Characterization of a distinct binding site for the prokaryotic

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 268, No. 6, Issue of February 25, pp. 4499-4503,1993 Printed in U. S. A .

Characterization of a Distinct Binding Site for the Prokaryotic Chaperone, GroEL, on a Human Granulocyte Ribonuclease*

(Received for publication, October 28, 1992)

Helene F. RosenbergSS, Steven J. Ackermanll, and Daniel G. Tenenn 11 From the $Laboratory of Host Defenses, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892 and the TDepartment of Medicine, Beth Israel Hospital, Boston, Massachusetts 02215

Although ribonucleases fold into correct tertiary conformation in vitro guided solely by information contained in the primary amino acid sequence (Sela, M., White, F. H., and Anfinsen, C. B. (1957) Science 124,691-693), it is not clear whether folding of these proteins proceeds unassisted in a complex intracellular environment. We describe here the specific and high affinity binding of groEL, the prokaryotic homolog of the heat shock protein 60 family of molecular chaperones, to recombinant eosinophil cationic protein and eosinophil-derived neurotoxin, two members of the human ribonuclease gene family. We have determined that groEL binds to a unique peptide sequence near the amino terminus of nascent eosinophil cationic protein that includes the first of eight cysteine residues. This binding site functions independently and can confer groEL binding activity on an unrelated carrier protein. GroEL dissociates from the binding site upon addition of ATP and M g + ; no other cations or cofactors are necessary. These findings suggest the possibility that interaction with a groEL-like molecular chaperone may be a requirement for correct folding andlor translocation of eukaryotic ribonucleases in vivo.

Until quite recently, it was assumed that most proteins fold spontaneously into their characteristic tertiary conformations (1,2). Although ribonucleases can fold spontaneously in dilute solution (2,3), it has never been clear how the nascent chains avoided folding errors during biosynthesis in vivo. Additional factors may be necessary to ensure that only correct structures are formed.

Mammalian ribonucleases are a large family of structurally related proteins that have two conserved histidines and a single conserved lysine residue in the catalytic site, and eight invariant cysteine residues paired in intrachain disulfide bonds (4, 5). Eosinophil cationic protein (ECP)’ and eosinophil-derived neurotoxin (EDN) are ribonucleases found in the specific granules of human eosinophilic leukocytes (6-8). ECP

* This work was supported in part by National Institutes of Health Grants HL02288 (to H. F. R.) and Grants AI22660, AI25230, and CA41456 (to D. G. T., and S. J. A.). The costs of publication of this article were defrayed in part by the payment of page charges, This article must therefore be hereby marked “aduertisernent” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

Defenses, Bldg. 10, Rm. llN114, NIAID/NIH, 9000 Rockville Pike, Q To whom correspondence should be addressed Lab. of Host

Bethesda, MD 20892. 11 Scholar of the Leukemia Society of America. The abbreviations used are: ECP, eosinophil cationic protein;

EDN, eosinophil-derived neurotoxin; hsp, heat shock protein; Rub- isco, ribulose-bisphosphate carboxylase/oxygenase; PCR, polymerase chain reaction; CLC, Charcot-Leyden crystal protein; PAGE, poly- acrylamide gel electrophoresis.

is a powerful helminthotoxin, cytotoxin, and neurotoxin (6) and has been shown to have anti-bacterial activity in vitro (9). Despite their high degree of sequence homology (lo), EDN shares only neurotoxic and ribonuclease activity with ECP (6,8) and shows little to no toxicity against other targets tested (6).

GroEL (monomer molecular mass 57 kDa (11-13)) is a member of the heat shock protein (hsp) 60, or “chaperonin” group of molecular chaperones (14). The groEL locus and gene product were first characterized as essential for the assembly of bacteriophages (15-17). GroEL interacts with several bacterial export proteins (18-21) and maintains them in an unfolded state appropriate for translocation through the outer membrane (18).

Several eukaryotic homologs of groEL have been identified, including the chloroplast Rubisco small-subunit binding protein (14, 22, 23) and mitochondrial hsp60s (24-28) that par- ticipate in protein folding and translocation (29-31). Esche- richia coli groEL can substitute for both the mitochondrial and chloroplast homologs in in vitro folding experiments (32- 36), and, analogous to our results with eukaryotic ribonucleases, groEL was found to copurify with a nascent Rubisco small-subunit fusion protein expressed in E. coli (37). Hom- ologs of groEL have also been identified on the surface of y/ 6 T lymphocytes (38) and on Daudi Burkitt lymphoma cells (39) whose functions are currently unknown. The T-complex- 1 protein has also been identified as a distant hsp60 homolog (40), and the recently described cytoplasmic chaperones be- longto the T-complex-l group (41-43).

We describe here the specific and high affinity binding of the molecular chaperone groEL to nascent chains of these two human granulocyte ribonucleases. A role for a groEL- like, hsp60 protein participating in ribonuclease biosynthesis is suggested.

MATERIALS AND METHODS

Fusion Protein Constructs-The glutathione-S-transferase fusion protein constructs used in these studies are diagrammed in Fig. 1. The amino acids are numbered beginning with the 1st residue of the mature, granule-derived forms of ECP and EDN (7). The signal sequences (10,44) have not been included. Coding sequences for EDN and ECP were amplified by polymerase chain reaction (PCR) from their corresponding cDNA sequences (12, 44) and inserted in-frame into the SmaI site of the pGEX3X (glutathione-S-transferase fusion protein) expression vector (Pharmacia LKB Biotechnology Inc.). The cDNA for the Charcot-Leyden crystal protein (CLC) was isolated by PCR amplification of the specific sequence from an HL-60 subline cDNA library (45) using primers specific to the 5’ and 3’ ends of the coding sequence (46). The product obtained was inserted in-frame into the EcoRI site of pGEX3X and confirmed by sequencing and by Western blotting of the fusion protein with polyclonal rabbit anti- CLC antisera. The gst-18ECP and gst-26ECP deletion constructs were generated by PCR amplification of the coding region of ECP minus the sequences encoding the first 18 or the first 26 amino acids,

4499

GroEL Binds to Human Ribonucleases 4500

A. gstEDN

8. gstECP

C. gst-18ECP

D gst-26ECP

E. gSt-33ECP

F . gstCLC

1 ~~~KPPQFTWAQWFETQHINMTSQQCTNAMQVINNYQRRCKNQNT . . .

{ ~ ~ ~ P P Q F T R A Q W F A I Q H I S L N P P R C T I A M R A I N N Y R W R C K N Q N T . . . <r(:PPRCTIAMRAINNYRWRCKNQNT. . .

~ ~ ( t & A I N N Y R W R C K N Q N T . . . d T $ W R C K N Q N T . . .

35 0. gstl9/35ECLC i ~ $ P P R C T I A H R A I N N Y R W ~ CLC FIG. 1. Glutathione-S-transferase (gs t ) fusion protein constructs used in experiments described in this report. n in the

deletion constructs refers to the number of amino-terminal residues deleted from the ECP polypeptide sequence. 19/35ECLC includes amino acids 19-35 ligated in-frame to the amino terminus of CLC.

respectively. These truncated coding regions were cloned in-frame into the BamHI and EcoRI sites of the pGEX2T expression vector (Pharmacia). To create gst-33ECP, the gstECP construct was re- stricted with ClaI and EcoRI, and the insert was isolated, blunt ended, and ligated in-frame into the SmaI site of pGEX3X. The 19/35ECLC fusion between ECP and CLC was made as follows. The cDNA sequence coding amino acids 19 through 35 was amplified by PCR and ligated in-frame via a primer-encoded EcoRI site to the 5' end of the coding sequence of CLC. This fusion was then ligated in-frame into the BamHI site of pGEX2T. All PCR products and fusion constructs were sequenced.

Production and Purification of Fusion Proteins-Constructs prepared as described were used to transform E. coli strains DH5a or HB101. Transformants were grown overnight at 37 "C in LB medium + 50 pg/ml ampicillin to stationary phase, then diluted 1:lOO in LB medium + ampicillin, and grown at 37 "C for 3 h with vigorous shaking. Three hours later, recombinant fusion protein synthesis was induced at 37 "C by addition of isopropyl-thio-galactoside (Bio-Rad) to a final concentration of 1 mM. After a 2-h induction period, the bacteria were harvested, washed in cold phosphate-buffered saline, frozen in dry ice/ethanol, and stored as a pellet at -80 "C prior to use. To harvest proteins, frozen pellets were resuspended in phosphate-buffered saline + 1 mM phenylmethylsulfonyl fluoride (Sigma) and sonicated briefly on ice. Triton X-100 (RPI, Elk Grove Village, IL) was added to a final concentration of 1%, and the debris removed by centrifugation at 40,000 X g at 4 "C. Preswelled glutathione- Sepharose matrix (Pharmacia) was added to the supernatant and equilibrated end-over-end for 15 min at 4 "C. The beads were washed four times with phosphate-buffered saline at 4 "C (total of 400 X gel bed volume). Bound protein was eluted with 50 mM Tris + 25 mM reduced glutathione (Sigma), pH 8.0, and evaluated by SDS-PAGE and Western blotting. For the dissociation experiments (Fig. 51, recombinant protein was not eluted. Beads with protein bound were diluted 1:1 in 2 X reducing sample buffer and evaluated as above.

Amino-terminal Sequencing-Purified proteins separated by SDS- PAGE were transferred to an Immobilon membrane (Millipore Corp., Marlboro, MA) and stained with Coomassie Blue as per manufactur- er's instructions to localize bands. The -60 kDa band copurifying with gstEDN was cut from the Immohilon membrane and analyzed at the microsequencing facility at Brigham and Womens' Hospital, Boston, MA. From 10 pmol of Immobilon-bound protein, 25 amino- terminal residues were identified (Fig. 2B), enabling conclusive identification of this -60-kDa copurifying protein as groEL.

Preparation of Anti-groEL Antiserum-The complete cDNA for the coding region of groEL was obtained by PCR amplification using primers corresponding to known nucleotide sequence (14) and purified E. coli chromosomal DNA as template. The amplification product was ligated in-frame in the EcoRI site of pGEX3X and confirmed as groEL by nucleotide sequence. The fusion protein was induced and purified as described above (omitting Triton X-100). Recombinant groEL was cleaved from the gst portion by addition of 1% weight/ weight Factor Xa (Boehringer Mannheim). Rabbits were injected with 150 pg of denatured/reduced recombinant groEL in complete Freund's adjuvant and boosted at 2-week intervals with 150 pg of denatured/reduced recombinant groEL in incomplete Freund's adjuvant. The antiserum obtained was tested by Western blotting against both injected antigen and the -60-kDa protein copurifying with gstEDN known to be groEL by amino-terminal sequencing. At 1:500 dilution, the anti-groEL antiserum reacted specifically with both the injected antigen and the protein copurifying with gstEDN and gstECP, but not with other transferred proteins. No proteins were detected with a 1500 dilution of preimmune serum.

Western Blotting-Standard procedures were followed (47). Briefly, nitrocellulose blots were immersed in 5% non-fat dry milk in Tris-buffered saline (TBS) with 0.5% Tween-20 to eliminate nonspe- cific binding. First antibody concentrations included anti-groEL at 1500 and anti-CLC, anti-EDN, anti-ECP, at 1:300 in TBS + Tween. Alkaline phosphatase conjugated goat anti-rabbit IgG (Bio-Rad) at a 1:lOOO dilution was.used as second antibody. Blots were developed with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate (Bio-Rad).

Dissociation Experiments-The gstl9/35ECLC protein containing the groEL-binding site at the amino terminus of the carrier protein (Fig. 1) was prepared as described above and isolated (along with copurifying groEL) on glutathione-Sepharose beads. After extensive washing, the beads were resuspended in dissociation buffer (20 mM Tris, pH 8.0 + 100 mM NaCl + 0.1% NaN3) and divided into aliquots. ATP (Sigma), MgS04, and/or KC1 were added to each aliquot to final concentrations of 5 mM. Samples were rotated end-over-end for 30 min at room temperature, after which the matrix was washed five times with dissociation buffer, diluted 1:l with 2 X reducing sample buffer, and evaluated by SDS-PAGE and Western blotting. Serial dilutions of the original isolate were prepared in order to estimate the extent of groEL dissociation.

GroEL Binds to Human Ribonucleases 4501 A.

a b c -. . 66 - 45 - 31- - " -K ::E

CIS'

22 -

6. a. -60 koa unknown XXAXDVKFGNDARVXMLRGVNVLADAVXXTL

1 31

b. groEL MAAKDVKFGNDARVKMLRGVNVLADAVKVTL ......... NH2

C. D. a b c a b c

66- m 66-

45 - -! 45 -

I

I 31" ~ 31 -

1 .

22- _ ' " , - 1

I 22-

FIG. 2. A , Coomassie Blue-stained SDS-PAGE of gst-fusion proteins recovered on glutathione-agarose mstrix. Lane a, gstEDN; lane b, gstECP; lane c, gst alone. The unexpected copurifying -60-kDa protein is visible in lanes a and b. B, amino-terminal sequence of the -60-kDa copurifying protein ( a ) compared to groEL from E. coli ( b ) (14). The X symbol indicates amino acids that were not identified by the sequencing procedure. Panels C and D show Western blotting with polyclonal rabbit anti-ECP/anti-EDN and anti-groEL antiserum, respectively. Lunes a, gst alone; lanes b, gstEDN; lanes c, gstECP.

RESULTS

The SDS-PAGE analysis of the ribonuclease fusion proteins produced in E. coli and purified from the bacterial sonicate on glutathione-agarose resin is shown in Fig. 2 A . In each case, an unexpected -60-kDa protein copurified with the gst-ribonuclease fusion proteins (lanes a and b) , but not with gst alone (lane c ) . The -60-kDa protein was not recognized by anti-ECP/EDN antiserum (Fig. 2C). This -60-kDaprotein was identified as groEL by amino-terminal sequencing (Fig. 2B) and by Western blotting with anti-groEL Antiserum (Fig. 20). The binding of groEL to these ribonuclease fusion proteins persisted despite the addition of detergent (1% Triton X-100) and reducing agent (10 mM dithiothreitol) to the buffers used in the purification procedure. In related studies, groEL and gstECP eluted as a single irregular peak from both anion- and cation-exchange gels (MonoQ at pH 8.0 or Monos at pH 4.0 both with 10 mM dithiothreitol) despite the large disparity in their respective PI values (groEL PI = 4.5, ECP PI = 10.8, gst PI = 6.2, predicted values).*

The binding site for groEL within gstECP was determined by creating a series of in-frame deletions from the amino terminus of ECP, designated gst-l8ECP, gst-PBECP, and gst- 33ECP. The value of n preceding ECP indicates the number of amino-terminal residues removed from the ECP sequence before in-frame ligation to gst (Fig. 1). While groEL copurified with gst-18ECP (Fig. 3, A and B, lanes b) , there was almost no groEL copurifying with gst-26ECP or gst-33ECP (lanes c and d) . The trace amounts of binding seen to the gst-26ECP

* H. F. Rosenberg, unpublished observations.

and gst-33ECP fusion proteins may represent a second minor binding site. The Western blot in Fig. 3C (polyclonal anti- ECP antiserum) compares protein loading and confirms the correct reading frame of each deletion construct.

The results shown in Fig. 4 demonstrate that the ECP polypeptide-binding site identified by deletion analysis rep- resents a complete and independent groEL-binding site. The nucleotide sequence encoding amino acids 19-35 of ECP was ligated in-frame to the amino terminus of the open reading frame of a cDNA encoding the eosinophil Charcot Leyden crystal protein (46). This construct was then expressed as a fusion protein with gst (gstl9/35ECLC). While groEL did not copurify with gstCLC alone (lane b), the addition of the groEL

A. a b c d e

97-

66

45 - gstECP

::;:;:E:; 31 - gst-33ECP

B. a b c d e

kDa 97- 66-

4 5

C.

66- - a b c d e

45- I

31-

FIG. 3. Coomassie Blue-stained SDS-PAGE ( A ) and West- ern blotting with rabbit anti-groEL antiserum ( B ) and anti- ECP antiserum (C). Lanes a, gstECP; lanes b, gst-1SECP; lanes c, gst-26ECP; lanes d, gst-33ECP; lanes e, gst alone.

A. a b c

97 6€

45

31

,-

B. a b c

66

kDa 45

31

C. a b c

66 45

31

FIG. 4. Coomassie Blue-stained SDS-PAGE ( A ) and West- ern blotting with anti-groEL ( B ) and anti-CLC antiserum (C). Lanes a, gstECP; lanes b, gstCLC; lanes c, gstl9/35ECLC.

4502 GroEL Binds to Human Ribonucleases

binding sequence to the the amino terminus of the CLC protein (lune c ) was sufficient to confer groEL binding activity on this unrelated carrier protein.

The results in Fig. 5 show that groEL dissociates from the binding site upon addition of ATP and Mg+. No further cations or cofactors are necessary. The dissociated groEL can be detected in the supernatant (data not shown). A compari- son with the intensities of anti-groEL staining of serial dilutions of the original isolate, gstl9/35ECLC (Fig. 5C), suggests that -90-95% of the groEL originally copurifying with gstl9/ 35ECLC dissociated from the complex under these conditions.

DISCUSSION

We have presented data describing the specific and high affinity binding of the prokaryotic chaperone groEL to nascent polypeptide chains of two granulocyte ribonucleases, ECP and EDN. The binding site for groEL on ECP is a unique peptide segment located near the amino terminus of ECP that includes the first of eight cysteine residues that are characteristic of all mammalian ribonucleases. The site is both necessary and sufficient to mediate binding of groEL. Transfer of the binding site to the amino terminus of an unrelated carrier protein confers on it the ability to bind

A. B.

a b c d e f g h a b c d e f g h

66 -. . .

-____I__* 66-.

45 - 45 - 4

kDa 31 - 31 -.

22 - 22 - C.

a b c d e f g h 6 6 - w - .-

45- . 1 . '

FIG. 5. Coomassie Blue-stained SDS-PAGE ( A ) and West- ern blotting with anti-groEL antiserum ( B ) . In each lane, matrix-bound gstl9/35ECLC with copurifying groEL was incubated at room temperature for 30 min with the following additions: lanes a, no addition; lanes b, + M e ; lanes c, +ATP; lanes d, +K'; lanes e, + M e and +ATP; lanes f, +K+ and +ATP; lanes g, +Me and +K'; lanes h, + M e , +ATP, and +K'. C, Western blot of a serial dilution of matrix-bound gstl9/35ECLC with copurifying groEL (A, lane a ) with anti-groEL antiserum. Lune a, no dilution; lane b, 1:l (50%); lane c, 1:4 (20%); lane d, 1:9 (90%); lane e, 1:19 (95%); lane f, 1:49; lane g, 1:99; lane h, 1:199. Values in parentheses represent the per- centage of groEL remaining in the diluted sample when compared to the original in lane a.

binding site

."""" - -,--

groEL. We have demonstrated that groEL dissociates from its binding site upon addition of ATP and M P ; no other cations or cofactors are necessary. GroEL has been found to bind to many unfolded proteins (48-50). The significance of these observations remains unclear. It should be noted that we are reporting an association between groEL and nascent polypeptides, not with polypeptides that have been subjected to post-translational denaturation and unfolding. The interaction between groEL and the granulocyte ribonucleases is analogous to that reported between groEL and nascent chains of recombinant Rubisco small-subunit (37), a protein known to associate with the chloroplast hsp60 homolog in its native locale (51,52).

The factors involved in the ability of groEL to recognize target proteins have not been completely clarified. Landry and Gierasch (53) reported that groEL favors sequences that have a propensity to form a-helices, while Schmidt and Buch- ner (54) described the interaction of groEL with an all /3 protein. Martin and colleagues (55) noted the recognition of a molten globule intermediate. To the best of our knowledge, our data represent the first identification of a defined binding site within the context of a potential target protein. We do not as yet know what features of this site are crucial for recognition by groEL. The Chou-Fasman prediction for this region of ECP is unremarkable. Amino acids 19-22 form a helix breaking turn, followed by a short, potentially a-helical region (Fig. 6). There is no significant amino acid sequence homology between the binding site identified on ECP and the sequences of known target proteins of groEL or of other hsp60 homologs. Although most bacterial ribonucleases are structurally dissimilar to the mammalian ribonucleases (56-58), E. coli ribonuclease I (RNase I) has eight cysteine residues, similar to those of its mammalian counterparts (59). There is a small area of sequence conservation between residues 22- 26 (RCTIAM) within the groEL-binding site of ECP, to amino acids 14-19 (RYVLAL) near the amino terminus of RNase I. RNase I is exported to the bacterial periplasmic space (60) and might be an as yet unrecognized target of groEL.

Our results may also have implications with respect to granule protein biosynthesis. Many features of protein synthesis are conserved throughout evolution. Recent work has shown that many aspects of post-translational processing of nascent polypeptides likewise occur by similar mechanisms in prokaryotes and eukaryotes. For example, eukaryotic signal sequences function appropriately in bacteria (61-63). A func- tional homolog of the eukaryotic signal recognition particle has been described in E. coli (64). The heat shock proteins, which are essential for protein folding and translocation in bacteria (18, 65-68), have numerous highly conserved eukar-

1 I I 19 35 53")

J

FIG. 6. Amino-terminal sequence of ECP (omitting signal sequence) (13). The binding site is included within the brackets. The numbers above the sequence refer to the order of amino acids. The assignments (@-forming, &breaking, a-forming, a-breaking, and turn) were made according to Chou-Fasman algorithms in "pepplot," GCG Sequence Analysis Software, University of Wisconsin, Madison, WI.

GroEL Binds to Human Ribonucleases 4503

yotic homologs that are involved in protein assembly and translocation (44-51, 68).

Bainton and colleagues (69, 70) have shown that the biosynthesis of eosinophil and neutrophil granule proteins resem- bles the biosynthesis of other eukaryotic secretory proteins. Nascent chains are polymerized on endoplasmic reticulum- bound ribosomes, processed in the endoplasmic reticulum, and ultimately packaged into specialized secretory granules. The chaperone proteins that have been found in the endoplasmic reticulum of mammalian cells include a member of the hsp7O family, BiP (71), and the hsp9O member, grp94 (72). Although hsp60 homologs exist in the mitochondria (24- 28), chloroplasts (14, 22-23), and the cytoplasm (41-43) of eukaryotic cells, no hsp60 homologs have been found in the ER, nor has a role for an hsp60 homolog in processing of granule/secretory proteins been presented. We have not yet determined whether groEL alone can facilitate the folding of these granulocyte ribonucleases in vitro. However, we have shown that a granulocyte ribonuclease contains an amino- terminal-binding site for groEL and may represent a target for an hsp60 homolog in its native locale.

Acknowledgments-We thank Dr. Peter F. Weller for the gift of anti-CLC protein antiserum and Margaret Ehrhardt of the Brigham and Womens' Microsequencing Facility for assistance with this work. We also thank Drs. Philip M. Murphy, John I. Gallin, and Harry L. Malech for critical review of the manuscript and Dr. Steven M. Holland for helpful discussions.

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