Abstract
In mammals, small heat-shock proteins (sHSPs) typically assemble into interconverting, polydisperse oligomers. The dynamic exchange of sHSP oligomers is regulated, at least in part, by molecular interactions between the α-crystallin domain and the C-terminal region (CTR). Here we report solution-state nuclear magnetic resonance (NMR) spectroscopy investigations of the conformation and dynamics of the disordered and flexible CTR of human HSP27, a systemically expressed sHSP. We observed multiple NMR signals for residues in the vicinity of proline 194, and we determined that, while all observed forms are highly disordered, the extra resonances arise from cis-trans peptidyl-prolyl isomerization about the G193-P194 peptide bond. The cis-P194 state is populated to near 15% at physiological temperatures, and, although both cis- and trans-P194 forms of the CTR are flexible and dynamic, both states show a residual but differing tendency to adopt β-strand conformations. In NMR spectra of an isolated CTR peptide, we observed similar evidence for isomerization involving proline 182, found within the IPI/V motif. Collectively, these data indicate a potential role for cis-trans proline isomerization in regulating the oligomerization of sHSPs.
Highlights
Small heat-shock proteins exist in all kingdoms of life and exhibit a diverse range of cellular roles from preventing protein aggregation, to upholding cytoskeletal integrity, and regulating apoptosis (Kampinga et al 2015)
Using [U-13C,15N]-HSP27, we recorded a two-dimensional (2D) 1H-15N heteronuclear singlequantum coherence (HSQC) spectrum, which correlates the chemical shifts of 1H nuclei that are covalently attached to 15N nuclei (Fig. 2a)
In order to assign the observed resonances to specific HSP27 residues, we recorded three-dimensional (3D) nuclear magnetic resonance (NMR) spectra from [U-13C, 15N]-labeled HSP27 that correlate 13C, 15N, and 1HN chemical shifts of neighboring amino acids whose nuclei are linked through a network of covalent bonds (Sattler et al 1999)
Summary
Small heat-shock proteins (sHSPs) exist in all kingdoms of life and exhibit a diverse range of cellular roles from preventing protein aggregation, to upholding cytoskeletal integrity, and regulating apoptosis (Kampinga et al 2015). Despite relatively small molecular masses, sHSPs typically assemble into large, dynamic oligomers of varying sizes and degrees of polydispersity (Mchaourab et al 2009; Hilton et al 2013b; Haslbeck and Vierling 2015). Crystal structures of non-mammalian sHSPs revealed that the IXI/V motif docks into a hydrophobic groove in the ACD of a neighboring subunit (Mchaourab et al 2009; Hilton et al 2013b; Haslbeck and Vierling 2015). Crystal structures of truncated constructs suggest that similar contacts are made in oligomeric mammalian sHSPs, and that docking of the CTR can in principle occur bi-directionally (Laganowsky et al 2010) and intra- or inter-monomer (Laganowsky and Eisenberg 2010) (Fig. 1b). It is evident that in solution these interactions are transient a Full-length HSP27
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