limited proteolysis of recombinant human soluble interleukin-2

0 1989 by The American Society for Biochemistry and Molecular B i o l o g y , Inc. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 264, No. 35, Issue of December 15, pp. 21097-21105,1989 Printed in U.S.A.

Limited Proteolysis of Recombinant Human Soluble Interleukin-2 Receptor IDENTIFICATION OF AN INTERLEUKIN-2 BINDING CORE*

(Received for publication, June 27, 1989)

May C. Miedel, Jeffrey D. Hulmes, and Yu-Ching E. Pan$ From the Department of Protein Biochemistry, Roche Research Center, Hofjmann-La Roche Inc., Nutley, New Jersey 071 10

Limited proteolysis of a recombinant, soluble form of the Tac protein, a human interleukin-2 receptor (rIL-2R), was performed using trypsin, Staphylococ- cus aureus V8 protease and proteinase K to study the structural requirements of interleukin-2 receptor (IL- 2R) for interleukin-2 (IL-2) binding. Sensitive proteolytic sites were found to be clustered in the regions of the polypeptide encoded by exons 3, 5, and 6, with a few semi-sensitive sites located within the two homologous domains encoded by exons 2 and 4. A number of nicked and truncated rIL-2R species generated by proteolysis were assayed for IL-2 binding using recombinant IL-2 (rIL-2) affinity gel and then structurally characterized. The results demonstrated that only the species that consist of the regions encoded by exons 2 and 4, joined by five disulfide bonds, are capable of binding IL-2 and that the presence of semi-sensitive cleavage sites within the two homologous domains had no apparent effect on IL-2 binding. These results sug- gest that the pattern of the sensitive cleavage sites in rIL-2R is closely related to the structural requirements for IL-2 binding. Based on the experimental results, a highly symmetrical core structure of IL-2R with a total of 135 amino acid residues was identified. This is the smallest protein moiety so far known to be capable of binding IL-2.

Interleukin-2 (IL-2)’ is a lymphokine secreted by T cells upon stimulation with antigen or mitogen (1). Activation of T lymphocytes also induces the synthesis of the receptors for IL-2 (IL-2R) (2). The interaction between IL-2 and its receptors is a key regulatory event during an immune response (3). The receptor exists in three forms with different IL-2 binding affinities and subunit compositions. The high affinity form consists of two distinct polypeptide chains with molecular weights of 70 and 55 kDa. The intermediate affinity form consists of the 70-kDa protein and the low affinity form

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

5040. $ T o whom correspondence should be addressed. Tel.: 201-235-

The abbreviations used are: IL-2, interleukin-2; IL-SR, interleukin-2 receptor; rIL-2R, recombinant soluble human interleukin-2 receptor; rIL-2, recombinant interleukin-2; HPLC, high performance liquid chromatography; SDS-PAGE, sodium dodecyl sulfate-poly- acrylamide gel electrophoresis; PIR, Protein Identification Resources; PTH, phenylthiohydantoin; PVDF, polyvinylidene difluoride; T, trypsin; V8, S. aureus V8 protease; PK, proteinase K; and the single- letter system for amino acid abbreviation is according to IUPAC-IUB recommendation.

consists of the 55-kDa protein (Tac) (4). The N-terminal amino acid sequence of Tac was partially defined from the purified protein, and the complete primary structure was deduced from the nucleotide sequence of the cDNA clone (5- 7). Subsequently, the organization of the gene structure of Tac was determined to be encoded by eight exons located in chromosome 10 (8, 9). This 251-amino acid protein consists of an extracellular domain of 219 amino acids encoded by exons 2-6, a transmembrane region, and a short carboxyl- terminal cytoplasmic tail encoded by exons 7 and 8. Amino acid sequence homology was detected between the peptide regions encoded by exon 2 and exon 4 having conserved cysteine residues (8). Recently, the cDNA encoding the 70- kDa protein (p70) was cloned (10).

Limited proteolysis with trypsin and endoproteinase Lys-C has shown that the natural Tac molecule, a glycoprotein, is formed by two disulfide-bonded domains connected by a hydrophilic segment (11, 12), and the disulfide arrangement of this protein was partially determined (13). A naturally occur- ring soluble form of the Tac protein, which was missing its normal transmembrane and cytoplasmic segments, was also isolated and partially characterized (10, 14, 15).

Earlier studies including molecular cloning, site-directed mutagenesis, and antibody recognition revealed some structure/function relationships of IL-2R and its interactions with 1L-2. Deletions of the C-terminal region of the receptor encoded by exons 5 , 6, 7, and some of exon 4 resulted in no apparent loss in IL-2 binding ability (13, 16-18). However, when a larger segment of the exon 4-encoded region was removed, the IL-2 binding ability was lost (13, 17). The shortest fragment of the molecule capable of binding IL-2 was found to be composed of amino acid residues 1-163 which includes the first 10 cysteine residues, and any mutations in these 10 cysteines resulted in an inactive molecule (13). These experiments show the essential role of the disulfides for IL-2 binding. In addition, antibody and ligand-receptor cross-link- ing experiments have demonstrated that the N-terminal portions of both the Tac molecule and IL-2 are important for IL- Z/IL-ZR binding (12, 19, 20). Recently, mutational analysis has revealed that two N-terminal segments in IL-2R (residues 1-6 and 35-43) are at or near IL-2/IL-2R contact sites (21). The three-dimensional structure of IL-2 has been determined, and the N-terminal region of this protein was found to be mainly a-helices (22).

TO facilitate the study of the IL-Z/IL-ZR interaction, a recombinant soluble form of the human Tac protein (rIL-ZR), which lacks the carboxyl-terminal cytoplasmic and most of the transmembrane regions, has been expressed in Chinese hamster ovary cells (23). The primary structure of this 224- amino acid recombinant glycosylated protein with an apparent molecular mass of 43 kDa was established, and the com-

21097

21098 Limited Proteolysis of IL-2 Receptor and the IL-2 Binding Core

plete disulfide arrangement was determined (24). Based on the assignment of the disulfide bonds, a structural model of the IL-2R was proposed (24). Since this recombinant protein was demonstrated to have the same biochemical and func- tional features as the natural Tac (17, 23), structural information obtained from this rIL-2R should be representative of the natural protein. The availability of large quantities of rIL- 2R has permitted us to study the detailed structural requirements of IL-2R for IL-2 binding. These results should provide a better understanding of the structure-function relationships of this molecule and aid in designing an effective IL-2 antag- onist (25).

In this study, we provide evidence that the sensitive cleavage sites are clustered in the regions encoded by exons 3, 5, and 6. Subsequently, several nicked and truncated rIL-2R species, with and without IL-2 binding capability, were generated for studying the structure/function relationships of IL- 2/IL-2R system. These results have led to the identification of a basic IL-2 binding core with a total of 135 amino acid residues representing the smallest protein moiety so far known to bind IL-2. It is smaller than the model we predicted previously based on exon structures (24). The symmetrical nature of this core structure and its role in IL-2/IL-2R binding will be discussed.

EXPERIMENTAL PROCEDURES AND RESULTS*

Limited Proteolysis and Elucidation of the Sensitive Cleavage Sites

As in the case of the natural Tac protein (12), limited proteolysis of rIL-2R produced stable digestion products by removing some exposed segments. In order to detect all of the exposed regions of the protein molecule, proteases with different specificities were used so that various sensitive cleavage sites could be detected. Aliquots of proteolytic digests of rIL- 2R generated by trypsin, Staphyloccocus aureus V8 protease, and proteinase K were taken at various time intervals and analyzed by 1) SDS-PAGE under reduced and nonreduced conditions, 2) HPLC peptide mapping, and 3) N-terminal sequence analysis.

SDS-PAGE-SDS-PAGE was used to monitor the disappearance of the intact rIL-2R (43 kDa) and the appearance of stable digestion products (Fig. 1). At earlier stages of digestion, under nonreducing conditions, three tryptic products of 41, 38, and 33 kDa were detected. Later, these were further digested to the 23-kDa and several lower molecular mass species. In the case of V8 protease and proteinase K digestions, stable products of 29 and 30 kDa were detected, respectively. Under reducing conditions, all the major cleavage products were reduced to lower molecular weight fragments, suggesting that disulfide bonds are present. Reduced intact rIL-2R is a single polypeptide chain, and therefore no change in M, was observed.

HPLC Peptide Mapping-The release of small peptides, the appearance of major digestion products, and consequently the disappearance of the intact protein were monitored by reversed-phase HPLC. The peptides recovered in pure form and in sufficient amounts were identified (Fig. 2, left panel), and the amount released of several key peptides was plotted against digestion time (Fig. 2 , right panel). The peptides released completely within 1 h of digestion are defined as

Portions of this paper (including “Experimental Procedures,” part of “Results,” Figs. S1-S3, and Tables Sl-S4) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

“fast-releasing” peptides, and their corresponding cleavage sites are defined as sensitive sites. From the three time course experiments, all of the sensitive sites were located within the exon 3,5, and 6 encoded regions. These sites were determined to be at the C terminus of Lys71, and A r e by trypsin, G1u8’, Glus7, G1u’68, and Glu18’ by V8 protease, and Thr86, GlnBg, Ser“’, Thrlgo, Serlgl, SerZ1*, and Alazz1 by proteinase K (see Table I). Some peptides (e.g. 72-83 from tryptic digest, 169- 224 from V8 protease digest) were modified or further degraded after they were released. Two slow releasing peptides, 32-36 and 68-71, in the tryptic digest were recovered in poor yields and therefore were not plotted. Additional cleavage results are discussed in the legend of Fig. 2.

The broad peaks in the HPLC maps shown in Fig. 2 corresponded to the major protein bands observed on the nonreduced SDS-PAGE (Fig. 1, left panels). The peak inten- sity of the intact protein eluting at 64 min decreased as the amount of the major digestion products increased. In the tryptic digest (Fig. 2, top), the early products (41-, 38-, and 33-kDa species) had the same retention times as that of the intact rIL-2R, since only small peptides from residues 72-83 were released. No peptides were released from the C-terminal portion, because all of the potential tryptic sites in this region were followed by proline. However, as incubation continued, the size of the protein peak (at 64 min) decreased and that of the irregularly shaped peak at 53 min, corresponding to the 23-kDa and several smaller species, increased, suggesting that the early protein species were further degraded. In contrast, V8 protease and proteinase K both removed a major portion of the C-terminal region, and the resulting products became more hydrophobic and eluted later than the intact protein (Fig. 2, center and bottom). V8 protease digestion products were very stable and the proteinase K products were slowly further digested.

N-terminal Sequencing-New N-terminal residues generated by cleavages at sensitive sites were also identified by N- terminal sequencing of the 1 h digests. In addition to the N terminus (Glu) of the intact protein and the N termini expected from the HPLC data, additional N-terminal residues were identified (Table 1). In the limited tryptic digest, three additional cleavage sites were detected at the C terminus of Arg6, Lysffl, and Arg14’. The lower sequence yields that were obtained indicated incomplete cleavage, thus these sites are defined as semi-sensitive sites. Lysffl was derived from the exon 3-encoded region, and A r e and Arg14’ were derived from the exon 2 and 4 encoded region^.^ The detection of Arg6, as a semi-sensitive site, is consistent with the observation (HPLC mapping) of a small amount of peptide corresponding to residues 32-36 which was released within 1 h. However, the peptide from the Arg14’ cleavage was not detected by HPLC, because it was still linked to the rest of the protein through disulfide bonds. The presence of semi-sensitive sites within the exon 2- and 4-encoded regions suggests a very important aspect of the structure-function relationships of IL-2R and will be discussed later. Another N terminus, expected from a semi-sensitive site at Arg67 which was detected by HPLC (Fig. 2), was not observed by N- terminal sequencing, because this site was glycosylated. In the limited proteinase K digest, a new N terminus resulting from the cleavage at the C-terminal side of Glng7 was detected in high yield. However, the expected peptide (90-97) corresponding to this sensitive site was not recovered in pure form

Another semi-sensitive site in exon 2 was identified at Arg35 during the characterization of cleavage products, see Miniprint Table S3 and Fig. S2 for details.

Limited Proteolysis of IL-2 Receptor and the IL-2 Binding Core 21099

41-

33- 38-

2 3-

I

FIG. 1. Proteolysis of rIL-2R monitored by reduced and nonreduced SDS-PAGE. Trypsin ( top) , V8 protease (center), or proteinase K (bot- torn). 200-300-pmoI aliquots of samples 29- were removed a t various time intervals, and 5-1076 of these aliquots were used for SDS-PAGE under nonreducing ( le f t ) and reducing (right) conditions. The re- maining portions of the digests were used for HPLC mapping and sequence analy- SIS.

I c "

0 0.5 1 2 4 6 E *

I4

21 1

I

. .

.. .

- ""- o 0.5 I z -" " 4 6 0 22hr

0 0 . 5 I 2 4 6 8 2 4 r - .

by HPLC. No additional cleavage sites were detected in the V8 protease digest by N-terminal sequencing.

Sensitive Cleavage Sites on rIL-2R Molecules-Table I sum- marizes some of the key results obtained from the limited proteolysis of rIL-2R. The cleavage sites are also marked in the structural model of IL-2R proposed previously (24) (Fig. 3). It clearly shows that the sensitive cleavage sites are clustered in regions encoded by exons 3, 5, and 6 with a set of semi-sensitive sites in each of the exon 2 and 4 regions.

Generation of Nicked and Truncated rIL-2R Species and Their IL-2 Binding and Structural Characteristics

Large scale limited proteolysis generated various rIL-2R digestion products (see "Experimental Procedure" for details) which were used to study the structure-function relationships of the IL-2/IL-2R binding and to search for the basic IL-2 binding structure. They were generated from single digestion of rIL-2R with trypsin, V8 protease, or proteinase K; double

digestion with V8 protease and trypsin; and triple digestion with V8 protease, proteinase K, and trypsin. All digestion products were subjected to the IL-2 binding assay using a rIL- 2 affinity gel (26) and to structural characterization (see below and Miniprint section for details). Since the majority of these fragments were nicked a t various sites and truncated at the C terminus, the term "nicked and truncated rIL-2R species" will be used throughout this manuscript.

IL-2 Binding Assay-The digestion products were mixed with rIL-2 affinity gel, and the species which did not bind rIL-2 remained in the supernatant while the bound molecules were desorbed by increasing acid and salt concentrations. Samples collected at the binding and desorption stages of the experiments were then analyzed for the presence of nicked and truncated rIL-2R species by SDS-PAGE. The results of the assays of the intact rIL-2R and the nicked and truncated rIL-2R species generated by limited tryptic digestion are shown in Fig. 4A. The intact rIL-2R and nicked and truncated


FIG. 2. Proteolysis of rIL-2R monitored by HPLC peptide mapping at 2 15 nm. 100-250-pmol aliquots of tryptic (top), V8 protease (center), or proteinase K (bottom) digestion were used for analysis (left panels). The peptide peaks, recovered in pure form, were identified and quantitated by amino acid analysis and plotted against incubation times (right panels). The conversion of glutamine into pyroglutamate at the N terminus of peptides 72-83 in tryptic digest ( top) and 170-190 in proteinase K digest (bottom) resulted in the appearance of a second peptide peak (desig- nated bya ' I " ' ) which eluted 1.5 min later with a concomitant decrease of the first peak. In the V 8 protease maps (center), all of the peptides released were derived from the C-terminal region, with the exception of peptide 83-87 from the exon 3 encoded region. The fast-releasing C- terminal peptide covering residues 169 to 224 was further cleaved and many of its partial breakdown products co-eluted between 35 and 40 min; thus, the rate of release of many peptides could not be monitored. Further cleavage of peptide 211-224 by V8 protease a t GIuz1' was responsible for the disappearance of this peptide. Due to the nonspecific nature of proteinase K, many sites were cleaved, and as a result, a large number of peptides were released (bottom). Those peptides purified in large quantities were characterized and were found to be derived from the exon 3-, 5- , and 6-encoded regions.

' 28.88 38.00 48.88 58.88 68.88 78.80 I I I I I 1 1

TIME (MINUTES) TIME (HOURS)

41-, 38-, and 33-kDa tryptic species were detected only in the samples collected in the desorption steps, indicating that they retained their IL-2 binding characteristics. In contrast, the 23-kDa and two smaller molecular mass species (17 and 3 kDa) were detected in the supernatant, indicating that they did not bind IL-2. The SDS-PAGE patterns of other nicked and truncated rIL-2R species which exhibited IL-2 binding are shown in Fig. 4B. They included the 29-kDa species generated by V8 protease, the 30-kDa species generated by proteinase K, the 28-, 26-, and 20-kDa species generated by double digestion with V8 protease and trypsin, and the 28-, 26-, and 20-kDa species generated by triple digestion with V8 protease, proteinase K, and trypsin. It is of interest to note that all of the early nicked and truncated species generated by limited proteolysis involving trypsin were detected as a triplet on nonreduced SDS-PAGE. The analyses of their primary structures indicated that this is due to partial removal of a glycopeptide by trypsin and maybe carbohydrate hetero- geneity (see Miniprint).

The IL-2 binding property of the nicked and truncated rIL- 2R species were also compared with that of the intact rIL-2R using rIL-2 affinity column chromatography. Nicked and truncated species and intact rIL-2R bound to the rIL-2 affinity column were eluted with a gradient of acid and salt.

Comparison was made between the elution profiles of the intact rIL-2R and the nicked and truncated species generated by triple digestion with V8 protease, proteinase K, and trypsin (Fig. 4C). The fact that the two protein peaks both eluted at the same position indicated that the intact and the nicked and truncated rIL-2R species have similar IL-2 binding prop- erties. In fact, all the major nicked and truncated rIL-2R species showed the same IL-2 binding characteristics as the intact protein with the exception of the 3-, 17-, and 23-kDa species from extended tryptic digestion which did not bind to IL-2.

Structural Characterization-The primary structures of the nicked and truncated rIL-2R species purified by rIL-2 affinity gel, as well as the 3-, 17-, and 23-kDa tryptic species which did not bind to rIL-2 affinity gel, were characterized (see Miniprint). The results of the IL-2 binding assay and structural characterization (Table 11) revealed that only the species with intact disulfides were capable of binding IL-2. The results also allowed identification of some additional small peptides and amino acids (ie. amino acids 167-168 and 36), which were not required for IL-2 binding, but were not previously detected by HPLC and/or N-terminal sequence analyses (Fig. S2, Table S3). Elucidation of the structures in relation to their IL-2 binding function will facilitate the understanding

Limited Proteolysis of IL-2 Receptor and the IL-2 Binding Core

TABLE 1 ( ' lmcwgc~ sitcs and major digestion products grnrrated by limited

proteolytic digrstion of rlL-2R T. trvnsin. V8. S. aureus V8 motease: PK. nroteinase K.

Nicked and truncated r1L"LR

"Cleavage sites are at the C terminus of the amino acid residue listed. These sites are cleaved completely by proteolytic enzymes within 1 h. Some sites are discussed only in the structural characterization section in the Miniprint.

" Detected as semi-sensitive sites of which the corresponding peptides were not completely released within 1 h. ' Detected hy N-terminal sequencing. 'I Detected hy HPLC peptide mapping. " This is from extended digestion. See Fig. S8 and Table S4 in the

Miniprint.

43 a

31 -

14

C

._ c-"'

FIG. 3. Structural model of rIL-2R showing the distribution of sensitive proteolytic sites. -t denotes the sensitive sites which were completelv cleaved within 1 h; - - + denotes semi-sensitive sites which were less than 40% cleaved within 1 h. + denotes glycosylation sites. 7: trvpsin; V8, S . aurrus V8 protease; PK, proteinase K.

E 1 2 3 tD.

97

66 /

of the IL-2/IL-2R interaction. Structural models of the various nicked and truncated rIL-2R species were constructed based on these results (see Miniprint).

DISCUSSION

We have demonstrated that limited proteolysis can be used to probe the structure-function relationships of IL-2R and its interaction with IL-2. In conjunction with HPLC peptide mapping, amino acid, and sequence analyses, limited proteolysis has provided detailed information on sensitive cleavage sites and the core structure involved in the IL-2 binding activity. The sensitive cleavage sites were located outside of the two disulfide-linked homologous domains (Fig. 3). Cleav- age a t these sites resulted in nicked and truncated rIL-2R species which still maintained the IL-2 binding conformation around the disulfide bridges.

The hydropathic profile of IL-2R shows that the exon 3 and 5 regions are very hydrophilic and likely to be exposed on the surface of the molecule (8). As presented in our structural model (Fig. 3), the sensitive cleavage sites were

tDa

91 66 - 43 . 31

21

11

4

21101

k Do -23

" 1 7

-3

5 6

21 / 4 "20

W " ! w

TIME (MINUTES)

FIG. 4. IL-2 binding experiments of intact and nicked and truncated rIL-2R species. A, SDS-PAGE was used to monitor the binding of intact ( / e f t ) and nicked and truncated species generated by tr.yptic digestion after 1 h (center) and 6 h (right). Supernatants collected at different stages of the binding experiments were monitored. SI, supernatant after binding sample to rIL-2 affinity gel, S,, supernatant from the wash with hinding huffer. E , , extraction with acid and salt. 14, SDS-PAGE patterns of nicked and truncated rIL- 2R species generated by single and multi-enzyme systems which bind 11,-2. Lunr I , protein markers; lnnr 2, intact rIL-2; lane 3 , 29-kDa species generated by V8 protease; lane 4, 30-kDa species generated by proteinase K; lune 5, species generated by V8 protease and tp-ptic digestions; lane 6, species generated by V8 protease, proteinase K, and tryptic digestions. C, rIL-2 affinity chromatography of intact rIL- 2R (boltom pane0 and the digestion product generated hy multi- enzymatic system using V8 protease, proteinase K, and trypsin ( top pond) . The column was equilihrated with huffer A (phosphate-huff- ered saline), then the digestion products were loaded. The bound species were eluted with a gradient of huffer R (0.1 N acetic acid, 0.1 M NaCI). The elut,ion was monitored with a postcolumn fluorescamine detection system.


TABLE 2 IL-2 binding and structural characteristics of major nicked and truncated rIL-2R species

For detailed experimental procedures, see "Experimental Procedures" section, and for structural models, see Fig. S1, S2 and S3. T, trypsin; V8, S. aureus V8 protease; PK, proteinase K; y, yes; n, no.

Intact rIL-2R, 43kDa 41 kDa 38kDa 33 kDa 23 kDa 17 kDa 3 kDa 29 kDa 28kDa 26kDa 21 kDa 28kDa 26 kDa 21 kDa

Binds IL-2 No. of disulfide 5

Y Y n n n Y Y Y Y 5

Y 5

Y 5 1

Y 3 1 5

Y 5

Y Y

bonds" 5 5 5 5 5 5

Total no. of 224 (206-212) 88 61 24 (157-163) 159 (145-152) (135-142) amino acids *

T V8, PK, V8 and T V8, PK, and T

Based on the assumption that there are two intra-domain bonds in exon 2. 'Some of the species contain the amino acids represented by dotted circles in the structural models (Figs. SI-

S3).

A

B

ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRAlKSGSLYMLCTGNSSHSSWD-NOCOCTSSATR H H H T T T C C T H U H H H H H H H H H H H H H H H H H H H H H H H H H S T T T C T T l T - l l T T l T T T C T T

d "'

HTCCTTTTTCCCTCHHHHHHUHUnHUSTTSSSSSSSSSSSSCTCCUnHHHHHHUTTTCTTCClTSCCCC ASLPGHCREPPPWENEATERIYHFVVQOMVYYOCV~GYRALHRQPAESVCKMTHGKTRWTOPOLICTGE

.' T

FIG. 5. IL-2 binding core of the IL-2R. A, proposed molecular model. The partially released amino acids, which were not required for binding, represented by dotted circles in Fig. S2 were excluded. A nick in the polypeptide chain at semi-sensitive site Arg14" is also marked. B, alignment of the two homologous domains and the secondary structure as determined by the algorithm proposed by Delphi e t al. (27). The areas with similar structures are shaded. H , a-helix; S , p sheet; T, turn; C, coil. Three semi-sensitive sites are also marked.

found within these regions which are distinct from the disulfide-linked homologous domains. These results raise the ques- tion of the roles of these hydrophilic regions in IL-2R. One possible role of the exon 3-encoded region is to bring the two domains in close proximity to assure the proper formation of disulfide bonds. Once the disulfide bonds are established, the exon 3-encoded region is no longer required. Refolding experiments involving the exon 2 and 4 regions without the exon 3 region are currently in progress to confirm this possibility. The C-terminal segment of approximately 50 amino acids probably provides proper spacing between the IL-2 binding domain and the cell membrane and is not crucial for IL-2 binding.

Cleavage at the semi-sensitive sites (Arg35, Arg36, and Argl4O) in the two disulfide-linked domains did not disrupt the disulfide conformation or effect IL-2 binding. However, upon extended tryptic digestion, cleavage at additional sites caused disruption of this conformation and generated the 23-, 17-, and 3-kDa species (see Miniprint) (Fig. S3). Although each of the three species had portions of the overall disulfide conformation, none could bind IL-2 (Fig. 4A). This suggested that both homologous domains held by the proper disulfides are essential for IL-2 binding. Some of the nicked and truncated rIL-2R species have less than 140 amino acid residues and are still capable of IL-2 binding (Figs. S1 and S2).

A model of the smallest protein moiety capable of binding to IL-2 is given in Fig. 5A. It represents the nicked and truncated rIL-2R species consisting of two homologous do-

mains with 135 amino acid residues generated by the triple enzymatic system. In this nicked and truncated rIL-ZR, the two homologous domains form two independent loops through intra-domain disulfide bonds (e.g. Cys28/30-Cys59/61 for the exon 2 region and for the exon 4 region). These two loops, oriented in an inverse relationship, are linked by two inter-domain disulfide bonds ( C y ~ ~ - C y d ~ ~ and Cys4'j- Cyslo4) to form a symmetrical structure. This model closely resembles the structural model predicted previously based on the exon structures (24).

The two sets of semi-sensitive sites in the two domain structures ( A r p , and Arg36 in one and Arg14' in the other) are also distributed in a symmetrical fashion. They are near the center of each domain (Fig. 5B) and occupy opposite positions within this two-dimensional structural model (Fig. 5A) . Analysis by Delphi algorithm (27) reveals that the secondary structures of the two domains display a high degree of similarity near the beginning and the end, but not in the central regions of the exon 2- and 4-encoded domains where the three semi-sensitive sites reside (Fig. 5B) . This suggests that the areas with nonconserved secondary structure are probably not directly involved in IL-2 binding. This is not entirely consistent with the conclusion derived from the mutational analysis of IL-2R from which direct contact sites between IL-2 and IL-2R have been assigned at or near two short segments, residues 1-6 and 35-43 (21). Our observation demonstrated that at least part of the 35-43 segment is not directly involved in IL-2 binding, since it can be cleaved without affecting binding. It also supports the alternative suggestion that the amino acid substitution in this segment by site-directed mutagenesis may simply influence the folding of the exon 2 encoded region (21).

On the basis of the symmetrical nature of the IL-2 binding core, one would expect that the two homologous domains share the same IL-2 binding characteristics. However, although deletion.of the exon 4 region resulted in a protein which lacks binding ability (5 , 6, 17, 18), only residues in the N-terminal region (i.e. exon 2 region) had been found to be the contact sites for IL-2 (12, 19-21). Thus, portions of both domains held in the proper orientation by the disulfide bonds form the essential IL-2 binding core. As demonstrated by site- directed mutagenesis and selective antibody binding, one possible role of the exon 4-encoded region has been speculated to be involved in binding a second subunit of IL-2 receptor (p70) (21). Recently, crystallization, crystal analysis, and prelimi- nary x-ray diffraction of a complex between rIL-2 and rIL- 2R was reported (28). However, the understanding of the interaction between IL-2 and the IL-2R subunit awaits the detailed analysis of the three-dimensional structures of the IL-2R and their IL-2 complexes.

Limited Proteolysis of IL-2 Receptor and the IL-2 Binding Core 21103

In conclusion, by using limited proteolysis and structural analyses, we have provided direct evidence for the IL-2R structure involved in the IL-2 binding. Using various proteases, a series of nicked and truncated rIL-2R species were generated. Those fragments which were still capable of binding IL-2 were shown to contain the intact disulfide arrangement. Based on the results, a highly symmetrical IL-2 binding core consisting mainly of the two homologous domains encoded by exons 2 and 4 with a total of 135 amino acids has been defined.

Acknowledgments-We would like to thank D. Weber and P. Bailon for the supply of rIL-2R and rIL-2 affinity gel, D. Tighe for drawing the models presented in this work, Drs. C. Y. Lai, T. Mowles, E. Heimer, and A. Felix for review of the manuscript, Dr. J. Smart for his support and advice, and F. Cuevas and L. Nelson for preparation of this manuscript.

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Continued on n e x t page.


fXPERlMENlM FWCEOURES

m: r l L -2 , c loned and expressed i n L. rpll. (29). imnob i l l zed an d e r i v a t i z e d s i l i c a 130) and T I L - Z R . cloned and expressed i n Chinese hamster ovary c e l l s (231, p u r l f i e d by rll-2 a f f i n l t y g e l (261, wew provided by 0. Yebber and P. Ba i lon . Chemicals were purchased from the f o l l m l n g IOUIC~S: HPLC grade water. and a c e t o n i t r i l e ( F i s h e r , Spr ing f ie ld . NJ); t r l f l U r O d C e t i C a d d (TFP) (Pierce, Rockford, It); fluorescarnine (Hoffmann-La Roche. Nutley, NJ); trypsin (Yorthlngton Biomedical. Freehold, NJ); z; m V8 protease, and pro te inare K (Boehr inger-Rnnhei l , Indlanapal i r , I N ) ; ammiurn bicarbonate

CA). Other mater ia ls *eve o f t h e h i g h e s t p w l t y ava i lab le . (Sigma, St. Louis. W ) ; Iequencel reagents and Iolventf (Appl ied Biosysteml, Foster Ci ty ,

kU.&: Llmi ted p ro teo lys is of r l L - 2 R w i t h t r y p s i n or V8 protease was performed i n 0.2W a m n i m bicarbonate at 37'C w i t h a substrate-to-enzyme r a t i o o f 30:l (u/u). Diges t ion wi th prote inase K was performed i n 50d4 t r i l - H C I , pH 8.0 at 25'C w i t h 1 substrate-to-enzyme P a t i o o f 1OO:l I*/*). Time C O U P S ~ study o f t h e p r o t e o l y t i c d i g e s t i o n s was p e r f o r m e d b y t a k i n g a l i q w t l o f the d lgest ion nlxture a t v a r i o u s tlme i n t e r v a l s . The

d iges t ions and by a c l d i f y i n g With TFA fo r p ro te lnare K d iges t ion . reac t ion$ were stopped by heat ing at llO°C f o r 5 m i n u t e r f o r t r y p t i c and V8 protease

Mul t ip le enzymat ic d iges t ion o f r lL -2R were pwformed i n o.2M ammiurn bicarbonate. Oouble digestion us ing V8 proteinase and t r y p s i n was performed at 37'C by f i r s t adding V8 protease at subs t ra te - to -enzyme ra t io o f 30 : l ( M u ) f o r 3 hours and then t ryps in at a subs t ra te - to -enzyme ra t io o f 50 : l (w/w) fov one hour. T r i p l e d i g e s t i o n w i t h V8 protease, proteinase K and t r y p s i n was per fo r red by t r e a t i n g w i t h V8 protease f o r 3 hours at 37% proteinase K f o r 1/2 h a w at 2S°C and t h e n t l y p s i n far 1 hour at 25% a: enzyme- to -subs t ra te ra t ios o f ]:IO. 1:lOO. and 1:30, respec t ive ly . The nicked and truncated rlL-ZR species were purified i m e d i a t e l y b y rll-2 a f f i n i t y g e l .

d iges t ions were compared f a r t h e i r I t - 2 b l n d l n g p r o p e r t y u s i n g rlL-2 a f f i n i t y g e l . The Intact rIL-2R and nicked-and-truncated TIL-2R species produced by the l i m i t e d p r o t e o l y t i c

p r o t e i n or the d iges t ion p roduc t was f i r s t a d j u s t i n g to pH 8 with mnwnium bicarbonate be fo re m ix ing w i th a f f i n i t y ge l f op a t l e w t IO minuter i n d batch PIIOCIII. The supernatant (8 was removed and the ge l was washed twlce with 0 .21 ammiurn b icarbonate ( 5 ) to remv!' any "on-bound species. The bound mater ia l was then desorbed from r l L ~ 2 a f f i n i t y g e l w t h 0.21 acetic a d d (pH 4) conta in ing 0.5M NaCl (El). A l i q u o t 3 o f sampler

Column a f f i n i t y Chromatography was 1110 performed to compare the It-2 b ind ing p roper ty o f co l lec ted at d i f f e r e n t stager of the b lndlng exper iments were then analyzed by SDS-PAGE.

vmious d iges t ion p roduc ts . One ml o f rlL-2 a f f l n l t y gel was packed i n t o a 3 nnr x 15 cm column (Omni, Hetuchen, NJ) then equibrated wl th phosphate buffer sal ine (PBS), pH 7. The

the bound proteln was e l u t e d w t h a l i n e a r g r a d i e n t o f 0 . lM acet ic ac id , 0 . lH NaCl a t a sample was loaded Onto the column. which *as then Mashed w i t h t h e e q u i l i b r a t i n g b u f f e r . and

f l o w r a t e o f 30 nl /hr . A poI tco1um f luarescarnine detect ion system was used fw moni to r ing column e f f l u e n t s (311.

Data obtained from amino ac rd ma lays i r , sequence ana lys is and HPLC were col lected. stored. and analyzed with a Hevlett-Pachard computer (Sunnyvale, CAI us ing a Nelson analyt ical ser ies 4400 XTRACHRW raftware system (Cupert ino. CA).

RESULTS

S t r u c t u r a l a n a l v r i r o f n i c k e d ~ d - t r u n c a t e d r l i - Z R m e c i e r t h a t b l n d 11-2 - To understand t h e s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p o f I t - Z R , the structures of vsrious nicked-and-truncated r lL -2R species dercr lbed in the prev ious sect ions were determned. A l l o f t h e s p e c i e s t h a t e x h i b i t 1 L - 2 b i n d i n g were f i r s t p u r i f i e d by rlL-2 a f f l n l t y gel p r i o r to f u r the r ana ly re r . The HPLC maps and t h e s t r u c t u r a l models are shown ~n Figs.

Tables 51-83 . 81~52. The r e s u l t s o f the amino a c l d and N-termlnal sequence analyrer are sumanzed r n

Spectes generated by l i m t e d t r y p t i c digestion - The a f f i n i t y p u r l f i e d l l m l t e d t r y p t i c rIL-ZR species were reduced by mercaptoethanol and the fragments were p u n f l e d by reversed-phase HPLC pept ide mapping (F ig . SI, top l e f t p a n e l ) . The rerultr of the a m n o ;;;;heyal&s "Ylh v ~ ~ ~ ~ ~ p ~ ~ ~ ~ ~ d e " ~ ~ ~ ~ ~ 5 ( T ~ ~ ~ ~ ~ ~ ~ ~ "y:~:j5 ~ ~ p ~ ~ ~ ~ r ~ ~ ~ ~ ~ ~ ~ ~ s ~ ~ ~ ~ ~ ~ ~

expected minor peptide fragments, 1-36, 37-68 and 37-71. wh l th were generated i n l o w y i e l d s from residues 1-68. 1-71. 81-224, 84-110, and 141-221 were I d e n t i f i e d on the map. and three

by cleavage at l e m i - r e n r l t i Y e r i t e s , were not recovered. The s t r u c t u r a l nwdel i s given I n Fig . S I ( tap r i gh t pane l ) .

A$ shown i n F i g . 1, th ree major TIL-ZR species o f 11, 38 and 33 kDa were detected by SDS-PAGE w i t h i n t h e first hour of proteo lys is . At tempts to separate them using mversed~phase HPLC and rll-2 a f f i n i t y g e l w i t h g r a d i e n t e l u t i o n * w e UnPUCCelsfUl. U l t m a t e l y , t h e y were e lec t rob lo t ted f rom 80s-PAGE Onto PVDF membrane, and the individual bands were subjected to N-terminal sequencing. The resu l t s l nd l ca ted t ha t t hey a l l had @e ;;$ ne;yolitermrni refybtlng from t r y p t i c cleavages at the C- termrnal r ider o f Arg ,

and Arg but v a r i e d i n t e r m O f t h e i r relative i n l t i a l y i e l d s . This obreAat ion iugger ted tha t the mo lecu la r we igh t d r f fe rencer wele poss ib l y re la ted to the extent o f t r y p t i c cleavage at c e r t a i n r i t e s . The absence o f the g lycopept ide cover ing residues 68-71 accounted for the 33 kDa species. Upon Its emo oval, the apparent M of the too two bands draDoed to 3 3 koa. This was i n aareement with the Obsewat10n in'the tlme cbume t r y p t i c d i g e s t i o n ( F i g . Z), I" which a p o r t i o n of peptide 68-71 was removed w i t h i n one h o w . In addit lon, endoproteinare Lys-C. a protease whlch c b y e s on ly at the C - t e l m i n d l r i d e o f l y s i n e . was used to d iges t r lL -2R. A s expected, Arg was not cleaved and t h e w f o r e no 33 koa species YIB detected by SDS-PAGi (data not shown) suggesting the 33 koa species was Indeed a r e s u l t o f cleavage a t Arg I, The d i f fe rence between the 4 1 and 38 XOa species i s not c l e w . I t may be due to carbohydrate heteragenelty.

Species generated by l i m t e d V8 pro teare 01 proteinase K d l g e r t i o n - The determination of the primary structures of the n lcked-and~truncated rlL-ZR species genented by V8 protease or proteinase K #a$ s t r a i g h t f o w a r d . As expected from the reduced SOS-PAGE (F ig . I ) , each

fragments by reversed-phase HPLC (F ig . SI, center and bottom, l e f t panels). The V8 ~ p e c l e s . when reduced by mercaptaethanol, cou ld be separated i n t o two maJor peptide

protease species o f 29 COa was reduced i n t o a 21 koa and a 10 hDa fragment correrpondlng t o residues 1-78 and 88-168, respec t ive ly . The potelnase K species o f 30 koa was reduced l n t o I 26 CDa and a 9 kDa fragment correrponding to r e r i p e r 1-86 nd 98.170, r e r p e c t i v e l y

apparent M for pept ide f ragments 1-78 and 1~86. The deduced I t rUCtUra l models are given (Table SI). Two N-l inked carbohydrate lites a t A m and A d c o n t r i b u t e to the h igh

~n F i g 81 (Fight center and bottom panels).

Specler generated by l i m t e d p r o t e o l y r l r w i t h multl-enzymatic ry r temr ~ Two sets of fur ther nlched-and-truncated rlL-2R rpecler generated urrng the cornb7nation o f 1) V8 Protedle and t r y p l l n or 2) V8 protease prote lnare K and t r y p s i n were a l s o p u n f i e d by TIL-2 a f f i n l t y gel and structural ly charact ; r lzed. The SDS-PAGE pat terns o f the two sets glven i n F i g . 4b shaved t l i p l e t bands o f 28. 26 and 21 koa an non-reduced IDS-PAGE wi th d i f ferences I "

cleavage e f f i c i e n c y of rem,-sensit ive s l t e l are tempemtwe dependent (Table 521. T r i p l e relative 7 n t e n s i t i e r . The i n i t i a l y i e l d s of the N-terminal requencer revealed t h a t t h e

digest ion w3th V8 protease. proteinase K and t r y p s i n was pexJormed at C to avoid over -d iges t ion by pra&inare K.Argl#a I l e s u l t , t h e y l e l d s of Ile and GlyLi70weW loner Ind>ca t ing t ha t Arg and w e ~ e cleaved at $lower rater at the lower

temperature. The d lges t lon p roduc ts were also reduced wlth nercaptoethanol and the

The fragments which *ere recovered i n g o d y l e l d r were i den t l f l ed by am lno 'ac ld ana lys i s r e s u l t i n g p e p t l d e fragments were p u r l f l e d by reversed-phase HPLC (Flg. 82 l e f t p a n e l ) .

(Table 53) and marked on the maps. Peptides derived from the C-temin+l regions o f t h e rIL-ZR were not present, because they were cleaved by V8 protease or pmte lnase K and renwved by a f f i n i t y p u r l f i c a t i o n p r l o r to analysis. The d i f fe rence between the top two bands (28 and 26 koa) and t h e l m e r band (21 koa) i s again due to the presence and absence O f the g lycopept lde 68-71 (Table 531. The deduced structural m d e l r a m g iven I n F l g . 82 ( r i gh t pane l ) .

2 t r u c t u r a l a n a l v r i r of nicked and t r u n u r IL -2R soecles t h a t do not blnd U better understand the requl rewntr for 11-2 b ind ing t he 3 I 7 and 23 kDa rpecles generated

- - TO

by extended t r y p t i c d i g e s t i o n (Flg. 1A1, which no' longer b ind It-2, were also characterized. The 6 - h o u r t r y p t i c d i g e s t was flrrt m i x e d w l t h r l L - 2 a f f i n i t y g e l to remove any I t - 2 bound species. and the f low- through Inc lud lng unabsorbed peptide fragments and the major r lL-2R species were purif ied by reversed-phase HPLC under nom-reducing conditions

resolved and t h e i r I d e n t i t l e s were establ ished (Table S I ) . The 23 koa species was the (F ig . 531. Three sets of d i s u l f i d e l i nked pep t ide segments and two m a l l peptides were C - t e r n l m l h a l f o f t h e p a l y p e p t j p cP@ frm 156-221 I l n k e d to pept lde segment 118.136 t h r o u g h t h e d i s u l f i d e p a i l Cy$ -Cy$ The I 7 kDa species consis r e g y p s Ibf2-32, 37-68 and 81.105) link;d by d l w l f l d e p a i n cy,"/ps.:::a'/gePtl~; CYS -Cy5 . It contains one of the Puta t ive 11-2 b ind lng rlter residues 35-43 (21) and matcher near ly hal f of the n icked-and- twncated rpeder genera ied by the t r ip le enz&atiC d i g e r t l o n . 111-148) l i n k e d by Cys'-Cyr . It a lso contrlnr one SDeula ted It-2 contact r l t e .

he Fkjrd ret conllsts o f two small peptide segments 11-16 and

. .

residues 1-6 (22). Molt O f the small peptides except 17-21 and 72-83 were not re ta ined in the columo under the condi t ions used f o r t h i s p e p t i d e map. The s t r u c t u r a l n o d e l i s given i n Fig. 83 ( r i gh t pane l ) .

Limited Proteolysis of IL-2 Receptor and the IL-2 Binding Core 21105 hble S4 - Inino acid cmpsitional anslyis o f nicked-and-truncated rlL-2R

species generatad by extended tryptic digestion purified by HPLC

Portion 17-21 72-03 1-16 and 22-32. 37-60" 110-135 and 141-148 139-681 and 156-224

Mobility * 3* 111 23*

I-terminal * Sequence

:MY also contain s m of the peptide in [I. I too small to be detected on SDS-PAGE or sequence not necessary.

NO not done. see Fig. 53 for structural nodel.

() expected values are in parentheses.

V8-PK-T __

, -10

dotted circler and arrows, respectively.

limited proteolysis of recombinant human soluble interleukin-2

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