In silico identification and biochemical characterization of the human dicarboxylate clamp TPR protein interaction network

Dicarboxylate clamp tetratricopeptide repeat (dcTPR) motif‐containing proteins are well‐known partners of the heat shock protein (Hsp) 70 and Hsp90 molecular chaperones. Together, they facilitate a variety of intracellular processes, including protein folding and maturation, protein targeting, and protein degradation. An extreme C‐terminal sequence, the EEVD motif, is identical in Hsp70 and Hsp90, and is indispensable for their interaction with dcTPR proteins. However, almost no information is available on the existence of other potential dcTPR‐interacting proteins. We searched the human protein database for proteins with C‐terminal sequences similar to that of Hsp70/Hsp90 to identify potential partners of dcTPR proteins. The search identified 112 proteins containing a Hsp70/Hsp90‐like signature at their C termini. Gene Ontology enrichment analysis of identified proteins revealed enrichment of distinct protein classes, such as molecular chaperones and proteins of the ubiquitin–proteasome system, highlighting the possibility of functional specialization of proteins containing a Hsp70/Hsp90‐like signature. We confirmed interactions of selected proteins containing Hsp70/Hsp90‐like C termini with dcTPR proteins both in vitro and in situ. Analysis of interactions of 10‐amino‐acid peptides corresponding to the C termini of identified proteins with dcTPR proteins revealed significant differences in binding strength between various peptides. We propose a hierarchical mode of interaction within the dcTPR protein network. These findings describe a novel dcTPR protein interaction networks and provide a rationale for selective regulation of protein–protein interactions within this network. dcTPR proteins are known partners of molecular chaperones Hsp70 and Hsp90. Interaction is mediated by C‐terminal EEVD sequence of Hsp70/Hsp90. Potential partners of dcTPR proteins that contain Hsp70/Hsp90‐like C termini were identified. Most of the identified partners belong to distinct functional groups. Selected protein–protein interactions via a dicarboxylate clamp mechanism were confirmed and a hierarchical mode of interaction within this network is proposed.