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Structural studies of complexes of kallikrein 4 with wild-type and mutated forms of the Kunitz-type inhibitor BbKI
Acta Crystallographica Section D ( IF 2.2 ) Pub Date : 2021-08-04 , DOI: 10.1107/s2059798321006483
Mi Li 1 , Jaroslav Srp 2 , Michael Mareš 2 , Alexander Wlodawer 1 , Alla Gustchina 1
Affiliation  

Structures of BbKI, a recombinant Kunitz-type serine protease inhibitor from Bauhinia bauhinioides, complexed with human kallikrein 4 (KLK4) were determined at medium-to-high resolution in four crystal forms (space groups P3121, P6522, P21 and P61). Although the fold of the protein was virtually identical in all of the crystals, some significant differences were observed in the conformation of Arg64 of BbKI, the residue that occupies the S1 pocket in KLK4. Whereas this residue exhibited two orientations in the highest resolution structure (P3121), making either a canonical trypsin-like interaction with Asp189 of KLK4 or an alternate interaction, only a single alternate orientation was observed in the other three structures. A neighboring disulfide, Cys191–Cys220, was partially or fully broken in all KLK4 structures. Four variants of BbKI in which Arg64 was replaced by Met, Phe, Ala and Asp were expressed and crystallized, and their structures were determined in complex with KLK4. Structures of the Phe and Met variants complexed with bovine trypsin and of the Phe variant complexed with α-chymotrypsin were also determined. Although the inhibitory potency of these variant forms of BbKI was lowered by up to four orders of magnitude, only small changes were seen in the vicinity of the mutated residues. Therefore, a totality of subtle differences in KLK4–BbKI interactions within the fully extended interface in the structures of these variants might be responsible for the observed effect. Screening of the BbKI variants against a panel of serine proteases revealed an altered pattern of inhibitory specificity, which was shifted towards that of chymotrypsin-like proteases for the hydrophobic Phe and Met P1 substitutions. This work reports the first structures of plant Kunitz inhibitors with S1-family serine proteases other than trypsin, as well as new insights into the specificity of inhibition of medically relevant kallikreins.

中文翻译:

激肽释放酶 4 与野生型和突变型 Kunitz 型抑制剂 BbKI 复合物的结构研究

BbKI 是一种来自紫荆的重组 Kunitz 型丝氨酸蛋白酶抑制剂,与人激肽释放酶 4 (KLK4) 复合,以中高分辨率测定了四种晶型(空间群P 3 1 21、P 6 5 22、P 2 1P 6 1 )。尽管所有晶体中蛋白质的折叠实际上是相同的,但在 BbKI 的 Arg64(占据 KLK4 中 S1 口袋的残基)的构象中观察到一些显着差异。尽管该残基在最高分辨率结构 ( P 3 1 21) 中表现出两个方向,从而与 KLK4 的 Asp189 形成典型的胰蛋白酶样相互作用或替代相互作用,但在其他三个结构中仅观察到单个替代方向。所有 KLK4 结构中相邻的二硫键 Cys191–Cys220 均部分或完全断裂。表达并结晶了其中Arg64被Met、Phe、Ala和Asp取代的BbKI的四种变体,并确定了它们与KLK4复合物的结构。还确定了与牛胰蛋白酶复合的 Phe 和 Met 变体以及与 α-胰凝乳蛋白酶复合的 Phe 变体的结构。尽管这些 BbKI 变体形式的抑制效力降低了四个数量级,但在突变残基附近仅观察到很小的变化。因此,这些变体结构中完全扩展的界面内 KLK4-BbKI 相互作用的总体微妙差异可能是造成观察到的效果的原因。针对一组丝氨酸蛋白酶筛选 BbKI 变体揭示了抑制特异性模式的改变,该模式向胰凝乳蛋白酶样蛋白酶的疏水性 Phe 和 Met P1 取代转变。这项工作报告了植物 Kunitz 抑制剂与胰蛋白酶以外的 S1 家族丝氨酸蛋白酶的第一个结构,以及对医学相关激肽释放酶的抑制特异性的新见解。
更新日期:2021-08-04
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