Institute of Marine and Environmental Technology, Department of Microbiology and Immunology, University of Maryland School of Medicine, 685 West Baltimore Street, Baltimore, MD 21201, United States.
Biochem Biophys Res Commun. 2011 Sep 23;413(2):389-94. doi: 10.1016/j.bbrc.2011.08.112. Epub 2011 Aug 27.
Group II chaperonins (Cpns) are essential mediators of cellular protein folding in eukaryotes and archaea. They consist of two back-to-back rings forming symmetrical cavities in which non-native substrates undergo appropriate folding, but the primary structural basis for the double ring formation remains unclear. To address this, we carried out systematic mutagenesis on the Cpn from the hyperthermophilic archaeon Pyrococcus furiosus, which is assembled from identical subunits. In our study, (21)GRDAQRMNIL(30) was found to be a critical domain for double ring formation. Deletion of this section stepwise beyond residue 20 resulted in failure to assemble double-ring oligomers and the progressive loss of chaperone function. A key domain spanning the residues 21-50 that is essential for the formation of tetramers that appear to be the intermediates for double ring assembly. Mutation of either Arg22 to Ala22 or Glu37 to Ala37 resulted in similar defects in double-ring assembly and functional deficits. A mutant with Arg22 and Glu37 switched assembled double rings efficiently and exhibited chaperone functions similar to the wild-type. Therefore, Arg22 and Glu37 could form inter-ring salt bridges critical for double ring formation. In addition, Asn28 and Ile29 were found to contribute significantly to ring formation. Sequence alignment revealed that these four residues are highly conserved among group II Cpns. This is the first report of a comprehensive N-terminal mutational analysis for elucidating the oligomerization of group II Cpns.
II 类分子伴侣（Cpns）是真核生物和古菌中细胞蛋白质折叠的重要介质。它们由两个背对背的环组成，形成对称的腔，非天然底物在其中进行适当的折叠，但双环形成的主要结构基础仍不清楚。为了解决这个问题，我们对来自嗜热古菌 Pyrococcus furiosus 的 Cpn 进行了系统的突变，该 Cpn 由相同的亚基组装而成。在我们的研究中，（21）GRDAQRMNIL（30）被发现是双环形成的关键结构域。在残基 20 之后逐步缺失该部分会导致无法组装双环寡聚物，并逐渐丧失伴侣功能。一个跨越残基 21-50 的关键结构域，对于形成似乎是双环组装中间体的四聚体至关重要。将 Arg22 突变为 Ala22 或 Glu37 突变为 Ala37 都会导致双环组装和功能缺陷类似的缺陷。Arg22 和 Glu37 突变的突变体能够有效地组装双环，并且表现出与野生型相似的伴侣功能。因此，Arg22 和 Glu37 可以形成对双环形成至关重要的环间盐桥。此外，发现 Asn28 和 Ile29 对环形成有重要贡献。序列比对显示，这四个残基在 II 类 Cpns 中高度保守。这是首次对 II 类 Cpns 的寡聚化进行全面的 N 端突变分析的报告。
