Odunuga Odutayo O, Hornby Judith A, Bies Christiane, Zimmermann Richard, Pugh David J, Blatch Gregory L
Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, Grahamstown 6140, South Africa.
J Biol Chem. 2003 Feb 28;278(9):6896-904. doi: 10.1074/jbc.M206867200. Epub 2002 Dec 13.
Murine stress-inducible protein 1 (mSTI1) is a co-chaperone that is homologous with the human Hsp70/Hsp90-organizing protein (Hop). Guided by Hop structural data and sequence alignment analyses, we have used site-directed mutagenesis, co-precipitation assays, circular dichroism spectroscopy, steady-state fluorescence, and surface plasmon resonance spectroscopy to both qualitatively and quantitatively characterize the contacts necessary for the N-terminal tetratricopeptide repeat domain (TPR1) of mSTI1 to bind to heat shock cognate protein 70 (Hsc70) and to discriminate between Hsc70 and Hsp90. We have shown that substitutions in the first TPR motif of Lys(8) or Asn(12) did not affect binding of mSTI1 to Hsc70, whereas double substitution of these residues abrogated binding. A substitution in the second TPR motif of Asn(43) lowered but did not abrogate binding. Similarly, a deletion in the second TPR motif coupled with a substitution of Lys(8) or Asn(12) reduced but did not abrogate binding. These results suggest that mSTI1-Hsc70 interaction requires a network of interactions not only between charged residues in the TPR1 domain of mSTI1 and the EEVD motif of Hsc70 but also outside the TPR domain. We propose that the electrostatic interactions in the first TPR motif made by Lys(8) or Asn(12) define part of the minimum interactions required for successful mSTI1-Hsc70 interaction. Using a truncated derivative of mSTI1 incapable of binding to Hsp90, we substituted residues on TPR1 potentially involved in hydrophobic contacts with Hsc70. The modified protein had reduced binding to Hsc70 but now showed significant binding capacity for Hsp90. In contrast, topologically equivalent substitutions on a truncated derivative of mSTI1 incapable of binding to Hsc70 did not confer Hsc70 specificity on TPR2A. Our results suggest that binding of Hsc70 to TPR1 is more specific than binding of Hsp90 to TPR2A with serious implications for the mechanisms of mSTI1 interactions with Hsc70 and Hsp90 in vivo.
小鼠应激诱导蛋白1（mSTI1）是一种共伴侣蛋白，与人热休克蛋白70/热休克蛋白90组织蛋白（Hop）同源。在Hop结构数据和序列比对分析的指导下，我们使用定点诱变、共沉淀分析、圆二色光谱、稳态荧光和表面等离子体共振光谱，对mSTI1的N端四肽重复结构域（TPR1）与热休克同源蛋白70（Hsc70）结合以及区分Hsc70和Hsp90所需的相互作用进行了定性和定量表征。我们发现，在第一个TPR基序中赖氨酸（8）或天冬酰胺（12）的取代并不影响mSTI1与Hsc70的结合，而这两个残基的双重取代则消除了结合。在第二个TPR基序中天冬酰胺（43）的取代降低了但并未消除结合。同样，第二个TPR基序中的缺失与赖氨酸（8）或天冬酰胺（12）的取代相结合，降低了但并未消除结合。这些结果表明，mSTI1-Hsc70相互作用不仅需要mSTI1 的TPR1结构域中的带电残基与Hsc70的EEVD基序之间的相互作用网络，还需要TPR结构域之外的相互作用。我们提出，由赖氨酸（8）或天冬酰胺（12）在第一个TPR基序中形成的静电相互作用定义了成功的mSTI1-Hsc70相互作用所需的部分最小相互作用。使用无法与Hsp90结合的mSTI1截短衍生物，我们取代了TPR1上可能参与与Hsc70疏水接触的残基。修饰后的蛋白与Hsc70的结合减少，但现在显示出对Hsp90的显著结合能力。相反，在无法与Hsc70结合的mSTI1截短衍生物上进行拓扑等效取代，并未赋予TPR2A对Hsc70的特异性。我们的结果表明，Hsc70与TPR1的结合比Hsp90与TPR2A的结合更具特异性，这对mSTI1在体内与Hsc70和Hsp90相互作用的机制具有重要意义
