The absolute structural requirement for a proline in the P3′-position of Bowman-Birk protease inhibitors is surmounted in the minimized SFTI-1 scaffold
The absolute structural requirement for a proline in the P3′-position of Bowman-Birk protease inhibitors is surmounted in the minimized SFTI-1 scaffold
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DOI：
10.1074/jbc.m601426200
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发表时间：
2006-08-18
影响因子：
4.8
通讯作者：
Craik, David J.
中科院分区：
文献类型：
--
作者：
Daly, Norelle L.;Chen, Yi-Kuang;Craik, David J.
SFTI-1是一种来自向日葵种子的小环肽，是任何天然存在的肽中最有效的胰蛋白酶抑制剂之一，与Bowman-Birk家族抑制剂（BBI）有关。BBI参与植物的防御机制，也具有作为癌症化学预防剂的潜力。SFTI-1的大小只有14个氨基酸，被认为是BBI活性位点区域的高度优化的支架，因此研究其重要的结构和功能特征是有意义的。本研究合成了12个SFTI-1丙氨酸突变体，并对其结构和活性进行了测定。SFTI-1包含一个结合环，该结合环与二硫键和构成环状骨架的二级肽环扣在一起。我们在这里表明，二级循环稳定的结合环的序列变异的后果。特别是，全长BBI具有保守的顺式脯氨酸，其先前已被证明是明确定义的结构和有效活性所需的，但我们在这里表明，SFTI-1支架可以容纳该残基的突变，并且仍然具有明确定义的天然样构象和纳摩尔抑制胰蛋白酶的活性。在Ala突变体中，当Asp（14）突变时发生最显著的结构扰动，并且似乎该残基在稳定Pro（13）之前的反式肽键中是重要的，因此是维持SFTI-1的高度受限结构的关键残基。该天冬氨酸残基被认为参与与SFTI-1从其58个氨基酸前体切除相关的环化机制。总的来说，SFTI-I的突变分析清楚地定义了SFTI-I支架的优化性质，并证明了二级环在维持结合环的活性构象中的重要性。
SFTI-1 is a small cyclic peptide from sunflower seeds that is one of the most potent trypsin inhibitors of any naturally occurring peptide and is related to the Bowman-Birk family of inhibitors (BBIs). BBIs are involved in the defense mechanisms of plants and also have potential as cancer chemopreventive agents. At only 14 amino acids in size, SFTI-1 is thought to be a highly optimized scaffold of the BBI active site region, and thus it is of interest to examine its important structural and functional features. In this study, a suite of 12 alanine mutants of SFTI-1 has been synthesized, and their structures and activities have been determined. SFTI-1 incorporates a binding loop that is clasped together with a disulfide bond and a secondary peptide loop making up the circular backbone. We show here that the secondary loop stabilizes the binding loop to the consequences of sequence variations. In particular, full-length BBIs have a conserved cis-proline that has been shown previously to be required for well defined structure and potent activity, but we show here that the SFTI-1 scaffold can accommodate mutation of this residue and still have a well defined native-like conformation and nanomolar activity in inhibiting trypsin. Among the Ala mutants, the most significant structural perturbation occurred when Asp(14) was mutated, and it appears that this residue is important in stabilizing the trans peptide bond preceding Pro(13) and is thus a key residue in maintaining the highly constrained structure of SFTI-1. This aspartic acid residue is thought to be involved in the cyclization mechanism associated with excision of SFTI-1 from its 58-amino acid precursor. Overall, this mutational analysis of SFTI-1 clearly defines the optimized nature of the SFTI-1 scaffold and demonstrates the importance of the secondary loop in maintaining the active conformation of the binding loop.