Tsunemi M, Matsuura Y, Sakakibara S, Katsube Y
Peptide Institute Inc., Protein Research Foundation, Osaka, Japan.
Biochemistry. 1996 Sep 10;35(36):11570-6. doi: 10.1021/bi960900l.
The crystal structure of a stoichiometric complex between an elastase-specific inhibitor elafin and porcine pancreatic elastase (PPE) has been determined and refined to a crystallographic R-factor of 19.7% at 1.9 A resolution. The polypeptide chain of elafin has a planar spiral shape with an exposed external part and an internal core part which resembles both the crystal structure of human seminal plasma inhibitor (HUSI-1) [Grütter, M. G., Fendrich, G., Huber, R., & Bode, W. (1988) EMBO J. 7, 345-351] and the solution structure of Na+,K(+)-ATPase inhibitor (SPAI-1) revealed by NMR analysis [Kozaki, T., Kawakami, Y., Tachibana, S., Hatanaka, H., & Inagaki, F. (1994) Pept. Chem., 405-408]. The external region containing the primary binding loop is interconnected by four disulfide bonds to the internal part composed of a beta-sheet and a hairpin loop. The scissile peptide bond Ala24i(P1)-Met25i(P1') in the primary binding site is intact, and its carbonyl carbon is in van der Waals contact with O gamma of the active site Ser195 of PPE. The seven residues of Leu20i(P5)-Leu26i(P2') of the primary binding loop and the three residues of Ser48i, Cys49i, and Ala52i of the adjacent hairpin loop are in contact with PPE by hydrogen bonds and/or van der Waals interactions in a manner similar to that observed for other serine protease-inhibitor complexes. Electron densities of the N-terminal residues Ala1i-Ser10i which are not responsible for the elastase inhibitory activity were not visible, probably due to disordered conformation. The guanido group (N eta 1, N eta 2) of Arg61 in the complex interacts with S delta of Met25i(P1') by possible hydrogen bonds between N and S atoms, accompanying a large positional shift of the side chain of Arg61-(S1') between the complexed and free forms of PPE. The primary binding site is stabilized by hydrogen bonds between the guanido group (N eta 1, N eta 2) of Arg22i(P3) and the carbonyl group of Met25i(P1') across the scissile bond, as well as by a hydrogen bond between the amino group of Cys23i(P2) and the carbonyl group of Ser48i in the internal core. This intramolecular hydrogen bond network and the network of four disulfide bonds might play a significant role in stabilizing the conformation of the binding site for expressing the potent specific inhibitory activity.
已确定弹性蛋白酶特异性抑制剂elafin与猪胰弹性蛋白酶（PPE）之间化学计量复合物的晶体结构，并在1.9 Å分辨率下精修至晶体学R因子为19.7%。elafin的多肽链呈平面螺旋形状，有一个暴露的外部部分和一个内部核心部分，这既类似于人精浆抑制剂（HUSI-1）的晶体结构[格鲁特，M. G.，芬德里希，G.，胡贝尔，R.，& 博德，W.（1988年）《欧洲分子生物学组织杂志》7卷，345 - 351页]，也类似于通过核磁共振分析揭示的Na⁺，K⁺ - ATP酶抑制剂（SPAI-1）的溶液结构[小崎，T.，川上，Y.，立花，S.，畑中，H.，& 稻垣，F.（1994年）《肽化学》，405 - 408页]。包含主要结合环的外部区域通过四个二硫键与由一个β - 折叠和一个发夹环组成的内部部分相连。主要结合位点中的可裂解肽键Ala24i（P1）- Met25i（P1'）完整无损，其羰基碳与PPE活性位点Ser195的Oγ处于范德华接触。主要结合环的Leu20i（P5）- Leu26i（P2'）的七个残基以及相邻发夹环的Ser48i、Cys49i和Ala52i的三个残基通过氢键和/或范德华相互作用与PPE接触，其方式类似于在其他丝氨酸蛋白酶 - 抑制剂复合物中观察到的情况。对弹性蛋白酶抑制活性无作用的N端残基Ala1i - Ser10i的电子密度不可见，可能是由于构象无序。复合物中Arg61的胍基（Nη1，Nη2）通过N和S原子之间可能的氢键与Met25i（P1'）的Sδ相互作用，同时伴随着Arg61 - （S1'）侧链在PPE的复合形式和游离形式之间的较大位置移动。主要结合位点通过横跨可裂解键的Arg22i（P3）的胍基（Nη1，Nη2）与Met25i（P1'）的羰基之间的氢键以及内部核心中Cys23i（P2）的氨基与Ser48i的羰基之间的氢键得以稳定。这种分子内氢键网络和四个二硫键网络可能在稳定结合位点的构象以表达强效特异性抑制活性方面发挥重要作用。
