Abstract
Hsp70 chaperones bind to various protein substrates for folding, trafficking, and degradation. Considerable structural information is available about how prokaryotic Hsp70 (DnaK) binds substrates, but less is known about mammalian Hsp70s, of which there are 13 isoforms encoded in the human genome. Here, we report the interaction between the human Hsp70 isoform heat shock cognate 71-kDa protein (Hsc70 or HSPA8) and peptides derived from the microtubule-associated protein Tau, which is linked to Alzheimer's disease. For structural studies, we used an Hsc70 construct (called BETA) comprising the substrate-binding domain but lacking the lid. Importantly, we found that truncating the lid does not significantly impair Hsc70's chaperone activity or allostery in vitro Using NMR, we show that BETA is partially dynamically disordered in the absence of substrate and that binding of the Tau sequence GKVQIINKKG (with a KD = 500 nm) causes dramatic rigidification of BETA. NOE distance measurements revealed that Tau binds to the canonical substrate-binding cleft, similar to the binding observed with DnaK. To further develop BETA as a tool for studying Hsc70 interactions, we also measured BETA binding in NMR and fluorescent competition assays to peptides derived from huntingtin, insulin, a second Tau-recognition sequence, and a KFERQ-like sequence linked to chaperone-mediated autophagy. We found that the insulin C-peptide binds BETA with high affinity (KD < 100 nm), whereas the others do not (KD > 100 μm). Together, our findings reveal several similarities and differences in how prokaryotic and mammalian Hsp70 isoforms interact with different substrate peptides.
Highlights
Hsp70 chaperones bind to various protein substrates for folding, trafficking, and degradation
To further develop BETA as a tool for studying Hsc70 interactions, we measured BETA binding in NMR and fluorescent competition assays to peptides derived from huntingtin, insulin, a second Tau-recognition sequence, and a KFERQ-like sequence linked to chaperone-mediated autophagy
Because the full-length 70-kDa protein is too large for detailed NMR studies, we set out to design a truncated form (Ͻ30 kDa) that would bind to TAU1 and be representative of the WT protein
Summary
The main focus of the current study is the interaction of the human Tau-derived peptide 590KVQIINKK, which we will refer to as TAU1, with human Hsc. It is known that substrate binding to prokaryotic DnaK enhances ATP hydrolysis but that this allostery is lost in human Hsc70 [11] Consistent with this idea, we found that the ATPase activity of wt Hsc and Hsc 508 are not/hardly affected by addition of NR peptide, whereas DnaK is strongly stimulated (Fig. 4D). The 70 kDa full-length Hsc does not give rise to good NMR spectra, because of dominance of the resonances of the unstructured tail [38], so we used a construct that includes NBD, BETA, and LID, without the TAIL This construct was enabled for substrate binding by including a mutation (L543Y) as suggested by the Lab of Dr. TAU1, GKVQIINKKG; insulin C-peptide (C-peptide); TAU2, VQIVYK; huntingtin, MATLEKLMKAFESLKSF. The inhibitory constant KI1 was computed from numerical integration of kinetic equations (see “Experimental procedures”); KI2 was computed from Equation 3 in Ref. 54
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