Terminal Regions Confer Plasticity to the Tetrameric Assembly of Human HspB2 and HspB3

Abstract

Heterogeneity in small heat shock proteins (sHsps) spans multiple spatiotemporal regimes—from fast fluctuations of part of the protein, to conformational variability of tertiary structure, plasticity of the interfaces, and polydispersity of the inter-converting, and co-assembling oligomers. This heterogeneity and dynamic nature of sHsps has significantly hindered their structural characterization. Atomic coordinates are particularly lacking for vertebrate sHsps, where most available structures are of extensively truncated homomers. sHsps play important roles in maintaining protein levels in the cell and therefore in organismal health and disease. HspB2 and HspB3 are vertebrate sHsps that are found co-assembled in neuromuscular cells, and variants thereof are associated with disease. Here, we present the structure of human HspB2/B3, which crystallized as a hetero-tetramer in a 3:1 ratio. In the HspB2/B3 tetramer, the four α-crystallin domains (ACDs) assemble into a flattened tetrahedron which is pierced by two non-intersecting approximate dyads. Assembly is mediated by flexible “nuts and bolts” involving IXI/V motifs from terminal regions filling ACD pockets. Parts of the N-terminal region bind in an unfolded conformation into the anti-parallel shared ACD dimer grooves. Tracts of the terminal regions are not resolved, most likely due to their disorder in the crystal lattice. This first structure of a full-length human sHsp heteromer reveals the heterogeneous interactions of the terminal regions and suggests a plasticity that is important for the cytoprotective functions of sHsps.

Sequence characteristics responsible for protein‐protein interactions in the intrinsically disordered regions of caseins, amelogenins, and small heat‐shock proteins

“…recently determined that the ACD of the dissociated HspB1 monomer is partially unfolded, that is, conformationally labile, and concomitantly, is more chaperone active. The conformational dynamism of sHsp NTRs is apparent from the inability to observe this region in crystal structures of sHsps, except for, and only to some extent, in the HspB2/B3 tetramer …”

“…HO‐SLiMs were identified in the NTR of seven out of the nine human sHsps listed in Supporting Information A Table S5 and in four of the six soy sHsps. Equation (13) identified the sequence 4‐IIL‐6 in HspB3, which was found to patch the hydrophobic pocket formed by β‐strands 4 and 8 of its HspB2 dimer partner in the crystalline 3:1 HspB2:HspB3 heterotetramer . Table S5 lists another potential IXI motif at a similar position in the sequence of HspB5 (αB‐crystallin) with two flanking His residues, 3‐IAIHH‐7.…”

“…The sHsps are intracellular chaperones that are present in all organisms where they are the first line of defense to prevent protein unfolding and aggregation due to the destabilizing effects of aging and stress conditions such as elevated temperature . They have three functional motifs: (a) a long, disordered, NTR, (b) a shorter, disordered, charged and mobile CTR, and (c) a central, conserved β‐sheet‐rich domain called the α‐crystallin domain (ACD) . The sHsps exhibit large sequence variations within their NTR and CTR.…”

“…The sHsps exhibit large sequence variations within their NTR and CTR. The structure of the ACD has been determined for a range of species and comprises an immunoglobulin fold . In its action as a molecular chaperone, the NTR likely makes contact with unfolding proteins and stabilizes them to prevent their aggregation .…”

“…The unstructured and highly dynamic C‐terminal extension in mammalian sHsps is similar in length and polarity to the highly flexible C‐terminal sequence in amelogenins. As in amelogenins, removal of this region leads to destabilization and large‐scale aggregation of sHsps …”