©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Hsc70-binding Peptides Selected from a Phage Display Peptide Library that Resemble Organellar Targeting Sequences (*)

(Received for publication, February 15, 1995; and in revised form, May 23, 1995)

Ivone M. Takenaka (1)(§) Sau-Mei Leung (2) Stephen J. McAndrew (1) Joseph P. Brown (3) Lawrence E. Hightower (2)

From the  (1)Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543, the (2)Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269-3044, and (3)Pathogenesis, Inc., Seattle, Washington 98119

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A 15-mer phage display random peptide library was screened with purified bovine Hsc70, and nucleotide sequence analysis of the selected clones showed a large enrichment for peptides containing basic sequences with at least KK, KR, or RR. Binding affinity for Hsc70 of representative peptides increased dramatically for heptamers compared with hexamers. The peptide NIVRKKK had the highest affinity for Hsc70, and substitution analyses showed that hydrophobic residues followed by basic residues play important roles in maintaining this affinity. In contrast, NIVRKKK was a weaker stimulator of the Hsc70 ATPase activity compared with pigeon cytochrome c peptide and FYQLALT, a peptide optimized for binding to Hsc70. FYQLALT effectively blocked the binding of NIVRKKK to Hsc70, possibly by causing a conformational change that masked Hsc70's binding site for the basic peptide. Two hypotheses are offered to explain the two different peptide motifs. First, it is proposed that Hsc70 recognizes two different amino acid sequence motifs in its dual roles of chaperoning proteins to organelles (NIVRKKK-like sequences) and facilitating protein folding (FYQLALT-like sequences). Second, the NIVRKKK motif may be used to bind certain folded proteins with which Hsc70 interacts, such as itself, p53, and Dnaj2.


INTRODUCTION

The 70-kilodalton heat shock cognate protein (Hsc70) is a constitutively expressed member of a family of peptide/unfolded protein-stimulated ATPases, the Hsp70 family of molecular chaperones. It can distinguish between native and unfolded forms of the same protein(1, 2) . Recent biochemical studies of a Hsc70 fragment generated using recombinant DNA technology have located the domain that binds unfolded proteins between residues 384 and 543(3) . This is the same region that was previously suggested to contain a human class I histocompatibility antigen-like peptide-binding domain based on secondary structure analyses and computer modeling (reviewed in (4) ).

Pelham (5) suggested that Hsp70 family proteins may distinguish unfolded from folded proteins based on the display of aliphatic amino acid residues normally buried inside folded proteins. Studies of peptides selected from a pool of random sequences by BiP (^1)(Grp78), the family member located in the endoplasmic reticulum, indicate that the peptide-binding site holds aliphatic residues in an environment energetically equivalent to the hydrophobic interior of folded proteins and that the minimum sequence length for effective binding to BiP is seven amino acid residues(6) . Analyses of two-dimensional NMR spectra indicate that DnaK, the bacterial homologue of Hsp70, binds peptides in an extended conformation with two large aliphatic residues having the strongest interactions with DnaK(7) .

Two groups have used affinity panning of f1 phage peptide display libraries to obtain more detailed information on the binding specificity of Hsp70 family members. Gragerov et al.(8) used DnaK to screen a library and found that peptides such as NRLLLTG containing internal aliphatic residues are preferred substrates, that inclusion of a basic residue is beneficial, and that peptides with acidic residues are disfavored. The high affinity BiP-binding peptides selected from display libraries by Gething and co-workers (9) contained a minimum of seven residues and had the motif Hy(W/X)HyXHyXHy where Hy is a large hydrophobic residue, W is tryptophan, and X is any amino acid except charged residues, which are not favored in BiP-binding peptides. Subsequently, both groups compared the binding of DnaK, Hsc70, and BiP to sets of peptides. Gragerov and Gottesman (10) found that DnaK and Hsc70 are more similar to each other in their relative affinities for a set of three peptides than to BiP. They have the highest affinities for a peptide (NLLRLTG) containing two large hydrophobic internal residues followed by a basic residue, which fits the same pattern as the highest affinity peptide for Hsc70 (NIVRKKK) described herein. Of the three Hsp70s, BiP was most tolerant of acidic residues. Fourie and co-workers (11) extended this comparison by showing that these three Hsp70 family proteins can have both common and exclusive binding specificities depending on the peptide sequence. A convincing example of this was a comparison of the peptide KYLMFKT, which binds all three proteins with similar relative affinities, and peptide KKLMFKT, which binds with affinities in the order DnaK > Hsc70 BiP.

The fact that all three Hsp70 family proteins require substrates containing large hydrophobic internal residues fits well with their proposed roles as molecular chaperones capable of distinguishing unfolded and native conformations of the same protein. However, several kinds of chaperoning have been proposed that may require these proteins to recognize different binding sites. For example, Hsp70 family proteins may very transiently bind hydrophobic sequences in nascent polypeptides to delay folding until the entire polypeptide is synthesized or to prevent aggregation of a denatured protein. A different function, the chaperoning of proteins from cytoplasmic sites of synthesis to organelles such as the endoplasmic reticulum, mitochondria, lysosomes, and nuclei, may require more stable associations with Hsp70 family proteins requiring binding to different amino acid sequences. Indeed, mitochondrial Hsp70 can bind to both the mitochondrial targeting sequence and to mature segments of unfolded precursor proteins during membrane translocation(12) . And apart from chaperoning, the binding sites for Hsp70 family proteins on folded proteins such as p53 are not hydrophobic and must involve a different type of interaction. Herein, we identify sequences with both hydrophobic and basic properties selected by affinity panning of f1 phage peptide display libraries using purified bovine Hsc70.


EXPERIMENTAL PROCEDURES

Library Construction

Hexamer and 15-mer phage display random peptide libraries were constructed using the fUSE-5 vector, kindly provided by G. Smith (University of Missouri)(13, 14, 15) . fUSE-5 replicative form DNA was purified from K802 Escherichia coli and linearized by SfiI digestion, and the resulting vector DNA was ligated to synthetic DNA fragments containing complementary BglI termini. These duplex oligonucleotides contained (NNM)(6) or (NNM) triplets in the coding strand, where N corresponds to equivalent amounts of A, T, G, and C and where M corresponds to equivalent amounts of G and T (minimizes occurrence of translational stop codons). The noncoding strand consisted of 18 or 45 residue stretches of the inosine nucleotide analog(16) . Recombinant DNA was electroporated into MC1061 E. coli electrocompetent cells, spread on 94 plates (24 24 cm) with NZamine-yeast extract (NZY) agar plus 40 µg/ml tetracycline (tet) and incubated overnight at 37 °C. Phage were collected by polyethylene glycol precipitation and purified on cesium chloride gradients. The number of transducing units (TU) was determined by infecting K91Kan^R cells with an aliquot from each library and plating on tet plates. fUSE5-vector DNA containing an insert restores the reading frame of the pIII gene product, enabling the phage to infect K91Kan^R cells and produce colonies on tet plates.

DNA Sequencing

Phage particles were prepared from individual clones, and DNA was routinely extracted using the automated Autogen 540 machine (ISS-Autogen). DNA was either manually sequenced using Sequenase 2.0 (U. S. Biochemical Corp.) or submitted to automated sequence analysis (Applied Biosystems Inc.) using an oligonucleotide primer corresponding to a region 100 nucleotides upstream of the fUSE-5 cloning site. DNA sequences were analyzed using MacVector software (Kodak Scientific Imaging Systems), and the deduced amino acid sequences were obtained.

Panning of Random Peptide Libraries

Enrichment and amplification of the library using a target protein was carried out as described previously with minor modifications(13) . Briefly, 30 µg of bovine Hsc70, kindly provided by S. Sadis (2) or 20 µg of streptavidin (Life Technologies, Inc.) were added to a 60-mm plate containing 1 ml of 0.1 M NaHCO(3), pH 7.0 or 9.2, for Hsc70 or streptavidin, respectively. The plate was gently rocked and incubated overnight at 4 °C. Unbound protein was removed by rinsing with TBS (150 mM NaCl, 50 mM Tris-HCl, pH 7.5), and the plate was incubated with blocking solution containing 30 mg/ml bovine serum albumin (BSA), 0.02% sodium azide in 0.1 M NaHCO(3) pH 7.0 for 1 h at room temperature. After a quick rinse, 1 ml of TBST (TBS plus 0.5% Tween 20) containing 5 10^9 TU of the original random peptide library was added to the plate and incubated for 30 min at room temperature followed by extensive washes with TBST. Bound phage were eluted with 0.1 N glycine-HCl, pH 2.5, containing 1 mg/ml BSA. The pH of the eluate was immediately neutralized with 1 M Trizma and then incubated for 15 min at room temperature with an equal volume of freshly prepared competent K91Kan^R cells. Luria broth containing 0.2 µg/ml of tet was added to the cultures and incubated for 1 h at 37 °C. Infected cultures were then spread on 24 24-cm NZamine-yeast extract tet plates and incubated overnight at 37 °C for amplification. This enriched library was subjected to two additional rounds of pannings using about 10 TU/round. Competent K91Kan^R cells were freshly prepared as follows(15) . 20 ml of NZamine-yeast extract broth containing 100 µg/ml kanamycin were inoculated with 100 µl of an overnight K91Kan^R cell culture and grown to A = 0.45. Cells were pelleted at 1000 g for 10 min and gently resuspended in 20 ml of 80 mM NaCl. Cells were gently shaken at 37 °C for 40 min, pelleted as described above, and gently resuspended in 1 ml of 80 mM NaCl, 50 mM NH(4)H(2)PO(4), pH 7.0.

Binding Specifity of Peptide-bearing Phage to a Target Protein

Phage samples were prepared from single tet-resistant colonies obtained by infecting K91Kan^R cells with an aliquot of the Hsc70-enriched library. These phage were tested for binding to Hsc70, as well as to several different proteins in a 96-well plate using the procedure described for panning but in a small scale. Briefly, about 1 µg of protein (Hsc70, streptavidin, BSA, actin, or polymerized tubulins) were added to respective wells of a 96-well plate containing 100 µl of 0.1 M NaHCO(3), pH 7.0, and incubated overnight at 4 °C. Following blocking and rinsing, 10^8 TU of phage were added to each well and incubated for 30 min at room temperature. Washes and elutions were performed as described for panning procedures. 20 µl of K91Kan^R cells, prepared as described above, were incubated with 20 µl of eluted phage solution for 15 min at room temperature. Infected cells were diluted to 200 µl with Luria broth plus 0.2 µg/ml tet and incubated as described above. 20 µl of each culture were spotted on tet plates. Hsc70-binding phage were also tested for competition with apocytochrome c protein and by specific elution with 1 µM ATP and 0.5 µM deoxyspergualin(17) . Qualitative results were determined by the relative number of colonies on tet plates.

In Vitro Peptide Competition Assays

The binding specificity of synthetic peptides to Hsc70 was determined as described with minor modifications(18) . Briefly, a pigeon cytochrome c peptide (Pc) (IFAGIKKKAERADLIAYLKQATAK) was radiolabeled with NaB[^3H](4) (15 Ci/mmol) by reductive methylation. Competition assays were carried out using highly purified (>95%) Hsc70 (1 µM) in a reaction mixture containing binding buffer (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1 mM EDTA), 0.9 µM of [^3H]Pc (K = 8 µM; specific activity, 182 µCi/mmol), (^2)5 µg of BSA, and various concentrations (0-1 mM or otherwise specified) of nonlabeled synthetic peptide in a total volume of 50 µl and incubated at 37 °C for 30 min. The complexes were then passed through Sephadex G-50 spin columns (Pharmacia Biotech Inc.). Quantitative binding of the radiolabeled peptide to Hsc70 present in the eluate was determined by scintillation counting. The relative binding affinities of these peptides were determined as the concentration of peptide necessary to inhibit 50% of the binding of [^3H]Pc to Hsc70 (IC).

ATPase Activity

Peptide-stimulated ATPase activity of Hsc70 was determined by measuring the release of radioactive inorganic phosphate (P(i)) from [-P]ATP (2) in the presence of increasing concentrations of peptides. An additional assay (peptide without Hsc70) was set up to determine the background P(i) release. All the samples were immediately transferred from ice to 37 °C. After 30 min of incubation, an 8-µl aliquot was withdrawn from each sample, and the radioactivity present was determined by liquid scintillation counting. The cpm values were subtracted from the background and converted to pmol of P(i) released into the supernatant. The pmol of P(i) released value was then converted to specific ATPase activity (pmol/min/µg), which was used to represent the rate of ATP hydrolysis at each peptide concentration. Fold increase of Hsc70 ATPase activity at each peptide concentration was calculated by normalizing the specific ATPase activity at each peptide concentration to the basal ATPase activity (Hsc70 without peptide). All assays were done at least twice, and the error bars on data points were less than 7%, except for Pc, which was less than 12%.

Synthetic Peptides

Peptides were synthesized by solid phase synthesis using the Fmoc (N-(9-fluorenyl)methyloxycarbonyl)-t-butyl amino acid system (Keck Foundation, Yale University). Synthetic peptides were analyzed by high performance liquid chromatography and mass spectroscopy. Concentrations of peptide stock solutions were determined by amino acid analyses.

Gel Electrophoresis and Silver Staining

Nonreducing, nondenaturing continuous polyacrylamide gel electrophoresis (native gel) was done using a modified procedure of Laemmli(19) . Gel sample buffer was added to samples to a final concentration of 62.5 mM Tris-HCl, pH 6.8, 10% (v/v) glycerol. Samples were then analyzed in a 6% (w/v) acrylamide, 0.16% (w/v) bis-acrylamide mini-slab gel at a constant voltage (200 V) for 60 min at room temperature in precooled run buffer (25 mM Tris base, 192 mM glycine) using a Bio-Rad mini-gel apparatus (Bio-Rad Laboratories). The gels were then fixed in 30% methanol, 10% acetic acid and stained according to a published procedure(20) .


RESULTS

Library Characterization

Both the 6-mer and the 15-mer phage display random peptide libraries were constructed and fully characterized. The 6-mer library contained 8.6 10^8 clones and 2.5 10 TU/ml. The 15-mer library contained 5.7 10^8 clones and 2.5 10 TU/ml. Because the HPQ sequence had been selected from several different peptide libraries when panned with streptavidin(16, 21) , we decided to use this system as a control for our libraries. Indeed, following three rounds of enrichment and amplification, the 15-mer library was enriched for peptides containing the HPQ epitope in a sequence such as LAYWEFVFHPQGDDL and the epitope PWAWLT(I). This latter sequence was also enriched in our 6-mer streptavidin-selected library and by others (22) . (^3)

Amino Acid Distribution in the 15-mer Original Library

Because only the 15-mer phage display random peptide library was used for the pannings described in this report, only its extended characterization is described. In order to closely approximate the frequency of amino acids encoded in this library, DNA from 100 individual tet-resistant clones derived from K91Kan^R cells infected with an aliquot of this library was analyzed, and peptide sequences were determined as described under ``Experimental Procedures.'' Fig. 1A shows the distribution of amino acids as a percentage of total. Uncharged, polar amino acids such as serine and glycine or hydrophobic amino acids such as leucine and valine were the most abundant amino acids in this library (geq8%). Glutamine and lysine were very rare (<2%). In order to eliminate the bias inherent in codon usage, the frequency of each amino acid was divided by the number of different codons encoding that particular amino acid. As shown in Fig. 1B, after this adjustment, the apparent bias for hydrophobic amino acids encoded by this library was eliminated. Except for glutamine and lysine, which still held as the least abundant amino acids at 0.4 and 0.6%, respectively, all other amino acids were uniformly distributed (ranging from 1 to 3%) in this library.


Figure 1: Amino acid distribution in the original 15-mer random peptide library. A, a total of 100 peptide sequences from the original library were obtained as described under ``Experimental Procedures'' and analyzed. Out of 1500 possible amino acids, 170 were not determined. The percentage of total for each amino acid was determined. B, the frequency of each amino acid was divided by its respective codon redundancy.



Hsc70-binding Peptides

About 30 µg of highly purified bovine Hsc70 was used for panning the 15-mer phage display random peptide library. Table 1shows 15-mer peptides most frequently isolated after three rounds of selection for binding to Hsc70 and amplification. Out of 97 clones sequenced, peptides containing short highly basic sequences with at least KK, KR, or RR were greatly enriched. Taking into account codon usage, lysines, histidines, and aspartic acid were highly enriched in the Hsc70 15-mer library. On the other hand, the ratio between the frequencies of amino acids in the Hsc70-enriched library to those in the original library indicated that lysine was extremely enriched in the Hsc70 library (Fig. 2). In order to confirm the binding specificity for Hsc70, phage preparations from 12 individual colonies obtained from infections of K91Kan^R cells with Hsc70-enriched library were tested for binding to Hsc70 and to other proteins, such as BSA, actin, and streptavidin, as well as for ATP release and competition by deoxyspergualin from Hsc70, as described under ``Experimental Procedures.'' Based on the number of colonies on tet plates, qualitative results clearly showed that these phage bound very poorly to other proteins (10-20%) relative to Hsc70 and that 70-80% of TU were released by 1 µM ATP or 0.5 µM deoxyspergualin. (data not shown).




Figure 2: Amino acid distribution in the Hsc70-selected 15-mer library. Results were determined based on 97 Hsc70 binding 15-mer peptide sequences. The bar graph shows the ratios of the amino acid frequencies in the Hsc70-selected library to those in the original library.



Based on these results, we synthesized some peptides derived from a representative of the Hsc70 binding sequences most frequently present in this enriched library. These peptides were then tested for binding to Hsc70 in competition experiments with [^3H]Pc peptide (K = 8 µM) as described under ``Experimental Procedures.'' The effect of the peptide length was tested because previous reports showed that heptamers bound better to BiP protein than did smaller peptides(6) . As shown in Table 2, the first set of hexamers, P1, P2 and P9, had low binding affinities to Hsc70 with an IC in the millimolar range. Comparisons between the binding affinities of Hsc70 for P1 versus P8 and P2 or P9 versus P7 clearly indicated the preference of Hsc70 for heptamers over hexamers. Overall, Hsc70-binding heptamers showed binding affinities ranging from about 3 to 250 µM.



The influence of positively charged amino acids on the binding affinity of these peptides to Hsc70 was tested using synthetic peptides in which lysines were replaced by negatively charged amino acids (P3, P4, and P11; Table 2). These peptides (control peptides) showed either none or very poor competition against Pc peptide. Changing basic amino acids to negatively charged residues in heptamers such as P10 and P14 substantially decreased the affinity of these peptides for Hsc70. Similar results were obtained by others(10, 11) . Unrelated peptide sequences containing bulky residues, such as P6, were also unable to compete against Pc for binding to Hsc70.

Among the synthetic peptides tested, P5 (NIVRKKK) was the highest affinity Hsc70-binding peptide. In order to evaluate the string of basic residues in P5 for binding to Hsc70, we synthesized a series of peptides having P5 lysines substituted by alanines. The binding results of this series of peptides (P17, P18, P19, and P20) to Hsc70 are shown in Table 2. A decrease of 6-20-fold in the binding of the modified peptides to Hsc70 was observed relative to P5. The largest loss of affinity (6-fold) occurred when arginine at position 4 was replaced with alanine. Sequential replacements of lysine by alanine resulted in smaller incremental losses in affinity (<2.5-fold). This result suggests that the binding affinity of peptides for Hsc70 can be finely tuned by changing the number of basic residues in the binding sequence, a potentially useful property for controlling the affinities of therapeutic peptides and peptide-mimetic drugs for molecular chaperones like Hsc70.

The amino-terminal portion of NIVRKKK contains two hydrophobic residues. Substituting lysine for isoleucine (P12) in this peptide considerably decreased its binding affinity (>70-fold). In P21, the same isoleucine was substituted with tyrosine resulting in decreased affinity to Hsc70. The change of lysine to alanine at the carboxyl terminus of P21 made only a small contribution to the loss of affinity as shown by the alanine scan. To test how tyrosines at the amino terminus would affect binding, peptides P7 and P8 were synthesized. Peptide P7 showed dramatic improvement over related hexamer P9 as expected from the peptide length effect. The restoration of the two hydrophobic residues at positions 2 and 3 in P8 resulted in a peptide with a very good binding affinity, but the presence of tyrosine instead of asparagine at the amino terminus contributed to a 10-fold weaker affinity compared with P5. Comparison of P21 with P5 further indicated that a hydrophobic residue, like isoleucine, is a key amino acid in the second position for good binding. Therefore, we propose that sequences of a nominal length of seven amino acids containing a combination of two hydrophobic residues like isoleucine and valine followed by basic amino acids is an important sequence for Hsc70 recognition.

We also tested the influence of several structural modifications of peptide P5 on the binding to Hsc70, such as constraining the peptide into cyclic forms by adding cysteines to the ends of its respective hexamer or heptamer. Cyclization was confirmed by mass spectroscopy. As shown by the results obtained with P15 and P16 (Table 2), their affinity for Hsc70 substantially decreased.

Peptide-stimulated ATPase Activity of Hsc70

Because P5 peptide had the highest binding affinity to Hsc70, we tested the ability of this peptide to stimulate its ATPase activity. As shown in Fig. 3, P5 stimulated Hsc70-ATPase activity 2-fold over the basal level at the highest concentration tested. Control peptides such as P3 and P10 were unable to stimulate any ATPase activity even at the highest concentration of 2.0 mM. The series of peptides with Lys Ala substitutions (P17-19) also were tested for their ability to stimulate Hsc70 ATPase activity. In contrast to binding affinities (Table 2), loss of basic residues did not strongly affect the stimulatory activity of these peptides.


Figure 3: Concentration-dependent effects of different peptides on Hsc70 ATPase activity. The fold increase of Hsc70 ATPase activity (pmol/min/µg) for P18 (NIVAAKK, open squares), P5 (NIVRKKK, solid circles), P17 (NIVAKKK, open circles), P19 (NIVAAAK, closed triangles), P10 (YVEDESG, solid squares), and P3 (VEDESG, open triangles) at various peptide concentrations was determined.



Because the highest affinity peptides found in previous library screens of Hsp70 family proteins are quite different from those selected from our library, peptide P5 was compared with one of these peptides, FYQLALT(11) . As shown in Fig. 4, in which lower concentrations of peptides were used than in Fig. 3, the concentration dependence of ATPase stimulation by P5 and FYQLALT are very different. Peptide FYQLALT was similar to the COOH-terminal fragment of Pc, used in our competition assays, in stimulating ATPase activity at much lower concentrations of peptide than P5. These results along with those from previous work (11) suggest that FYQLALT and Pc are both high affinity binders and efficient stimulators of Hsc70 ATPase, whereas NIVRKKK binds Hsc70 with high affinity but is an inefficient stimulator of its ATPase activity.


Figure 4: Concentration-dependent effects of different peptides on Hsc70 ATPase activity. The rate of ATP hydrolysis (pmol/min/µg) of Hsc70 for FYQLALT (open circles), Pc (solid triangles), and P5 NIVRKKK (solid circles) at various peptide concentrations was determined.



Competition Binding of NIVRKKK and FYQLALT to Hsc70

To determine whether these two peptides bound Hsc70 competitively, we took advantage of the fact that Hsc70 with bound NIVRKKK migrates more slowly on native gels than unbound Hsc70 monomer, whereas Hsc70 with bound FYQLALT migrates faster. 2 µg of Hsc70 (0.3 µM) was incubated with 250 µM of NIVRKKK in the presence of 0, 3, 6, or 10 µM of FYQLALT at 37 °C for 30 min in buffer C (20 mM Hepes-KOH, pH 7.0, 25 mM KCl, 10 mM (NH(4))(2)SO(4), 2 mM magnesium acetate, 0.1 mM EDTA, 1 mM dithiothreitol) containing 0.15 µM of ADP added to bind nucleotides to all of the Hsc70. After incubation, the proteins were separated in a 6% nonreducing, nondenaturing polyacrylamide gel, and the proteins were visualized by silver staining (Fig. 5). In the absence of the second peptide, the Hsc70-NIVRKKK complexes were detected as a major and a minor band migrating between the Hsc70 monomer and dimer (Fig. 5, lane 2). Upon addition of increasing concentrations of FYQLALT, these two complexes diminished and two complexes corresponding to Hsc70-FYQLALT appeared (Fig. 5, lanes 3-5). These complexes migrated faster than Hsc70 monomer, characteristic of Hsc70-FYQLALT (Fig. 5, lane 6), suggesting that they were in more compact conformations and that there may be a least two different conformations of Hsc70 with bound FYQLALT.


Figure 5: The competition binding of NIVRKKK and FYQLALT to Hsc70. Lane 1, Hsc70 alone; lanes 2-5, Hsc70 with 250 µM NIVRKKK in the presence of 0, 3, 6, or 10 µM of FYQLALT; lane 6, Hsc70 in the presence of 10 µM FYQLALT. The migration of Hsc70 monomer, Hsc70-NIVRKKK, and Hsc70-FYQLALT complexes are indicated. Hsc70 migrated as a doublet in the presence of NIVRKKK or FYQLALT.




DISCUSSION

The 6-mer and, more specifically, the 15-mer phage display random peptide libraries described in this report have been fully characterized. Detailed analyses of the 15-mer library showed that all amino acids, with the exception of glutamine and to a lesser extent lysine, were uniformly represented. For a complete characterization of these libraries, we tested our ability to select peptide sequences known to bind to streptavidin, as described previously. As expected, peptides containing the HPQ sequence were highly enriched in the streptavidin-selected 15-mer library. The sequence PWAWLT(I) was highly enriched in both libraries.

Our protocol for screening the 15-mer library differs from those used by Gething and colleagues (9) and Gragerov et al.(8) in the conditions used to release phage bound to Hsp70 family proteins. The previous studies used ATP in low salt buffers to release bound phage. Exchange of ADP for ATP on Hsp70 family proteins is sufficient to release them from unfolded proteins and subsequent hydrolysis of bound ATP is thought to return Hsp70 to a high affinity, peptide-binding conformation(23) . Alternatively, Greene and co-workers (24) have shown that Hsc70bulletATP is the form that preferentially binds clathrin-coated vesicles and that clathrin is released during uncoating as a stable Hsc70-ADP-clathrin complex for transport or perhaps temporary storage of clathrin subunits. This approach has resulted in the identification of primarily hydrophobic sequences. In the present study, an acidic buffer without ATP was used to release phage-displaying peptides bound to Hsc70 primarily by electrostatic interactions resulting in the identification of a combined hydrophobic and basic peptide motif for Hsc70 substrates.

Herein, radioactively labeled Pc fragment was used in competition binding assays with peptides. Scatchard plots of the binding of Hsc70 to Pc fragment are consistent with a single binding site on Hsc70 monomer(24) . Competition binding assays done in the same study indicated that clathrin, peptide C, RNase S peptide, and Pc fragment all recognize the same site on Hsc70 but with widely different affinities. The peptide FYQLALT is an example of a hydrophobic sequence that binds with high affinity to DnaK, BiP, and Hsc70. Its IC for Hsc70 competing with reduced carboxymethylated lactalbumin is about 5 µM(11) . The highest affinity basic peptide in our study, NIVRKKK, had an IC for Hsc70 competing with Pc fragment of about 2 µM, so both of these peptides bind with relatively high affinities; however, they differ dramatically in their abilities to stimulate Hsc70 ATPase activity (Fig. 4). Stimulation of ATPase activity by FYQLALT occurred in the micromolar range, and stimulation by NIVRKKK occurred in the millimolar range. Sequential replacement of Arg^4, Lys^5, and Lys^6 with Ala did not significantly affect the ability of NIVRKKK to stimulate the Hsc70 ATPase. All of these peptides were relatively weak stimulators. The peptide FYQLALT effectively blocked the binding of NIVRKKK to Hsc70, suggesting that these two peptides may bind to the same or overlapping sites. However, consideration of the gel analysis (Fig. 5) suggests another possibility. Hsc70-FYQLALT complexes migrated faster than Hsc70 monomers in native gels, consistent with a conformational change in Hsc70 to a more compact form upon binding of the hydrophobic peptide. Such a conformational change may mask a separate NIVRKKK binding site. Taken together, these observations suggest that the interactions of these two peptides with Hsc70 may be fundamentally different. Where on Hsc70 would they bind? The only hydrophobic regions in the COOH-terminal domain of Hsc70 suitable for interactions with peptides like FYQLALT are the predicted beta strands (amino acids 392-504). Based on computer modeling of the peptide-binding domain of Hsc70(4) , it has been suggested that negatively charged residues may be clustered in predicted alpha helical regions such as residues 512-536 of Hsc70. Such a region could bind basic sequences such as NIVRKKK, and a site with both acidic and hydrophobic character could be formed by hydrophobic beta strands and acidic helical segments.

We offer several hypotheses for the possible significance of these different interactions. First, the interactions involved in folding pathways may be different from those used in transport or chaperoning of proteins within the cell. For example, hydrophobic segments of proteins may be bound and released in relatively rapid cycles of nucleotide exchange and hydrolysis as part of protein folding, whereas transport may require longer-lived associations governed more by accessory proteins than by nucleotides, and NIVRKKK-like binding sites may be used for these. For example, Hsc70 binds to the nuclear localization sequences of both SV40 large T antigen and nucleoplasmin using a cluster of negatively charged residues on Hsc70(25, 26) . Nuclear localization sequences can be either contiguous runs of basic residues flanked by prolines like PKKKRKV of the SV40 large T antigen or more complex, bipartite signals as found in nucleoplasmin (RPAATKKAGEAKKKKLDKEDE). These sequences have the basic part of the NIVRKKK motif but often lack the preceding hydrophobic residues, a consideration that makes the nuclear localization sequences less appealing as representatives of this motif.

Another possibility is that NIVRKKK may be related to mitochondrial targeting signals. It has been reported that these sequences have the capacity to form amphipathic alpha helices in which basic and hydrophobic residues are arranged on opposite faces of these helices(27, 28) . Kiebler et al.(29) have speculated that the negatively charged cytosolic domain of Mom22, a component of a mitochondrial receptor complex, may transiently bind the clusters of basic residues in mitochondrial signal sequences. A possible scenario could be the transfer of basic signal sequences from acidic binding sites on Hsc70 to Mom22.

A second hypothesis is that Hsc70 binds to unfolded proteins via hydrophobic sequences for chaperoning both in folding pathways and for transport but that the NIVRKKK motif is used to bind certain folded proteins with which Hsc70 interacts. For example, the last 28 amino acids at the highly basic COOH terminus of the p53 tumor suppressor protein (SSHLKSKKGQSTSRHKKLMFKTEGPDSD) contain binding site(s) for Hsc70(30) . Another possibility is a role in the self-association of Hsc70 into multimers, because bovine Hsc70 contains a sequence in the ATPase domain (NKRAVRRLR) that may serve as an NIVRKKK-like binding site. And finally, NIVRKKK was used to search for sequence similarity in the GenBank and Swiss Protein data bases using the FastA method (31) . Interestingly, DnaJ2, a DnaJ homolog from humans(32) , ranked sixteenth among the matches with the sequence KIVREKK (amino acid residues 200-207).


FOOTNOTES

*
This work was supported by the Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, CT and the University of Connecticut Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Bristol-Myers Squibb Pharmaceutical Research Institute, P. O. Box 4000, Princeton, NJ 08543. Tel.: 609-252-3291; Fax: 609-252-3307.

(^1)
The abbreviations used are: BiP, immunoglobulin-binding protein; tet, tetracycline; TU, transducing unit(s); BSA, bovine serum albumin; Pc, pigeon cytochrome c peptide.

(^2)
S. Nadler, unpublished data.

(^3)
J. Scott, personal communication.


ACKNOWLEDGEMENTS

We thank Joan Carboni and Steve Nadler for helpful discussions and Sandro Arufo for critical review of this manuscript.


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