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. 2013 Apr 26;288(17):11897-906.
doi: 10.1074/jbc.M113.450437. Epub 2013 Mar 13.

Small heat shock protein IbpB acts as a robust chaperone in living cells by hierarchically activating its multi-type substrate-binding residues

Xinmiao Fu  1 , Xiaodong Shi, Linxiang Yin, Jiafeng Liu, Keehyoung Joo, Jooyoung Lee, Zengyi Chang
Affiliations

Small heat shock protein IbpB acts as a robust chaperone in living cells by hierarchically activating its multi-type substrate-binding residues

Xinmiao Fu et al. J Biol Chem. .

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.

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Figures

FIGURE 1.
FIGURE 1.
Sites of Bpa incorporation in IbpB and types of substrate-binding residues. The N-terminal arm (amino acids 1–39), the α-crystallin domain (amino acids 40–120), and the C-terminal extension (amino acids 121–142) of IbpB are indicated by solid, dotted, and dashed lines, respectively. The 71 amino acid positions into which Bpa was incorporated and examined are indicated by the boldface red letters. The residues that failed to bind substrate proteins are labeled 0, and the type I–III residues are labeled 1–3, respectively.
FIGURE 2.
FIGURE 2.
The chaperone activity of IbpB is barely affected by incorporation of the unnatural amino acid Bpa at 71 selected individual residue positions. A and B, immunoblot results with EF-Tu and IbpB present in the soluble (S) and pellet (P; insoluble) fractions of the whole cell extract (W) of ΔibpB cells expressing His-tagged Bpa variant (A) or wild-type (B) proteins of IbpB, with the whole cell extract incubated at 50 °C for 1 h before centrifugation at 15,000 × g for 10 min. C, SDS-PAGE analysis results with the purified cross-linked products of IbpB mediated by formaldehyde (for 30 min) in living cells cultured at 50 °C, with the cross-linkage broken by boiling the sample for 20 min (second lane) before loading on the gel. The two most predominant cross-linked proteins (second lane) were identified as EF-Tu and TnaA by mass spectrometry. D, immunoblot results with endogenous IbpA and recombinant His-tagged wild-type IbpB proteins from ΔibpB cells cultured at 50 °C using anti-IbpB polyclonal antibody, which cross-reacts with endogenous IbpA, an IbpB-homologous protein in E. coli. Their comparable expression levels demonstrate that recombinant wild-type IbpB and the Bpa variant proteins were expressed in ΔibpB cells at a level comparable with endogenous IbpB because the protein levels of endogenous IbpB and IbpA were highly comparable with each other, as reported previously (52).
FIGURE 3.
FIGURE 3.
Examination of the level of in vivo photo-cross-linked products of the 71 Bpa variants of IbpB from cells cultured at 30 and 50 °C. A–C, immunoblot results with the in vivo photo-cross-linked products of the IbpB variants with Bpa incorporated in the N-terminal arm (A), the α-crystallin domain (B), and the C-terminal extension (C) using the anti-His tag antibody. Cells were grown at 30 °C and treated at the same temperature (b lanes) or 50 °C (c lanes) for 30 min before photo-cross-linking. The Bpa variant samples with no UV irradiation (a lanes) and wild-type IbpB (lanes 10a, 10b, and 10c in B) were used as controls. The dotted lines in B and C indicate the region (Glu-104–Ala-139) within which the IbpB dimers and trimers appear as doublets, most likely reflecting one or two cross-linkages within the dimers and two or three within the trimers. D, SDS-PAGE analysis results with the in vivo photo-cross-linked (30 °C) products of Y45Bpa (lane 4), which were purified before loading onto the gel. Purified Y45Bpa with (lane 3) or without (lane 2) in vitro photo-cross-linking was included as a control. The dimeric and trimeric bands in lane 4 were later cut and identified as IbpB by mass spectrometry.
FIGURE 4.
FIGURE 4.
Semiquantification of relative substrate-binding levels for each of the 71 Bpa variants of IbpB. The relative substrate-binding levels for each Bpa variant at 30 °C (^) and 50 °C (▿) were calculated as described under “Experimental Procedures” based on the immunoblot data from Fig. 3 (A–C). The N-terminal region that corresponds to the structurally disordered N-terminal region of Hsp16.9 ends at Phe-32 in IbpB (as indicated by the dashed line).
FIGURE 5.
FIGURE 5.
Substrate binding of IbpB in living cells starts at 25 °C and further increases upon temperature elevation. A–C, immunoblot results with the in vivo photo-cross-linked products of Bpa variants of IbpB using the anti-His tag antibody, with Bpa incorporated at Phe-8, Phe-16, and Gln-24 (type I residues), Phe-4 and Trp-13 (type II residues), or Phe-32 and Tyr-45 (type III residues), respectively. Cells expressing P8Bpa, F16Bpa, and Q24Bpa were cultured overnight at 20, 25, or 30 °C before being subjected to UV irradiation for photo-cross-linking. Cells expressing F4Bpa, W13Bpa, F32Bpa, or Y45Bpa were grown at 30 °C and treated at the indicated temperatures (30, 37, 42, 45, and 50 °C) for 30 min before being subjected to UV irradiation. D, immunoblot results with formaldehyde-mediated in vivo cross-linked products of IbpB, with the cells grown overnight at 20, 25, or 30 °C before incubation at 25 °C and addition of formaldehyde (lanes 1–6) or with the cells grown at 30 °C, treated at 30, 42, or 50 °C for 30 min, and pre-cooled to 30 °C for 30 s before addition of formaldehyde (lanes 7–10). HMW, high molecular weight.
FIGURE 6.
FIGURE 6.
Mapping type I–III residues into the modeled dodecameric structure and schematically illustrating the temperature-dependent hierarchical activation of the substrate-binding residues of IbpB. A, the dodecameric structure of IbpB was modeled by reference to the three-dimensional structure determined for Hsp16.9, with the N-terminal arm, α-crystallin domain, and C-terminal extension colored in blue, white, and green (first panels), respectively. The amino-terminal 31 residues in half of the 12 subunits of IbpB were assumed to be structurally disordered by referring to Hsp16.9 (13) and were thus disregarded during modeling. Type I–III residues (all colored red) were mapped into the modeled IbpB structure (second, third, and fourth panels, respectively). B, schematic illustration of the hierarchical activation of type I–III substrate-binding residues of IbpB at particular temperatures. Inactivated residues are colored dark gray, and activated residues are colored blue. The activation of type III residues involves certain oligomeric reorganization. It is also illustrated here that multiple species of substrate proteins may simultaneously bind to each IbpB oligomer and that each substrate molecule may bind to multiple residues in IbpB.
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