Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy.
PLoS One. 2013;8(2):e56254. doi: 10.1371/journal.pone.0056254. Epub 2013 Feb 15.
Acyl aminoacyl peptidases are two-domain proteins composed by a C-terminal catalytic α/β-hydrolase domain and by an N-terminal β-propeller domain connected through a structural element that is at the N-terminus in sequence but participates in the 3D structure of the C-domain. We investigated about the structural and functional interplay between the two domains and the bridge structure (in this case a single helix named α1-helix) in the cold-adapted enzyme from Sporosarcina psychrophila (SpAAP) using both protein variants in which entire domains were deleted and proteins carrying substitutions in the α1-helix. We found that in this enzyme the inter-domain connection dramatically affects the stability of both the whole enzyme and the β-propeller. The α1-helix is required for the stability of the intact protein, as in other enzymes of the same family; however in this psychrophilic enzyme only, it destabilizes the isolated β-propeller. A single charged residue (E10) in the α1-helix plays a major role for the stability of the whole structure. Overall, a strict interaction of the SpAAP domains seems to be mandatory for the preservation of their reciprocal structural integrity and may witness their co-evolution.
酰基氨基酸肽酶是由 C 端催化 α/β-水解酶结构域和 N 端β-三叶状螺旋结构域通过连接结构元件组成的双结构域蛋白，该连接结构元件在序列上位于 N 端，但参与 C 结构域的三维结构。我们使用整个结构域缺失的蛋白变体和α1-螺旋上有取代的蛋白研究了嗜冷菌 Sporosarcina psychrophila （SpAAP）中两种结构域和桥结构（在这种情况下是一个名为α1-螺旋的单螺旋）之间的结构和功能相互作用。我们发现，在这种酶中，结构域间的连接对整个酶和β-三叶状螺旋的稳定性有显著影响。α1-螺旋对于完整蛋白的稳定性是必需的，就像在同一酶家族的其他酶中一样；然而，仅在这种嗜冷酶中，它会使分离的β-三叶状螺旋不稳定。α1-螺旋中的一个带电荷的残基（E10）对整个结构的稳定性起着重要作用。总的来说，SpAAP 结构域之间的严格相互作用对于保持它们相互结构完整性是强制性的，并且可能见证了它们的共同进化。
