The N-terminal arm of small heat shock proteins is important for both chaperone activity and substrate specificity.
Small heat shock proteins (sHSPs) are a ubiquitous class of molecular chaperones that interacts with substrates to prevent their irreversible insolubilization during denaturation. How sHSPs interact with substrates remains poorly defined. To investigate the role of the conserved C-terminal alpha-crystallin domain versus the variable N-terminal arm in substrate interactions, we compared two closely related dodecameric plant sHSPs, Hsp18.1 and Hsp16.9, and four chimeras of these two sHSPs, in which all or part of the N-terminal arm was switched. The efficiency of substrate protection and formation of sHSP-substrate complexes by these sHSPs with three different model substrates, firefly luciferase, citrate synthase, and malate dehydrogenase (MDH) provide new insights into sHSP/substrate interactions. Results indicate that different substrates have varying affinities for different domains of the sHSP. For luciferase and citrate synthase, the efficiency of substrate protection was determined by the identity of the N-terminal arm in the chimeric proteins. In contrast, for MDH, efficient protection clearly required interactions with the alpha-crystallin domain in addition to the N-terminal arm. Furthermore, we show that sHSP-substrate complexes with varying stability and composition can protect substrate equally, and substrate protection is not correlated with sHSP oligomeric stability for all substrates. Protection of MDH by the dimeric chimera composed of the Hsp16.9 N-terminal arm and Hsp18.1 alpha-crystallin domain supports the model that a dimeric form of the sHSP can bind and protect substrate. In total, results demonstrate that sHSP-substrate interactions are complex, likely involve multiple sites on the sHSP, and vary depending on substrate.
Basha
Eman
E
Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721, USA.
Friedrich
Kenneth L
KL
Vierling
Elizabeth
E
D000595:Amino Acid Sequence; D002950:Citrate (si)-Synthase / Q000378:metabolism; D019281:Dimerization; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006358:Hot Temperature; D008156:Luciferases / Q000378:metabolism; D008291:Malate Dehydrogenase / Q000378:metabolism; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D050506:Mutant Chimeric Proteins / Q000138:chemical synthesis / Q000378:metabolism / Q000502:physiology; D018532:Pisum sativum / Q000737:chemistry / Q000235:genetics; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D012995:Solubility; D013379:Substrate Specificity; D014908:Triticum / Q000737:chemistry / Q000235:genetics; D038201:alpha-Crystallins / Q000502:physiology
C101303:HSP16.9 protein, Triticum aestivum; C489905:HSP18 protein, plant; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D050506:Mutant Chimeric Proteins; D010446:Peptide Fragments; D010940:Plant Proteins; D038201:alpha-Crystallins; D008291:Malate Dehydrogenase; D008156:Luciferases; D002950:Citrate (si)-Synthase