The structure of the tetratricopeptide repeats of protein phosphatase 5: implications for TPR-mediated protein-protein interactions

Abstract

The tetratricopeptide repeat (TPR) is a degenerate 34 amino acid sequence identified in a wide variety of proteins, present in tandem arrays of 3-16 motifs, which form scaffolds to mediate protein-protein interactions and often the assembly of multiprotein complexes. TPR-containing proteins include the anaphase promoting complex (APC) subunits cdc16, cdc23 and cdc27, the NADPH oxidase subunit p67 phox, hsp90-binding immunophilins, transcription factors, the PKR protein kinase inhibitor, and peroxisomal and mitochondrial import proteins. Here, we report the crystal structure of the TPR domain of a protein phosphatase, PP5. Each of the three TPR motifs of this domain consist of a pair of antiparallel α-helices of equivalent length. Adjacent TPR motifs are packed together in a parallel arrangement such that a tandem TPR motif structure is composed of a regular series of antiparallel α-helices. The uniform angular and spatial arrangement of neighbouring α-helices defines a helical structure and creates an amphipathic groove. Multiple-TPR motif proteins would fold into a right-handed super-helical structure with a continuous helical groove suitable for the recognition of target proteins, hence defining a novel mechanism for protein recognition. The spatial arrangement of α-helices in the PP5-TPR domain is similar to those within 14-3-3 proteins.

mSmile is Necessary for Bronchial Smooth Muscle and Alveolar Myofibroblast Development

“…TPR repeats are found in proteins with various cellular functions, including cell cycle control, protein transport, and protein dephosphorylation (Blatch and Lassle, 1999). TPR domains participate in protein-protein interactions with TPR-containing and SH2 domain-containing proteins (Das et al, 1998).…”

Yeast ortholog of the Drosophila crooked neck protein promotes spliceosome assembly through stable U4/U6.U5 snRNP addition

“…The first crystal structure of a TPR protein, a three repeat segment of the PP5 protein phosphatase, was recently solved (Das et al+, 1998)+ The authors of this study (Das et al+, 1998) predict that clustered TPR elements interact to form a right-handed super-helix well suited to act as a platform for the assembly of protein complexes+ Based on its highly repetitive TPR structure and the experimental data presented here, we propose that Clf1p serves as such a multifaceted platform and drives splicing complex assembly by binding multiple spliceosomal proteins+ While unique in its repetitive simplicity, Clf1p serves as a valuable model for how other TPR proteins might punctuate spliceosome assembly through the parallel recruitment of transacting factors to their multiple TPR surfaces+…”

“…The Clf1p binding site is within the amino terminus of Prp40p+ Two WW domain elements are present in the amino terminus of Prp40p (Kao & Siliciano, 1996) and have been suggested as possible sites of BBP/SF1 interaction (Abovich & Rosbash, 1997)+ This region of Prp40p is unnecessary for Clf1p interaction, however (Table 1)+ Clf1p does not interact with the isolated WW domain (Prp40p-WW)+ In addition, TPR elements are believed to bind alpha helical surfaces in their target proteins (e+g+, see Tzamarias & Struhl, 1995;Das et al+, 1998), three of which are predicted in the N-terminal half of Prp40p downstream of the WW region+ Removal of the WW domain from the amino half of Prp40p only slightly reduces the level of lacZ reporter gene transactivation when these three helical segments are present (Prp40p-HI, II, III)+ However, deletion of the first of these helices (Prp40p-HII,III), significantly reduces the level of transactivation, suggesting that helix I may form a portion of the Clf1p recognition domain+ Both halves of Clf1p interact with Prp40p, although not as well as the full-length Clf1p+ Presumably Prp40p binds Clf1p through contacts that fall on either side of (and perhaps FIGURE 6. Affinity purification of Clf1p-defective splicing complexes+ Biotin-substituted RP51A (lanes 1-4) or RP51A without biotin (lane 5) was incubated for 45 min under standard splicing conditions in the Clf1p-complete extract (lane 4) or in an extract depleted of Clf1p prior to preparation by transcriptional repression of GAL1::CLF1 (lanes 1-3, 5)+ Splicing complexes (Bound) were recovered by streptavidin chromatography and then assayed by Northern blot for their snRNA contents+ In addition, the total unfractionated snRNA (Total) and the snRNAs remaining unselected after streptavidin chromatography (Unbound) were assayed for the Clf1p-depleted samples+ The location of the spliceosomal snRNAs are indicated by arrow heads on the left+ The high level of reporter gene transactivation suggests, but does not prove, that the interactions of Clf1p with Mud2p and Prp40p are direct+ Additional support for this interpretation is provided by the observation that in vitro-synthesized Mud2p and Prp40p bind a hemagglutinin-tagged version of Clf1p (i+e+, Clf1HAp)+ An antihemagglutinin specific antibody, HA+11, precipitates CLF1HAp but not Mud2p, Prp40p, or a luciferase control protein (Fig+ 8A)+ However, when the in vitro translated proteins are mixed prior to immune precipitation, Mud2p and Prp40, but not the luciferase control coimmune precipitate with the Clf1HAp protein+ The Mud2p/Prp40p/Clf1HAp coprecipitation was maximal at 100 mM NaCl; at 200 mM NaCl, lowered levels of all FIGURE 8.…”

Two mutant forms of the S1/TPR-containing protein Rrp5p affect the 18S rRNA synthesis in Saccharomyces cerevisiae

“…Rrp5p TPR motifs could be implicated in protein-protein interactions (Lamb et al+, 1995)+ The crystal structure of the TPR repeats was recently resolved (Das et al+, 1998) and revealed a spatial arrangement of one TPR unit in two antiparallel a-helices encompassing the A and B domains reported in Figure 2D+ The a-helices A and B were shown to constitute the inside and outside faces of the TPR repeats respectively, thereby indicating that the conserved residues of the B domain are probably essential residues to mediate protein-protein interactions (Das et al+, 1998)+ The rrp5⌬6 allele encodes a polypeptide deleted for two amino acids in TPR unit 1 (alanine 1494 and arginine 1495) which are conserved residues of the B domain of the TPR motif (Fig+ 2D)+ Therefore, the rrp5⌬6 allele could encode a defective protein-binding factor at restrictive temperature (37 8C) that would be impaired in the recruitment of important component(s) of the maturation process+ As already mentioned, a large number of trans-acting factors other than Rrp5p are required for the same rRNA-processing step (Venema & Tollervey, 1995)+ The Rok1p protein is an attractive candidate to interact with Rrp5p since it was isolated in this study as an extragenic multicopy and low-copy suppressor of the rrp5⌬6 temperature-sensitive mutation, and was also previously isolated with the same synthetic lethality screening used to isolate RRP5 (Venema & Tollervey, 1996;Venema et al+, 1997)+ Rrp5p and Rok1p tightly participate in rRNA processing at step A 2…”