Kumar Sanjit, Singh Nagendra, Mishra Biswajit, Dube Divya, Sinha Mau, Singh S Baskar, Dey Sharmistha, Kaur Punit, Sharma Sujata, Singh Tej P
Department of Biophysics, All India Institute of Medical Sciences, New Delhi, India.
BMC Struct Biol. 2010 Nov 20;10:41. doi: 10.1186/1472-6807-10-41.
Plants produce a wide range of proteinaceous inhibitors to protect themselves against hydrolytic enzymes. Recently a novel protein XAIP belonging to a new sub-family (GH18C) was reported to inhibit two structurally unrelated enzymes xylanase GH11 and α-amylase GH13. It was shown to inhibit xylanase GH11 with greater potency than that of α-amylase GH13. A new form of XAIP (XAIP-II) that inhibits α-amylase GH13 with a greater potency than that of XAIP and xylanase GH11 with a lower potency than that of XAIP, has been identified in the extracts of underground bulbs of Scadoxus multiflorus. This kind of occurrence of isoforms of inhibitor proteins is a rare observation and offers new opportunities for understanding the principles of protein engineering by nature.
In order to determine the structural basis of the enhanced potency of XAIP-II against α-amylase GH13 and its reduced potency against xylanase GH11 as compared to that of XAIP, we have purified XAIP-II to homogeneity and obtained its complete amino acid sequence using cloning procedure. It has been crystallized with 0.1 M ammonium sulphate as the precipitating agent and the three-dimensional structure has been determined at 1.2 Å resolution. The binding studies of XAIP-II with xylanase GH11 and α-amylase GH13 have been carried out with surface plasmon resonance (SPR).
The structure determination revealed that XAIP-II adopts the well known TIM barrel fold. The xylanase GH11 binding site in XAIP-II is formed mainly with loop α3-β3 (residues, 102 - 118) which has acquired a stereochemically less favorable conformation for binding to xylanase GH11 because of the addition of an extra residue, Ala105 and due to replacements of two important residues, His106 and Asn109 by Thr107 and Ser110. On the other hand, the α-amylase binding site, which consists of α-helices α6 (residues, 193 - 206), α7 (residues, 230 - 243) and loop β6-α6 (residues, 180 - 192) adopts a stereochemically more favorable conformation due to replacements of residues, Ser190, Gly191 and Glu194 by Ala191, Ser192 and Ser195 respectively in α-helix α6, Glu231 and His236 by Thr232 and Ser237 respectively in α-helix α7. As a result, XAIP-II binds to xylanase GH11 less favorably while it interacts more strongly with α-amylase GH13 as compared to XAIP. These observations correlate well with the values of 4.2 × 10(-6) M and 3.4 × 10(-8) M for the dissociation constants of XAIP-II with xylanase GH11 and α-amylase GH13 respectively and those of 4.5 × 10(-7) M and 3.6 × 10(-6) M of XAIP with xylanase GH11 and α-amylase GH13 respectively.
植物产生多种蛋白质抑制剂以保护自身免受水解酶的侵害。最近有报道称，一种属于新亚家族（GH18C）的新型蛋白质XAIP可抑制两种结构不相关的酶——木聚糖酶GH11和α-淀粉酶GH13。研究表明，它对木聚糖酶GH11的抑制效力比对α-淀粉酶GH13的更强。在多花虎眼万年青地下鳞茎提取物中已鉴定出一种新形式的XAIP（XAIP-II），其对α-淀粉酶GH13的抑制效力比XAIP更强，而对木聚糖酶GH11的抑制效力比XAIP更低。抑制剂蛋白同工型的这种情况很少见，为理解自然蛋白质工程原理提供了新机会。
为了确定XAIP-II相对于XAIP对α-淀粉酶GH13抑制效力增强及其对木聚糖酶GH11抑制效力降低的结构基础，我们已将XAIP-II纯化至同质，并使用克隆程序获得了其完整氨基酸序列。它已以0.1 M硫酸铵作为沉淀剂结晶，并在1.2 Å分辨率下确定了三维结构。已通过表面等离子体共振（SPR）对XAIP-II与木聚糖酶GH11和α-淀粉酶GH13的结合进行了研究。
结构测定表明XAIP-II采用了众所周知的TIM桶状折叠。XAIP-II中木聚糖酶GH11结合位点主要由环α3-β3（残基102 - 118）形成，由于额外添加了一个残基Ala105以及两个重要残基His106和Asn109分别被Thr107和Ser110取代，该环获得了对木聚糖酶GH11结合不利的立体化学构象。另一方面，由α-螺旋α6（残基193 - 206）、α7（残基230 - 243）和环β6-α6（残基180 - 192）组成的α-淀粉酶结合位点，由于α-螺旋α6中残基Ser190、Gly191和Glu194分别被Ala191、Ser192和Ser195取代，α-螺旋α7中Glu231和His236分别被Thr232和Ser237取代，采用了更有利的立体化学构象。结果，与XAIP相比，XAIP-II与木聚糖酶GH11的结合不太有利，而与α-淀粉酶GH13的相互作用更强。这些观察结果与XAIP-II与木聚糖酶GH11和α-淀粉酶GH13的解离常数分别为4.2×10⁻⁶ M和3.4×10⁻⁸ M以及XAIP与木聚糖酶GH11和α-淀粉酶GH13的解离常数分别为4.5×10⁻⁷ M和3.6×10⁻⁶ M的值很好地相关。
