Small Heat Shock Protein IbpB Acts as a Robust Chaperone in Living Cells by Hierarchically Activating Its Multi-type Substrate-binding Residues

Xinmiao Fu,Xiaodong Shi,Linxiang Yin,Jiafeng Liu,Keehyoung Joo,Jooyoung Lee,Zengyi Chang

doi:10.1074/jbc.m113.450437

Abstract

As ubiquitous molecular chaperones, small heat shock proteins (sHSPs) are crucial for protein homeostasis. It is not clear why sHSPs are able to bind a wide spectrum of non-native substrate proteins and how such binding is enhanced by heat shock. Here, by utilizing a genetically incorporated photo-cross-linker (p-benzoyl-l-phenylalanine), we systematically characterized the substrate-binding residues in IbpB (a sHSP from Escherichia coli) in living cells over a wide spectrum of temperatures (from 20 to 50 °C). A total of 20 and 48 residues were identified at normal and heat shock temperatures, respectively. They are not necessarily hydrophobic and can be classified into three types: types I and II were activated at low and normal temperatures, respectively, and type III mediated oligomerization at low temperature but switched to substrate binding at heat shock temperature. In addition, substrate binding of IbpB in living cells began at temperatures as low as 25 °C and was further enhanced upon temperature elevation. Together, these in vivo data provide novel structural insights into the wide substrate spectrum of sHSPs and suggest that sHSP is able to hierarchically activate its multi-type substrate-binding residues and thus act as a robust chaperone in cells under fluctuating growth conditions.

Highlights

Small heat shock proteins are ubiquitous molecular chaperones
It is striking that of the 48 residues, about half are polar residues. These residues generally appear to be hierarchically activated in a temperature-dependent manner, with a number of them switching from mediating oligomerization to mediating substrate binding upon temperature elevation. These features collectively allow IbpB, or small heat shock proteins (sHSPs) in general, to function as a robust molecular chaperone to act upon a large diversity of substrate proteins in living cells growing under fluctuating conditions
For the convenience of immunoblot analysis and protein purification, these Bpa variants were all expressed as C-terminally His-tagged forms; to avoid the interference of endogenous IbpB, the Bpa variants were expressed in ibpB-deleted E. coli cells

Summary

Background

Small heat shock proteins are ubiquitous molecular chaperones. Results: A total of 20 and 48 substrate-binding residues in IbpB were identified to function at 30 and 50 °C, respectively, in living cells. Significant but unresolved scientific questions regarding the functions of sHSPs in living cells include which residues of sHSPs mediate the binding of the wide spectrum of natural substrate proteins [17] and how they play a role in the normally observed ( under in vitro conditions) heat shock-enhanced substrate-binding capacity of sHSPs [23] To address these questions, we chose the inclusion bodybinding protein IbpB, a sHSP from Escherichia coli (24 –30), as a model to investigate the substrate-binding features of sHSPs in living cells. These residues generally appear to be hierarchically activated in a temperature-dependent manner, with a number of them switching from mediating oligomerization to mediating substrate binding upon temperature elevation Together, these features collectively allow IbpB, or sHSPs in general, to function as a robust molecular chaperone to act upon a large diversity of substrate proteins in living cells growing under fluctuating conditions

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION