X-ray Crystal Structure Analysis of Prolyl Endopeptidase - Inhibitor Complex

脑膜败血症黄杆菌的脯氨酸内肽酶（PEPase）是一种丝氨酸蛋白酶，它可以在脯氨酸残基的羧基侧切割肽键，其中肽的长度可达约30个氨基酸。在15℃的温度下，通过液滴的气相扩散得到PEPase晶体。将蛋白滴液与含22%PEG4000的储层溶液平衡，并在pH7.5下用0.1M Hepes缓冲。坐滴液中含有等体积的8mg/mL酶和贮液。晶体属于空间群P4_1，晶胞尺寸为a=b= 3.17Å， c=169.02Å。不对称单元中有两个分子。在Rigaku R-Axis IV上采集100K下至3.2 Åresolutions的x射线强度数据，并使用CrystalClear进行处理。确定和细化的起始模型为猪脑模型（PDB代码1H2W）。用AMORE/CCP4程序确定其结构，再用CNS程序对其进行细化。当最终晶体r因子和Rfree分别低于26%和32%时，加入溶剂分子。整体折叠与不对称两分子相同，α-碳原子位置的均方根偏差为0.04Å。该单体呈圆柱形，高约70Å，直径约50 Å，由一个肽酶结构域及其催化三元组Ser536/Asp620/His655组成。该结构域具有典型的α/β水解酶折叠，并在6个α-螺旋旁包含8条β-链。Ser536位于链和螺旋之间的转弯处。这种排列称为亲核弯管。因此，Ser536的OH基团很容易接近催化咪唑基团和亲核试剂攻击乙醛碳原子。Asp620位于His655的咪唑环平面，His655位于中间环。

The Proly Endopeptidase (PEPase) from Flavobacterium meningosepticum is one of the serine protease that cleaves peptide bonds at the carboxyl side of proline residue within which peptides are up to approximately 30 amino acids long. Crystals of PEPase were obtained at 15℃ by vapor diffusion in sitting drops. The protein drops were equilibrated against reservoir solutions containing 22%PEG4000 and buffered at pH7.5 by 0.1M Hepes. The sitting drops contained equal volumes of 8mg/mL enzyme and reservoir solutions. The crystals belong to space group P4_1 and the unit cell dimensions are a=b= 3.17Å, c=169.02Å. There are two molecules in the asymmetric unit. X-ray intensity data to 3.2 Åresolutions were collected at 100K on Rigaku R-Axis IV and were processed with the CrystalClear. The starting model for determination and refinement was that of the porcine brain (PDB code 1H2W). The structure was determined by molecular replacement method with the program AMORE/CCP4 and then refined with the program CNS. Solvent molecules were added once the final crystallographic R-factor and Rfree was lower than 26% and 32%, respectively. The overall fold is the same as that of the asymmetric two molecules, with rms deviations between α-carbon atom positions of 0.04Å. The monomer has a cylindrical shape of an approximate hight of 70Å and diameter of 50 Å, consists of a peptidase domain with its catalytic triads Ser536/Asp620/His655. The domain exhibits a characteristic α/β hydrolase fold and contains a central eight β-strands and is beside the six α-helices. Ser536 is found at the tip of a turn between strand and helix. Such an arrangement referred to as nucleophile elbow. Consequently, the OH group of Ser536 is well accessible to the catalytic imidazole group and nucleophile attacks the aldehyde carbone atom. Asp620 is in the plane of the imidazole ring of His655 which is located in the middle loop.