Cell polarity oscillations in motility are driven by a prokaryotic small Ras-like GTPase, mutual gliding protein A (MglA), which switches from one cell pole to the other in response to extracellular signals. MglA dynamics is regulated by MglB, which functions both as a GTPase activating protein (GAP) and a guanine nucleotide exchange factor (GEF) for MglA. With an aim to dissect the asymmetric role of the two MglB protomers in the dual GAP and GEF activities, we generated a functional MglAB complex by coexpressing MglB with a linked construct of MglA and MglB. This strategy enabled us to generate mutations of individual MglB protomers (MglB or MglB linked to MglA) and delineate their role in GEF and GAP activities. We establish that the C-terminal helix of MglB, but not MglB, stimulates nucleotide exchange through a site away from the nucleotide-binding pocket, confirming an allosteric mechanism. Interaction between the N-terminal β-strand of MglB and β of MglA is essential for the optimal GEF activity of MglB. Specific residues of MglB which interact with Switch-I of MglA, partially contribute to its GAP activity. Thus, the role of the MglB protomer in the GAP activity of MglB is limited to restricting the conformation of MglA active site loops. The direct demonstration of the allosteric mechanism of GEF action provides us new insights into the regulation of small Ras-like GTPases, a feature potentially present in many uncharacterized GEFs.

中文翻译：

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不对称相互作用 MglB 二聚体对原核小 Ras 样 GTPase MglA 的 GTPase 激活机制

运动中的细胞极性振荡是由原核小 Ras 样 GTP 酶、相互滑动蛋白 A (MglA) 驱动的，它响应细胞外信号从一个细胞极切换到另一个细胞极。 MglA 动力学受 MglB 调节，MglB 既充当 MglA 的 GTP 酶激活蛋白 (GAP) 又充当鸟嘌呤核苷酸交换因子 (GEF)。为了剖析两个 MglB 原体在 GAP 和 GEF 双重活性中的不对称作用，我们通过将 MglB 与 MglA 和 MglB 的连接结构共表达来生成功能性 MglAB 复合物。该策略使我们能够生成单个 MglB 原体（MglB 或 MglB 与 MglA 连接）的突变，并描述它们在 GEF 和 GAP 活动中的作用。我们确定 MglB 的 C 端螺旋（而非 MglB）通过远离核苷酸结合口袋的位点刺激核苷酸交换，证实了变构机制。 MglB 的 N 端 β 链和 MglA 的 β 之间的相互作用对于 MglB 的最佳 GEF 活性至关重要。与 MglA 的 Switch-I 相互作用的 MglB 的特定残基部分有助于其 GAP 活性。因此，MglB原体在MglB的GAP活性中的作用仅限于限制MglA活性位点环的构象。 GEF 作用的变构机制的直接证明为我们提供了对小型 Ras 样 GTP 酶调节的新见解，这一特征可能存在于许多未表征的 GEF 中。