In Silico Identification of Carboxylate Clamp Type Tetratricopeptide Repeat Proteins in Arabidopsis and Rice As Putative Co-Chaperones of Hsp90/Hsp70

Abstract

The essential eukaryotic molecular chaperone Hsp90 operates with the help of different co-chaperones, which regulate its ATPase activity and serve as adaptors to recruit client proteins and other molecular chaperones, such as Hsp70, to the Hsp90 complex. Several Hsp90 and Hsp70 co-chaperones contain the tetratricopeptide repeat (TPR) domain, which interacts with the highly conserved EEVD motif at the C-terminal ends of Hsp90 and Hsp70. The acidic side chains in EEVD interact with a subset of basic residues in the TPR binding pocket called a ‘carboxylate clamp’. Since the carboxylate clamp residues are conserved in the TPR domains of known Hsp90/Hsp70 co-chaperones, we carried out an in silico search for TPR proteins in Arabidopsis and rice comprising of at least one three-motif TPR domain with conserved amino acid residues required for Hsp90/Hsp70 binding. This approach identified in Arabidopsis a total of 36 carboxylate clamp (CC)-TPR proteins, including 24 novel proteins, with potential to interact with Hsp90/Hsp70. The newly identified CC-TPR proteins in Arabidopsis and rice contain additional protein domains such as ankyrin, SET, octicosapeptide/Phox/Bem1p (Phox/PB1), DnaJ-like, thioredoxin, FBD and F-box, and protein kinase and U-box, indicating varied functions for these proteins. To provide proof-of-concept of the newly identified CC-TPR proteins for interaction with Hsp90, we demonstrated interaction of AtTPR1 and AtTPR2 with AtHsp90 in yeast two-hybrid and in vitro pull down assays. These findings indicate that the in silico approach used here successfully identified in a genome-wide context CC-TPR proteins with potential to interact with Hsp90/Hsp70, and further suggest that the Hsp90/Hsp70 system relies on TPR co-chaperones more than it was previously realized.

The Arabidopsis TETRATRICOPEPTIDE THIOREDOXIN-LIKE Gene Family Is Required for Osmotic Stress Tolerance and Male Sporogenesis

“…Recently, TTL proteins have been proposed as potential interactors of the Hsp90 and Hsp70 chaperone complexes (Prasad et al, 2010). This is based on the finding that TTLs, like confirmed Hsp90 cochaperones, such as Hsp70-Hsp90 organizing protein, high-M r immunophilins (cyclophilin 40 and FK506-binding proteins 51 and 52), protein phosphatase 5, and the C terminus of Hsc70-interacting protein, contain a subset of basic residues in the TPR-binding pocket known as the carboxylate clamp.…”

“…This is based on the finding that TTLs, like confirmed Hsp90 cochaperones, such as Hsp70-Hsp90 organizing protein, high-M r immunophilins (cyclophilin 40 and FK506-binding proteins 51 and 52), protein phosphatase 5, and the C terminus of Hsc70-interacting protein, contain a subset of basic residues in the TPR-binding pocket known as the carboxylate clamp. The carboxylate clamp domain interacts with the acidic side chains of the highly conserved EEVD motif at the C-terminal ends of Hsp90 and Hsp70 (Carrigan et al, 2006;Prasad et al, 2010).…”

“…In Arabidopsis, more than 235 proteins containing TPR motifs, usually arranged in tandem repeats of three to 16, have been identified (D'Andrea and Regan, 2003;Prasad et al, 2010). Many of these proteins show a modular structure containing additional domains that mediate interactions with downstream proteins acting as cochaperones or have enzymatic activities that amplify and transduce signals (D'Andrea and Regan, 2003;Davies and Sánchez, 2005).…”

“…Hsp90 lies at the center of a multiprotein chaperone complex that facilitates the folding of client proteins into their stable or active conformations (Krishna and Gloor, 2001). In Arabidopsis, TTL proteins have been identified as putative chaperone receptors based on the identification of conserved amino acids in their TPR motifs known as carboxylate clamps, opening the possibility that TTL proteins act in concert with Hsp90 chaperone complexes (Prasad et al, 2010). In addition, TTL3 was identified as an interacting partner of the activated (phosphorylated) cytoplasmic domain of VASCULAR HIGHWAY1/BRI1-LIKE RECEPTOR KINASE2, a receptor-like kinase of the BRI1 family with a role in vascular development (Ceserani et al, 2009).…”

AtTPR7 is a chaperone docking protein of the Sec translocon in Arabidopsis

“…In this study we describe a TPR domain-containing plantspecific protein, termed AtTPR7 (At5g21990), which was previously identified by Prasad et al (Prasad et al, 2010). The protein was previously described as a protein of the outer envelope in chloroplast [OEP61 (von Loeffelholz et al, 2011)] and proposed to function as a chaperone receptor for preproteins.…”