The co-chaperones Hop and Hip have, in addition to Hsp-binding TPR domains, a similar C-terminal region that is marked by a DP-repeat motif (). Mutations to this region functionally disrupt either Hop or Hip, but little is known about how the DP-repeat region participates in co-chaperone function. To gain a better understanding of the structural context of the DP-repeat region, we generated partial proteolytic profiles (Konigsberg 1995
) of wild-type and DP-mutant proteins.
Fig 1. Diagram of major Hop and Hip domains and shared C-terminal sequences. The Hop domains tetratricopeptide repeat 1 (TPR1) and TPR2a are important binding sites for heat shock protein 70 (Hsp70) and Hsp90, respectively. A binding partner for TPR2B has not (more ...)
Wild-type Hop and the AP2 mutant, which contains alanine substitutions for the charged DP-repeat residues at positions 492, 494, and 501 (Chen and Smith 1998
), were digested over a time course with a limiting concentration of protease. The fragments generated were separated on SDS gels and visualized using Coomassie staining. No differences in fragment patterns were observed with tryptic digests (not shown); however, parallel digestions of Hop and AP2 with subtilisin () or chymotrypsin () resulted in fragment patterns that differed at the early proteolytic time points (1 and 5 minutes) but merged into a common profile after 15 minutes. In the subtilisin-digested samples (), several large and small Hop fragments were generated after 1–5 minutes that are absent in the corresponding AP2 lanes. A similar decrease in complexity was observed when comparing Hop and AP2 chymotryptic patterns from early time points (). The reduced complexity of the early AP2 fragment profile, as seen with both subtilisin and chymotrypsin, suggests that partially resistant regions of Hop are more accessible to protease in the DP mutant. Thus, the C-terminal DP repeat may normally interact with other sequences to form a more compactly folded polypeptide.
Fig 2. Partial proteolytic digests of wild-type Hop and the AP2 mutant. Purified recombinant Hop forms were subjected to proteolysis with either subtilisin (A) or chymotrypsin (B). After the times indicated below each panel, digestion was stopped, and the resulting (more ...)
To examine the DP-repeat region more directly, we first established that the epitope for anti-Hop monoclonal antibody F5 (Smith et al 1993
) lies near the C-terminus immediately downstream from the DP repeats (). A series of Hop C-terminal truncation mutants, generated by introducing stop codons into Hop/pSPUTK, were individually expressed in rabbit RL to generate radiolabeled products for F5 immunoprecipitation trials. Samples from each synthesis reaction were separated by gel electrophoresis, and radiolabeled Hop forms were detected by autoradiography (middle panel). Equal aliquots from the synthesis mixtures were added to RL, and immunoprecipitation with F5 antibody resin was performed. Washed immunoprecipitates were separated by gel electrophoresis and stained to visualize total protein (upper panel). As indicated, the stained bands represent the endogenous Hop-Hsp90-Hsp70 complex that is abundantly precipitated from RL (Smith et al 1993
) and the F5 heavy chain (HC). The stained gel was dried and autoradiographed to detect radiolabeled Hop forms that bind to F5 (bottom panel). Truncations shorter than N537 completely lacked binding to F5, indicating that critical aa in the F5 epitope reside in the C-terminus of Hop, downstream of the DP repeats.
We next probed chymotryptic digests of Hop and AP2 with F5 to detect fragments containing this epitope (). With wild-type Hop, a protease-resistant fragment of approximately 9–10 kDa (indicated by arrow on the left) was observed after 60 minutes of digestion. In contrast, there was only a minimal recovery of F5-reactive bands after the briefest exposure of AP2 to chymotrypsin. The mutations in AP2 do not alter the F5 epitope because the full-length wild-type and mutant proteins are recognized equally by F5 immunostaining (compare 0-minute lanes in ). Therefore, the DP-repeat region, which is resistant to proteolysis in wild-type Hop becomes highly sensitive in the AP2 mutant.
Similar to the Hop studies presented in , the DP-repeat region of Hip was probed by limited proteolytic mapping (). The Hip mutant APAV2, which contains alanine substitutions for the glutamic and aspartic acid residues in 2 DPEV sequences (see ) and has altered Hsp70-binding properties (Prapapanich et al 1998
), was compared with wild-type Hip in protease assays. The chymotrypsin digestion patterns for Hip and APAV2 () are mostly similar and closely resemble the Hip chymotryptic pattern recently reported by Velten et al (2002)
. Two differences are noted when comparing wild-type and mutant Hip-digestion patterns. First, the undigested APAV2 resolves as a doublet, whereas wild-type Hip is a single band. As will be demonstrated below, the lower band of the APAV2 doublet probably represents protein in which the C-terminal DP-repeat region was cleaved away by bacterial proteases before purification of recombinant protein. A second distinction in the Hip fragment pattern is a 12-kDa fragment generated during the brief digestions (1- and 5-minute lanes). Overall, there are fewer differences in the Hip wild-type and mutant protease patterns when compared with the Hop major fragment patterns (). Similar results were obtained when Hip and APAV2 were digested with subtilisin or trypsin (results not shown).
Fig 3. Partial proteolytic digests of wild-type Hip and the APAV2 mutant. Purified recombinant proteins were digested as in the previous figure. (A) Chymotryptic fragment patterns were generated by gel separation and Coomassie staining of samples. The anti-Hip (more ...)
We identified 2G6, one of our previously developed anti-Hip monoclonal antibodies (Prapapanich et al 1996a
), as having an epitope near the C-terminus of Hip. A truncation mutant, N334, which retains the DP repeats but lacks downstream sequences, was not recognized by 2G6 (data not shown). The 2G6 and F5 epitopes are fully contained within the respective C-terminal regions of Hip and Hop because each antibody cross-reacts with the corresponding tail-swap chimera we describe in later experiments (results not shown). We used 2G6 in Western immunoblots to probe digests for proteolytic fragments containing C-terminal Hip sequences. In a chymotryptic time course (), a 12-kDa fragment was immunostained that corresponds to the fragment noted in . The 12-kDa fragment degrades further to a fragment of less than 10 kDa (indicated by arrow) after 15 minutes of digestion. No 2G6-reacting fragments were detected in any of the digested APAV2 samples, indicating that this region is rendered more sensitive to proteolysis by mutation of the DP repeats. Also, note that in the undigested APAV2 lane 2G6 reacts exclusively with the upper band of the doublet, supporting our previous contention that the lower band of the doublet lacks the C-terminal region.
Immunoblots of tryptic digests provide further support for a proteolytically resistant fragment containing the DP repeat (). Although the major tryptic fragments from Hip and APAV2 are similar (Coomassie-stained gel not shown), 2G6 detected a C-terminal Hip fragment of approximately 9–10 kDa size (indicated by arrow), which was detected at the 1-minute– through 60-minute–digestion time points. This fragment was completely absent in the APAV2 digests.
The results in and suggest that the DP repeats of Hop and Hip exist in protease-resistant conformations that could reflect structural domains. To help determine the boundaries of these putative DP-domains, we obtained partial aa sequences from chymotryptic fragments. Samples of Hop and Hip were individually digested with chymotrypsin for 15 or 60 minutes, respectively, after which fragments were separated by gel electrophoresis, transferred to PVDF membrane, and detected by Coomassie staining. Bands corresponding to the minimally sized fragments detected in and were excised and submitted for automated N-terminal sequencing. The partial sequence from the Hop fragment (NRHDSPED) matched the sequence beginning at position 477 that is 15 residues upstream from the initial DPEV. The predicted size of a Hop fragment beginning at 477 and extending to the C-terminus is 7687 Da; this correlates well with the apparent size of the chymotryptic Hop fragment. Whereas the resistant fragment retains the F5 epitope that maps within 10 aa of the C-terminus, the putative DP-domain appears to extend from immediately before the DP repeats to or very near the C-terminus. Partial aa sequence for the protease-resistant Hip chymotryptic fragment (GSFPGGFP) begins at residue 279; this sequence is at the beginning of the GGMP-repeat region (Prapapanich et al 1996a
) that immediately precedes the DP repeats. Whether the unusual GGMP repeat is itself resistant to proteolysis or falls within a putative DP-repeat domain is unclear. The predicted size of a Hip fragment extending from residue 279 to the C-terminus is 9217 Da; as in the case of the Hop fragment, this corresponds well with the apparent size of the protease-resistant Hip DP fragment and is consistent with a putative Hip DP-domain that extends to or near the C-terminus.
Truncation of the entire DP-repeat region or DP-point mutation inhibits the ability of Hop to bind to Hsp70 and to support assembly of PR complexes (Chen and Smith 1998
). To map out more precisely the extent of C-terminal aa that are functionally important for Hop, we used a series of C-terminal truncation mutants (see ) to compare coimmunoprecipitation patterns with PR, Hsp70, and Hsp90 (). Mutant cDNAs were expressed in vitro to generate radiolabeled products (as in ), and equal amounts of each product were added to RL mixtures for receptor assembly (), Hsp70 binding (), or Hsp90 binding (). Removal of 6 aa (N537) had little effect on assembly of Hop in PR complexes or in Hsp interactions. However, truncating 5 additional aa (N532) inhibited assembly with PR complexes. Defective assembly with PR correlates precisely with the loss of Hsp70 binding (); this finding is similar to our previous observations on Hsp70 binding and PR assembly by mutant AP2 (Chen and Smith 1998
). As we had observed previously with AP2, the DP region is not required for Hsp90 binding, and all truncation mutants were recovered in Hsp90 complexes at a level identical to that of full-length Hop (). The results in link sequences near the C-terminus with the upstream DP repeats in a common functional role for Hsp70 binding and PR assembly. Thus, the DP repeats contribute to a functional domain that corresponds to the protease-resistant domain encompassing the C-terminus of Hop.
Fig 4. Protein interactions of Hop C-terminal truncation mutants. (A) Radiolabeled Hop forms were synthesized by in vitro expression, and an equal amount of each form was added to reticulocyte lysate (RL) used for assembly of PR complexes. The assembly mixtures (more ...)
A single Hip C-terminal truncation mutant (N334) was tested for Hsp70 and PR interactions (results not shown). The recovery of N334 in Hsp70 complexes was similar to wild-type Hip, although there was a reduced recovery of N334 in PR complexes. This resembles what we had observed previously with the APAV2 point mutant (Prapapanich et al 1998
) and with N303 (Prapapanich et al 1996b
), a more extended C-terminal truncation mutant.
Although the Hop and Hip DP regions have significant sequence similarity and both localize to proteolytically stable fragments, we wondered whether these putative domains would be functionally interchangeable between the 2 proteins. Tail-swap chimeras, as described in the Materials and Methods, were generated and compared with wild-type proteins for recovery in Hsp and PR complexes (). To facilitate the generation of the chimeras, first it was necessary to introduce a couple of point mutations in the wild-type cDNAs, as described in the Materials and Methods. It was possible that these intermediate mutations would alter Hip or Hop function, but we observed no difference between wild-type and intermediate mutant interactions with Hsp70, Hsp90, or PR (results not shown). The Hop chimera (HopTS) bound as well as wild-type Hop to Hsp90 (). The Hip forms were not examined in this assay because Hip does not directly bind Hsp90. Both Hop and Hip bind Hsp70, so these were compared alongside the respective tail-swap chimeras for Hsp70 binding (). Relative to wild-type proteins, both chimeras are recovered at a reduced level in Hsp70 complexes. We next compared recovery of Hop, Hip, and tail-swap chimeras with PR complexes assembled in vitro (). Reflecting their reduced interactions with Hsp70, the tail-swap chimeras are impaired in their ability to assemble into PR complexes. Therefore, the DP domains of Hip and Hop are not functionally equivalent despite their structural similarities.
Fig 5. Interactions by Hop and Hip tail-swap chimeras with heat shock protein 90 (Hsp90) (A), Hsp70 (B), and progesterone receptor complexes (C). Radiolabeled test proteins were added to reticulocyte lysate (RL) before immunoprecipitation reactions. For each (more ...)
Our final analysis focuses on sequence differences within the DP repeats of Hip and Hop. As illustrated in , the first unit of the DP repeat in both proteins consists of DPEV, but the second unit is DPEV in Hip and DPAM in Hop. To address the significance of these differences, we generated mutants in which the EV and AM amino acids of the second unit were exchanged (). HopEV and wild-type Hop bound identically to Hsp90 (results not shown); however, HopEV bound Hsp70 much more weakly than wild-type Hop (). Conversely, there was little difference in binding of Hip and HipAM to Hsp70. Paralleling this observation, recovery of HopEV in PR complexes was greatly reduced, whereas HipAM behaved much like its wild-type counterpart ().
Fig 6. Binding of Hop, Hip, and corresponding DP–point mutants to heat shock protein 70 (A) and to PR complexes (B). As in the previous figures, the upper panel in each set is a Coomassie-stained gel of total proteins in the immunoprecipitates, and the (more ...)