Egloff M P, Sarda L, Verger R, Cambillau C, van Tilbeurgh H
Laboratoire de Cristallisation et Cristallographie des Macromolécules Biologiques, URA 1296-CNRS, Faculté de Médecine Nord, France.
Protein Sci. 1995 Jan;4(1):44-57. doi: 10.1002/pro.5560040107.
Colipase (Mr 10 kDa) confers catalytic activity to pancreatic lipase under physiological conditions (high bile salt concentrations). Previously determined 3-A-resolution X-ray structures of lipase-colipase complexes have shown that, in the absence of substrate, colipase binds to the noncatalytic C-terminal domain of pancreatic lipase (van Tilbeurgh H, Sarda L, Verger R, Cambillau C, 1992, Nature 359:159-162; van Tilbeurgh et al., 1993a, Nature 362:814-820). Upon lipid binding, conformational changes at the active site of pancreatic lipase bring a surface loop (the lid) in contact with colipase, creating a second binding site for this cofactor. Covalent inhibition of the pancreatic lipase by a phosphonate inhibitor yields better diffracting crystals of the lipase-colipase complex. From the 2.4-A-resolution structure of this complex, we give an accurate description of the colipase. It confirms the previous proposed disulfide connections (van Tilbeurgh H, Sarda L, Verger R, Cambillau C, 1992, Nature 359:159-162; van Tilbeurgh et al., 1993a, Nature 362:814-820) that were in disagreement with the biochemical assignment (Chaillan C, Kerfelec B, Foglizzo E, Chapus C, 1992, Biochem Biophys Res Commun 184:206-211). Colipase lacks well-defined secondary structure elements. This small protein seems to be stabilized mainly by an extended network of five disulfide bridges that runs throughout the flatly shaped molecule, reticulating its four finger-like loops. The colipase surface can be divided into a rather hydrophilic part, interacting with lipase, and a more hydrophobic part, formed by the tips of the fingers. The interaction between colipase and the C-terminal domain of lipase is stabilized by eight hydrogen bonds and about 80 van der Waals contacts. Upon opening of the lid, three more hydrogen bonds and about 28 van der Waals contacts are added, explaining the higher apparent affinity in the presence of a lipid/water interface. The tips of the fingers are very mobile and constitute the lipid interaction surface. Two detergent molecules that interact with colipase were observed in the crystal, covering part of the hydrophobic surface.
辅脂酶（分子量10 kDa）在生理条件下（高胆汁盐浓度）赋予胰脂肪酶催化活性。先前确定的胰脂肪酶-辅脂酶复合物的3-A分辨率X射线结构表明，在没有底物的情况下，辅脂酶与胰脂肪酶的非催化性C末端结构域结合（范·蒂尔伯格H、萨尔达L、韦尔热R、坎比洛C，1992年，《自然》359:159 - 162；范·蒂尔伯格等人，1993年a，《自然》362:814 - 820）。脂质结合后，胰脂肪酶活性位点的构象变化使一个表面环（盖子）与辅脂酶接触，为该辅因子创造了第二个结合位点。用膦酸酯抑制剂对胰脂肪酶进行共价抑制可得到胰脂肪酶-辅脂酶复合物衍射效果更好的晶体。根据该复合物2.4-A分辨率的结构，我们对辅脂酶进行了准确描述。它证实了先前提出的二硫键连接（范·蒂尔伯格H、萨尔达L、韦尔热R、坎比洛C，1992年，《自然》359:159 - 162；范·蒂尔伯格等人，1993年a，《自然》362:814 - 820），这些连接与生化归属不一致（沙伊兰C、凯费莱克B、福格利佐E、沙皮斯C，1992年，《生物化学与生物物理研究通讯》184:206 - 211）。辅脂酶缺乏明确的二级结构元件。这种小蛋白质似乎主要通过贯穿扁平状分子的五个二硫键的扩展网络来稳定，使它的四个手指状环相互交织。辅脂酶表面可分为与脂肪酶相互作用的相当亲水的部分和由手指末端形成的更疏水的部分。辅脂酶与脂肪酶C末端结构域之间的相互作用通过八个氢键和大约80个范德华接触得以稳定。盖子打开后，又增加了三个氢键和大约28个范德华接触，这解释了在脂质/水界面存在时更高的表观亲和力。手指末端非常灵活，构成脂质相互作用表面。在晶体中观察到两个与辅脂酶相互作用的去污剂分子，覆盖了部分疏水表面。
