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Identification of substrate binding sites on a small heat shock protein reveals a unique mode of interaction between differentially aggregating substrates
Author(s) -
Jaya Nomalie,
Garcia Victor,
Vierling Elizabeth
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.673.6
Subject(s) - substrate (aquarium) , chemistry , proteolysis , biochemistry , protein–protein interaction , biophysics , linker , biology , enzyme , ecology , computer science , operating system
Small heat shock proteins (sHSPs) function as molecular chaperones by preventing irreversible protein aggregation. The mechanism of sHSP‐substrate interaction is not clearly understood, due to the difficulty of studying the heterogeneous complex formed between sHSPs and denaturing substrates. Using the photoactivatable cross‐linker, p‐benzoyl‐L‐phenylalanine (Bpa), site‐specifically incorporated into the well‐characterized sHSP from pea, PsHsp18.1, we show for the first time direct interaction between a sHSP and substrate in sHSP‐substrate complexes. Model substrates firefly luciferase (Luc), malate dehydrogenase (MDH) and a putative native substrate fructose bisphosphate aldolase (FBPA) were used in these experiments. Our results show that the N‐terminal arm of PsHsp18.1 makes direct contacts with all three substrates strengthening the relationship between this evolutionary variable region and substrate protection. Interestingly, Luc, a fast aggregating substrate, makes more contacts with the N‐terminal arm and limited contacts in the structurally conserved α‐crystallin region. However, MDH and FBPA, slow aggregating substrates, make direct contacts not only with the N‐terminal domain but also with the α‐crystallin region of the sHSP. These data lead us to propose a model in which the N‐terminal arm makes intial contacts with the hydrophobic regions of the unfolding substrate, but over time those contacts can be altered to include new contacts in the α‐crystallin region. This rearrangement may be substrate dependent and critical to the architecture of the soluble sHSP‐substrate complex. We are using chemical modification and limited proteolysis to gain further insight into the organization of sHSP and susbtrate within the complex.