(Received for publication, February 15, 1995; and in revised form, May 23, 1995)
From the
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.
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 ()(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.
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.
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 [H]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.
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.
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.
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 Hsc70ATP 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
,
Lys
, and Lys
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
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
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
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 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).