Czapinska H, Otlewski J, Krzywda S, Sheldrick G M, Jaskólski M
Department of Protein Engineering, Institute of Biochemistry and Molecular Biology, University of Wroclaw, Tamka 2, Wroclaw, 50-137, Poland.
J Mol Biol. 2000 Feb 4;295(5):1237-49. doi: 10.1006/jmbi.1999.3445.
A mutant of bovine pancreatic trypsin inhibitor (BPTI) has been constructed and expressed in Escherichia coli in order to probe the kinetic and structural consequences of truncating the binding loop residues to alanine. In addition to two such mutations (Thr11Ala and Pro13Ala), it has a conservative Lys15Arg substitution at position P(1) and an unrelated Met52Leu change. In spite of the binding loop modification, the affinity for trypsin is only 30 times lower than that of the wild-type protein. At pH 7.5 the protein can be crystallized on the time-scale of hours, yielding very stable crystals of a new (tetragonal) form of BPTI. Conventional source X-ray data collected to 1.4 A at room temperature allowed anisotropic structure refinement characterized by R=0.1048. The structure reveals all 58 residues, including the complete C terminus, which is in a salt-bridge contact with the N terminus. The Cys14-Cys38 disulfide bridge is observed in two distinct chiralities. This bridge, together with an internal water molecule, contributes to the stabilization of the binding loop. The Ala mutations have only an insignificant and localized effect on the binding loop, which retains its wild-type conformation (maximum deviation of loop C(alpha) atoms of 0.7 A at Ala13). Four (instead of the typical three) additional water molecules are buried in an internal cleft and connected to the surface via a sulfate anion. Three more SO(4)(2-) anions are seen in the electron density, one of them located on a 2-fold axis. It participates in the formation of a dimeric structure between symmetry-related BPTI molecules, in which electrostatic and hydrogen bonding interactions resulting from the mutated Lys15Arg substitution are of central importance. This dimeric interaction involves direct recognition loop-recognition loop contacts, part of which are hydrophobic interactions of the patches created by the alanine mutations. Another 2-fold symmetric interaction between the BPTI molecules involves the formation of an antiparallel intermolecular beta-sheet that, together with the adjacent intramolecular beta-hairpin loops, creates a four-stranded structure.
构建了牛胰蛋白酶抑制剂（BPTI）的一个突变体，并在大肠杆菌中进行表达，以探究将结合环残基截短为丙氨酸所带来的动力学和结构影响。除了两个这样的突变（Thr11Ala和Pro13Ala）外，它在P(1)位置有一个保守的Lys15Arg替换以及一个不相关的Met52Leu变化。尽管结合环发生了修饰，但对胰蛋白酶的亲和力仅比野生型蛋白低30倍。在pH 7.5时，该蛋白可在数小时的时间尺度上结晶，得到一种新的（四方晶系）形式的BPTI的非常稳定的晶体。在室温下收集到1.4 Å的常规光源X射线数据，使得以R = 0.1048为特征的各向异性结构精修成为可能。该结构揭示了所有58个残基，包括完整的C末端，它与N末端形成盐桥接触。观察到Cys14 - Cys38二硫键存在两种不同的手性。这个二硫键与一个内部水分子一起，有助于结合环的稳定。丙氨酸突变对结合环只有微不足道的局部影响，结合环保留其野生型构象（在Ala13处环Cα原子的最大偏差为0.7 Å）。四个（而非典型的三个）额外的水分子被埋在一个内部裂隙中，并通过一个硫酸根阴离子与表面相连。在电子密度图中还可见另外三个SO(4)(2-)阴离子，其中一个位于二重轴上。它参与了对称相关的BPTI分子之间二聚体结构的形成，其中由突变的Lys15Arg替换产生的静电和氢键相互作用至关重要。这种二聚体相互作用涉及直接的识别环 - 识别环接触，其中一部分是由丙氨酸突变产生的区域的疏水相互作用。BPTI分子之间的另一种二重对称相互作用涉及形成一个反平行的分子间β - 折叠片，它与相邻的分子内β - 发夹环一起形成一个四链结构。
