Dijkstra B W, Renetseder R, Kalk K H, Hol W G, Drenth J
J Mol Biol. 1983 Jul 25;168(1):163-79. doi: 10.1016/s0022-2836(83)80328-3.
The previously published three-dimensional structure of porcine pancreatic prophospholipase A2 at 3 A resolution was found to be incompatible with the structures of bovine phospholipase A2 and bovine prophospholipase A2. This was unexpected because of the very homologous amino acid sequences of these enzymes. Therefore, the crystal structure of the porcine enzyme was redetermined using molecular replacement methods with bovine phospholipase as the parent model. The structure was crystallographically refined at 2.6 A resolution by fast Fourier transform and restrained least-squares procedures to an R-factor of 0.241. The crystals appeared to contain phospholipase A2 and not prophospholipase A2. Apparently the protein is slowly converted under the crystallization conditions employed. Our investigation shows that, in contrast to the previous report, the three-dimensional structure of porcine phospholipase A2 is very similar to that of bovine phospholipase A2, including the active site. Smaller differences were observed in some residues involved in the binding of aggregated substrates. However, an appreciable conformational difference is in the loop 59 to 70, where a single substitution at position 63 (bovine Val leads to porcine Phe) causes a complete rearrangement of the peptide chain. In addition to the calcium ion in the active site, a second calcium ion is present in the crystals; this is located on a crystallographic 2-fold axis and stabilizes the interaction between two neighbouring molecules.
先前发表的猪胰原磷脂酶A2在3埃分辨率下的三维结构被发现与牛磷脂酶A2和牛原磷脂酶A2的结构不兼容。由于这些酶的氨基酸序列非常同源，这一结果出乎意料。因此，以牛磷脂酶为母体模型，采用分子置换法重新测定了猪酶的晶体结构。通过快速傅里叶变换和约束最小二乘法在2.6埃分辨率下对该结构进行晶体学精修，R因子为0.241。晶体似乎含有磷脂酶A2而非原磷脂酶A2。显然，在所用的结晶条件下，蛋白质会缓慢转化。我们的研究表明，与先前的报道相反，猪磷脂酶A2的三维结构与牛磷脂酶A2非常相似，包括活性位点。在参与聚集底物结合的一些残基中观察到较小的差异。然而，在59至70位的环区存在明显的构象差异，其中63位的单个取代（牛的缬氨酸变为猪的苯丙氨酸）导致肽链完全重排。除了活性位点中的钙离子外，晶体中还存在第二个钙离子；它位于晶体学二重轴上，稳定了两个相邻分子之间的相互作用。
