Abstract
Small heat shock proteins (sHSPs) and the related alpha-crystallins are ubiquitous chaperones linked to neurodegenerative diseases, myopathies, and cataract. To better define their mechanism of chaperone action, we used hydrogen/deuterium exchange and mass spectrometry (HXMS) to monitor conformational changes during complex formation between the structurally defined sHSPs, pea PsHsp18.1, and wheat TaHsp16.9, and the heat-denatured model substrates malate dehydrogenase (MDH) and firefly luciferase. Remarkably, we found that even when complexed with substrate, the highly dynamic local structure of the sHSPs, especially in the N-terminal arm (>70% exchange in 5 s), remains unchanged. These results, coupled with sHSP-substrate complex stability, indicate that sHSPs do not adopt new secondary structure when binding substrate and suggest sHSPs are tethered to substrate at multiple sites that are locally dynamic, a feature that likely facilitates recognition and refolding of sHSP-bound substrate by the Hsp70/DnaK chaperone system. Both substrates were found to be stabilized in a partially unfolded state that is observed only in the presence of sHSP. Furthermore, peptide-level HXMS showed MDH was substantially protected in two core regions (residues 95-156 and 228-252), which overlap with the MDH structure protected in the GroEL-bound MDH refolding intermediate. Significantly, despite differences in the size and structure of TaHsp16.9-MDH and PsHsp18.1-MDH complexes, peptide-level HXMS patterns for MDH in both complexes are virtually identical, indicating that stabilized MDH thermal unfolding intermediates are not determined by the identity of the sHSP.
Highlights
26634 JOURNAL OF BIOLOGICAL CHEMISTRY ones associated with diverse cellular activities [1, 2]
Nothing is known about sHSP structural rearrangements that must accompany formation of sHSP-substrate complexes. sHSP subunit exchange can continue in the presence of bound substrate [12, 20], and the chaperone remains protease-accessible [21, 22], a recent study indicates that protease sites on the sHSP N-terminal arm are protected in substrate complexes [23]
malate dehydrogenase (MDH) has been used extensively as a model substrate for the chaperone GroEL [41,42,43,44] and for sHSPs, including TaHsp16.9 and PsHsp18.1 [16, 18]. sHSP-MDH complexes were formed by incubating either 24 M PsHsp18.1 or TaHsp16.9 with MDH at an sHSP/MDH molar ratio of 2.4:1 or 3:1, respectively, at 45 °C for 0 –120 min (Fig. 1, a and b). These sHSP/MDH ratios were chosen because they afford complete protection of MDH from heat insolubilization, and on a molar basis PsHSP18.1 is more effective than TaHsp16.9 [18]
Summary
26634 JOURNAL OF BIOLOGICAL CHEMISTRY ones associated with diverse cellular activities [1, 2]. Of the sHSP-substrate interaction is challenging, because of the difficulties associated with investigating heterogeneous protein mixtures exemplified by sHSP-substrate complexes [1, 2]. Both the N-terminal arm and -4 strand of the ␣-crystallin domain have been implicated as sHSP substrate binding sites, but overlap of proposed binding sites with structural elements required for sHSP oligomerization (and structural integrity) complicates data interpretation [18, 19]. Results generally agree that substrates bind when in an aggregation-prone, partially unfolded molten globule form, both early and late unfolding intermediates have been identified as binding structures
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