Giese Kim C, Vierling Elizabeth
Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, 85721, USA.
J Biol Chem. 2004 Jul 30;279(31):32674-83. doi: 10.1074/jbc.M404455200. Epub 2004 May 19.
Oligomerization is an essential property of small heat shock proteins (sHSPs) that appears to regulate their chaperone activity. We have examined the role of conserved hydrophobic residues that are postulated to stabilize sHSP oligomers. We identified a mutation of Synechocystis Hsp16.6 that impairs function in vivo and in vitro. The V143A mutation is in the C-terminal extension, a region predicted to form an oligomeric interaction with a hydrophobic region that includes the site of a previously characterized mutation, L66A. Both mutants were dimeric, but V143A had a stronger oligomerization defect than L66A. However, V143A protected a model substrate better than L66A. This suggests that although the two regions both play a role in oligomerization, they are not equivalent. Nevertheless, the addition of either dimeric sHSP enhanced the in vitro chaperone activity of wild type Hsp16.6, consistent with models that the sHSP dimers initiate interactions with substrates. Suppressor analysis of V143A identified mutations in the N terminus that restored activity by restabilizing the oligomer. These mutants were allele-specific and unable to suppress L66A, although they suppressed a dimeric C-terminal truncation of Hsp16.6. Conversely, suppressors of L66A were unable to suppress either V143A or the truncation, although they, like suppressors of V143A, stabilize the Hsp16.6 oligomer. We interpret these data as evidence that the mutations V143A and L66A stabilize two different dimeric structures and as further support that sHSP dimers are active species.
寡聚化是小分子热休克蛋白(sHSPs)的一项基本特性,似乎能调节其伴侣活性。我们研究了假定可稳定sHSP寡聚体的保守疏水残基的作用。我们鉴定出集胞藻Hsp16.6的一个突变,该突变在体内和体外均损害其功能。V143A突变位于C末端延伸区,该区域预计会与一个疏水区域形成寡聚相互作用,该疏水区域包括一个先前已表征突变L66A的位点。两个突变体均为二聚体,但V143A的寡聚化缺陷比L66A更强。然而,V143A对模型底物的保护作用优于L66A。这表明尽管这两个区域在寡聚化中均起作用,但它们并不等同。尽管如此,添加任何一种二聚体sHSP均增强了野生型Hsp16.6的体外伴侣活性,这与sHSP二聚体启动与底物相互作用的模型一致。对V143A的抑制子分析鉴定出N末端的突变,这些突变通过重新稳定寡聚体来恢复活性。这些突变体具有等位基因特异性,无法抑制L66A,尽管它们抑制了Hsp16.6的二聚体C末端截短。相反,L66A的抑制子无法抑制V143A或截短体,尽管它们与V143A的抑制子一样,能稳定Hsp16.6寡聚体。我们将这些数据解释为V143A和L66A突变稳定了两种不同二聚体结构的证据,并进一步支持sHSP二聚体是活性物种。