Tertiary structure of an amyloid immunoglobulin light chain protein: a proposed model for amyloid fibril formation.
Tertiary structure of an amyloid immunoglobulin light chain protein: a proposed model for amyloid fibril formation.
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DOI：
10.1073/pnas.92.21.9490
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发表时间：
1995-10
影响因子：
11.1
通讯作者：
Norbert Schormann;Jill R Murrell;J. Liepnieks;Merrill D. Benson
中科院分区：
文献类型：
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作者：
Norbert Schormann;Jill R Murrell;J. Liepnieks;Merrill D. Benson
从1例系统性淀粉样变性(BRE)患者的尿液中分离到一种免疫球蛋白轻链蛋白。轻链(VL)蛋白可变区的完整氨基酸序列确定其为kappa I，与其他kappa I淀粉样蛋白相关蛋白相比，它具有独特的残基，包括Ile-34、Leu-40和Tyr-71。为了研究BRE VL的三级结构，利用从患者BRE的骨髓DNA中扩增出的PCR产物，在大肠杆菌中表达BRE VL。将扩增产物连接到热诱导复制载体pCZ11中。以硫酸铵为沉淀剂，对重组BRE VL进行分离、纯化、结晶。得到了两种晶型。在晶型I中，BRE VL kappa结构域结晶为二聚体，其单位晶胞常数与先前发表的kappa蛋白结构相似。与来自患者REI的非淀粉样蛋白VL kappa结构域相比，发现高变量片段中残基的位置以及框架区域(FR)片段40-46(FR2)和66-67(FR3)的差异显著。此外，在单体-单体和二聚体-二聚体界面上，可以看到两种类型的局部二聚体的位置差异。根据堆积图，提出了一个基于伪六角螺旋结构的淀粉样轻链(AL)原纤维模型，每360度旋转上升约两个二聚体的宽度。这种螺旋结构可能与电子显微镜所确定的淀粉样原纤维的尺寸一致。
An immunoglobulin light chain protein was isolated from the urine of an individual (BRE) with systemic amyloidosis. Complete amino acid sequence of the variable region of the light chain (VL) protein established it as a kappa I, which when compared with other kappa I amyloid associated proteins had unique residues, including Ile-34, Leu-40, and Tyr-71. To study the tertiary structure, BRE VL was expressed in Escherichia coli by using a PCR product amplified from the patient BRE's bone marrow DNA. The PCR product was ligated into pCZ11, a thermal-inducible replication vector. Recombinant BRE VL was isolated, purified to homogeneity, and crystallized by using ammonium sulfate as the precipitant. Two crystal forms were obtained. In crystal form I the BRE VL kappa domain crystallizes as a dimer with unit cell constants isomorphous to previously published kappa protein structures. Comparison with a nonamyloid VL kappa domain from patient REI, identified significant differences in position of residues in the hypervariable segments plus variations in framework region (FR) segments 40-46 (FR2) and 66-67 (FR3). In addition, positional differences can be seen along the two types of local diads, corresponding to the monomer-monomer and dimer-dimer interfaces. From the packing diagram, a model for the amyloid light chain (AL) fibril is proposed based on a pseudohexagonal spiral structure with a rise of approximately the width of two dimers per 360 degree turn. This spiral structure could be consistent with the dimensions of amyloid fibrils as determined by electron microscopy.