Insights into small heat shock protein and substrate structure during chaperone action derived from hydrogen/deuterium exchange and mass spectrometry.
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.
Cheng
Guilong
G
Department of Chemistry, University of Arizona, Tucson, Arizona 85721, USA.
Basha
Eman
E
Wysocki
Vicki H
VH
Vierling
Elizabeth
E
D000818:Animals; D018834:Chaperonin 60 / Q000737:chemistry / Q000378:metabolism; D003903:Deuterium / Q000737:chemistry* / Q000378:metabolism; D041961:Deuterium Exchange Measurement / Q000379:methods; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000378:metabolism; D018532:Pisum sativum / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013552:Swine; D014908:Triticum / Q000737:chemistry
D018834:Chaperonin 60; D029968:Escherichia coli Proteins; C101303:HSP16.9 protein, Triticum aestivum; C489905:HSP18 protein, plant; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D046912:Multiprotein Complexes; D010940:Plant Proteins; D011994:Recombinant Proteins; D003903:Deuterium; D008291:Malate Dehydrogenase; C063842:dnaK protein, E coli