Pol E, Björk I
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala Biomedical Center, Box 575, SE-751 23 Uppsala, Sweden.
Biochemistry. 1999 Aug 10;38(32):10519-26. doi: 10.1021/bi990488k.
The importance of residues in the second hairpin loop and the C-terminal end of mammalian cystatin B for binding of proteinases was elucidated by mutagenesis of the bovine inhibitor. Bovine cystatin B was modeled onto the crystal structure of the human inhibitor in complex with papain with minimal structural changes. Substitution of the two deduced contact residues in the second hairpin loop, Leu-73 and His-75, with Gly resulted in appreciably reduced affinities for papain and cathepsins H and B. These losses indicated that the two residues together contribute 20-30% of the free energy of binding of cystatin B to these enzymes and that Leu-73 is responsible for most of this contribution. In contrast, the small decrease in the affinity for cathepsin L suggested that the second hairpin loop is less important for inhibition of this proteinase. Replacement of the contact residue in the C-terminal end, Tyr-97, with Ala resulted in losses in affinity for papain and cathepsins L and H that were consistent with Tyr-97 contributing 6-12% of the energy of binding of cystatin B to these enzymes. However, this substitution minimally affected the affinity for cathepsin B, indicating that the C-terminal end is of limited importance for binding of this proteinase. All affinity decreases were due predominantly to increased dissociation rate constants. These results show that both the second hairpin loop and the C-terminal end of cystatin B contribute to anchoring the inhibitor to target proteinases, each of the two regions interacting with a different domain of the enzyme. However, the relative contributions of these two interactions vary with the proteinase.
通过对牛胱抑素B进行诱变，阐明了哺乳动物胱抑素B第二个发夹环和C末端的残基在蛋白酶结合中的重要性。将牛胱抑素B模拟到与木瓜蛋白酶复合的人抑制剂的晶体结构上，结构变化最小。将第二个发夹环中两个推导的接触残基Leu-73和His-75替换为甘氨酸，导致对木瓜蛋白酶、组织蛋白酶H和B的亲和力明显降低。这些亲和力的损失表明，这两个残基共同贡献了胱抑素B与这些酶结合自由能的20%-30%，且Leu-73对这一贡献起主要作用。相比之下，对组织蛋白酶L亲和力的小幅下降表明，第二个发夹环对该蛋白酶的抑制作用不太重要。将C末端的接触残基Tyr-97替换为丙氨酸，导致对木瓜蛋白酶、组织蛋白酶L和H的亲和力损失，这与Tyr-97贡献了胱抑素B与这些酶结合能量的6%-12%一致。然而，这种取代对组织蛋白酶B的亲和力影响最小，表明C末端对该蛋白酶的结合重要性有限。所有亲和力的降低主要是由于解离速率常数增加。这些结果表明，胱抑素B的第二个发夹环和C末端都有助于将抑制剂锚定到靶蛋白酶上，这两个区域分别与酶的不同结构域相互作用。然而，这两种相互作用的相对贡献因蛋白酶而异。
