Abnormal Assemblies and Subunit Exchange of αB-Crystallin R120 Mutants Could Be Associated with Destabilization of the Dimeric Substructure

Abstract

Mutation of the Arg120 residue in the human alphaB-crystallin sequence has been shown to be associated with a significant ability to aggregate in cultured cells and have an increased oligomeric size coupled to a partial loss of the chaperone-like activity in vitro. In the present study, static and dynamic light scattering, small-angle X-ray scattering, and size exclusion chromatography were used to follow the temperature and pressure induced structural transitions of human alphaB-crystallin and its R120G, R120D, and R120K mutants. The wild type alphaB-crystallin was known to progressively increase in size with increasing temperature, from 43 to 60 degrees C, before aggregating after 60 degrees C. The capacity to increase in size with temperature or pressure, while remaining soluble, had disappeared with the R120G mutant and was found to be reduced for the R120K and R120D mutants. The R120K mutant, which preserves the particle charge, was the less impaired. The deficit of quaternary structure plasticity was well correlated with the decrease in chaperone-like activity previously observed. However, the mutant ability to exchange subunits, measured with a novel anion exchange chromatography assay, was found to be increased, suggesting subtle relationships between structural dynamics and function. From molecular dynamic simulations, the R120 position appeared critical to conserve proper intra- and intersubunit interactions. In silico mutagenesis followed by simulated annealing of the known small heat shock protein 3D structures suggested a destabilization of the dimeric substructure by the R120 mutations. The whole of the results demonstrated the importance of the R120 residue for structural integrity, both static and dynamic, in relation with function.

The pivotal role of the β7 strand in the intersubunit contacts of different human small heat shock proteins

“…It is worthwhile to mention that mutation of the neighboring residues often leads to significant changes in the structure and properties of sHsp. For instance, mutation R120G results in the change of oligomeric structure, chaperone-like activity, and intersubunit interaction of αB-crystallin (Kumar et al 1999;Perng et al 1999;Simon et al 2007;Michiel et al 2009;Bova et al 1999). Similar effects were observed in the case of HspB8 where mutations K137E and/or K141E were also accompanied by significant changes in the structure and properties (Kasakov et al 2007;Kim et al 2006).…”

“…In order to check this suggestion, we introduced Cys residues into the structure of human αB-crystallin, HspB6 and HspB8 in a position homologous to that of Cys141 of mouse Hsp25 (or Cys137 of human HspB1). The human wild-type αB-crystallin does not contain intrinsic Cys residues and the multiple alignment of human sHsp (Kasakov et al 2007;Michiel et al 2009;Bagneris et al 2009) indicates that Glu117 of αB-crystallin is in a position homologous to that of Cys137 of HspB1. Therefore, we introduced the single-mutation E117C into the structure of human αB-crystallin (Fig.…”

“…1a). HspB6 contains the single Cys46 in the variable N-terminal end and Glu116 in a position homologous to Cys137 of HspB1 (Kasakov et al 2007;Michiel et al 2009;Bagneris et al 2009). To exclude the effect of Cys46 of HspB6 on the subunit interaction we replaced this residue by Ser and mutated Glu116 to Cys, thus obtaining the so-called Cysmutant (C46S/E116C) of HspB6 (Fig.…”

“…Destabilization of intersubunit contacts of HspB8 can be due to at least two factors. Firstly, this protein is lacking conservative hydrophobic stretch I-X-I/ V in the variable C-terminal tail, and secondly the multiple alignment (Michiel et al 2009;Bagneris et al 2009) indicates that the primary structure of HspB8 makes impossible formation of β2 strand of α-crystallin domain. Both the hydrophobic stretch in the C-terminal tail and β2 strand might participate in formation of intersubunit contacts (Stamler et al 2005;Bagneris et al 2009).…”

“…Up until now, the structure of only three sHsp, namely Methanococcus jannaschii Hsp16.5 (Kim et al 1998), wheat Triticum aestivum Hsp16.9 (van Monfort et al 2001) and Tsp36 of parasitic flatworm Taenia saginata (Stamler et al 2005) was described in the literature. Although the primary structure of human sHsp is similar to that of sHsp isolated from other species, there are certain important differences both in the conserved α-crystallin domain and in the variable N-and C-terminal ends (van Monfort et al 2001;Mchaourab et al 2009;Kasakov et al 2007;Michiel et al 2009). This complicates direct comparison of the structure of already crystallized sHsp (Kim et al 1998;van Monfort et al 2001;Stamler et al 2005) with their human counterparts.…”