site in domains V and VI

The EMBO Journal vol. 13 no.8 pp. 1790 - 1798, 1994

Integrin LFA-1 Ol subunit contains an ICAM-1 bindingsite in domains V and VI

Paula Stanley, Paul A.Bates', Joanna Harvey,Robert l.Bennett and Nancy Hogg2Leukocyte Adhesion Laboratory and IBiomolecular ModellingLaboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields,London WC2A 3PX, UK2Corresponding author

Communicated by M.Fried

In order to identify a binding site for ligand intercellularadhesion molecule-1 (ICAM-1) on the (2 integrinlymphocyte function-associated antigen-1 (LFA-1),protein fragments of LFA-1 were made by in vitrotranslation of a series of constructs which featureddomain-sized deletions starting from the N-terminus ofthe a subunit of LFA-1. Monoclonal antibodies andICAM-1 were tested for their ability to bind to theseprotein fragments. Results show that the putative divalentcation binding domains V and VI contain an ICAM-1binding site. A series of consecutive peptides coveringthese domains indicated two discontinuous areas asspecific contact sites: residues 458-467 in domain V andresidues 497-516 in domain VI. A three-dimensionalmodel of these domains of LFA-1 was constructed basedon the sequence similarity to known EF hands. The tworegions critical for the interaction of LFA-1 with ICAM-1lie adjacent to each other, the first next to the non-functional EF hand in domain V and the secondcoinciding with the potential divalent cation binding loopin domain VI. The binding of ICAM-1 with the domainV and VI region in solution was not sensitive to divalentcation chelation. In short, a critical motif for ICAM-1binding to the a subunit of LFA-1 is shared between tworegions of domains V and VI.Key words: adhesion/cation binding site/ICAM-l/integrin/LFA-1

IntroductionLymphocyte function-associated antigen-I (LFA- 1) is aleukocyte adhesion receptor belonging to the (2 family ofintegrins (Springer, 1990). It is expressed on all leukocytesand, by binding to ligand intercellular adhesion molecule-I(ICAM-1), provides the major adhesive force betweenleukocytes and their target cells. Integrins are oa( hetero-dimers which require bound divalent cations in order tofunction (Hynes, 1992). At the N-terminus of the ae subunitare seven homologous tandemly repeated domains (I - VII)of which the last three or four are suggested to contain EF-hand-type divalent cation binding sequences (Tufty andKretsinger, 1975). The integrin divalent cation binding motifdiffers from classical EF hand sequences in that it lacks the

crucial -z coordinating residue at position 12. Whether thesesequences form structurally complete metal binding sites hasbeen questioned, leading to the suggestion that ligandscontaining peptide motifs such as RGD may supply a crucialaspartate residue to complete the coordination geometry ofthe divalent cation (Corbi et al., 1987; Humphries, 1990).Alternatively, such a residue could be contributed fromelsewhere in the integrin as in galactose binding protein(Vyas et al., 1987). Several studies have now providedevidence for the divalent cation binding capabilities of theintegrins. The covalent coupling of 58Co(III) to vitronectinreceptor is a direct demonstration of the binding of metalions (Smith and Cheresh, 1991). This is further supportedby the binding of Ca2+ to a protein fragment spanning theEF hand-type domains (IV -VII) of the platelet integringpIIbIlla (Gulino et al., 1992). Finally, a modelling studyof hybrid integrin -calmodulin EF hands predicts the integrinloop to be a divalent cation chelation site (Tuckwell et al.,1992).In general, there has been little information about the

ligand binding sites on integrins with the exception of the33 integrin, gpIIbllIa. Specifically, the binding site of an1 lImer peptide from the fibrinogen 'y chain has been mappedto domain V of the gplbIJEla ca subunit (D'Souza et al., 1990,1991). Similarly, cross-linking an RGD-containing peptideto domains II-VI of the vitronectin receptor oa subunit hasalso indicated the importance of the repeated domains forligand recognition by this second (3 integrin (Smith andCheresh, 1990). In addition to these repeated domains, the(2 integrins such as LFA-1 (and (31 integrins VLA-1 andVLA-2) contain a - 200 residue sequence called the'inserted' or 'I' domain (positioned between domains II andIII) (Larson et al., 1989). Monoclonal antibodies (mAbs)which affect 32 integrin function have now been localizedto the 'I' domain which suggests that this domain participatesin the process of ligand binding and in fact might containa ligand binding site (Diamond and Springer, 1993; Landiset al., 1993).LFA- 1 would be predicted to interact with ICAM- 1

differently from the way in which the (3 integrins interactwith their ligands as human ICAM-I does not contain anRGD sequence (Simmons et al., 1988; Staunton et al., 1988)and RGD analogues apparently do not interfere with LFA- Ibinding to ICAM-I (Marlin and Springer, 1987). In addition,it is known that the LFA-I 'binding footprint' on ICAM-1is broad, covering the first and possibly part of the seconddomain of ICAM-1 (Staunton et al., 1990; Berendt et al.,1992). Analysis of hybrid receptors indicates that recognitionof ICAM-1 resides with the LFA-1 a subunit (Johnstonet al., 1990). In this study, we show that one binding sitefor human ICAM-1 is located within domains V and VI onthe LFA-1 at subunit.

1790 © Oxford University Press

ICAM-1 binding site in LFA-1 domains V and VI

L.A.\- i1.N I

I I it 1 Porn1 III IVVI V II

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,. .. .. ,. .- .- ,r ,r ,-.- .l' .-' ." ." .- ."

Al !'

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:AS.E('!77-

Fig. 1. LFA-l a subunit deletion series: cDNAs consist of a series of five fragments deleted sequentially from the N-terminus. The fragments arenamed according to the first expressed domain, namely I, II, 'I' Dom, HI and IV, and terminate at the beginning of domain VII. Fragmentscontaining domains V - VI, V - VII and a fragment covering the rest of the a subunit to the C-terminus (C) were also made. The position of the 'I'domain is indicated as 2 and the putative divalent cation binding sequences as E.

ResultsIn vitro expression of a nested series of domain-deleted protein fragments from the LFA- 1 a subunitand monoclonal antibody bindingThe aim of this study was to define the binding site forICAM-1 on the LFA-1 a subunit. The rationale was to testthe ICAM- 1 binding activity of a series of protein fragmentsfrom the LFA-1 a subunit which featured domain-sizeddeletions, starting from the N-terminus and ending at domainVII. In addition, two further cDNAs were made which codedfor domains V -VII and the region spanning the region fromthe end of domain VII to the transmembrane region(C-terminal fragment, C). This series of fragments isillustrated in Figure 1. The cloned PCR constructs weretranscribed and translated in vitro; the translation bands ineach case corresponded to proteins of the expected sizes asshown in Figure 2B.

In order to establish whether the protein fragmentsresembled their domain counterparts in native LFA-1, wemapped two specific mAbs prior to testing the fragmentsfor their ability to interact with ICAM-1. The mAbs 38(Dransfield and Hogg, 1989) and MHM24 (Hildreth et al.,1983), specific for the LFA-1 a subunit and able to inhibitthe LFA-1 -ICAM-1 interaction, were tested for their abilityto bind to the protein fragments. mAbs 38 (Figure 2A) andMHM24 (data not shown) both immunoprecipitated the threeprotein fragments which contained the 'I' domain, namelyI, II and 'I' Dom. As they did not immunoprecipitatefragments from which the 'I' domain was absent, namelyIII, IV and C, it was concluded that these mAbs recognizedepitopes within the 'I' domain. Non-specific binding wasminimal as indicated by immunoprecipitation with a controlCD8 mAb 14 (and other control mAbs, data not shown).

In all experiments, the translation efficiency of eachfragment was checked by SDS -PAGE in order to ensurethat equivalent amounts of each fragment were beingtranslated (Figure 2B). As the fragments were recognizedin a specific manner the translated protein fragments wereconsidered to retain conformational features of native LFA- 1oa subunit.

ICAM-1 binds to a fragment which includes domainsV and VI of the LFA-1 a subunitThe translated LFA-1 a subunit protein fragments were thentested for their ability to be precipitated by ICAM-1 usingan ICAM-lFc construct consisting of the first three domainsof ICAM-1 fused to human Fc as previously described(Berendt et al., 1992). As the Fc component was of humanIgGl isotype, the IgGI myeloma protein CRI was used forcontrol precipitations. ICAM-lFc bound reproducibly toseven of the LFA-1 a subunit fragments, namely I, II, 'I'Dom, III, IV, V-VI and V-VII but not to C fragment(Figure 3A-C). Control precipitations with protein CRI andSepharose -protein A alone (results not shown) gavebackground binding. Thus the minimum sequence to whichICAM-1 specifically bound consisted of domains V and VIwhich indicated that a binding site was located within these132 residues.

Further localization of the ICAM- 1 binding site withindomains V and VIFurther characterization of the ICAM-1 binding site wascontinued through the use of a series of consecutive peptidescorresponding to the sequence of the LFA- 1 ae subunit fromdomain IV to domain VII. Figure 4 illustrates the peptideboundaries which were designated in accordance with thepredicted secondary structure of domains IV - VII in anattempt to preserve peptide conformation in solution(Eliopoulos et al., 1982). As well as demonstrating bindingof ICAM- 1 to LFA- 1 fragments in solution, it wasconsidered important to confirm and extend the precedingobservations in a more physiological context by testing theeffect of the peptides on the binding of T cell LFA-1 topurified ICAM-1.The two peptides, dom 5 (428-467: GTQIGSYFG-

GELCGVDVDQDGETELLLIGAPLFYGEQRGG) anddom 6.1 (497-516: GEAITALTDINGDGLVDVAV)showed significant inhibition at 1 mg/ml. Dom 5.3(458-477: PLFYGEQRGGRVFIYQRRQL) also signifi-cantly inhibited T cell binding to ICAM-1 at 0.5 mg/mlwhich was the maximum concentration at which this peptidewas fully soluble (Table IA). The remaining nine peptides

1791

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A

C9 7

69-

w46-

46 -

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30-

I_ .I

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B C

97-

69-97-

46 -

69- *30-

30-46 -

30 -

21 5-

i;!

I 11 1'V III f i I1 1' 111 I\ t

Fig. 2. Anti-LFA-1 ca mAb 38 precipitates 'I' domain-containingfragments. (A) A series of LFA-1 a protein fragments obtained by invitro transcription and translation in the presence of [35S]methioninewere precipitated with mAb 38 (+). Control precipitations were

carried out with CD8 mAb 14 (-). (B) The translation efficiency of a

sample of each of the fragments was checked by SDS-PAGE. Proteinfragments corresponding to the expected molecular size for full-lengthtranslation product are indicated by arrowheads. Molecular weightmarkers in kDa are indicated on the ordinate.

and an unrelated 39mer control had no effect on ICAM-1

binding of T cells at 1 mg/ml (Table IA), and even whentested at 5 mg/mi (data not shown), indicating that positiveblocking activity was not simply a non-specific effect ofpeptides in solution. Figure 5 illustrates dose-responsecurves for two of the inhibitory peptides, dom 5 and dom6.1 with both peptides showing characteristically steeptitration curves. Titration of peptide dom 5.3 also showeda steep dose -response curve with a loss of activity by 125jig/ml (50 jtM) (data not shown).

Reversed sequences of the blocking peptides, namely domSrev, dom 6. lrev and dom 5.3rev, were also tested and

Fig. 3. ICAM-l precipitates LFA-l a subunit fragments containingdomains V and VI. Using protein fragments as described in the legendto Figure 2, ICAM-lFc (+) precipitated fragments I, II, 'I' Dom, mI,(A) III, IV but not C-terminal fragment (C) (B) and also precipitatedfragments V-VI and V-VII (C). The second of each pair of lanesshows control precipitations (-) using IgGI protein CRI, all of whichare negative.

DOMAIN

LFA-la

IV V VI VIl

367 421 483 542 604

dom dom dom dom dom dom dom dom dom dom dom4.1 4.2 4.3 5.1 5.2 5.3 5/6 6.1 6.2 6/7 7

dom5

Poptlde

Cation Binding Motif

Blocking PopUde

Fig. 4. Designation of LFA-l a subunit peptides and their locationwithin domains IV-VII. The precise amino acid boundaries of

individual peptides are given in Table I. Peptides which blocked the

LFA-1-ICAM-1 interaction are indicated.

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P.Stanley et al.

A

97--

69 -

46-


Table L. Inhibition of the binding of T cell LFA-1 to ICAM-I by LFA-I peptides

Peptide Sequence % Control (z SD) Molarity at 1 mg/ml (mM)

(A)dom 4.1 Asn367-Ser388 104.3 (6.8) 0.406dom 4.2 Thr384-Met4O5 98.5 (16.6) 0.396dom 4.3 Pro400-Ser421 94.0 (14.0) 0.195dom 5 Gly428-Gly467 48.8 (11.1) 0.236dom 5.2 Glu438-Ala457 106.3 (6.9) 0.486dom 5.3 Pro458-Leu477 57.5 (15.3) 0.201dom 5/6 Gly478-Gly497 116.8 (5.8) 0.464dom 6.1 Gly497-Val516 61.2 (10.8) 0.515dom 6.2 GlyS17-LeuS36 107.6 (6.4) 0.480dom 6/7 Ser537-PheSS6 92.0 (14.9) 0.44dom 7 Val563 -Met582 101.8 (7.8) 0.499mAb 38 (anti LFA-1 a) 3.4 (1.78)mAb 15.2 (anti ICAM-1) 10.2 (11.1)(B)dom S Gly428-Gly467 38.5 (3.9)dom Srev Gly467-Gly428 128.3 (12.5)dom 5.3 Pro458-Leu477 57.8 (6.6)dom 5.3rev Leu477-Pro458 114.5 (23.3)dom 6.1 Gly497-Val516 55.7 (6.7)dom 6.1rev Val516-Gly497 107.3 (9.6)mAb 38 (anti LFA-1 a) 4.4 (1.9)

The effect of each peptide on the LFA-1 -ICAM-1 interaction is presented as a percentage of the control level of T cell binding obtained in theabsence of peptide (% control + SD) (A). Consecutive peptides from domains IV-VII were tested in triplicate at 1 mg/ml, with the exception ofdom 4.3 and dom 5.3 which were fully soluble only at 0.5 mg/ml (n = 4). (B) blocking peptides dom 5, dom 5.3 and dom 6.1 were tested withtheir 'reversed' sequences at 2 mg/ml in a separate set of experiments (n = 3). Inhibition by peptides dom 5, dom 5.3 and dom 6.1 is significant atthe concentrations shown: dom 5, P < 0.005; dom 5.3, P = 0.006; dom 6.1, P = 0.018 using a t-test for independent samples. Monoclonalantibodies against LFA-la (mAb 38, CD1la) and ICAM-1 (mAb 15.2, CDS4) are used as positive controls for the inhibition of T cell binding. Allnon-blocking peptides also failed to inhibit at 5 mg/ml.

consistently had no blocking activity at 2 mg/ml (0.5 mg/mlfor dom 5.3 and dom 5.3rev) (Table IB) showing thatinhibition was due to specific sequences, and not simply toa combination of residues.Therefore in a second assay system measuring

LFA-1/ICAM-1 binding, domains V and VI of LFA-1 areshown to be involved in ICAM-1 recognition and the useof blocking peptides has highlighted two particular areaswithin these domains as contact sites. As dom 5 and dom5.3 peptides are both active in blocking, the shared residues458-467 must contain the 'blocking site'. Additionally,residues 497-516 in domain VI (covered by dom 6.1peptide) also form part of the ICAM-1 binding site on theLFA-1 ox subunit.

Blocking of ICAM-1 interaction with protein fragment111 by LFA-1 peptidesIn order to show that both the T cell and solution based assayswere measuring the same event, peptides corresponding tothe two areas which blocked binding of T cell LFA-1 toICAM-1, were tested for their ability to inhibit ICAM-1binding of in vitro translated LFA-1 protein fragments.Precipitation of fragment III (domains III-VI) by ICAM-lFcwas performed in the presence of dom 5, dom 6.1 andcontrol peptides. Dom 5 peptide reduced the precipitationof fragment III compared with ICAM-lFc alone and thecontrol peptide at 1 mg/ml (Figure 6). Neither dom 6.1 northe dom 5/6 (and dom 7 data not shown) control peptides(all at 1 mg/ml) blocked the precipitation of ICAM-lFc byfragment IH (even at 5 mg/ml). The fact that the dom 5

120-

e.-100-

U 8080

a'Z 60

0-

.0

-4

i_.40

Concentration (mg/ml)

* control- 39mer- dom 5

-X9*- dom 7- dom 6.1

2

Fig. 5. Dose-response curves for inhibition of LFA-l-mediated T cellbinding via LFA-1 to ICAM-1 by peptides dom 5, dom 6.1, dom 7and a 39mer control peptide (see text). Error bars represent thestandard deviation for three separate experiments where eachconcentration was tested in triplicate. Results are given as a percentageof binding in the absence of peptide.

peptide was able to interfere with the ICAM-1 precipitationof LFA-1 a subunit fragments in solution confirms itsblocking activity by a second method. Why peptide dom 6.1did not block the solution based assay is uncertain; one

possibility is that it is less potent than dom S (as seen in theT cell assay).

1793

Precipitation

AM-.

Control

I

L

11

i

. )

Irr,r-

0

OS

ciL

CrI

Fig. 6. Peptide dom 5 blocks binding of ICAM-1 to in vitro translatedprotein fragment III (domains HI-VI). (A) ICAM-lFc wascoincubated with peptides dom 5, dom 5/6, dom 6.1 and 39mercontrol. Only peptide dom 5 (lane 3) interfered with ICAM-lFc-mediated precipitation of the LFA-1 a fragment compared withICAM-lFc alone (lane 1). CRI protein served as a control (lane 2).(B) Control represents an aliquot of each sample, showing equivalenttranslation in each reaction.

Binding of ICAM- 1 to LFA- 1 domain fragment isnot divalent cation dependentThe divalent cation Mg2+ is required for LFA-1 to functionand as LFA-1 domains V-VII are EF hand-like in structure,they have been implicated in metal binding. We thereforeinvestigated whether the observed ICAM-1 binding to thisregion was dependent upon divalent cation. The ability ofICAM-1 to bind to the protein fragment III (domains mII-VI)was assessed in the presence and absence of 2.5, 5 and10 mM EDTA with concentrations compensating for thedivalent cation present in the transcription/translationmixture. It can be seen that even the highest concentrationof EDTA had no effect on ICAM-1 binding (Figure 7).Therefore the divalent cation does not appear to be requiredfor ICAM-1 binding to domains V and VI of the LFA-letsubunit in solution.

Molecular modelling of LFA- 1 a subunit domains Vand VIIn order to have a structural representation of the regionsof LFA-1 to which ICAM-l binds, domains V and VI were

modelled. The homologous sequences of the 60-63 residuesin LFA-1 domains V, VI and VII have been suggested tocontain EF hand motifs (Larson et al., 1989). Multiplesequence alignment indicates that each domain contains twoEF hand-like motifs (Figure 8). The first half of each domainbears strong sequence similarity to the classical EF hand(helices A and B). However, the second half of each LFA-ldomain shows less conservation of this motif in that the metalbinding residues are only weakly conserved although the twohelices (C and D) are predicted to remain. This suggests thatthe overall fold of each of domains V and VI resembles theEF hand-containing domains of several solved crystal

Fig. 7. Divalent cation chelation fails to interfere with ICAM-lbinding to protein fragment IH (domains HII-VI). (A) EDTA (2.5, 5,10 mM) (lane 3-5) fails to inhibit ICAM-1 binding to fragment HIcompared with ICAM-lFc alone (lane 1). Control peptide CRI plusfragment Ill (lane 2). (B) Control samples of the precipitationsshowing equivalent translation.

structures in containing a pair of EF hands (with onepotentially functional and the other non-functional inintegrins).A ribbon diagram illustrating the packing of the two

domains V and VI is shown in Figure 9. The two domainswere packed together in the same orientation as the domainsin calmodulin with helix D running smoothly into helix A'.The two peptides which interfered with the LFA-l -ICAM-1interaction occupy adjacent positions on the same face ofthe model, specifically interacting with the loop between the'functional' and 'non-functional' EF hand in domain V andwith the loop of the putative metal binding EF hand indomain VI. Although speculative, this modelling exerciseprovides a structural perspective to the in vitro experimentsby suggesting that ICAM-l forms contacts with LFA-l alongone face of domains V and VI.

DiscussionLittle is known about the physical interaction of the 32integrin LFA-1 with its ligand ICAM-1. In this study wehave mapped a binding site for ICAM-1 to domains V andVI of the a subunit. Initially the site was localized to thedomain V-VI subregion using in vitro translated proteinfragments, which were shown to resemble features of nativeLFA-1 in that they reacted with specific monoclonalantibodies (also see Landis et al., 1993; R.C.Landis,personal communication). As a 'domain subtraction'procedure was used to localize this ICAM-1 binding site,further ICAM-1 binding sites may exist which are N-terminalto the domain V-VI site.

Fine mapping of the ICAM-l binding site using short 'sub-domain' peptides showed that two discontinuous regions ofdomains V and VI contributed to the binding site. Thedomain V region (by extrapolation 458-467: PLFYG-

1794

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A

B

A

B

Precipitation

Control

_____b

f .,

ICr: o0

21 1Ti, 0

t

_ _4I

f. fx n

4 + 4±

H H H

,: H ;N


VttLFA-1 (V) (435) FGGE LCGV -LFA-1 (VI) (496) FGEAI TALTLFA-1 (VIl) (556) FGRSAI-HGVKVLA-2 (V) (462) FGSVLCSV,-VLA-2 (VI) (525) FGSAIAALSVLA-2 (VII) (589) FGRSLDGYG

4CPV(1)(43) VKKAFA I -3CLN (1) (12) FKEAFSLF-3CLN (2) (85) I R E ATF R VF-5TNC (1) (22) F K A AF DMF -5TNC (2) (98) L E D CF R IF -

SS F- H (A)M2+

I X

D GE TG D G L VG D G LAKDT TM D G F NGD]S TKS GFGDGTGUNGYGGGDA[gG F

* * * * *

1 3 5 9 12

LLLIGAPLFYGE-QRGGRVFIYQRROQL.GFE EV-VAVGAPLEE---QGAVYIFNGRHGGLSPOPS-VAVG,AESQMI--VLSSRPVVDMVTL,MSFSPAVLL.VGAPMYMS--DLKKEEGRVY.LFT.ILKKGIL-VIVG-SPLENQN-SGAVYIYNGHQGT l.RTKYS-VSIGAFG:QVVQLWSQSIADVA I EASF.TPEKIEDEL:KL-FLONFKADARALTDGETKTFLKAGDC STKIELGTVMRS --- LGaNPTEAELODM I NEV DjAAAIELRHVMTN -- -LGEKLTDEE.VDEMWIREA NTKIELG.TVMRM---LGQNPTKEElL=:DAII.EEV D EIE E LGEILRA-- - TGEHVTEEDI-EDLUMKDSDJ K

H (B) H (C)

1 3

S E - -- L GDPGGYPLGR (495)QjR--IEETQVLSGI OW (555)E IPVHEVECSYSTnSNI( (618)GQHOFLL GPEG.IE.E N TR (524)OKILGSDGAFRSHLQY (588)T L -VNK N AO IL.KLC.F (652)D --GIDIGKIGV,D[EFT (103)D ---- G N GT C F'P E F:L`(68)D -- -G

DGQ EIEY,IEEFV. (142)D ---- GSGTIDFE E FIL (79)N - ND G RI D F:D ElF L (155)

H (D)-* * * *

5 9 12

Fig. 8. Multiple sequence alignments of the putative divalent cation binding domains V-VII from integrins LFA-1 and VLA-2 plus the EF handcation binding domains from the X-ray crystal structures for carp parvalbumin (4CPV 1) (Kumar et al., 1990) [names given are the Brookhavendatabase codes for the X-ray crystal structures used (Bernstein et al., 1977)] and the two domains of calmodulin (3CLN 1 and 3CLN 2) (Babu et al.,1988), and of turkey troponin-C (5TNC 1 and 5TNC 2) (Herzberg and James, 1988). Each domain consists of a pair of EF hands. The averagesecondary structure (SS) for the crystal structures is shown. Residues involved in direct binding of divalent cation are denoted by M2+ with thepositions of Ca2+ coordinating residues indicated by numbered asterisks. Sequence similarities between the integrins and the crystal structures areboxed, hydrophobic residues (F, W, L, V, I, M, A, G, P, R and K) involved in tertiary interactions (shaded boxes) (residues R and K areconsidered to belong to the hydrophobic set if their long hydrophobic CH2 chains are involved in hydrophobic interactions with other members of theset and their hydrophilic heads point out towards the solvent) and residues that have the potential to bind M2+ (D, E, N, Q, S, T) (open boxes).Lines connecting columns of residues (generally conserved hydrophobics) indicate the stronger tertiary interactions within the model for the LFA-1domain repeats as well as within the crystal structures. The overall topology is expected to be the same for all domains, that is a pair of EF handsper domain. The divalent cation binding potential of the second EF hand (helices C and D) is obviously diminished considerably as there is onlylimited sequence similarity with the cation binding residues (i.e. essentially only the second and third M2+ residues, in positions 3 and 5, showsequence similarities). The second repeat is therefore only distantly related to the first more classical EF hand motif.

EQRGG) follows immediately on from the putative EF handin domain V, whereas the domain VI sequence (497-516:GEAITALTDINGDGLVDVAV) encompasses the divalentcation binding motif of domain VI (see bold residues).Neither the reversed sequences nor peptides from domainsIV and VII, including those within the putative metal bindingsequence, were inhibitory. Thus the peptide blocking databoth supported and extended the initial localization of thebinding site carried out with the in vitro translated a subunitprotein fragments. Although relatively high concentrationsof peptides were required to achieve blocking ( - 200 ItM),this is in line with recent measurements of the weak bindingaffinity of one other pair of adhesion partners, CD2 andCD48 (van der Merwe et al., 1993).The most immediate comparison can be made with the

03 integrin gpIIblla binding site for fibrinogen oy chainpeptide which maps to a single site in domain V (D'Souzaet al., 1990, 1991). The gpIlbEla ligand binding site differsfrom that described for LFA- 1 in that it covers the EF handsite (residues 296-306) next to the LFA-l binding site, i.e.analogous to the dom 5.2 region. However, it is of interestthat a ,32 and (3 integrin utilize similar areas of the a subunitto interact with their respective ligands in spite of the verydissimilar nature of these ligands. ICAM-1 which is amember of the immunoglobulin superfamily and a cellmembrane protein, shares no obvious sequence similaritywith fibrinogen which is a soluble plasma protein. Thesequences corresponding to the ICAM- 1 binding region indomain V are only weakly conserved in other integrins. Thisarea has been highlighted in rat VLA-1 as a potential ligandbinding site because of its variant and extended sequence(Ignatius et al., 1990). As another method of characterizingthe means by which ICAM-l interfaces with LFA-1, we havemodelled domains V and VI. The model suggests for thefirst time that each domain contains not one but a pair of

dom5/5.3

do

DOM VlI

DOM V

Fig. 9. Ribbon diagram for the model of domains V and VI ofLFA-1. Plus signs indicate the positioning of the peptides that interferewith LFA-l -ICAM-1. The two regions are located on the same sideof the two domain model but are interrupted by the cavity betweenthe two domains. The diagram was produced with the aid of thedisplay programme MOLSCRIPT (Kraulis, 1991). Helices are labelledA to D for the first repeat and A' to D' for the second. The dashedline indicates the boundary between the domain repeats. Hatchedcircles suggest positioning of the putative divalent cations (their exactposition was not modelled).

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P.Stanley et al.

EF hand motifs, only one of which has retained the potentialto bind divalent cation. This pairing of EF hands in whichone partner is redundant is seen in other divalent cationbinding molecules and is thought to maximize the bindingaffinity of the 'active' partner (van Eerd and Takahashi,1976; Cook et al., 1991). In the model the two stretchesof sequence representing the blocking peptides are juxtaposedalong one face of domains V and VI. Although such amodelling exercise must be considered to be speculative, thederived structure does illustrate how both peptides mightcompose parts of a single binding site on LFA-1.

In solution, the binding of ICAM-1 to this region wasfound not to be dependent on the presence of divalent cation.Thus bound cation may make little difference to the tertiarystructure of the immediate site as shown for annexin V(P.Freemont and H.Driessen, personal communication) buthave its effect on longer range interactions as has also beenseen for annexin V (Lewit-Bentley et al., 1992) and otherCa2+ binding proteins (Gariepy and Hodges, 1983). It maybe relevant that a theoretical modelling study with a chimericintegrin-calmodulin EF hand suggests the Ca2+ bindingloops of annexin V to resemble those of integrin (Tuckwellet al., 1992). In seeming conflict with this finding is the factthat the intact LFA-1 heterodimer requires Mg2+ to bindICAM-1 in a stable complex (Marlin and Springer, 1987;Dransfield et al., 1992). A possible resolution of theseobservations is that divalent cation may be required for ligandbinding not to the isolated a subunit, but in order to alterthe tertiary relationship between at and ,3 subunits in the intactheterodimer to facilitate ligand binding. In support of thishypothesis, the 24 epitope which is a marker of activatedLFA- 1, requires Mg2 + for expression on intact LFA- 1molecules but is exposed on isolated a subunits in the absenceof Mg2+ (Dransfield and Hogg, 1989; Dransfield et al.,1990).Another possibility is that initial binding of ligand might

facilitate divalent cation binding by contributing acoordinating residue such as the aspartate residue of RGD-containing ligands (Humphries, 1990). However, asdiscussed in the Introduction, recent studies with the (3integrins suggest that the sites are structurally complete interms of divalent cation binding (Smith and Cheresh, 1991).For LFA-1, the number and identity (e.g. Mg2+ binding)of occupied sites is not known and it is possible that thedomain VI (and domain V) site does not normally binddivalent cation. However, studies carried out on a mixtureof ,B2 integrins suggest the potential for full occupancy ofthe sites at least by Ca2+ (Gahmberg et al., 1988). Recentlythe role of divalent cation in integrin function has becomemore complex with the suggestion that the 'I' domaincontains a metal binding site and is involved in an undefinedmanner in ligand binding (Michishita et al., 1993).

In summary we have defined an ICAM-l binding site onthe LFA-1 a subunit with contact points within domains Vand VI. More information will be required about thestructure of intact LFA-l in order to understand further howthis site participates in ligand binding during the process ofLFA-l activation.

Materials and methodsPCR amplification of fragmentsTo construct the LFA-1 (x subunit deletion series, fragments were amplifiedusing PCR strategy from a cDNA clone, 3R1 with domain boundaries

assigned according to the designation of Larson and colleagues (Larson et al.,1989). The 5' PCR primers were designed such that they contained a BamHIrestriction enzyme site, followed by a Kozak sequence to maximizetranslational efficiency (Kozak, 1987), an in-frame initiating methionine anda 24 bp hybridizing sequence. The 3' primer consisted of the hybridizingsequence plus a HindUI restriction site. The primers were as follows (withrestriction enzyme sites given in bold type): 11-5', 5'-CGGGA-TCCCATGCCAGTCACCCTGAGAGGTTCCAAC-3'; 'I' Dom-5',5'-CGGGATCCCATGGACCTGGTATTTCTGTTTGATGGT-3'; III-5',5'-CGGGATCCCATGAAACAGGACCTGACTTCCTrCAAC-3'; IV-5',5'-CGGGATCCCATGAATGAACCATTGACACCAGAAGTG-3'; V-5',5 '-CGGGATCCCATGCAGGTCCAGACAATCCATGGGACC-3';Common-3', 5'-CCCAAGCTTAATTCCTGAGAGCACTTGGGT-CCC-3'.The V-VII and C fragments were amplified from a cDNA library,

HPB.ALL. The primers for the C fragment PCR differed from all of theothers by containing a 5' EcoRI site and a 3' Clal site. Primers for theV-VII and C fragment were as follows: V-VII-5', 5'-CGGGATCC-CATGCAGGTCCAGACAATCCATGGGACC-3'; V-VII-3', 5'-CCC-AAGCTTCAACCCCCGTCTTCTGGTCCGGTG-3'; C-5', 5'-CGGAA-TTCCCATGTTCCCAGGAGGGAGACATGAACTC-3'; C-3', 5'-CCAT-CGATCTGCTTCTCATACACCACGTCAAC-3'.PCR amplifications were carried out using a GeneAmp DNA kit with

native Taq DNA polymerase (Perkin Elmer Cetus). All reactions wereperformed according to the manufacturer's instructions. Thirty amplificationcycles consisting of 2 min at 94°C, 2 min at 590C and 9.9 min at 70°Cwere used, followed by a 9.9 min chase at 70°C.

All PCR fragments were cloned into the phagemid vector pBluescriptII KS+ (Stratagene). Sequencing was carried out by dideoxy chaintermination analysis using the Sequenase version 2.0 sequencing kit (USB).

In vitro transcription and translationTranscription and translation was carried out using the TNT T7- coupledrabbit reticulocyte lysate system (Promega) according to the manufacturer'sinstructions. 1 Ag of cloned DNA in Bluescript KS+ phagemid was addedto each reaction in a total volume of 50 1d containing 50% reticulocyte lysate.Reactions were carried out at 30°C for 90 min in the presence of[35S]methionine (Amersham). Initial experiments were performed usingseparate transcription (Stratagene) and translation (Promega) reactionsyielding comparable results.

Immunoprecipitation and SDS - PAGEFollowing transcription and translation, 50 Al of 2 x EIA buffer (500mMNaCl, 100mM HEPES, pH 7.0) containing 0.2% NP40 was added to each50 41 translation reaction and the mixture was divided equally. 3 ,ug ofICAM- IFc were added to one half of the reaction mixture and 3 ,Ig of CRImyeloma protein to the other half. mAbs were used either in the formof tissue culture supernatant at 150 t1 per sample or purified protein at 3 ygper sample. The total volume of the reaction was made up to 200 1.l with1 x EIA (250 mM NaCl, 50 mM HEPES pH 7.0). Reactions wereincubated at 4°C for 2 h and then added to 20 ,ul packed volume of proteinA-Sepharose CL-4B (Pharmacia) which had been pre-washed with1 x EIA. The reactions were incubated at 4°C overnight with constantagitation. The protein A-Sepharose was pelleted by brief centrifugationand 10 u1 of supernatant were removed for SDS-PAGE analysis. Theremaining supernatant was discarded and the protein A -Sepharose washedthree times with 1 x EIA + 0.1% NP40 and twice with 1 x EIA. 20 /Ilof SDS-PAGE reducing buffer were added to the protein A beads andboiled, and samples were electrophoresed on 9% polyacrylamide gels. Gelswere treated with EN3HANCE (NEN) prior to autoradiography. In thepeptide blocking experiments, peptides were added to a final concentrationof mg/mil. Peptide dom 6.1 was tested up to 5 mg/nil. In other experimentsthe divalent cation chelator EDTA was added to final concentrations of 2.5,5 and 10 mM.

Preparation and purification of ICAM- lFc and mAbsThe ICAM-lFc fragment contains the first three domains of ICAM-1 fusedto a human IgGI Fc tail and was prepared as previously described (Berendtet al., 1992), using serum-free medium (ADC 1, Biological Industries Ltd)to exclude the possibility of contamination with bovine immunoglobulin.The purified human IgGI myeloma protein CRI was a gift from Dr RoyJefferis, University of Birmingham. The mAbs used in this study were LFA-la specific mAbs 38 (Dransfield and Hogg, 1989), MHM24 (Hildreth et al.,1983), an ICAM-1 (CD54) specific mAb 15.2 and a control mAbl4 specificto CD8 (Dransfield et al., 1992). mAbs were purified by protein Achromatography (Ey et al., 1978).

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Synthetic peptidesPreparation and characterization of synthetic peptides. The peptides weresynthesized on a model 430A Applied Biosystems Solid Phase Synthesizeron 4-hydroxymethylphenoxymethyl resin using 9-fluorenylmethyloxy-carbonyl for temporary a-amino group protection (Carpino and Han, 1992).Each amino acid was coupled as a hydroxybenzatriazole active ester, auto-matically formed immediately prior to use. Cleavage from the resin anddeprotection of the peptide were achieved with trifluoroacetic acid containingphenol, ethanediol, thioanisole and water at 20'C for 2 h. The purity andmolecular weight of individual peptides were analysed by plasma desorptionmass spectrometry and the sequences by gas phase amino acid sequencing.A peak corresponding to the expected molecular weight was found for eachpeptide and in all cases peptides were found to contain the correct sequence.Synthesized peptides were desalted using Sephadex G25 (Pharmacia) toremove potentially cytotoxic compounds and were stored for short periodsprior to use as lyophilized powders under desiccating conditions at roomtemperature.

Peptide solubility. Amino acid analysis (Applied Biosystems Model 420H)was used to establish the solubility of all peptides which were tested atconcentrations where they were known to be fully soluble. For most peptidesthis was at least 5 mg/ml, but for dom 6.1 it was 2 mg/ml, and for dom4.3 and dom 5.3 it was 0.5 mg/ml. Peptide dom 5.1 was essentially insoluble.As a further precaution, all peptides were either centrifuged at 60 g or filteredusing low protein binding filters (0.22 /m, Millipore) prior to use in thebinding assays. Thus any non-specific inhibition by insoluble peptide wasavoided. No difference in experimental results was observed between filteredand centrifuged peptides.

T cell binding to ICAM- 1T lymphoblastoid cells were expanded from unstimulated PBMC by 1-2weeks' culture in rIL-2 (20 ng/ml; Cletus) with details as previously described(Dransfield et al., 1992). Purified ICAM-lFc protein (40 l1 of aconcentration of 20 ,tg/ml in PBS-A) was added to each well of flat-bottomed96-well plates (Immulon 1, Dynatech) before overnight incubation at 4°C.Prior to the assay, the plates were saturated with 2.5% BSA in PBS-A(lacking in Ca2+ and Mg2+) (100 1l/well) by incubation for 2 h at roomtemperature and finally washed four times with PBS-A and once in HEPESbuffer (20 mM HEPES, 140 mM NaCl, 2 mg/ml glucose, pH 7.4).

Detection of T cell binding to ICAM-lFc was carried out using thetechnique described previously (Cabanias and Hogg, 1991). Briefly,5 x 107 cultured T cells were labelled with 200 ACi 5ICrO42-, for 90 minand resuspended in HEPES buffer containing 4 mM MgCl2, 4 mM EGTAand 100 nM PdBu (2 x final concentration). 50 ul of T cells plus 50 1lof peptide or mAb at designated concentrations were added to each wellof an ICAM-lFc-coated 96-well plate. Plates were incubated on ice for20 min, centrifuged at 30 g for 1 min and then incubated for a further 35min at 37°C. T cells which remained bound after five washes in warmedRPMI were then lysed in 1% Triton X-100 in water and the incorporatedradioactivity measured using a Betaplate counter (LKB Instruments).Monoclonal antibodies against LFA-1 ax subunit (mAb 38) and ICAM-1(mAb 15.2) were included in assays as controls for assessingLFA-1/ICAM-1-mediated adhesion.

Cell viability assaysAll peptides used in these experiments were tested for cytotoxic activityusing an MTT assay (Plumb et al., 1989) with modification (Ross et al.,1992). Briefly T cells at 50 Id per well, were plated out in 96-well flat-bottomed plates at a concentration of 5 x 105 cells/ml in RPMI plus 10%FCS. 50 1l of peptide in RPMI were added to the T cells to give a finalpeptide concentration of 5 mg/ml (the highest peptide concentration usedin the assays). 20 11 of MTT (at 5 mg/mi in RPMI) were then added toeach well and the plate incubated at 37°C for 2 h (note: T cell assays werefor 55 min only). The plate was then centrifuged at 30 g for 1 min andthe MTT and medium were carefully removed. 200 yl of dimethyl sulfoxideplus 25 1d of Sorensen's glycine buffer were then added to each well andthe optical density (OD) was read at 570 nm (Titertek Multiskan, FlowLaboratories). A titration of cell concentration was included to establishthe expected OD reading under non-toxic conditions. None of the peptidesused gave OD readings lower than the expected value for the equivalentconcentration of cells in the absence of peptide.

Procedures for molecular modellingConserved features of the metal binding domains V-VII of LFA-lIa wereidentified by comparison with the equivalent domains of a second integrin,VLA-2 (Takada and Hemler, 1989) (Figure 8). The alignment shows that

the first half of each domain bears strong sequence similarity to an EF hand(helices A and B) in that four of the five divalent cation and all hydrophobicresidues of the first amphipathic helix are conserved. However, the secondhalf of each LFA-1 domain shows less conservation of this motif in thatthe metal binding residues are only weakly conserved although the two helices(C and D) are predicted to remain. These sequences were then comparedwith the EF hand-containing domains of three calcium binding proteins forwhich X-ray crystal structures are known. Each X-ray structure has onepair of Ca2+ binding EF hands per domain, a feature which appears tomaximize Ca2+ binding affinity over individual EF hands (Sekharudu andSundaralingam, 1988). Such EF hand pairing appeared also to be a featureof the integrins as key tertiary interactions are predicted to be conservedbetween the hydrophobic residues of helices A and B of the first half ofthe domain with predicted helices C and D of the second half of the domain.Each domain was modelled from the X-ray crystal coordinates of carp

parvalbumin (Kumar et al., 1990) using the technique of replacing non-regular regions of the template, usually the loop regions, by databasefragment searches (Bates and Stemnberg, 1992). Helices B and B' are bentdue to the proline residue midway along each helix. Although proline residuesare rarely found in this position (MacArthur and Thornton, 1991) they dooccur (Dempsey et al., 1991; Dekker et al., 1993). A similar distortionnot involving proline is seen in the B helix of parvalbumin which has beenattributed to the requirements of the EF hand to readjust following thedynamic binding of calcium (Kumar et al., 1990). It is possible that theproline residue in the B helix of some of the integrin repeats serves thesame purpose.The domains are linked together in the same way as the domains in

calmodulin (Babu et al., 1988) except that the long helix between eachglobular domain has been substantially truncated such that the last helixof each repeating domain is made to run directly into the first helix of thenext. This packing arrangement is the most logical in terms of maintaininga self-avoiding protein chain and an absence of 'linker residues' betweendomains, i.e. the residues of helix D/A' must contribute to the tertiarycontacts within each of the two domains. The complete model was energy-minimized using the CHARMM program (Brooks et al., 1983) with defaultvalues as incorporated into QUANTA in the POLYGEN software.

Several points validate the model presented here. There is substantialsequence similarity within the first half of each domain to a classical EFhand and EF hands usually occur in pairs. There are no steric clashes inthe model and conserved hydrophobic residues pack well within and betweeneach domain. There are no unbalanced charges that lack the potential tobe solvated. Finally, the model, which was independently derived, allowsa simple interpretation of the peptide analysis.

AcknowledgementsWe are extremely grateful to Nicola O'Reilly for the synthesis of the peptidesused in this study. We thank Dr David Simmons (Oxford) for the HPB.ALLcDNA library and Dr T.S.Springer (Boston) for LFA-1 ax subunit construct3R1; Drs Alister Craig and Tony Berendt (Oxford) for the ICAM-lFcconstruct; Alison McDowall and Dr Paul Hessian for help with the T cellassays and the preparation and purification of the ICAM-lFc; Drs AlexLaw and A.McMichael (Oxford) for mAb MHM24, Dr Roy Jefferis(Birmiingham) for myeloma protein CRI and Dr Fiona Watt for control 39merpeptide; Dr Mike Fried for discussion about in vitro transcription/translationsystems; Dr M.J.E.Stemberg for discussion about the modelling procedures.We thank our colleagues Paul Freemont, Paul Hessian, Clive Landis, MikeStemnberg and Mairi Stewart for their many helpful comments on themanuscript and Louise Dewhurst for her assistance with its preparation.

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Received on July 28, 1993; revised on February 1, 1994

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site in domains V and VI