Human Hsp70 Substrate-Binding Domains Recognize Distinct Client Proteins

The 13 Hsp70 proteins in humans act on unique sets of
substrates
with diversity often being attributed to J-domain-containing protein
(Hsp40 or JDP) cofactors. We were therefore surprised to find drastically
different binding affinities for Hsp70-peptide substrates, leading
us to probe substrate specificity among the 8 canonical Hsp70s from
humans. We used peptide arrays to characterize Hsp70 binding and then
mined these data using machine learning to develop an algorithm for
isoform-specific prediction of Hsp70 binding sequences. The results
of this algorithm revealed recognition patterns not predicted based
on local sequence alignments. We then showed that none of the human
isoforms can complement heat-shocked DnaK knockout Escherichia coli cells. However, chimeric Hsp70s
consisting of the human nucleotide-binding domain and the substrate-binding
domain of DnaK complement during heat shock, providing further evidence
in vivo of the divergent function of the Hsp70 substrate-binding domains.
We also demonstrated that the differences in heat shock complementation
among the chimeras are not due to loss of DnaJ binding. Although we
do not exclude JDPs as additional specificity factors, our data demonstrate
substrate specificity among the Hsp70s, which has important implications
for inhibitor development in cancer and neurodegeneration.