Hsp21

From Proteopedia

Current revision

'Structure of small heat shock protein (Hsp21)'

Introduction

Small heat-shock proteins (sHSPs) constitute a highly conserved family of molecular chaperones that prevent stress-induced protein misfolding and aggregation, particularly during heat shock. Their expression can increase dramatically, reaching up to 1% of total cellular protein, highlighting their critical role in thermotolerance and in the regulation of cellular stress responses. sHSPs bind to denaturing proteins in an ATP-independent manner, thereby preventing irreversible aggregation. Rather than directly refolding substrate proteins, they act as a first line of defense by stabilizing unfolded or partially folded intermediates and subsequently coordinating with ATP-dependent chaperone systems, such as HSP70 and the HSP100/Clp chaperone machinery, to facilitate substrate disaggregation and refolding.

Small heat-shock proteins can assemble into oligomers with diverse subunit stoichiometries. Each sHSP monomer has a tripartite domain architecture. This includes an N-terminal region (NTR), a conserved α-crystallin domain (ACD), and a C-terminal region (CTR). The ACD is composed of antiparallel β-strands that form a structured immunoglobulin-like β-sandwich fold. The ACD is flanked by variable N-terminal and C-terminal regions, which are involved in oligomerization. The NTR and CTR show extensive sequence variation.

Hsp21

Hsp21 is a chloroplast-localized small heat-shock protein present in all photosynthetic plants and has a crucial role in multiple developmental and stress-related processes, including chloroplast development, fruit ripening, thermomemory regulation, and protection of the photosynthetic apparatus during heat stress.

Structure of Hsp21 Revealed by Cryo-EM

HP 21 forms a dodecamer arranged in a tetrahedral symmetry, which means it has 12 subunits organised around 4 axes of 3-fold rotational symmetry.

Stabilization interfaces

The oligomerisation of Hsp21 is mediated by the dimeric and non-dimeric interfaces. At the dimeric interface, the β5–β7 loop interacts with β-strands 2 and 3 of the opposing α-crystallin domain (ACD) through a limited number of hydrogen bonds. In addition, the unmodeled residue Ala127 from the opposite monomer is positioned in proximity to the β5–β7 loop and may further contribute to dimer stabilisation.

Hsp21 lacks the canonical β6 strand, conserved in many small heat-shock proteins, resulting in an unconventional mode of dimerisation, which makes it structurally weaker and more dynamic, likely promoting monomerisation of Hsp21 at elevated temperatures and during substrate engagement. The non-dimeric interface involves interactions mediated by conserved residues Val181 and Ile183 located within the C-terminal region. Because Hsp21 contains only a weak dimerisation motif in its C-terminus, oligomer assembly is further supported by contacts involving β4–β8 strand interactions between neighbouring monomers.

Additionally, the NTRs are associated with the β7 strand of each unit, which stabilizes the dodecamer.

Substrate of Hsp21

Previous studies identified 1-deoxy-D-xylulose-5-phosphate synthase (DXPS) as a key substrate of Hsp21.

Hsp21 Dodecamer Disassembly

The chemical crosslinking and electron microscopy analysis revealed the following:

Native State: At physiological temperatures (≤37°C), Hsp21 remains primarily in its dodecameric state.

Heat Activation: At higher temperatures, a significant amount of the monomeric Hsp21 was produced.

Incubation with DXPS: An Hsp21-DXPS complex was observed by SDS-PAGE and confirmed by mass spectrometry.

Electron Microscopy: Size reduction and structural perturbation of the Hsp21 oligomer at high temperatures were visually confirmed by electron microscopy.

DXPS induces partial unfolding of Hsp21 upon binding

Conformational Change in the α-Crystallin Domain (ACD)

​The ACD in the unbound state, Hsp21 dodecamer structure, contains seven β-strands. The ACD in the Hsp21-DXPS complex structure only contains six β-strands.

Hsp21 partly unfolds its ACD to "peel away" the first β-strand (β2) during its interaction with DXPS. The detached β2 strand connected to the highly flexible N-terminal region (NTR) becomes very mobile and is not clearly resolved in the final structure. This confirms its dynamic nature.

​Link to Monomerization

​The loss of the β2 strand is expected to destabilize the dimeric interface, suggesting that this conformational change is coupled to the monomerization of Hsp21 from the dodecamer.

​Change in β-Sheet Alignment

Substrate binding induces a major conformational transition in the β-sandwich core of the ACD. The domain switches from a natural "twisted" state (unbound) to an "aligned" state (bound), where the two β-sheets are nearly parallel to each other. This structural plasticity is essential for the chaperone to function.

​Conformational Changes in Other Regions

​Both the β5-β7 loop and the C-terminal region (CTR)assume different conformations in the DXPS-bound form compared to the free form, indicating that the entire Hsp21 monomer adjusts its shape to accommodate the substrate.

BI3323-Aug2025

doi.org/10.1038/s41467-021-23338-y