Rall S C, Ye P, Bu G, Wardell M R
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 1998 Sep 11;273(37):24152-7. doi: 10.1074/jbc.273.37.24152.
The 39-kDa receptor-associated protein (RAP), a specialized chaperone for endocytic receptors of the low density lipoprotein receptor gene family, is a triplicate repeat sequence (residues 1-100, 101-200, and 201-323, respectively), with the three repeats having different functional roles. The goal of the present study was to use a combination of protease sensitivity and guanidine denaturation analyses to investigate whether human RAP correspondingly contained multiple structural domains. Protease sensitivity analysis using six proteolytic enzymes of varying specificity showed that RAP has two protease-resistant regions contained within repeat 1 (residues 15-94) and repeat 3 (residues 223-323). Guanidine denaturation analysis showed that RAP has two phases in its denaturation, an early denaturation transition at 0.6 M guanidine HCl, and a broad second transition between 1.0 and 3.0 M guanidine HCl. Analysis of the denaturation of the individual repeats showed that, despite the similarity in sequence and protease sensitivity between repeats 1 and 3, repeat 1 was a stable structure, with a sharp transition midpoint at 2.4 M guanidine HCl, while repeat 3 was relatively unstable, with a transition midpoint at 0.6 M guanidine HCl. Repeat 2 had a denaturation profile almost identical to that of repeat 3. Denaturation analysis of the contiguous repeats 1 and 2 (residues 1-210) indicated that repeats 1 and 2 probably interact to form one structural domain represented by the broad transition, while repeat 3 constitutes a separate domain represented by the early transition. A two-domain model of RAP three-dimensional structure is proposed that integrates both structural and functional information, in which a helical segment from repeat 2 interacts with the known three-helix bundle of repeat 1 to form a four-helix bundle structural domain, while repeat 3 forms the other structural domain.
39 kDa受体相关蛋白（RAP）是低密度脂蛋白受体基因家族内吞受体的一种特殊伴侣蛋白，它是一个三联重复序列（分别为残基1 - 100、101 - 200和201 - 323），这三个重复序列具有不同的功能作用。本研究的目的是结合蛋白酶敏感性和胍变性分析，以研究人RAP是否相应地包含多个结构域。使用六种具有不同特异性的蛋白水解酶进行的蛋白酶敏感性分析表明，RAP在重复序列1（残基15 - 94）和重复序列3（残基223 - 323）中包含两个蛋白酶抗性区域。胍变性分析表明，RAP的变性有两个阶段，在0.6 M盐酸胍时出现早期变性转变，在1.0至3.0 M盐酸胍之间出现较宽的第二个转变。对各个重复序列变性的分析表明，尽管重复序列1和3在序列和蛋白酶敏感性上相似，但重复序列1是一个稳定结构，在2.4 M盐酸胍时具有尖锐的转变中点，而重复序列3相对不稳定，在0.6 M盐酸胍时具有转变中点。重复序列2的变性图谱几乎与重复序列3相同。对连续的重复序列1和2（残基1 - 210）的变性分析表明，重复序列1和重复序列2可能相互作用形成一个由宽转变代表的结构域，而重复序列3构成一个由早期转变代表的单独结构域。提出了一个RAP三维结构的双结构域模型，该模型整合了结构和功能信息，其中来自重复序列2的一个螺旋段与已知的重复序列1的三螺旋束相互作用形成一个四螺旋束结构域，而重复序列3形成另一个结构域。
