Dynamin-superfamily proteins (DSPs) are large self-assembling mechanochemical GTPases that harness GTP hydrolysis to drive membrane remodeling events needed for many cellular processes. Mutation to alanine of a fully conserved lysine within the P-loop of the DSP GTPase domain results in abrogation of GTPase activity. This mutant has been widely used in the context of several DSPs as a dominant-negative to impair DSP-dependent processes. However, the precise deficit of the P-loop K to A mutation remains an open question. Here, we use biophysical, biochemical and structural approaches to characterize this mutant in the context of the endosomal DSP Vps1. We show that the Vps1 P-loop K to A mutant binds nucleotide with an affinity similar to wild type but exhibits defects in the organization of the GTPase active site that explain the lack of hydrolysis. In cells, Vps1 and Dnm1 bearing the P-loop K to A mutation are defective in disassembly. These mutants become trapped in assemblies at the typical site of action of the DSP. This work provides mechanistic insight into the widely-used DSP P-loop K to A mutation and the basis of its dominant-negative effects in the cell.

中文翻译：

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在dynamin超家族蛋白Vps1的主要负P环赖氨酸突变的结构和功能表征。

动力蛋白超家族蛋白（DSPs）是大型的自组装机械化学GTP酶，可利用GTP水解来驱动许多细胞过程所需的膜重塑事件。DSP GTPase结构域的P环内完全保守的赖氨酸突变为丙氨酸会导致GTPase活性丧失。此突变体已在几种DSP的背景下广泛使用，以显着的负向削弱了依赖DSP的过程。然而，P环K突变为A的确切缺陷仍然是一个悬而未决的问题。在这里，我们使用生物物理，生化和结构方法来表征内体DSP Vps1中的此突变体。我们表明，Vps1 P环K突变体以与野生型相似的亲和力结合核苷酸，但在GTPase活性位点的组织中显示出缺陷，解释了水解的缺乏。在细胞中，带有P环K到A突变的Vps1和Dnm1在拆卸中有缺陷。这些突变体被困在DSP的典型作用位点中。这项工作为广泛使用的DSP P环K到A突变及其在细胞中显性负效应的基础提供了机械原理。