Szpikowska B K, Swiderek K M, Sherman M A, Mas M T
Division of Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
Protein Sci. 1998 Jul;7(7):1524-30. doi: 10.1002/pro.5560070705.
The eukaryotic cytosolic chaperonins are large heterooligomeric complexes with a cylindrical shape, resembling that of the homooligomeric bacterial counterpart, GroEL. In analogy to GroEL, changes in shape of the cytosolic chaperonin have been detected in the presence of MgATP using electron microscopy but, in contrast to the nucleotide-induced conformational changes in GroEL, no details are available about the specific nature of these changes. The present study identifies the structural regions of the cytosolic chaperonin that undergo conformational changes when MgATP binds to the nucleotide binding domains. It is shown that limited proteolysis with trypsin in the absence of MgATP cleaves each of the eight subunits approximately in half, generating two fragments of approximately 30 kDa. Using mass spectrometry (MS) and N-terminal sequence analysis, the cleavage is found to occur in a narrow span of the amino acid sequence, corresponding to the peptide binding regions of GroEL and to the helical protrusion, recently identified in the structure of the substrate binding domain of the archeal group II chaperonin. This proteolytic cleavage is prevented by MgATP but not by ATP in the absence of magnesium, ATP analogs (MgATPyS and MgAMP-PNP) or MgADP. These results suggest that, in analogy to GroEL, binding of MgATP to the nucleotide binding domains of the cytosolic chaperonin induces long range conformational changes in the polypeptide binding domains. It is postulated that despite their different subunit composition and substrate specificity, group I and group II chaperonins may share similar, functionally-important, conformational changes. Additional conformational changes are likely to involve a flexible helix-loop-helix motif, which is characteristic for all group II chaperonins.
真核细胞胞质伴侣蛋白是大型异源寡聚复合物，呈圆柱形，类似于同源寡聚的细菌对应物GroEL。与GroEL类似，利用电子显微镜在MgATP存在的情况下检测到了胞质伴侣蛋白形状的变化，但与GroEL中核苷酸诱导的构象变化不同，关于这些变化的具体性质尚无详细信息。本研究确定了胞质伴侣蛋白中当MgATP与核苷酸结合结构域结合时发生构象变化的结构区域。结果表明，在没有MgATP的情况下用胰蛋白酶进行有限的蛋白水解可将八个亚基中的每一个大约切成两半，产生两个约30 kDa的片段。通过质谱（MS）和N端序列分析发现，切割发生在氨基酸序列的一个狭窄范围内，对应于GroEL的肽结合区域以及最近在古菌II型伴侣蛋白底物结合结构域结构中确定的螺旋突出部。在没有镁的情况下，MgATP可阻止这种蛋白水解切割，但ATP、ATP类似物（MgATPyS和MgAMP-PNP）或MgADP则不能。这些结果表明，与GroEL类似，MgATP与胞质伴侣蛋白的核苷酸结合结构域结合会诱导多肽结合结构域发生长程构象变化。据推测，尽管I型和II型伴侣蛋白的亚基组成和底物特异性不同，但它们可能共享相似的、功能上重要的构象变化。额外的构象变化可能涉及一个灵活的螺旋-环-螺旋基序，这是所有II型伴侣蛋白的特征。
