利用三度空間結構與胺基酸定點突變定位β-乳球蛋白與維生素D在第二結合區之交互作用

Abstract

β-乳球蛋白(LG)約佔牛乳中蛋白質10~15％，關於加熱造成LG結構不穩定及破壞已被廣泛探討。LG最主要之生理功能為攜帶疏水性分子，如維生素A、D、棕櫚酸，此結合區域稱為Calyx。本實驗室利用同步輻射光繞射分析LG與維生素D複合體之結晶結構，首次發現LG具兩個維生素D結合區，第二結合區稱為exosite，位於LG分子胜肽C端之136至149胺基酸序列，由疏水性loop連接雙極性α-helix及strand I形成疏水性口袋。經實驗證實，LG於100℃加熱16分鐘，仍以exosite結合維生素D。雙極性α-helix由親水性胺基酸、疏水性胺基酸分別位於helix縱軸之兩側組成，獨特地，疏水性一側結合維生素D。為了深入瞭解exosite上α-helix結合維生素D之相關疏水性胺基酸，利用大腸桿菌表現LG重組蛋白(rLG)，利用胺基酸定點突變雙極性α-helix之疏水性胺基酸，探討其與維生素D結合之重要性。首先將LG基因序列構築於大腸桿菌表現質體pQE30上，經大量表現及純化rLG，發現其維生素D結合力與加熱LG相似，其值為33 nM。而後依序將Leu-133、Phe-136、Leu-140疏水性胺基酸置換為脯胺酸(Pro)，比rLG結合維生素D親和力降低2.7~3.78倍。Leu-140為雙極性α-helix與維生素D產生疏水性鍵結之唯一胺基酸(鍵結長度為3.8 Å)，置換為極性胺基酸之離胺酸(Lys)及麩胺酸(Glu)，比rLG降低3.11~3.21倍維生素D親和力。但置換為非極性胺基酸如苯丙胺酸(Phe)，只約略降0.63倍。印證雙極性α-helix於維持exosite之整體結構或其所包含之疏水性胺基酸，對於結合維生素D皆扮演不可或缺角色。LG可經由小腸表皮細胞以專一性受器進入血液循環系統，故可應用於LG攜帶維生素D類似物之相關藥物，促進藥物輸送，增加人體吸收。

Bovine β-lactoglobulin (LG) is one of the major milk proteins consisting approximately of 10–15% by weight and has been studied extensively for its thermally unstable and molten globule properties. A remarkable property of LG is its ability to bind hydrophobic molecules such as retinol, fatty acids, and vitamin D in the primary binding site (Calyx). Recently, we demonstrated that there are two vitamin D3 binding-sites in each LG molecule using synchrotron X-ray diffraction. The second binding site (exosite) is near the surface pocket (residues 136-149) between the α-helix and strand I linked by the hydrophobic loop. We further demonstrated that the exosite is heat resistant for vitamin D3 binding (100℃), but not the Calyx. The remarkable feature of the α-helix is that it forms an amphipathic orientation with one face of totally charged amino-acid residues and another face of totally hydrophobic residues (without exception). In the present study we investigate the role of essential residues of the amphipathic α-helix in the exosite that are involved in vitamin D3 binding using site-directed mutagenesis. First, we constructed and expressed recombinant LG (rLG) by expression vector pQE30 in E. coli. We demonstrated that rLG maintains only an exosite available for vitamin D binding and has the same binding affinity at 33 nM as heated LG. Second, we used Pro as a potential helix breaker to replace each respective hydrophobic residue, namely Leu-133, Phe-136, and Leu-140. It reveals that the vitamin D binding affinity of each mutant was drastically decreased by 2.7 to 3.78-fold. A marked decrease in binding affinity was seen when Leu-140 (within 3.8 Å distance with vitamin D) was substituted with Lys and Glu (3.11-3.21-fold), whose decrease is significantly higher than that with Phe (0.63-fold). Thus, the helix oriented as amphipathic configuration plays a key role in sustaining a hydrophobic pocket. Since LG is directly internalized into the epithelial lining of the intestine via a receptor, understanding the structure-function relationship may aid in designing vitamin D analogs for facilitating pharmaceutical delivery.