Biophysical Chemistry, Institute of Biochemistry, University of Greifswald, 17489 Greifswald, Germany.
Synthetic and Structural Biochemistry, Institute of Biochemistry, University of Greifswald, 17489 Greifswald, Germany.
Int J Mol Sci. 2022 Mar 23;23(7):3468. doi: 10.3390/ijms23073468.
(1) The serine protease inhibitor Kazal type 1 (SPINK1) inhibits trypsin activity in zymogen granules of pancreatic acinar cells. Several mutations in the gene are associated with acute recurrent pancreatitis (ARP) and chronic pancreatitis (CP). The most common variant is SPINK1 p.N34S. Although this mutation was identified two decades ago, the mechanism of action has remained elusive. (2) SPINK1 and human cationic trypsin (TRY1) were expressed in , and inhibitory activities were determined. Crystals of SPINK1-TRY1 complexes were grown by using the hanging-drop method, and phases were solved by molecular replacement. (3) Both SPINK1 variants show similar inhibitory behavior toward TRY1. The crystal structures are almost identical, with minor differences in the mutated loop. Both complexes show an unexpected rotamer conformation of the His63 residue in TRY1, which is a member of the catalytic triad. (4) The SPINK1 p.N34S mutation does not affect the inhibitory behavior or the overall structure of the protein. Therefore, the pathophysiological mechanism of action of the p.N34S variant cannot be explained mechanistically or structurally at the protein level. The observed histidine conformation is part of a mechanism for SPINK1 that can explain the exceptional proteolytic stability of this inhibitor.
(1) 丝氨酸蛋白酶抑制剂 Kazal 型 1（SPINK1）抑制胰腺腺泡细胞酶原颗粒中的胰蛋白酶活性。基因中的几个突变与急性复发性胰腺炎（ARP）和慢性胰腺炎（CP）有关。最常见的变体是 SPINK1 p.N34S。尽管该突变在二十年前就被发现了，但作用机制仍难以捉摸。(2) 在中表达 SPINK1 和人阳离子胰蛋白酶（TRY1），并测定抑制活性。使用悬滴法生长 SPINK1-TRY1 复合物的晶体，并通过分子置换法解决相问题。(3) 两种 SPINK1 变体对 TRY1 均表现出相似的抑制行为。晶体结构几乎相同，突变环略有差异。两个复合物均显示 TRY1 中 His63 残基的意想不到的旋转构象，该残基是催化三联体的成员。(4) SPINK1 p.N34S 突变不影响蛋白的抑制行为或整体结构。因此，在蛋白水平上，无法从机制或结构上解释 p.N34S 变体的病理生理作用机制。观察到的组氨酸构象是 SPINK1 机制的一部分，它可以解释该抑制剂异常的蛋白水解稳定性。
