3A分辨率绿豆胰蛋白酶抑制剂Lys活力碎片—牛胰蛋白酶(MBILF-BTRY)复合物的晶体结构分析和分子模型

CRYSTAL STRUCTURE ANALYSIS AND MOLECULAR MODEL OF MUNG BEAN TRYPSIN INHIBITOR LYS ACTIVE FRAGMANT BOVINE TRYPSIN (MBILFBTRY) COMPLEX AT 3.0 A RESOLUTION

用分子置换法成功地解得了MBILF-BTRY的晶体结构,并得到了它的立体结构模型。绿豆胰蛋白酶抑制剂属于絲氨酸蛋白酶抑制剂中结构最复杂的Bowman-Birk型抑制剂。这一类型抑制剂的立体结构以前尚未见报道。MBILF-BTRY复合物晶体的晶胞参数为a=62.99,b=63.54,c=69.70,α=β=γ=90°,空间群为P222,复合物分子量约为27500daltons。用理学电机的Ru-300型转靶X光机及AFC-5型四园衍射仪收集了分辨率为3范围内的独立衍射点5142个。

Crystal structure of MBILF-BTRY complex was successfully solved by molecular replacement method and its 3-dimensional molecular model was thereby derived. Mung bean trypsin inhibitor belongs to Bowman-Birk inhibitor group, which is by far the most complicated among the ten fundamental groups of serine protease inhibitor. Neither the 3-dimensional structure of Bowman-Birk inhibitor group nor the stereoscopic conformation of its complex with protease has ever been reported. The crystallographic data of MBILF-BTRY complex are found to be a=62.99A, b=63.54A, c=69.70A, α=β=γ=90°;space group P222;and Z=4. Its molecular weight is about 27500 dalton. On a Rigaku AFC-5 four-circle diffractometer attached to a RU-300 rotating anode generator, 5142 independent reflections of up to 3A resolution were collected. The model BTRY coordinates are those of the refined BTRY taken from Protein Data Bank. Relative orientation angles between the model BTRY and BTRY in our crystal, α=145°, β=46°andγ=75°(expressed in terms of Crowther angles), were obtained by using Crowther’s fast rotation function with diffraction data of 4A resolution. Translation function of three pairs of molecules related by three screw axes 2were calculated by applying Lattman’s program. From the implications revealed by the predominant peaks on the three Harker sections, the relative translation components between the model BTRY and that in our crystal were found to be △x=-10.1A, A.y=-3.0A and △z=-13.3A. Group refinement had improved the above rotational and translational parameters and gave the following rasults: α=146.4°, β=45.7°, γ=74.4° and △x=-9.81A, △y=-2.96A, △z=-13.08A. After the model BTRY molecule was oriented and located in the correct azimuth and position within the cell according to the refined parameters, the R-factor dropped to 0.390. An electron density map and a difference map both of 3A resolution were calculated with coefficients of 2|F|-|F|and|F|-|F| respectively and with Sim-weighted phases. Dense contour levels apparently attributable to the MBILF could be clearly seen near the active site of BTRY molecule. The molecular size of MBILF was approximately estimated at 15×15×25 A. It could be unambiguously discerned that the electron density corresponding to the long side chain of Lys 20 of MBILF molecule extends deeply into the specific pocket and terminates near Asp 189 OD1 of BTRY. The polypeptide folding could be traced in the Fourier map with reference to the difference Fourier. And a preliminary stereoscopic model of MBILF has been already constructed. Different from the usual protease inhibitors of known 3-dimensional structure, the striking feature of the MBILF is that its polypeptide folding does not include any secondary structure, such as a-helix and well defined β-pleated sheet structure. Three large distorted rings each composed of nine to ten amino acid residues are cross-linked by four disulphide bridges to form a wedge-shaped structure with the head part sharpened arround the residue Lys 20 of MBILF and flattened gradually backwards. This enables the MBILF molecule to approach easily the active site of the BTRY and to bind tightly with the latter. The conformation of the head part of MBILF is similar to that of Kazal inhibitor group. In comparison with the pancreatic trypsin inhibitor (PTI), Bowman-Birk inhibitor is somewhat more involved in forming hydrogen bonds. The refinement of the MBILF-BTRY structure at higher resolution is now in progress. The 3-dimensional structure of MBILF-BTRY complex reveals for the first time a stereoscopic pattern of Bowman-Birk type inhibitor which may well be conducive to the structure-function correlation studies.