Biechimka el Bi*Td~'.~'aAcla, i ! 18 ~199| J 25=35 ¢'~ l'4tJl ~sevier Science PllM~she~ B.V. All fights reser~'ed 0167-4B38/t~I/S03.5U
25
BBAPRO34~47
The role of arginine residues in interleukin 1 receptor binding V e n k a t a B. N a n d u r i 1, J e f f r e y D. H u l m e s I.,, Y u - C h i n g E. P a n t, Patricia L KJlian z a n d Alvin S. Stern s Deparma'm afPraWin Bieclu'm~tO'. R~che Research Center, Hoffinmm-La Roche hie.. Nutlo; NJ (U.S.A.~ and " lX,lmament of lo~nutwpl',armacdeg): Roche i~'search Center. Hoffmmm-La Roche Inc.. Nutle); NJ {U.S.A.) (Rece~,'cd Z'4 May 1991)
Ke~~rd~: |nterleukin i: |nter|eukin ! receptor. Phenylglyoxal:Protein modification:Receptorbindingasmy (EL-4ceil) Inlefleugin I (EL-I) is a family of polypeptide Otokines that plays an essential role in modulating immune and inflamm_atory responses. IL-i adiHly is mediated by either of two distinct proteins, IL, icz or IL-I/$, both of which bind to the same receptor fonnd on T-bmphocytes, fibroblasts and endothelial cells (Type I receptor). The effect of specific chemical modifimtinn d recombinant IL-la and IL-I/$ on receptor binding was examined. Modification of the proteins with phe~lglyoxaL an arginine-specific reagent, resulted in the loss of Type I IL-I receptor binding activity. The stoichiome~ of this modification revealed that a single arginine in either IL-I~ or IL-I# is responsible for the loss of activity. C y a ~ bromide cleavage of phenylglyoxal modified IL-la and IL-IIL follmmi by sequencing of the peptides, re~'ealed that arginineq2 in IL-la and arginine-4 in IL-ifl, which occupy the same topology in the respective co'stallographic smmmres, are the target of phenylglyoxal. These results suggest that an arginiue residue plays an important role in ligand-receptor interaction.
Introduction lnterleukin 1 (IL-i). secreted by activated macrophages and other cells, mediates a variety of inflammatory and immunological responses [1.2]. IL-I activi~ includes the ability to regulate a subset of th~mocytes [3A], stimulate B-ceil proliferation and differentiation [5.6] and stimulate proliferation of precursor hematopoietic cells [7.8]. In addition, it has been demonstrated that mice pretreated with IL-I prior to inoculation with bacteria have higher suwival rates [9.10] and IL-I affords protection to animals against normally lethal doses of radiation [11]. However. IL-I has also been associated v,ith what would appear to be injurious activities. For exam#e, IL-I affects synovial and dermal fibrobiasts to produce tissue-degrading
• Present address: Deparlmen! of Protein Chemistq'. American
Cyanamid Co_ Pearl R~,er. NY IIF~5. U.S.A. Abbreviations: |L-I. intefleukin I; riL-l, recombinant interlcukin |: PBS. Dulbecco's phosphate4~uffere_.d saline (~ilhoul calcium chloride or magnesium chloride~ BSA. tm~ine serum albumin: TCA.
ttiehloroac=tic acid: PTH. ph=n)'lthiohydantoin. Correspondence: AS_ Stern. Department of Protein Biochemist~'. Roche Research Center. Hoffmann-La Roche inc.. Nutley. NJ 07110-ll(~9. U.S.A.
proteinascs [12,13] and prostaglandin E2 i14]. These and other studies suggest that IL-I may be involved in certain immune- and inflammatory-based diseases such as rheumatoid arthritis. Two species of IL-1 have been identified and have been di;,inguished as human IL-la and human !1.-1/3 [ 15- i 7]. Both proteins are synthesized as larger precursor molecules of approx, similar size (31-33 kDa) with the mature ~ 17 kDa species deriving from the carboxy-terminal domain of their respective precursor. Although there is no more than 25% sequence identity between the two biologically active mature forms, both human IL-la and human IL-l/3 have similaL if not identical, activities. This is not surprising since they bind to the .same receptor proteins [18-20] with similar affinity on T-lymphocytes, fibroblasts and endothelial cells (Type I receptor) and B-lymphocytes and macrophages (Type 2 receptor) [21,22]. This may be explained by their similarities in tertiary structure. The X-ray diffraction structure of human IL-Ij0 [23-25] has been described as a tetrahedron with edges formed by antiparallel B-strands. The core of the human IL-la structure [26] is a capped/3-barrel that possesses 3-fold symmetry and displays a topology similar to that observed for 11.-1/3. However, the amino acids which interact with the Type I or Type 2 receptor have not been defined.
26 In understanding, on a rrmlecular le~'el, the range of bk~togical responses elicited ~" IL-1. it is necessaD to identify the act~,'e site(s) of the IL-1 molecule in hs interaction whh the IL-I receptor. Pre~ous[y it had been demonstrated that chetnica] modification of IL-1 arg[nine residues destr~ed th~,xnoc~e proliferation act~'it~" [2728] and p.'rogenici~" [~1. Based on t h e ~ earlier chemical nmd~f~atien results, pheny|glyoxal ~as chosen as the arg[nine-specific reagent to probe and compare the actk'e silos of human IL-la and human IL-lfl. These studies identi~' similar topographic regions of the ~'o molecules which are important for Type I receptor binding and subsequent actg'at/on of celts. Mater~ls and Methods
Proteins E.xpress~on of the human IL-I eDNA in E. colt ~ie|ded mature protein co~esponding to residues 117271 of the unprocessed pro-lL-la precursor molecule [I7| and residues [ ~7-269 of ~he unprocessed pro-lL-I precursor molecule [ 15]. Human rlL-la, human rlL-i and mouse rlL-[a ~ere purified as pre~Jously desenbed [17] excep~ that the proleins were prepared in mM Tris-HCl (pH 8.1). containing 0.4 M NaCL The specific act~,~' o[ the proteins ~as determined in the mouse DI0.G4.1 ce|l proliferation assay [30]. Human rlL-la, human rlL-l~ and mouse rlL-la had specific act~.'ities of 5.7- ~0'~. 6.1 - 1O: and 5.1- l0 s units/rag. respectively. Mouse rlL-l~ was radiolabeled ,,~'[th Nat'-~l as previously descnT~ed [31]. The labeled protein ~'as fully acti~'e in the D10.G4.1 cell proliferation assay ~hen compared to the unlabeled protein.
obtained a~ ~=C, using a sweep w~dth of 7000 Hz, with I28 to 1024 transients collected per spectrum, at a re~.c|e rate of 3.0 s. The residual water re,nonce was reduced ~ prk~rit~ng with the dccoupler.
Smicl~m~esO" of 17-1ZC/phenylglyoxal to IL-I T~'o nn~l of human rlL-l¢ or human rlL-lp were incubated ~ l h 6 mM [7-~C]phen~'lglyoxal (specific aet~-i~- 5 m ~ / m m o l (Amersham, Arlington Heights. IL~) in a 59 #l reaction ~ol. containing 50 mM phosphate buffer (pH 8.0) and 5% glycerol (v/v) at 3TC. At the end of 90 rain. l ~ u g of BSA was added as a nonspecifie prote~n cartier and the incorporation of [7~"C~hen.~l_~..~xa| into IL-J was determined ~ precipitartan of the protein ~ith the addition of 50% (w/v) TCA to aehie~-e a ! ~ final concentration. The precipitales ~,ere colleclcd ~" centrifuging the mixture at 8 ~ × g for 30 rain and v,~ashed (2-3-times) with 5% TCA foll~-od ~" ~'ater. The pellets were then resuspcnded ~n 8 M urea and transferred to a scintillalion x~iaI ,~-here the rad~oactK~- was measured in 3 ml of Ecos~nt H (Nafiona[ D~agn~ics. Somerville, NJ)
Soluble receptor binding assay for nwasuring IL-I
A stock solution (0.I M) of phenyl~yoxal (Sigma) was prepared by dissolving the cry'stats in 50% ethanol. M #g of human rIL-la or human rlL-l/~ were incubated ~'ith var~'ing concentrations of phenylgi~'oxal in a final reaction ~'d. of 50 t~l containing 50 mM phosphate buffer (pH 8.0) and 5% gl)'cerol at 3"FC. After t3O min of incubation, aliquots ~ere removed and diluted t00-fo[d with 50 mM phosphate buffer (pH 8.0) containing i% BSA. Aliquots were removed for the determination of IL-I binding activily, as de~ribed below, and the remaining protein was precipitated by the addition of 50% iwt./vol.) TCA (to achieve a 10% final concentration) and used for the preparation of peptide cleavage fragments. For ~H-NMR studies. D,O was exchanged into the modification reactions.
"[he receptor binding assay [31] is based on competition b e ~ e e n IL-I in a sarnp]e and radio[abeled mouse riL-la for binding to soluble IL-1 T~ept_or. Soluble Type I IL-I receptor was prepared from mouse EL4.lL-2 th~moma cells (ATCC No. TIB-181) as previously described [31]. -~ ng of rlL-! (modified and unmodified} and L~l-labeled mouse rlL-la (2.0-i04 cpm) was incubated whh soluble IL-! receptor for i h at 3TC in a total reaction ~'o!. of t59 #! (PBS containing 8 mM CHAPS, ~ mM NaCI and 0.1% BSA). After addition of bovine y-~obulin as a nonspecific protein carrier, receptor-b~und uzSl-mouse rlL-la was separated from free radiolabeled iigand by precipitation whh 12% imlvethylene glycol 8000 (Simna). The precipitated protein complex was collected onto Whatman G F / C glass fiber filters (Fisher Scientific). washed, and the filters were counted in a gamma counter. Nonspecific binding was determined ~" the addition of excess unlabeled human rlL-la (50 riM). Aeti~[~- was determined as percentage inh~ition of specific binding of labeled mouse rlL-la by IL-I in the test sample. The percentage inhibition was calculated by subtracting from 1 the difference bet,~-een sample binding and non.specific binding (cpm) divided by the difference between total binding and non.specific binding (cpm). This number was then muhiplied by 100 to obtain the percentage inhibition ~alue.
tH-AqffR umdysis
Protein analysis
NMR spectra were acquired at 500 MHz on a Varinn VXR-5~S spectrometer. Sample concentrations ranged from 0.2 to 0.5 mM in protein. Spectra were
In order to locate the site of arginine modification, phenylglyoxal-treated riL-i was cleaved with cyanogen bromide and the resulting pepfides sequenced and
Chemical modification reactiwzs
compared to pcptide fragments generated from the digestion of unmodified rlL-I, 2 nmol rlL-I was modified ~ t h 6 mM phenylglyoxal as described above and precipitated with 10~ T C A (final concentration). Unmodified protein was similarly precipitated. The pellets were washed t ~ c e with 5% T C A and finally with water. The precipitates were then dissolved in 100pl of 7 0 ~ formic a d d and cyanogen bromide was added in 100-fold molar excem over methionine residues. The digestion m ~ u r e s were incubated in the dark at room t e m ~ r a t u r e for 16-18 h and then diluted approx. 10-fold with water and dried under a stream of helium.
.^
1
:[ 0 o
0 ~B
0.1
1
i~! a
I I I
.5
The resulting digests of modified and unmodified protein underwent sequence analyses using an Applied Biosystems model 470A gas-phas~ sequencer [32], Phenylthiohydantoin (PTH) amino acid derivatives were identified 'on-line" with an Applied Biosystems model 120A PTH analyzer [33]. Results
Inactiration of IL-I by phenylglyoxal To examine the role of arginine in the binding of I L - l a and I L - I ~ to their receptor and to use this information in identifying common topological regions within the two IL-I molecules, the proteins were reacted with phenylglyoxal. Previously it had been demonstrated that phenylglyoxal-treated IL-1 was less active in a variety of assays [27-29] suggesting that a common active site of IL-I was essential for biological activity on diversified cell targets. However, at the time of those studies, the occurrence of two different IL-1 molecules and IL-I receptors was not known. Preincubutton of human r l L - l a and human r l L - l p with increasing concentrations of phenylglyoxal at 37°C for 90 min resulted in the loss of receptor binding activity. (Fig. IA and B) with a concomitant loss of T-cell proliferation activity (data not shown) in a dose-dependent manner. There was a linear loss of the above functions of I D I up to approx. 6 mM phenylglyoxal concentration. The ability of human r l L - l a to bind to its Type 1 receptor was found to decrease with time of reaction with a FLxcd concentration of phenylglyoxal (Fig. 2). A similar time-course of inactivation was found for human rlL-I//. Thus, at 90 min incubation time, 80-90% loss of receptor binding ability of the congeners was
90 m
fi0
40 i
4O
+6
10 0
100
0.I
~L-1 .S
(ng/rrO
Fig. I. Effecl o f p b e n y l ~ y o x a ! o n ~¢ceptor binding aclivi~, o f I L - l a
and IL-IH. (AI Human rlL-l~ (I nmol) and (B) human rlL-IH (i nrnol) were reacted gith 2 mM ! o). 4 mM (+). 6 mM( A ). 8 mM (o). 10 mM ( x ~and no ( A ) phem't~ml and binding act~.itT measured ~' soluble receptor binding a-.sa~-.Serial 5-fold dilutions starting at It'll ng/ml of the respecff,-eligands g ere added to duplicate tubes and the assay ~-as performed as described under Materials and Methods. D a t a r e p r e s e n t binding as p e r c e n t a g e o f that obser~ed in
the absence of any competing ligand (approx. 10th'h')cpm/assayL
JL
2O 10 0
.i
0
lb
T
I
I
ii
30
45
60
75
i 9O
T i m e (rain) Fig. 2. Time-course for effi:ct of phenylg[yoxal on inactivation of
receptor binding aclivily of IL-lu. IDle (I nmol) was reacted wilh (A) and without (o) 6 mM phenylgb'oxalat 37°C for various times. diluted in BSA 0%) and binding activity measured as described in Fig. I.
28 ii ~iii !i !ii~ ~i il !:
i
!!i '
il i! !:
iii:
ili l
!
ii ~i
ii
~ii i~i, ~
ii i!~ii ::i:!i
i
ii
i
i
-t-~'.
v,,;,r
, ,i
!~
V
i~, /
ij,,
i
'
tl
|
!liii !
Fig. 3. ~H-NMR spectra of m~.tified IAI and natite (B) IL-la. Ar, cxp'ar~ion of Ib¢ upfie|d porlh)n of each speclrum is sl'a~n to ihe righl. Spectra were acquired a.~ de~cn'bed under Malerials and Method-~. Chemical ~hi[b are referenced to external ..-,~l[um 4.4~dirnelhyl4-si|apenlane .,,uffonate_ Excepziona[|) large re,~onances pre~ent in the ~pectra are due to r~[dual H . O . g|)eero~, buffer tatnnp(ment.~, and excess phenvl£Ayoxal.
apparent with 6 mM phenyiglyoxal while under similar conditions of incubation of control rlL-I (without pheny'lglyoxalk no loss of receptor binding activity was observed. Identical inactivation results were obtained with modified compared to control protein when assayed for their ability' to stimulate T-lymphocyte proliferation (data not shov,~n). IH-NMR spectra of natize and modified IL-I The overall conformations of the native and modified IL-I molecules were assessed by NMR spectroscopy. For both IL-la and IL-IIL the nH spectrum
of the modified protein was generally similar to that of the native protein (Fi~. 3 and 4). In particular, the resonances with unusuaUy high upfield shifts retained their positions in the modified proteins (insets of Figs. 3 and 4). T h e ~ re.,~nances are predominantly methyl groups of leucine and isoleucine, whose unusual chemical shifts are probably caused ~" proximit2,' to aromatic groups in a hydrophobic core, and are therefore good indicators of a distinct globular structure. The modified proteins appear to retain the overaU tertiary fold of the native proteins. There are some small chemical shift differences apparent between the modified and
29 native molecules which suggests local structural pertubations. T h e ~ appear to be more extensive for IL-I~ (Fig. 4) than for I L - l a (Fig. 3). $loichiometry o f [7- :4C lphenylglyoxal modification o f IL-! Phenylglyoxal is known to form stable adducts with guanidyl groups of arginine residues in proteins [34.35]. Since it had been demonstrated that two molecules of phenylglyoxai react with one arginine residue [351. the number of tool of l~Cdabeled phenyiglyoxal bound per tool of IL-I was determined by TCA precipitation. These labeling studies found that approx. 2 mol of [TJ~C]phenrlglyoxal was bound per tool of human rlL-
TABLE I Stoichiomt'tn' o[ 17-~C]pholylglyoxal binding to IL-I " Addition
mol phenylglyoxal/molIL-I
IL-la IL-13
2.03 2.25
nmol of human rlDl. and human rIL-IL¢were incubatedwith 6 mM [TJ~C]phenylglyoxalfor 91J min at 37°C as described under Malcfials and Methods and the incorporation of label into IL-I determined by liquid ~intillation spectroscopy.
" 2
l a or human rlL-l/3 (Table I) when the proteins were inactivated to approx. 90%. This suggests that one of the three arginine residues in both I L - l a and 1L-IB is
i •
"
I 8
. . . .
I 7
"
~
"
~,""r '-~' 6
~
,
•
I 5
•
~
~
| 4
~"'~
,
,
i 3
. . . .
I 2
. . . .
I
. . . .
1
I -0
. . . . nora
"~mT"q"~T"l~"l'"lt'"l'"'l'"~'"'l"'l'"l 0
6
0
4
0.2
-0
I
-0
~
pl~
Fig. 4. ~H-NMR spectra of modified (A) and nalive (B) IL-i/L An expansion of the upfield portion of each spectrum is shown Io the right.
Conditionswere as that described in Fig.3.
3O
Human rlL-1 ~;~
:
CNBr
1
2O
......P~tid~ 1 ( ~ ) ...............................................~ ~ , ~ t i ~
...................................
21 ASh Asp AZa L~u A.snGIn Set lle lle FA~-]AIo ASh AspG~ Tyr L~u Thr / ~ . . . . . . . . . . . . ..o . ..o...,
4O A~ A~
. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .o . . . . .., ... . . . . . .. . . . .
CNBr ...........................................................................................~ i
.... ~ "
............................
~
61 8,0 AspA.k: LysBe ThrVd Re LeuFA-~-l~e Ser Lys ll'wCk~ LeuTyr Val Thr Aio Gln . . . . . . . . . . . . . . . . . ... ... , . . o . . . ,
. . . . . . . . . . . . . . . . . .. ... . . . . .
o...o,.o
. . . . . . . . . . . . . . . .. . . . . . . . . . . . ..o , . o . , o
.....
..
81 CNBrj. Asp Clu Asp Gin Pro Vd Leu Leu Lys Glu Met~Pro Glu h
. . . . . . . . . . . . . . . . . . . . o.o . , . . . , . o o o , o . . . ,
......
100 Pro Lys Thr Re Thr Gly
....................................................................................~p...Pe~tide 4 (,~,~).................................... t01 Se=- Glu Thr Ash Leu Leu Phe Pine Trp Gtu ~ ..o~
120 His Gly Thr Lys A.sn Tyr Pne Th~ Set
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
121
Vd Ak: His Pro A.sn Leu Phe Be Aia ~
. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. . . . . . . . . . . . . .. . .,
.......
......
Lys ~
. . . . . . . . . . . . . . . . . ... ... . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . .
140 Asp TyF Tip Vd Cys Leu Alo Gly . . . . . . . . . . . . . . . . . . . . . . . . . . .. o .o o .
141
.....
~..
. . . . . . . . . . . . . . . . . . . . . . . .
1,55
H.uman
rIL-lg
:
-- Peptide
CNBr . . . . . . . . . . . . . . . . . . . . . . . . . CNBr /
~..~,~
T~-)
-~IM--P,~,,~ s (.~)
......................................................................................
CNBr h
........................... I~i Hi ..... Peptide 4- ( , 8 )
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
BO 61 G!y Leu Ly~ Glu Lys Asn Leu Tyr Leu Set Cys V~I Leu L ~ Asp Asp Ly~ Pro Thr Leu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
, ..............
, ....................
o ..........
81
c~n
L ~ C~. S ~ Vd Asp P~o Ly~ As. TF Pro Lys Lys Lys ..................................................................................................................
10t Phe Ash Lys I~e Clu lie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.~=Br
,....,
......................
aet.Gtu Lys ~
100
Phe Vd
.I~i14.....Peptide 5 ( ~ ) ' . . .
120 Asn Asn Lys Leu Giu Phe Glu Set A]o Gin Phe Pro Ash Trp
, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
CNBr ~
121 ............................................. CNBr
141
, ......
o,,
o,,
140
Pep.tide 6 ( ~ } ..........................................
g
153
Gin Asp ,e Thr Asp Phe Thr Met.Gin Phe Vd 7S~;~.1~ ............................................................ I~,q ..... Peptide Fig..'3. The primary structures ~)f IL-h~ and IL-I/.J idemifying argininc rcsidu¢~ I [] ). sile of q,'anogen bromide cleavage ( .~) and the resultant generation ¢~f pcplidcs from IL-I~ [Peptid¢ I(~) --, Peptid¢ 4(a and IL-Ip [Pcplide l(~8)--* Peptide 7(/])t that were sequenced. )}
modified although variable and partial modification of the arginines could not be discounted by the above data.
the phenytglyoxal adducts [36] made a rapid analysis essential. 2 nmot of modified and unmodified human rlL-la and human riL-If3 were chemically digested with cyanogen bromide as described under Materials and Methods. The amino acid sequence of each mixture of cyanogen bromide-generated peptides of digested control and modified human riL-la and digested control and modified human rlL-l.8 was determined by automated Edman degradation. Data from the first 25 cycles of analysis of I D l e peptides indicated four sequences present, as would be expected from the number of methionine residues present in IL-la (Fig. 5). Arginine-12 was prominently missing from the first cycle of the sequence analysis of the digested modified human rlL-lot identifying this residue as the site of phenylglyoxai reaction in IL-la (Fig. 6). Similarly, data from 15 cycles of analysis of It-I/3 peptides indicated seven sequences present, as would be expected from the number of methionine residues present in IL-I~
ldemi#cation of tiw site of phenylglyoxal reaction Based on the above data, a strategy was designed to identify the arginine residuels) in It-in and IL-I/3 that is essential for iigand binding to the Type ! IL-! receptor. Modification and chemical cleavage by cyanogen bromide of the proteins would yield a number of fragments of predicted ~quence (Fig, 5). The cleavage products could be sequenced directly and an arginine residue conspicuously missing from a sequencing cycle would be identified as the site of phenylglyoral reaction. This simple procedure was chosen over purification of the Oeptides because it assured complete analysis of each of the generated peptides as opposed 1o the possibility of peptide loss during the resolution of peptide fragments upon reversed-phase HPLC separation, in addition, the known instability of
P=' E = .L,-
K~
,m~
T i,~. I=~ --r. G~'°°_,_,E'==T'"N'°"L'" L ' ' F , o ~
A~
'°s .... o~E.O
K~
'~"
Sm
L~m J~ l~ T=
l'~
L ~ R eg
i~o
S " K ~ T'~Q'-
EL
.._.., a
J
I'"
400
K-~
PEPTIDE 2 (¢4) Aal
"t
N = D = O~ y=
L~a
a s, N, ~1~11 1
I~ Ell ~ 2
3
4-
5
I~
~ 7
E:
L~ g
10
11
12
13
14-
15
16
17
1~
19
20
~'1
22
23
24-
2.~
CYCLES Fig. 6. Amino acid sequence analysis of cyanogen bromide peptides from IL-I~ (cross-hatched bar) and phenylgt~)xablnodified IL-I~ (~)lid bar). I D l a was modified with 6 mM phenyZglyoxaL After incubation for 90 rain at 37°C, the protein was precipitated wilh TCA (I(F,;~ final concentration) and fragmented with cyanogen bromide in 70% formic acid for 18 h. The pcptides thus obtain,'d were sequenced by gas.phase automated Edman degradation. No arginine peak (*) was observed at O~c]e 1 of the PTH amino acid analysis of tile modiEed It-Ice peptides indicating modification of arginine-12 of IL-la. ~. estimated yield based on the obtained wdue divided by the expected number of the same amino acid at that cycle (Fig. 5).
PEPTIDE 7 ( ~ ) 200
S ' ~'
~'v'~2F 1~ L ,:a
G 'a
8@oP•
K 1~' PEPI]DE 6 (.,~)
2®i
~a K g"
~
'1~i[' [~ Tl~ii"4'1~i"
PEPTIDE 5 (,6)
1
Fsa
F,,.o V..7 Q ~
E 600
E ' ~ I ' ~ N '~' N , K '°91_,, o
PEPriDE 4 (.,6)
G~
j 400 "' 200
E~'~
~-
N~ D~ S~'~i ~i
.
I~ [~
HI
v a A "~' PS"
I91BI
E'~7
Q-,,~V~ V+l F 4~ 200 Jl~!
S"~ .~
~
.
.
.
.
.
PEPTIDE 2 (,,6)
+.l==)2.3
A ~ p2
o ,..
1
.....
2
3
•
4
5
.,
6
7 8 CYCLES
m, 9
re*Ill'~° ~i' ~i
10 11 12 13 14 15
Fig. 7. Amim~ acid .,,equcnc~:anal).'.,is of ~.3'anogcn bromide pcptides from IL-Ifl (crt~.,,.,,-hatchedbar) and phenylglyoxal-modified iL-lp (.,~lid barL IL-I~ ~as m~}dified v,ith phenylgt.voxal and [ragmenled with ~'anogcn bromide as described in FiG. & The peptides thus obtained were sequencedh~ gas-phase automated Edman degradatk, n. Marked rcducti0n {*) of the arginine peak was ,.}bsc~x-cdat ~'¢1c 4 with slight reduction t "" ) at l.)'cle I I ~lf the PTEi amino acid anaL~sisof the modified IL-t/3 pepli.dcs. This indicates modification , t at[ininc-4 and partial modification of arginine-tl af IL-I/~. N I. not identified: ~. estimated yield based on the t)btained value db,Jded by the expected number of the same amino acid at that cycle {Fig. 5).
33 (Fig. 5). Argininc-4 was conspicuously reduced from the fourth cycle of the sequence analysis of peptides generated from modified IL-lfl when compared to control protein identifying this residue as the site of phenylg~yoxai reaction in IL-i/3 (Fig. 7). Furthermore, a partial modification may have occurred at arginine- I 1 since the sequence data are equivocal at this residue (Fig, 7). Subsequently. the intact molecules were sequenced which verified the extent of modification of these arginine residues in both proteins. Both arginine12 and arginine-4 were missing from the twelfth and fourth cycles of the sequence analyses of modified IL-hx and IL-I~ respectively {data not shown). Discussion The presence of arginine in the active site of 1L-I has been indicated by the sensitivity of these cytokines to phenylglyoxal treatment [27-29]. Since phenylglyoxai has been known to selectively react with arginine residues, identification of putative arginine residues in IL-1 that are reactive toward phenyiglyoxal was impor-
tant inasmuch as data recently obtained from mutagcnesis of IL-! eDNA denoted the importance of arginine residues. For example, truncation by removing amino acids from the amino-terminal end of IL-la showed biological activity markedly decreased with the removal of arginine-12 [37]. Truncations of IL-l~ showed homologous requirements for biological activity as truncation of IL-la in that activity was maintained in aminoterminal deletions until the arginine at position 4 was removed [37,38]. Furthermore, using combinatorial cassette mutagenesis, Yanofsky and Zurawski [39] have shown IL-la to have an abmlute requirement for a basic residue at arginine-12 although these studies could not determine if changes at this residue simply caused structural defects. Similarly, by site specific mutagenesis, arginine-4 of l L-l/3 had been shown to be one of the key residues in the function of IL-I/3 [38,40] although in these reports it had been concluded that the differences in activity of these recombinant proteins were due to either significant changes in the conformation [38] or folding of the mutant polypeptides [40].
\
• . •
.
/
..'
Fig. 8. Su~rposition of crystallographically determined structures of iL-la [26] and IL-I# (B.J. Graves, personal communication) showing the proximily of arginine-12 of lL-la [R-12(a)] and arginine-4 of IL-I/3 lR-4(/3)]. The guanidino head groups of thes,' residues are labeled in the lower central portion of the figure. The backbone is shown as a Ca tracing viewed approx, down the pseudo 3-fold axis as shc~wn in Fig. 2 of Graves et al. [26]. This close-up shows primarily the six ,B-strands which form the sides of the/3-barrel. The proteins were aligned using the common 81 a-carbons of the 1L-I fold 126]. The close fit of the central B-strands of IL-l~r ( ) to those of IL-I B ( - - - - - - ) is apparent.
e x t e n d the a b o v e o b s e r v a t i o n s to the ! a r g i n i n e residues in the I L - i p o l y p e p r r e s p e c t i v e T y p e 1 r e c e o t 3 r (i.e, by ng o r s a h - b r i d g e f o r m a t i o n ) . By sitem o d i f i c a t i o n o f the wild-type protein° c h a r a c t e r o f the a r g i n i n e r e s i d u e s w a s ; simply a r e q u i r e m e n t for the m a i n t e [I s t r u c t u r a l stability o r a t t a i n m e n t o f of the p r o t e i n since the global c o n f o r m o d i f i e d m o l e c u l e s w e r e essentially I t h a t o f the native p r o t e i n as a s s e s s e d roscopy. A l t h o u g h s o m e small, local rbations were apparent, the effects of tokincs a r e not c a u s e d by a large o v e r range in the m o l e c u l e s . T h e d a t a unltifies a r g i n i n c - 1 2 o f I L - l a a n d argias essential r e s i d u e s r e l a t e d directly to :don a n d the c o n c o m i t a n t induction o f I.v. h a d b e e n d e m o n s t r a t e d that t h e c o n i n e - l l o f I L - l / 3 to a glycine r e s i d u e (by l t a g e n e s i s ) yields an a n a l o g which has biological activity t h a n the wild-type [owever. the r e c e p t o r affinity o f the w a s d e c r e a s e d by only 25%. T h i s sugf i n c - l l m a y b c involved in a c t i v a t i n g :ion e v e n t s [41] b u t not in b i n d i n g o f :eptor. T h e r e f o r e , the p a r t i a l m o d i f i c a :-l l by p h e n y l g l y o x a l (Fig. 7) p r o b a b l y ate significantly to the loss o f r e c e p t o r consistent with t h e c o n c l u s i o n t h a t t h e t e r t i a r y s t r u c t u r e s of I L - l a a n d I L - l f l [ 2 3 - 2 6 A 2 - 4 4 ] . T h e e x a m i n a t i o n o f the :thine-12 a n d a r g i n i n e - 4 on the tertiary l a a n d I L - l f l respectively, l e a d s t o t h e e a c h b i n d the T y p e I r e c e p t o r in simir e g i o n s o f e a c h m o l e c u l e (Fig. 8). in o u r results, t h e s e r e s i d u e s a r e o n the ~roteins at the s t a r t o f / ] - s h e e t regions. :nee h a s d e m o n s t r a t e d that B cells conIL-! r e c e p t o r m o l e c u l e s ( T y p e 2) t h a n e l I L - I r e c e p t o r [21,22], It will b e o f line the a r g i n i n e - m o d i f i e d I L - I p r o t e i n s oint o f d i f f e r e n t i a l i n t e r a c t i o n o n T y p e us T y p e l r e c e p t o r a n d c o r r e l a t e d blocs. F u r t h e r m o r e , with a d d i t i o n a l d a t a ific c h e m i c a l m o d i f i c a t i o n a n d site-ditests, w e h o p e to m a p the b o u n d a r i e s o f e a n d to identify s u r f a c e r e s i d u e s t h a t t with the r e c e p t o r . Such results will b e cts for the design o f I L - I agonists a n d ng m o l e c u l a r m o d e l l i n g t e c h n i q u e s .
Acknowledgments W e t h a n k Drs. B r a d f o r d G r a v e s . M a r c o s H a t a d a a n d V i n c e n t M a d i s o n for useful discussions o n t h e s t r u c t u r e o f I L - l a a n d IL-i/3. W e also t h a n k Mr. J o s e p h Plocinski for c o n d u c t i n g the D I 0 . G 4 . 1 ceil p r o liferation assay. Dr. D a v i d Ft3' a n d Mr. D a v i d G r e e l e y for N M R s p e c t r o ~ o p y a n d Ms. J o A n n C a s t e l l a n o , Ms. Lisa N i e v e s a n d Mr. D e n n i s T i g h e for the p r e p a r a t i o n o f this m a n u s c r i p t .
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3 Lichtman, A.H., Chin. J.. Schmidt, J.A, and Abbas. A.K. (1988) Prta:. Natl. Acad. Sci, USA 85, t/t~t}9-9703, 4 Weaver, C.T.. Haw~'lowicz. C.M. and Unanue. E.R. t 1988) Prim. Natl. Acad. Sci. USA 85. 8181-8185. 5 Pike. B.L. and Nossal. G.J.V. (Iq85) Proc. Natl. Acad. So. USA 82. 8153-8157. 6 Chiplunkar, S-. Langhorne. J. and Kaufmann. S.H.E. (1986) J. Immuno[. i37. 3748-3752. 7 Mochizuki. D.Y.. Eisenman. J.R.. Conlon, P3.. Larscn. A.D. and Tushinski. R.J. (1987) Proc. Natl. Acad. ScL USA 84. 52{~7-5271. 8 Moore, M.A.S. and Warren. DJ. (It/87) Prim. Natl. Acad. Sci. USA 84. 7134-7138. 90zaki. Y.. Ohashi. T.. Minami. A. and Nakamura. S. (1987) Infect. lmmun. 55. 1436-1440. it) Czupp..'nski. CJ., Broxvn, J_F.. Ytmng. K.M.. Cooley. AJ. and Kurtz. R.S. (I988) J. lmmunol. 14t). 902-968. l I Neta. R., Douches. S.D. and Oppcnheim. JJ. (It~86) J. Immunot. 136, 2483-2485. 12 Mizel. S.B.. Dayer. J.-M., Krane, S.M, and Mergenhagen. S,E. (l~Sl) Proc. Natl. Acad. Sci. USA 78, 2474-2477. 13 Gowen, M.. Wi~d. D.D.. Ihrie, E J_ Meats. J.E. and Russell. R.G. (1984) Biochirn. Biophys. Aeta 707. I86-193. 14 Dayer, J.-M., Stephen,,gm. M.L. SchmidL E.. Karge, W, and Krane, S,M, (1981) FEBS Left. 124, ??_~3-L~6. 15 Auron. P.E.. Webh, A.C., Roscnwasser. LJ., Mucci, S,F.. Rich. A.. Wolff. S.M. and Dinarello. C.A. (Iq,'¢4) Proc, Natl. Acad. Sci. USA Ri. 7~17-7t)11. 16 March. C.J., Mosley, B., Larsen. A., Cerreni. D.P.. Braedt. G.. Price. V., Gillis, S., Henney. C.S.. Kronheim, S.R.. Grabstein, K_ Conlon, P.J.. Itopp, T.P. and Cosman, D. (19851 Nature 315. 041-647. 17 Gubler. U.. Chua. A.O.. Stern. A.S.. I-lellmann, C.P.. Vitek. M,P.. DeChiara. T.M.. Benjamin, W,R,, Collier. KJ.. Dukovich, M,, Familletti. P.C.. Fiedler-Na~'. C.. Jen~m, J.. Kaffka. K.. Kilian. P,L.. Stremto. D.. Wittreich. B.H-. ~,Voehle. O.. Mizd. S.B. and Lomedico, P,T. (1986) J. Immunol. 136. 2492-2497. i8 Dower, S.K.. Kronheim, S.R., Hopp, T.P.. Cantreil. M., Deeley, M.. Giilis. S.. Henney, C.S. and Urdal, D.L. (198fi) Nature 324. 266-268. Iq Dower, S.K.. Call. S.M.. Gillis. S. and Urdat. D.L (10861 Pro,:. Natl. Acad. Sci. USA 83. 1(1fi0-|(164~ 20 Kilian. P.L.. Kaffka. K.. Stern. A_S,, Woehle, D.. Benjamin. W.R., DcChiara. T.M.. Gubler, U., Farrar. J.J., Mizel. S.B. and Lomedico. P,I. (t98t~) J. Immunot. 136. 4509-4514.
2t Chizzonit¢. R , Truitt. T,, Kilian, P.L.. Stem, A.S., Nuncs, P, Parker, K.P., Kaflka. K.L, Chua. A.O.. Lugg. D.IC and Gubler. U. (1989) Prt¢. Natl. Acad. gel USA 86, 8029-8033. 22 Bomsztyk, K, Sims, J.E., Stanton. T.H., Slack, J., McMahan. CJ.. Valentine, M.A. and Do~'er, S.K. (1989) Proc. Natl. Acad. Sci. USA 86, 81134-8(B8. Z'~ Priestle. J.P.. Sh~ir. H.P. and Grfitter. M.G. (1988) EMBO J. 7. 339-343. 24 Priestle, J.P., Sch~ir~ H.-P. and Griittcr, M.G. (1989) Proc. Natl. Acad. Sci. USA 86. 9667-9671. Finzcl, B.C., Clarity, L.L, Holland, D,R.. Muchmorc. S,W., Watenpaugh, K.D. and Einspahr, H.M. 11989) .I. Mol. Biol. 20V, 770-791. 26 Graves. BJ.. Hatada. M.H.. Hendrickson. W.A.. Miller. J.IC. Madison. V.S. and Satow, Y. (19901 Biochemistry 29. 2679-2684. 27 Mizel. S.B. 11980) Mol. lmmunol. 17. 571-577. 28 Krakauer, T. (1985) J. Leukocl,le Bio. 37. 511-518. 29 Dinarello, C.A., Bendtzen, K. and Wolff. S. 11982) Inflammation 6. 72-78. 30 Kaye. J.. Porcelli. S.. Tile, J.. Jones, B. and Janev,'ay. C.A. (1983) J. Exp. Med. 158. 836-8Y6. 31 Riske. F.. Chizzonite. R.. Nunes. P. and Stem. A.S. (1990) Anal. Biochem. 185. 206-212. 32 Hewick. R.M.. Hunkapiller. MW.. H~md. L.E. and Dr~'er. WJ. (1981) J. Biol. Chem. 256, 7990-7997.
33 liunkapitler, M.W.. Granlund-Mnyer, K. and White]y, N.W. 119861 in Methods of Protein Characterization (Shively, J.E., ed.), pp. 315-327, The Humana Press, Clifhm. 34 Takahashi, K. 119681 J. Bk)l. Chem. 243, 6171-6179. 35 Takahashi, K. 11977) J. Biochem, (Tokyo) 81, 4113-414. 36 Mohan. P.M., Basu. A., Basu. S,. Abraham, K.I. and Modal MJ. 119881 Biochemistry 27. 226-233. 37 Mosley. B., Dower. S.K.. Gillis, S. and Cosman. D. (19871 Proc. Nail. Acad. Sci. USA 84. 4572-4-576. 3~: Kamogashira. T., Sakaguehi. M.. Ohmoto. Y.. Mizuno. K., Shimizu, R., Nagamura, K., Nakal, S., Masui, Y. and Hirai, Y, (1988l L Biochem. 11¼, 837-840. 39 Yanofsky. S.D. and Zurawski, G. (19901 J. Biol. Chem. 265. ! 31100-130o6. 40 Huang. JJ.. Ne~lon. R.C., ttoruk, R.. Matthew. J.B.. Covington, M.. Pczzclla, K. and Lin. Y. 119871 FEBS Lctt. 223. 294-298. 41 Gchrke. L.. Jobling. S,A.. Paik, L,S,K,, McDonald. B., Rosenwasser. LJ. and Auron, P.E. ( 19991J. Biol. Chem 265, 5922-5925. 42 Gmnenborn. A.M.. Clore. G.M.. Schmeissner. U. and Wingfictd, P. (1986) Eur, J. Biochem, 161.37-43. 43 Craig. S., Schmeissner. U.. Wingfield. P. and Pain. R.H. (I987) Biochemistry 26. 3570-3576. 44 Clor¢, G.M., Wingfield, P.T. and Gronenlxnn. A.M. 119911 Biochemistry 30, 2315-23Z3..
25
BBAPRO34~47
The role of arginine residues in interleukin 1 receptor binding V e n k a t a B. N a n d u r i 1, J e f f r e y D. H u l m e s I.,, Y u - C h i n g E. P a n t, Patricia L KJlian z a n d Alvin S. Stern s Deparma'm afPraWin Bieclu'm~tO'. R~che Research Center, Hoffinmm-La Roche hie.. Nutlo; NJ (U.S.A.~ and " lX,lmament of lo~nutwpl',armacdeg): Roche i~'search Center. Hoffmmm-La Roche Inc.. Nutle); NJ {U.S.A.) (Rece~,'cd Z'4 May 1991)
Ke~~rd~: |nterleukin i: |nter|eukin ! receptor. Phenylglyoxal:Protein modification:Receptorbindingasmy (EL-4ceil) Inlefleugin I (EL-I) is a family of polypeptide Otokines that plays an essential role in modulating immune and inflamm_atory responses. IL-i adiHly is mediated by either of two distinct proteins, IL, icz or IL-I/$, both of which bind to the same receptor fonnd on T-bmphocytes, fibroblasts and endothelial cells (Type I receptor). The effect of specific chemical modifimtinn d recombinant IL-la and IL-I/$ on receptor binding was examined. Modification of the proteins with phe~lglyoxaL an arginine-specific reagent, resulted in the loss of Type I IL-I receptor binding activity. The stoichiome~ of this modification revealed that a single arginine in either IL-I~ or IL-I# is responsible for the loss of activity. C y a ~ bromide cleavage of phenylglyoxal modified IL-la and IL-IIL follmmi by sequencing of the peptides, re~'ealed that arginineq2 in IL-la and arginine-4 in IL-ifl, which occupy the same topology in the respective co'stallographic smmmres, are the target of phenylglyoxal. These results suggest that an arginiue residue plays an important role in ligand-receptor interaction.
Introduction lnterleukin 1 (IL-i). secreted by activated macrophages and other cells, mediates a variety of inflammatory and immunological responses [1.2]. IL-I activi~ includes the ability to regulate a subset of th~mocytes [3A], stimulate B-ceil proliferation and differentiation [5.6] and stimulate proliferation of precursor hematopoietic cells [7.8]. In addition, it has been demonstrated that mice pretreated with IL-I prior to inoculation with bacteria have higher suwival rates [9.10] and IL-I affords protection to animals against normally lethal doses of radiation [11]. However. IL-I has also been associated v,ith what would appear to be injurious activities. For exam#e, IL-I affects synovial and dermal fibrobiasts to produce tissue-degrading
• Present address: Deparlmen! of Protein Chemistq'. American
Cyanamid Co_ Pearl R~,er. NY IIF~5. U.S.A. Abbreviations: |L-I. intefleukin I; riL-l, recombinant interlcukin |: PBS. Dulbecco's phosphate4~uffere_.d saline (~ilhoul calcium chloride or magnesium chloride~ BSA. tm~ine serum albumin: TCA.
ttiehloroac=tic acid: PTH. ph=n)'lthiohydantoin. Correspondence: AS_ Stern. Department of Protein Biochemist~'. Roche Research Center. Hoffmann-La Roche inc.. Nutley. NJ 07110-ll(~9. U.S.A.
proteinascs [12,13] and prostaglandin E2 i14]. These and other studies suggest that IL-I may be involved in certain immune- and inflammatory-based diseases such as rheumatoid arthritis. Two species of IL-1 have been identified and have been di;,inguished as human IL-la and human !1.-1/3 [ 15- i 7]. Both proteins are synthesized as larger precursor molecules of approx, similar size (31-33 kDa) with the mature ~ 17 kDa species deriving from the carboxy-terminal domain of their respective precursor. Although there is no more than 25% sequence identity between the two biologically active mature forms, both human IL-la and human IL-l/3 have similaL if not identical, activities. This is not surprising since they bind to the .same receptor proteins [18-20] with similar affinity on T-lymphocytes, fibroblasts and endothelial cells (Type I receptor) and B-lymphocytes and macrophages (Type 2 receptor) [21,22]. This may be explained by their similarities in tertiary structure. The X-ray diffraction structure of human IL-Ij0 [23-25] has been described as a tetrahedron with edges formed by antiparallel B-strands. The core of the human IL-la structure [26] is a capped/3-barrel that possesses 3-fold symmetry and displays a topology similar to that observed for 11.-1/3. However, the amino acids which interact with the Type I or Type 2 receptor have not been defined.
26 In understanding, on a rrmlecular le~'el, the range of bk~togical responses elicited ~" IL-1. it is necessaD to identify the act~,'e site(s) of the IL-1 molecule in hs interaction whh the IL-I receptor. Pre~ous[y it had been demonstrated that chetnica] modification of IL-1 arg[nine residues destr~ed th~,xnoc~e proliferation act~'it~" [2728] and p.'rogenici~" [~1. Based on t h e ~ earlier chemical nmd~f~atien results, pheny|glyoxal ~as chosen as the arg[nine-specific reagent to probe and compare the actk'e silos of human IL-la and human IL-lfl. These studies identi~' similar topographic regions of the ~'o molecules which are important for Type I receptor binding and subsequent actg'at/on of celts. Mater~ls and Methods
Proteins E.xpress~on of the human IL-I eDNA in E. colt ~ie|ded mature protein co~esponding to residues 117271 of the unprocessed pro-lL-la precursor molecule [I7| and residues [ ~7-269 of ~he unprocessed pro-lL-I precursor molecule [ 15]. Human rlL-la, human rlL-i and mouse rlL-[a ~ere purified as pre~Jously desenbed [17] excep~ that the proleins were prepared in mM Tris-HCl (pH 8.1). containing 0.4 M NaCL The specific act~,~' o[ the proteins ~as determined in the mouse DI0.G4.1 ce|l proliferation assay [30]. Human rlL-la, human rlL-l~ and mouse rlL-la had specific act~.'ities of 5.7- ~0'~. 6.1 - 1O: and 5.1- l0 s units/rag. respectively. Mouse rlL-l~ was radiolabeled ,,~'[th Nat'-~l as previously descnT~ed [31]. The labeled protein ~'as fully acti~'e in the D10.G4.1 cell proliferation assay ~hen compared to the unlabeled protein.
obtained a~ ~=C, using a sweep w~dth of 7000 Hz, with I28 to 1024 transients collected per spectrum, at a re~.c|e rate of 3.0 s. The residual water re,nonce was reduced ~ prk~rit~ng with the dccoupler.
Smicl~m~esO" of 17-1ZC/phenylglyoxal to IL-I T~'o nn~l of human rlL-l¢ or human rlL-lp were incubated ~ l h 6 mM [7-~C]phen~'lglyoxal (specific aet~-i~- 5 m ~ / m m o l (Amersham, Arlington Heights. IL~) in a 59 #l reaction ~ol. containing 50 mM phosphate buffer (pH 8.0) and 5% glycerol (v/v) at 3TC. At the end of 90 rain. l ~ u g of BSA was added as a nonspecifie prote~n cartier and the incorporation of [7~"C~hen.~l_~..~xa| into IL-J was determined ~ precipitartan of the protein ~ith the addition of 50% (w/v) TCA to aehie~-e a ! ~ final concentration. The precipitales ~,ere colleclcd ~" centrifuging the mixture at 8 ~ × g for 30 rain and v,~ashed (2-3-times) with 5% TCA foll~-od ~" ~'ater. The pellets were then resuspcnded ~n 8 M urea and transferred to a scintillalion x~iaI ,~-here the rad~oactK~- was measured in 3 ml of Ecos~nt H (Nafiona[ D~agn~ics. Somerville, NJ)
Soluble receptor binding assay for nwasuring IL-I
A stock solution (0.I M) of phenyl~yoxal (Sigma) was prepared by dissolving the cry'stats in 50% ethanol. M #g of human rIL-la or human rlL-l/~ were incubated ~'ith var~'ing concentrations of phenylgi~'oxal in a final reaction ~'d. of 50 t~l containing 50 mM phosphate buffer (pH 8.0) and 5% gl)'cerol at 3"FC. After t3O min of incubation, aliquots ~ere removed and diluted t00-fo[d with 50 mM phosphate buffer (pH 8.0) containing i% BSA. Aliquots were removed for the determination of IL-I binding activily, as de~ribed below, and the remaining protein was precipitated by the addition of 50% iwt./vol.) TCA (to achieve a 10% final concentration) and used for the preparation of peptide cleavage fragments. For ~H-NMR studies. D,O was exchanged into the modification reactions.
"[he receptor binding assay [31] is based on competition b e ~ e e n IL-I in a sarnp]e and radio[abeled mouse riL-la for binding to soluble IL-1 T~ept_or. Soluble Type I IL-I receptor was prepared from mouse EL4.lL-2 th~moma cells (ATCC No. TIB-181) as previously described [31]. -~ ng of rlL-! (modified and unmodified} and L~l-labeled mouse rlL-la (2.0-i04 cpm) was incubated whh soluble IL-! receptor for i h at 3TC in a total reaction ~'o!. of t59 #! (PBS containing 8 mM CHAPS, ~ mM NaCI and 0.1% BSA). After addition of bovine y-~obulin as a nonspecific protein carrier, receptor-b~und uzSl-mouse rlL-la was separated from free radiolabeled iigand by precipitation whh 12% imlvethylene glycol 8000 (Simna). The precipitated protein complex was collected onto Whatman G F / C glass fiber filters (Fisher Scientific). washed, and the filters were counted in a gamma counter. Nonspecific binding was determined ~" the addition of excess unlabeled human rlL-la (50 riM). Aeti~[~- was determined as percentage inh~ition of specific binding of labeled mouse rlL-la by IL-I in the test sample. The percentage inhibition was calculated by subtracting from 1 the difference bet,~-een sample binding and non.specific binding (cpm) divided by the difference between total binding and non.specific binding (cpm). This number was then muhiplied by 100 to obtain the percentage inhibition ~alue.
tH-AqffR umdysis
Protein analysis
NMR spectra were acquired at 500 MHz on a Varinn VXR-5~S spectrometer. Sample concentrations ranged from 0.2 to 0.5 mM in protein. Spectra were
In order to locate the site of arginine modification, phenylglyoxal-treated riL-i was cleaved with cyanogen bromide and the resulting pepfides sequenced and
Chemical modification reactiwzs
compared to pcptide fragments generated from the digestion of unmodified rlL-I, 2 nmol rlL-I was modified ~ t h 6 mM phenylglyoxal as described above and precipitated with 10~ T C A (final concentration). Unmodified protein was similarly precipitated. The pellets were washed t ~ c e with 5% T C A and finally with water. The precipitates were then dissolved in 100pl of 7 0 ~ formic a d d and cyanogen bromide was added in 100-fold molar excem over methionine residues. The digestion m ~ u r e s were incubated in the dark at room t e m ~ r a t u r e for 16-18 h and then diluted approx. 10-fold with water and dried under a stream of helium.
.^
1
:[ 0 o
0 ~B
0.1
1
i~! a
I I I
.5
The resulting digests of modified and unmodified protein underwent sequence analyses using an Applied Biosystems model 470A gas-phas~ sequencer [32], Phenylthiohydantoin (PTH) amino acid derivatives were identified 'on-line" with an Applied Biosystems model 120A PTH analyzer [33]. Results
Inactiration of IL-I by phenylglyoxal To examine the role of arginine in the binding of I L - l a and I L - I ~ to their receptor and to use this information in identifying common topological regions within the two IL-I molecules, the proteins were reacted with phenylglyoxal. Previously it had been demonstrated that phenylglyoxal-treated IL-1 was less active in a variety of assays [27-29] suggesting that a common active site of IL-I was essential for biological activity on diversified cell targets. However, at the time of those studies, the occurrence of two different IL-1 molecules and IL-I receptors was not known. Preincubutton of human r l L - l a and human r l L - l p with increasing concentrations of phenylglyoxal at 37°C for 90 min resulted in the loss of receptor binding activity. (Fig. IA and B) with a concomitant loss of T-cell proliferation activity (data not shown) in a dose-dependent manner. There was a linear loss of the above functions of I D I up to approx. 6 mM phenylglyoxal concentration. The ability of human r l L - l a to bind to its Type 1 receptor was found to decrease with time of reaction with a FLxcd concentration of phenylglyoxal (Fig. 2). A similar time-course of inactivation was found for human rlL-I//. Thus, at 90 min incubation time, 80-90% loss of receptor binding ability of the congeners was
90 m
fi0
40 i
4O
+6
10 0
100
0.I
~L-1 .S
(ng/rrO
Fig. I. Effecl o f p b e n y l ~ y o x a ! o n ~¢ceptor binding aclivi~, o f I L - l a
and IL-IH. (AI Human rlL-l~ (I nmol) and (B) human rlL-IH (i nrnol) were reacted gith 2 mM ! o). 4 mM (+). 6 mM( A ). 8 mM (o). 10 mM ( x ~and no ( A ) phem't~ml and binding act~.itT measured ~' soluble receptor binding a-.sa~-.Serial 5-fold dilutions starting at It'll ng/ml of the respecff,-eligands g ere added to duplicate tubes and the assay ~-as performed as described under Materials and Methods. D a t a r e p r e s e n t binding as p e r c e n t a g e o f that obser~ed in
the absence of any competing ligand (approx. 10th'h')cpm/assayL
JL
2O 10 0
.i
0
lb
T
I
I
ii
30
45
60
75
i 9O
T i m e (rain) Fig. 2. Time-course for effi:ct of phenylg[yoxal on inactivation of
receptor binding aclivily of IL-lu. IDle (I nmol) was reacted wilh (A) and without (o) 6 mM phenylgb'oxalat 37°C for various times. diluted in BSA 0%) and binding activity measured as described in Fig. I.
28 ii ~iii !i !ii~ ~i il !:
i
!!i '
il i! !:
iii:
ili l
!
ii ~i
ii
~ii i~i, ~
ii i!~ii ::i:!i
i
ii
i
i
-t-~'.
v,,;,r
, ,i
!~
V
i~, /
ij,,
i
'
tl
|
!liii !
Fig. 3. ~H-NMR spectra of m~.tified IAI and natite (B) IL-la. Ar, cxp'ar~ion of Ib¢ upfie|d porlh)n of each speclrum is sl'a~n to ihe righl. Spectra were acquired a.~ de~cn'bed under Malerials and Method-~. Chemical ~hi[b are referenced to external ..-,~l[um 4.4~dirnelhyl4-si|apenlane .,,uffonate_ Excepziona[|) large re,~onances pre~ent in the ~pectra are due to r~[dual H . O . g|)eero~, buffer tatnnp(ment.~, and excess phenvl£Ayoxal.
apparent with 6 mM phenyiglyoxal while under similar conditions of incubation of control rlL-I (without pheny'lglyoxalk no loss of receptor binding activity was observed. Identical inactivation results were obtained with modified compared to control protein when assayed for their ability' to stimulate T-lymphocyte proliferation (data not shov,~n). IH-NMR spectra of natize and modified IL-I The overall conformations of the native and modified IL-I molecules were assessed by NMR spectroscopy. For both IL-la and IL-IIL the nH spectrum
of the modified protein was generally similar to that of the native protein (Fi~. 3 and 4). In particular, the resonances with unusuaUy high upfield shifts retained their positions in the modified proteins (insets of Figs. 3 and 4). T h e ~ re.,~nances are predominantly methyl groups of leucine and isoleucine, whose unusual chemical shifts are probably caused ~" proximit2,' to aromatic groups in a hydrophobic core, and are therefore good indicators of a distinct globular structure. The modified proteins appear to retain the overaU tertiary fold of the native proteins. There are some small chemical shift differences apparent between the modified and
29 native molecules which suggests local structural pertubations. T h e ~ appear to be more extensive for IL-I~ (Fig. 4) than for I L - l a (Fig. 3). $loichiometry o f [7- :4C lphenylglyoxal modification o f IL-! Phenylglyoxal is known to form stable adducts with guanidyl groups of arginine residues in proteins [34.35]. Since it had been demonstrated that two molecules of phenylglyoxai react with one arginine residue [351. the number of tool of l~Cdabeled phenyiglyoxal bound per tool of IL-I was determined by TCA precipitation. These labeling studies found that approx. 2 mol of [TJ~C]phenrlglyoxal was bound per tool of human rlL-
TABLE I Stoichiomt'tn' o[ 17-~C]pholylglyoxal binding to IL-I " Addition
mol phenylglyoxal/molIL-I
IL-la IL-13
2.03 2.25
nmol of human rlDl. and human rIL-IL¢were incubatedwith 6 mM [TJ~C]phenylglyoxalfor 91J min at 37°C as described under Malcfials and Methods and the incorporation of label into IL-I determined by liquid ~intillation spectroscopy.
" 2
l a or human rlL-l/3 (Table I) when the proteins were inactivated to approx. 90%. This suggests that one of the three arginine residues in both I L - l a and 1L-IB is
i •
"
I 8
. . . .
I 7
"
~
"
~,""r '-~' 6
~
,
•
I 5
•
~
~
| 4
~"'~
,
,
i 3
. . . .
I 2
. . . .
I
. . . .
1
I -0
. . . . nora
"~mT"q"~T"l~"l'"lt'"l'"'l'"~'"'l"'l'"l 0
6
0
4
0.2
-0
I
-0
~
pl~
Fig. 4. ~H-NMR spectra of modified (A) and nalive (B) IL-i/L An expansion of the upfield portion of each spectrum is shown Io the right.
Conditionswere as that described in Fig.3.
3O
Human rlL-1 ~;~
:
CNBr
1
2O
......P~tid~ 1 ( ~ ) ...............................................~ ~ , ~ t i ~
...................................
21 ASh Asp AZa L~u A.snGIn Set lle lle FA~-]AIo ASh AspG~ Tyr L~u Thr / ~ . . . . . . . . . . . . ..o . ..o...,
4O A~ A~
. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .o . . . . .., ... . . . . . .. . . . .
CNBr ...........................................................................................~ i
.... ~ "
............................
~
61 8,0 AspA.k: LysBe ThrVd Re LeuFA-~-l~e Ser Lys ll'wCk~ LeuTyr Val Thr Aio Gln . . . . . . . . . . . . . . . . . ... ... , . . o . . . ,
. . . . . . . . . . . . . . . . . .. ... . . . . .
o...o,.o
. . . . . . . . . . . . . . . .. . . . . . . . . . . . ..o , . o . , o
.....
..
81 CNBrj. Asp Clu Asp Gin Pro Vd Leu Leu Lys Glu Met~Pro Glu h
. . . . . . . . . . . . . . . . . . . . o.o . , . . . , . o o o , o . . . ,
......
100 Pro Lys Thr Re Thr Gly
....................................................................................~p...Pe~tide 4 (,~,~).................................... t01 Se=- Glu Thr Ash Leu Leu Phe Pine Trp Gtu ~ ..o~
120 His Gly Thr Lys A.sn Tyr Pne Th~ Set
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
121
Vd Ak: His Pro A.sn Leu Phe Be Aia ~
. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. . . . . . . . . . . . . .. . .,
.......
......
Lys ~
. . . . . . . . . . . . . . . . . ... ... . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . .
140 Asp TyF Tip Vd Cys Leu Alo Gly . . . . . . . . . . . . . . . . . . . . . . . . . . .. o .o o .
141
.....
~..
. . . . . . . . . . . . . . . . . . . . . . . .
1,55
H.uman
rIL-lg
:
-- Peptide
CNBr . . . . . . . . . . . . . . . . . . . . . . . . . CNBr /
~..~,~
T~-)
-~IM--P,~,,~ s (.~)
......................................................................................
CNBr h
........................... I~i Hi ..... Peptide 4- ( , 8 )
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
BO 61 G!y Leu Ly~ Glu Lys Asn Leu Tyr Leu Set Cys V~I Leu L ~ Asp Asp Ly~ Pro Thr Leu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
, ..............
, ....................
o ..........
81
c~n
L ~ C~. S ~ Vd Asp P~o Ly~ As. TF Pro Lys Lys Lys ..................................................................................................................
10t Phe Ash Lys I~e Clu lie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.~=Br
,....,
......................
aet.Gtu Lys ~
100
Phe Vd
.I~i14.....Peptide 5 ( ~ ) ' . . .
120 Asn Asn Lys Leu Giu Phe Glu Set A]o Gin Phe Pro Ash Trp
, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
CNBr ~
121 ............................................. CNBr
141
, ......
o,,
o,,
140
Pep.tide 6 ( ~ } ..........................................
g
153
Gin Asp ,e Thr Asp Phe Thr Met.Gin Phe Vd 7S~;~.1~ ............................................................ I~,q ..... Peptide Fig..'3. The primary structures ~)f IL-h~ and IL-I/.J idemifying argininc rcsidu¢~ I [] ). sile of q,'anogen bromide cleavage ( .~) and the resultant generation ¢~f pcplidcs from IL-I~ [Peptid¢ I(~) --, Peptid¢ 4(a and IL-Ip [Pcplide l(~8)--* Peptide 7(/])t that were sequenced. )}
modified although variable and partial modification of the arginines could not be discounted by the above data.
the phenytglyoxal adducts [36] made a rapid analysis essential. 2 nmot of modified and unmodified human rlL-la and human riL-If3 were chemically digested with cyanogen bromide as described under Materials and Methods. The amino acid sequence of each mixture of cyanogen bromide-generated peptides of digested control and modified human riL-la and digested control and modified human rlL-l.8 was determined by automated Edman degradation. Data from the first 25 cycles of analysis of I D l e peptides indicated four sequences present, as would be expected from the number of methionine residues present in IL-la (Fig. 5). Arginine-12 was prominently missing from the first cycle of the sequence analysis of the digested modified human rlL-lot identifying this residue as the site of phenylglyoxai reaction in IL-la (Fig. 6). Similarly, data from 15 cycles of analysis of It-I/3 peptides indicated seven sequences present, as would be expected from the number of methionine residues present in IL-I~
ldemi#cation of tiw site of phenylglyoxal reaction Based on the above data, a strategy was designed to identify the arginine residuels) in It-in and IL-I/3 that is essential for iigand binding to the Type ! IL-! receptor. Modification and chemical cleavage by cyanogen bromide of the proteins would yield a number of fragments of predicted ~quence (Fig, 5). The cleavage products could be sequenced directly and an arginine residue conspicuously missing from a sequencing cycle would be identified as the site of phenylglyoral reaction. This simple procedure was chosen over purification of the Oeptides because it assured complete analysis of each of the generated peptides as opposed 1o the possibility of peptide loss during the resolution of peptide fragments upon reversed-phase HPLC separation, in addition, the known instability of
P=' E = .L,-
K~
,m~
T i,~. I=~ --r. G~'°°_,_,E'==T'"N'°"L'" L ' ' F , o ~
A~
'°s .... o~E.O
K~
'~"
Sm
L~m J~ l~ T=
l'~
L ~ R eg
i~o
S " K ~ T'~Q'-
EL
.._.., a
J
I'"
400
K-~
PEPTIDE 2 (¢4) Aal
"t
N = D = O~ y=
L~a
a s, N, ~1~11 1
I~ Ell ~ 2
3
4-
5
I~
~ 7
E:
L~ g
10
11
12
13
14-
15
16
17
1~
19
20
~'1
22
23
24-
2.~
CYCLES Fig. 6. Amino acid sequence analysis of cyanogen bromide peptides from IL-I~ (cross-hatched bar) and phenylgt~)xablnodified IL-I~ (~)lid bar). I D l a was modified with 6 mM phenyZglyoxaL After incubation for 90 rain at 37°C, the protein was precipitated wilh TCA (I(F,;~ final concentration) and fragmented with cyanogen bromide in 70% formic acid for 18 h. The pcptides thus obtain,'d were sequenced by gas.phase automated Edman degradation. No arginine peak (*) was observed at O~c]e 1 of the PTH amino acid analysis of tile modiEed It-Ice peptides indicating modification of arginine-12 of IL-la. ~. estimated yield based on the obtained wdue divided by the expected number of the same amino acid at that cycle (Fig. 5).
PEPTIDE 7 ( ~ ) 200
S ' ~'
~'v'~2F 1~ L ,:a
G 'a
8@oP•
K 1~' PEPI]DE 6 (.,~)
2®i
~a K g"
~
'1~i[' [~ Tl~ii"4'1~i"
PEPTIDE 5 (,6)
1
Fsa
F,,.o V..7 Q ~
E 600
E ' ~ I ' ~ N '~' N , K '°91_,, o
PEPriDE 4 (.,6)
G~
j 400 "' 200
E~'~
~-
N~ D~ S~'~i ~i
.
I~ [~
HI
v a A "~' PS"
I91BI
E'~7
Q-,,~V~ V+l F 4~ 200 Jl~!
S"~ .~
~
.
.
.
.
.
PEPTIDE 2 (,,6)
+.l==)2.3
A ~ p2
o ,..
1
.....
2
3
•
4
5
.,
6
7 8 CYCLES
m, 9
re*Ill'~° ~i' ~i
10 11 12 13 14 15
Fig. 7. Amim~ acid .,,equcnc~:anal).'.,is of ~.3'anogcn bromide pcptides from IL-Ifl (crt~.,,.,,-hatchedbar) and phenylglyoxal-modified iL-lp (.,~lid barL IL-I~ ~as m~}dified v,ith phenylgt.voxal and [ragmenled with ~'anogcn bromide as described in FiG. & The peptides thus obtained were sequencedh~ gas-phase automated Edman degradatk, n. Marked rcducti0n {*) of the arginine peak was ,.}bsc~x-cdat ~'¢1c 4 with slight reduction t "" ) at l.)'cle I I ~lf the PTEi amino acid anaL~sisof the modified IL-t/3 pepli.dcs. This indicates modification , t at[ininc-4 and partial modification of arginine-tl af IL-I/~. N I. not identified: ~. estimated yield based on the t)btained value db,Jded by the expected number of the same amino acid at that cycle {Fig. 5).
33 (Fig. 5). Argininc-4 was conspicuously reduced from the fourth cycle of the sequence analysis of peptides generated from modified IL-lfl when compared to control protein identifying this residue as the site of phenylg~yoxai reaction in IL-i/3 (Fig. 7). Furthermore, a partial modification may have occurred at arginine- I 1 since the sequence data are equivocal at this residue (Fig, 7). Subsequently. the intact molecules were sequenced which verified the extent of modification of these arginine residues in both proteins. Both arginine12 and arginine-4 were missing from the twelfth and fourth cycles of the sequence analyses of modified IL-hx and IL-I~ respectively {data not shown). Discussion The presence of arginine in the active site of 1L-I has been indicated by the sensitivity of these cytokines to phenylglyoxal treatment [27-29]. Since phenylglyoxai has been known to selectively react with arginine residues, identification of putative arginine residues in IL-1 that are reactive toward phenyiglyoxal was impor-
tant inasmuch as data recently obtained from mutagcnesis of IL-! eDNA denoted the importance of arginine residues. For example, truncation by removing amino acids from the amino-terminal end of IL-la showed biological activity markedly decreased with the removal of arginine-12 [37]. Truncations of IL-l~ showed homologous requirements for biological activity as truncation of IL-la in that activity was maintained in aminoterminal deletions until the arginine at position 4 was removed [37,38]. Furthermore, using combinatorial cassette mutagenesis, Yanofsky and Zurawski [39] have shown IL-la to have an abmlute requirement for a basic residue at arginine-12 although these studies could not determine if changes at this residue simply caused structural defects. Similarly, by site specific mutagenesis, arginine-4 of l L-l/3 had been shown to be one of the key residues in the function of IL-I/3 [38,40] although in these reports it had been concluded that the differences in activity of these recombinant proteins were due to either significant changes in the conformation [38] or folding of the mutant polypeptides [40].
\
• . •
.
/
..'
Fig. 8. Su~rposition of crystallographically determined structures of iL-la [26] and IL-I# (B.J. Graves, personal communication) showing the proximily of arginine-12 of lL-la [R-12(a)] and arginine-4 of IL-I/3 lR-4(/3)]. The guanidino head groups of thes,' residues are labeled in the lower central portion of the figure. The backbone is shown as a Ca tracing viewed approx, down the pseudo 3-fold axis as shc~wn in Fig. 2 of Graves et al. [26]. This close-up shows primarily the six ,B-strands which form the sides of the/3-barrel. The proteins were aligned using the common 81 a-carbons of the 1L-I fold 126]. The close fit of the central B-strands of IL-l~r ( ) to those of IL-I B ( - - - - - - ) is apparent.
e x t e n d the a b o v e o b s e r v a t i o n s to the ! a r g i n i n e residues in the I L - i p o l y p e p r r e s p e c t i v e T y p e 1 r e c e o t 3 r (i.e, by ng o r s a h - b r i d g e f o r m a t i o n ) . By sitem o d i f i c a t i o n o f the wild-type protein° c h a r a c t e r o f the a r g i n i n e r e s i d u e s w a s ; simply a r e q u i r e m e n t for the m a i n t e [I s t r u c t u r a l stability o r a t t a i n m e n t o f of the p r o t e i n since the global c o n f o r m o d i f i e d m o l e c u l e s w e r e essentially I t h a t o f the native p r o t e i n as a s s e s s e d roscopy. A l t h o u g h s o m e small, local rbations were apparent, the effects of tokincs a r e not c a u s e d by a large o v e r range in the m o l e c u l e s . T h e d a t a unltifies a r g i n i n c - 1 2 o f I L - l a a n d argias essential r e s i d u e s r e l a t e d directly to :don a n d the c o n c o m i t a n t induction o f I.v. h a d b e e n d e m o n s t r a t e d that t h e c o n i n e - l l o f I L - l / 3 to a glycine r e s i d u e (by l t a g e n e s i s ) yields an a n a l o g which has biological activity t h a n the wild-type [owever. the r e c e p t o r affinity o f the w a s d e c r e a s e d by only 25%. T h i s sugf i n c - l l m a y b c involved in a c t i v a t i n g :ion e v e n t s [41] b u t not in b i n d i n g o f :eptor. T h e r e f o r e , the p a r t i a l m o d i f i c a :-l l by p h e n y l g l y o x a l (Fig. 7) p r o b a b l y ate significantly to the loss o f r e c e p t o r consistent with t h e c o n c l u s i o n t h a t t h e t e r t i a r y s t r u c t u r e s of I L - l a a n d I L - l f l [ 2 3 - 2 6 A 2 - 4 4 ] . T h e e x a m i n a t i o n o f the :thine-12 a n d a r g i n i n e - 4 on the tertiary l a a n d I L - l f l respectively, l e a d s t o t h e e a c h b i n d the T y p e I r e c e p t o r in simir e g i o n s o f e a c h m o l e c u l e (Fig. 8). in o u r results, t h e s e r e s i d u e s a r e o n the ~roteins at the s t a r t o f / ] - s h e e t regions. :nee h a s d e m o n s t r a t e d that B cells conIL-! r e c e p t o r m o l e c u l e s ( T y p e 2) t h a n e l I L - I r e c e p t o r [21,22], It will b e o f line the a r g i n i n e - m o d i f i e d I L - I p r o t e i n s oint o f d i f f e r e n t i a l i n t e r a c t i o n o n T y p e us T y p e l r e c e p t o r a n d c o r r e l a t e d blocs. F u r t h e r m o r e , with a d d i t i o n a l d a t a ific c h e m i c a l m o d i f i c a t i o n a n d site-ditests, w e h o p e to m a p the b o u n d a r i e s o f e a n d to identify s u r f a c e r e s i d u e s t h a t t with the r e c e p t o r . Such results will b e cts for the design o f I L - I agonists a n d ng m o l e c u l a r m o d e l l i n g t e c h n i q u e s .
Acknowledgments W e t h a n k Drs. B r a d f o r d G r a v e s . M a r c o s H a t a d a a n d V i n c e n t M a d i s o n for useful discussions o n t h e s t r u c t u r e o f I L - l a a n d IL-i/3. W e also t h a n k Mr. J o s e p h Plocinski for c o n d u c t i n g the D I 0 . G 4 . 1 ceil p r o liferation assay. Dr. D a v i d Ft3' a n d Mr. D a v i d G r e e l e y for N M R s p e c t r o ~ o p y a n d Ms. J o A n n C a s t e l l a n o , Ms. Lisa N i e v e s a n d Mr. D e n n i s T i g h e for the p r e p a r a t i o n o f this m a n u s c r i p t .
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