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. 2016 Jan 20:7:10357.
doi: 10.1038/ncomms10357.

Protein unfolding as a switch from self-recognition to high-affinity client binding

Bastian Groitl  1 , Scott Horowitz  1   2 , Karl A T Makepeace  3 , Evgeniy V Petrotchenko  3 , Christoph H Borchers  3 , Dana Reichmann  1 , James C A Bardwell  1   2 , Ursula Jakob  1
Affiliations

Protein unfolding as a switch from self-recognition to high-affinity client binding

Bastian Groitl et al. Nat Commun. .

Abstract

Stress-specific activation of the chaperone Hsp33 requires the unfolding of a central linker region. This activation mechanism suggests an intriguing functional relationship between the chaperone's own partial unfolding and its ability to bind other partially folded client proteins. However, identifying where Hsp33 binds its clients has remained a major gap in our understanding of Hsp33's working mechanism. By using site-specific Fluorine-19 nuclear magnetic resonance experiments guided by in vivo crosslinking studies, we now reveal that the partial unfolding of Hsp33's linker region facilitates client binding to an amphipathic docking surface on Hsp33. Furthermore, our results provide experimental evidence for the direct involvement of conditionally disordered regions in unfolded protein binding. The observed structural similarities between Hsp33's own metastable linker region and client proteins present a possible model for how Hsp33 uses protein unfolding as a switch from self-recognition to high-affinity client binding.

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Figures

Figure 1
Figure 1. Identification of the Hsp33-client-binding site in vivo.
(a) Eighteen sites in Hsp33 were selected for the incorporation of unnatural amino acids as shown in the I-TASSER-based ribbon (left) and indicated in dark pink on the surface (right) models of reduced E. coli Hsp33. Hsp33 consists of an N-terminal domain (cyan), a metastable linker region (light pink) and a redox switch domain (purple), in which four thiolate anions arranged in a C232-X-C234-X31-C265-X-Y-C268 motif coordinate one zinc ion (red sphere) under reducing conditions. Only one monomer of the dimeric crystal structure is shown. In solution, reduced Hsp33 is monomeric. (b) E. coli cells overexpressing the Hsp33M172S-BPA variants were shifted from 30 °C to heat-shock conditions (43 °C for 5, 10 or 20 min) (HS). The cells were then either left untreated or exposed to UV irradiation for 10 min to induce crosslinking. Western blot analysis using anti-Hsp33 antibodies was used to visualize the crosslinking products (CL).
Figure 2
Figure 2. Monitoring conformational rearrangements in purified Hsp33M172-tFPA variants using 19F NMR.
(a) Chaperone activity of reduced, zinc-reconstituted or HOCl-activated wild-type Hsp33, Hsp33M172S or Hsp33M172S-tFPA variants. Chaperone activity was measured by testing the influence of a four-fold molar excess of Hsp33 on the aggregation of chemically unfolded CS at either 20 °C (blue bars) or 30 °C (orange bars) or on thermally unfolded CS at 43 °C (red bars). Chaperone activity of 0% is defined as the light-scattering signal 4 min after addition of CS in the absence of chaperones. Activity of 100% corresponds to the light-scattering signal of CS in the presence of a four-fold molar excess of wild-type Hsp33 that had been activated for 2 min in 200 μM HOCl at 30 °C. All experiments were conducted at least 3–5 times and the s.e.m. is shown. (b) Temperature dependence of the 19F NMR signal in select Hsp33M172S-tFPA mutants. 19F NMR spectra of tFPA alone or the indicated mutant variants were recorded at either 25 °C (blue), 35 °C (orange) or 45 °C (red). (c) N-terminal linker-docking surface (cyan) and the metastable linker region (pink) of E. coli Hsp33 in the inactive, closed state (left) (I-TASSER model) and in the activated, open state (right) (PDB 1HW7). The close proximity of F157 and F187 (and L202) to Arg155 and Arg159 in the closed state of Hsp33 is likely responsible for the distinctive down-field chemical shift change observed in the mutant variants under inactivating conditions.
Figure 3
Figure 3. Monitoring Hsp33-client-binding interactions using site-specific 19F NMR.
19F NMR spectra of select Hsp33M172StFPA variants in the absence (blue) or presence (magenta) of (a) NPY or (b) NPY labelled with the paramagnetic spin-label TEMPO. The incubation temperature was set to 35 °C. All experiments were conducted at least three times.
Figure 4
Figure 4. Determining the Hsp33-client interaction sites using in vitro crosslinking.
(a) In vitro crosslinks specifically between oxidized (activated) wild-type Hsp33 and (S)NPY-peptide are displayed in a linear representation created with Cross-Link Viewer (SNPY is the NPY peptide containing one additional serine residue at the N-terminus). Residues that crosslinked with more than one crosslinker are shown as dashed lines in the respective colours (ABAS: red; CDBPS: yellow; and EDC: blue). (b) In vivo and (c) in vitro crosslinking sites are indicated on the crystal structure of oxidized, domain-swapped E. coli Hsp331-255 (PDB 1HW7). Only one of the two subunits that are found in the crystal structure is shown. Zero-length in vitro crosslinks with EDC are indicated in red; long- and medium-range crosslinks (CBDPS and ABAS) are shown in dark pink.
Figure 5
Figure 5. The client-binding site in Hsp33.
(a) Structure of domain-swapped E. coli Hsp331-255 (PDB 1HW7) with one monomer in surface representation and the other one in cartoon depiction. All in vivo crosslinking-positive residues, all zero-length in vitro crosslinking sites, and 19F NMR-positive sites are highlighted in red. Long- and medium-range in vitro crosslinking sites are marked in dark pink, and sites previously suggested to be involved in client binding by limited proteolysis experiments are depicted in orange. Most identified client interaction sites overlap with interaction sites between Hsp33's linker-docking region and the linker region. (b) Docking model of NPY peptide (PDB 1RON) using the truncated oxidized Hsp33 model (residues 182–219 were removed from PDB 1HW7). The top ten docking models are shown in various colours. The Hsp33 subunits are depicted in green and gold, respectively.
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