Ipomoelin與多種醣類MMP、MGP或唾液酸複合物之結構分析

Abstract

Ipomoelin (IPO) 是一個甘藷(Ipomoea batatas cv. Tainung 57)中傷害誘導的防禦蛋白，IPO會被機械性傷害、茉莉酸甲酯(Methyl Jasmonate)以及乙烯等多種植物逆境與訊息分子所誘導表現，IPO屬於凝集素家族的一員，可與醣類分子結合的蛋白質之一。為了瞭解IPO與數種親和性高的醣類分子之結合情形，將原態IPO以及IPO與三種不同的醣受質分子：methyl α-D-mannopyranoside (MMP)、methyl α-D-glucopyranoside (MGP)或sialic acid (SA)所形成之複合物進行養晶條件的篩選與晶體的培養，其中IPO與醣類分子所形成之複合物以共結晶的方式進行養晶，拿到蛋白質晶體後，利用新竹國家同步輻射中心進行X光繞射數據收集。繞射數據經過初步的計算之後，發現這些晶體呈現不同的空間群，原態IPO之單位晶格為a=87.5 Å; b=139.5 Å; c=189.9 Å; α=β=γ=90°，空間群為I222。IPO的三維立體結構，利用分子置換法(molecular replacement)並以旋花凝集素(Calespa, pdb id: 1OUW)結構為模板，取得相位角，並進一步計算電子密度圖，建立並修正蛋白質分子結構，解析度為2.3 Å。IPO與醣類所形成之複合物晶體，IPO-MMP空間群為C2221，解析度為2.1 Å；IPO-MGP空間群為P21，解析度為2.1 Å；IPO-SA空間群為P41，解析度為1.9 Å。IPO由14個平行的β摺板，組成一稜柱狀(β-prism)的結構，並以四聚體的形式存在。醣類分子MMP、MGP及SA可以藉由電子雲密度圖清楚的在棱柱狀結構中，遠離N端的結合口袋區發現，由Gly21、Tyr97、Ser140、Gly141、Trp142、Tyr143及Asp145七個胺基酸組成一結合口袋區，其中IPO上的Gly21、Trp142及Asp145三個胺基酸各會與MMP、MGP和SA形成氫鍵。另外，MGP與SA兩者皆會與IPO上的Tyr97及Tyr143形成氫鍵，兩者不同的是，MGP會與Gly141形成氫鍵；SA會與Ser140形成氫鍵，這些結果顯示這些胺基酸可能與醣類分子結合的能力或特異性有關。

Ipomoelin (IPO) is a defense protein from sweet potato (Ipomoea batatas cv. Tainung 57) and will be induced by wounding, methyl jasmonate (MeJA), and ethylene. IPO belongs to Lectin family and it can bind various carbohydrates in different affinities. For investigating the binding modes of IPO for various carbohydrates, the purified proteins of native IPO, IPO in complex with methyl α-D-mannopyranoside (MMP), methyl α-D-glucopyranoside (MGP), sialic acid (SA) were respectively crystallized and resolved. They showed different space group after indexing and scaling. The native IPO is a space group I222 with unit-cell a=87.5 Å, b=139.5 Å, c=189.9 Å, α=β=γ=90º. The structure of native IPO was determined by molecular replacement method in CNS program using Calespa structure (pdb id: 1OUW) as search model. Finally, the crystal structure of IPO was resolved at a resolution 2.3 Å. The following complex structures of IPO with carbohydrates were crystallized in space group C2221 for IPO-MMP at 2.1 Å resolution, P21 for IPO-MGP at 2.1 Å resolution, and P41 for IPO-SA at 1.9 Å resolution, respectively. IPO consists of fourteen strands of β-sheet to form a β-prism structure and functions as tetramer. The carbohydrates MMP, MGP, and SA could be clearly observed in the binding pocket of IPO and contoured by the omitted map. In general, three carbohydrates, MMP, MGP and SA share three hydrogen-bonding interactions with Gly21, Trp142 and Asp145 of IPO. The atoms O3 and O4 of MMP form a network of four hydrogen bonds with IPO. Additionally, MGP and SA share two other hydrogen-bonding interactions with Tyr97 and Tyr143 of IPO. IPO have another hydrogen bond between Gly141 and MGP, and another hydrogen bond between Ser140 and SA. The results from IPO in complex with various carbohydrates will provide the further information of binding specificities in IPO from lower to higher affinity.