Sha B, Lee S, Cyr D M
Center for Macromolecular Crystallography, University of Alabama at Birmingham, 35294-0005, USA.
Structure. 2000 Aug 15;8(8):799-807. doi: 10.1016/s0969-2126(00)00170-2.
Molecular chaperone Hsp40 can bind non-native polypeptide and facilitate Hsp70 in protein refolding. How Hsp40 and other chaperones distinguish between the folded and unfolded states of proteins to bind nonnative polypeptides is a fundamental issue.
To investigate this mechanism, we determined the crystal structure of the peptide-binding fragment of Sis1, an essential member of the Hsp40 family from Saccharomyces cerevisiae. The 2.7 A structure reveals that Sis1 forms a homodimer in the crystal by a crystallographic twofold axis. Sis1 monomers are elongated and consist of two domains with similar folds. Sis1 dimerizes through a short C-terminal stretch. The Sis1 dimer has a U-shaped architecture and a large cleft is formed between the two elongated monomers. Domain I in each monomer contains a hydrophobic depression that might be involved in binding the sidechains of hydrophobic amino acids.
Sis1 (1-337), which lacks the dimerization motif, exhibited severe defects in chaperone activity, but could regulate Hsp70 ATPase activity. Thus, dimer formation is critical for Sis1 chaperone function. We propose that the Sis1 cleft functions as a docking site for the Hsp70 peptide-binding domain and that Sis1-Hsp70 interaction serves to facilitate the efficient transfer of peptides from Sis1 to Hsp70.
分子伴侣Hsp40能够结合非天然多肽，并在蛋白质重折叠过程中促进Hsp70发挥作用。Hsp40及其他伴侣蛋白如何区分蛋白质的折叠态与未折叠态以结合非天然多肽是一个基本问题。
为研究此机制，我们测定了酿酒酵母Hsp40家族重要成员Sis1的肽结合片段的晶体结构。2.7埃的结构显示，Sis1在晶体中通过一个晶体学二重轴形成同型二聚体。Sis1单体呈细长形，由两个具有相似折叠方式的结构域组成。Sis1通过一段短的C末端延伸区域形成二聚体。Sis1二聚体具有U形结构，在两个细长的单体之间形成一个大裂缝。每个单体中的结构域I包含一个疏水凹陷，可能参与结合疏水氨基酸的侧链。
缺少二聚化基序的Sis1（1 - 337）在伴侣活性方面表现出严重缺陷，但能够调节Hsp70的ATP酶活性。因此，二聚体形成对于Sis1的伴侣功能至关重要。我们提出，Sis1裂缝作为Hsp70肽结合结构域的对接位点，且Sis1与Hsp70的相互作用有助于促进肽从Sis1向Hsp70的有效转移。
