Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore, 117543, Singapore.
Mechanobiology Institute, National University of Singapore, Singapore, 117411, Singapore.
Cell Mol Life Sci. 2024 May 13;81(1):216. doi: 10.1007/s00018-024-05238-8.
p50RhoGAP is a key protein that interacts with and downregulates the small GTPase RhoA. p50RhoGAP is a multifunctional protein containing the BNIP-2 and Cdc42GAP Homology (BCH) domain that facilitates protein-protein interactions and lipid binding and the GAP domain that regulates active RhoA population. We recently solved the structure of the BCH domain from yeast p50RhoGAP (BCH) and showed that it maintains the adjacent GAP domain in an auto-inhibited state through the β5 strand. Our previous WT BCH structure shows that a unique kink at position 116 thought to be made by a proline residue between alpha helices α6 and α7 is essential for the formation of intertwined dimer from asymmetric monomers. Here we sought to establish the role and impact of this Pro116. However, the kink persists in the structure of P116A mutant BCH domain, suggesting that the scaffold is not dictated by the proline residue at this position. We further identified Tyr124 (or Tyr188 in BCH) as a conserved residue in the crucial β5 strand. Extending to the human ortholog, when substituted to acidic residues, Tyr188D or Tyr188E, we observed an increase in RhoA binding and self-dimerization, indicative of a loss of inhibition of the GAP domain by the BCH domain. These results point to distinct roles and impact of the non-conserved and conserved amino acid positions in regulating the structural and functional complexity of the BCH domain.
p50RhoGAP 是一种与小 GTP 酶 RhoA 相互作用并下调其活性的关键蛋白。p50RhoGAP 是一种多功能蛋白，包含 BNIP-2 和 Cdc42GAP 同源（BCH）结构域，该结构域促进蛋白-蛋白相互作用和脂质结合，以及 GAP 结构域，该结构域调节活性 RhoA 群体。我们最近解析了来自酵母 p50RhoGAP（BCH）的 BCH 结构域的结构，并表明它通过β5 链将相邻的 GAP 结构域保持在自动抑制状态。我们之前的 WT BCH 结构表明，在位置 116 处存在一个独特的弯曲，据认为是由α6 和α7 之间的脯氨酸残基形成的，这对于从不对称单体形成交织的二聚体至关重要。在这里，我们试图确定该 Pro116 的作用和影响。然而，在 P116A 突变 BCH 结构域的结构中，该弯曲仍然存在，这表明支架不是由该位置的脯氨酸残基决定的。我们进一步确定 Tyr124（或 BCH 中的 Tyr188）是关键β5 链中的保守残基。扩展到人类同源物，当取代为酸性残基 Tyr188D 或 Tyr188E 时，我们观察到 RhoA 结合和自身二聚化增加，表明 BCH 结构域对 GAP 结构域的抑制作用丧失。这些结果表明，非保守和保守氨基酸位置在调节 BCH 结构域的结构和功能复杂性方面具有不同的作用和影响。
