包含 GTP 酶结构域和束状信号元件（BSE）的无核苷酸和 GTP 类似物结合状态下的结构。DNM1L 的 GTP 酶结构域与 dynamin 结构相关，通过五个高度保守的基序与核苷酸 5'-鸟苷酰基-亚氨二磷酸（GMP-PNP）结合，而 BSE 则折叠成相对侧的口袋。基于这些结构，通过丙氨酸突变和动力学测量系统地绘制了 GTP 酶中心图。因此，鉴定了对 GTP 酶反应至关重要的残基，其中包括磷酸结合环中的 Lys38、Ser39 和 Ser40、来自开关 I 的 Thr59、来自开关 II 的 Asp146 和 Gly149、G4 元件中的 Lys216 和 Asp218 以及 G5 元件中的 Asn246。此外，DNM1L 独特的 16 残基插入区中的突变 Glu81 和 Glu82 显著影响活性。预测的组装界面中的 Gln34、Ser35 和 Asp190 突变干扰了过渡态类似物诱导的 GTP 酶结构域二聚化，并导致脂质刺激的 GTP 酶活性丧失。我们的数据表明 DNM1L 和 dynamin 的催化机制相关，涉及它们的 GTP 酶结构域的二聚化。
