Journal of Science 107, 2065-2070 (1994) 2065 Printed in Great Britain © The Company of Biologists Limited 1994

COMMENTARY Structural motifs for recognition and adhesion in members of the immunoglobulin superfamily

Claire L. Holness and David L. Simmons* Laboratory, Imperial Cancer Research Fund, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK *Author for correspondence

INTRODUCTION The Ig domain found in recognition/adhesion molecules conforms to the β barrel plan. Compared with V The immunoglobulin superfamily is the most abundant family domains, adhesion domains seem to have longer β strands and of cell surface molecules, accounting for 50% of leukocyte shorter connecting loop regions. The antigen-combining site of surface glycoproteins. This evolutionary success story is consists of the CDR (complementarity determining thought to be due to the stability of the Ig domain, which is regions) 1, 2 and 3, which are largely made from connecting able to resist the harsh proteolytic and oxidative environment loops. In contrast, adhesion binding surfaces seem to be made of the extracellular world. By mutation and selection, the Ig from the β strands themselves, arranged in flat faces. This is, domain has evolved to serve many different functions of course, a simplification and exceptions exist for both cases; including: receptors for growth factors (CSF-1 receptor, PDGF for antibodies the precise shape of the framework region of β receptor, FGF receptors); receptors for the Fc region of Ig (IgG strands is key to the organisation of the CDR loop regions. The receptors; CD16, CD32, CD64; CD89 IgA receptor); and as CDRs are not completely autonomous units; witness the trials adhesion molecules, which now seems to be the function of the of those presently engaged in humanising antibodies and gen- majority (CD2/CD58, CD28 and CTLA4, which bind to B7 erating in vitro antibody repertoires. For adhesion molecules, and B70; CD4/class II, CD8/class I, CD31/CD31, CD50 there are exceptions to the dominance of β strand face inter- (ICAM-3)/LFA-1, CD54 (ICAM-1)/LFA-1, CD102 (ICAM- actions, as some key residues involved in adhesion binding 2)/LFA-1, CD106 (VCAM)/VLA-4, NCAM/NCAM. , sites have been found in loops. MAG, TAG-1, CEA) (Springer, 1990). The packing and orientation of domains in adhesion The ideas and conventions defining the domain organisation molecules seems to be remarkably uniform. The contiguous Ig of this family were established by Alan Williams and Neil domains are arranged as an extended rod, with only small Barclay in a series of papers in the late 80s (Williams and angles of pitch between adjacent domains. For example, there Barclay, 1988; Williams et al., 1989). The basic Ig domain is is a common packing arrangement of domains 1 and 2 of CD2 built from a tightly packed barrel of β strands, either 7 strands and CD4, which is quite different from that seen in antibodies. in C1 and C2 domains (arranged in 2 layers of 3 and 4 strands So far, no antibody-like pattern, with heavy chain V domain designated ABE and CC′FG, respectively) or 9 strands in V bent at 90¡ to the contiguous CH1 domain, has been found. domains (arranged in two layers of 4 and 5 designated ABDE Although adhesin domains exist as an extended array, large and CC′C′′FG, respectively). There have been several recent rotational displacements between adjacent domains have been structural studies on adhesion molecules of the IgSF, including seen in the solved crystal structures of both CD2 and CD4 CD2 (solution NMR, Driscoll et al., 1991; Withka et al., 1993; (Jones et al., 1992; Ryu et al., 1990) and in the proposed model and crystallography at 2.8 Å resolution, Jones et al., 1992), CD4 for the N-terminal domains of ICAM-1 (Berendt et al., 1992). domains 1-2 (crystallography at 2.6 Å resolution, Garrett et al., For all these molecules adjacent domains are rotated by 160- 1993; Ryu et al., 1990; Wang et al., 1990) and domains 3-4 180¡ relative to each other. (crystallography at 2.8 Å resolution; Brady et al., 1993); the However, in spite of the common gross organisation of IgSF single V domain of CD8 (crystallography at 2.6 Å resolution; adhesins, there is a surprising diversity of modes of interaction Leahy et al., 1992); class II (crystallography at 2.8 Å resolu- amongst individual members. Fortunately, however, they can tion; Brown et al., 1993). In addition there have been extensive be grouped into a small number of patterns: (1) heterotypic mutagenesis screens of Ig domains involved in adhesion binding mediated by the N-terminal Ig domain(s); (2) including: CD2, which binds CD58 (LFA-3) (Somoza et al., homotypic binding mediated by ‘internal’ domain(s) and 1993; Arulanandam et al., 1993); CD4, which binds class II requiring anti-parallel alignment of interdigitated molecules. (Moebius et al., 1993); ICAM-1, which bind LFA-1 (Staunton et al., 1990; Berendt et al., 1992); and VCAM-1, which binds VLA-4 (Osborn et al., 1994; Vonderheide et al., 1994). Key words: Ig superfamily, adhesion, binding site

2066 C. L. Holness and D. L. Simmons

Although the focus of this Commentary will be IgSF Key to symbols in all Figs: members involved in adhesion/recognition, the Ig fold has also evolved to bind small ligands such as growth factors. Some of Basic immunoglobulin related domain the recent work mapping their binding sites, which tend to be discrete binding pockets in internal domains, will be discussed. Type III related domain

α1 and β1 peptide binding domains of class II HETEROTYPIC CAMs AND THE DOMINANCE OF D1 Binding sites and surfaces within domains The most detailed molecular picture of IgSF domain recogni- tion comes from heterotypic interactions involving leukocyte Tyrosine kinase domain CAMs; specifically CD2/CD58 CD4/class II and ICAM- 1/LFA-1. Combinations of mutagensis screens with modelling A and/or structural analysis have localised binding sites to par- CD2 CD2/CD58 ticular domains, faces and key residues. The results indicate that in these receptor/ligand pairs, the IgSF binding surface is D2 D1 localised to one face, the CFG face, and key residues are located within that face and at its edges (Fig. 1). A general model for how IgSF domains of adhesion molecules interact CFG face was proposed by Springer (1991) based on the NMR structure and mutagenesis studies of CD2, and the precedent of how the domains of antibody molecules pack together. ICAM-1 (CD54) For human CD2, residues clustered in the β strands C (K34 B D5 D4 D3 D2 D1 and E36) and C′ (R48 and K49) and also charged residues in AAA the connecting loop between the F and G strands (K91 and AAA N92) have been shown to be critical in this interaction (Somoza AAA et al., 1993; Arulanandam et al., 1993). Similarly located AAA charged residues have been identified as key components of Mac-1 LFA-1 the binding site on ICAMs 1 and 3 for the leukocyte LFA-1. For human ICAM-1 and ICAM-3, residues E37 (E34, ICAM-3 (CD50) ICAM-1) in the CC′ strand and Q75 (Q72, ICAM-1) within D5 D4 D3 D2 D1 domain 1 of ICAMs-1 and 3 are essential components of the LFA-1 binding site (Staunton et al., 1990; Fawcett et al., 1992; Vazeux et al., 1992; de Fougerolles et al., 1993; Holness et al., unpublished data). In addition, Li et al. (1993) have defined a peptide from domain 1 of ICAM-2 (covering residues 21-42; LFA-1 Staunton et al., 1989a), which inhibits binding of ICAM-2 to β ′ LFA-1. This peptide encompasses strands B, C and C and Class II dimer thus confirms the importance of this region for all the ICAMs C as they interact with LFA-1. The key residues, E34 and Q73, are conserved across species and are present in chimpanzee CD4 AAAAAAAA ICAM-1 (Hammond and McClelland 1993), rat ICAM-1 (Kita AAAAAAAA et al., 1992), mouse ICAM-1 (Horley et al., 1989) and mouse AAAAAAAA ICAM-2 (Xu et al., 1992). Indeed, this motif may be generally important for the inter- action of with members of the immunoglobulin super- family (IgSF). To date, there are three examples of Class II dimer IgSF/integrin interactions: ICAMs with LFA-1 and/or Mac-1 (Simmons et al., 1988; Staunton et al., 1988, 1989a; Diamond Fig. 1. Binding sites in heterotypic CAMs. (A) CD2, and the interaction of CD2 with CD58; (B) ICAMs 1 and 3; (C) CD4 et al., 1991); VCAM with VLA-4 (α4β1) and α4β7 integrins (Osborn et al., 1992; Elices et al., 1990; Ruegg et al., 1992); interacting with a dimer of MHC class II molecules. and MadCAM-1 with the α4β7 integrin (Berlin et al., 1993). A tripeptide motif LDV containing a charged residue (D) at an ICAM-1 can also bind to the major group of human rhi- equivalent location on the adhesion domains of VCAM-1 (D1 noviruses and to an unidentified receptor expressed on the and D4) (i.e. within the C-C′ loop region) is also important for surface of -infected erythrocytes. In binding to its cognate integrin VLA-4 (α4β1), (Osborn et al., both cases the interaction surfaces are distinctly different from 1994; Vonderheide et al., 1994; and John Clements, British Bio- the LFA-1 binding face. ICAM-1 is thought to fit into a deep technology, personal communication). Thus, a structural motif canyon on the surface of the rhinovirus and the binding site consisting of two elements, an acidic residue (either D or E) encompasses a large area of the ICAM (Staunton et al., 1989b, located in a CC′ loop and additional residues in the FG loop 1990). Similarly, the P. falciparum receptor makes multiple region, is emerging as a theme in IgSF interactions. contacts on the opposite face (the CDR-2 like loop linking the Immunoglobulin structural motifs 2067

C and E strands) to the LFA-1 contact area (the CFG surface; Oligosaccharides Berendt et al., 1992; Ockenhouse et al., 1992). Before leaving this type of interaction, some mention should be made of the potential influence that oligosaccharide chains Extended faces have on binding. Many IgSF members are very heavily glyco- Recent work has revealed that in addition to the major inter- sylated and are especially rich in N-linked glycans, e.g. half of action face located in the N-terminal domain, the contiguous the mass of ICAM-3 is due to glycan decoration and there are domain, D2, also contributes to the binding site. For CD2, it seven putative N-linked glycan sites in the N-terminal domains has long been known that combinations of mAbs that map to that contain the LFA-1 binding site. There is little experimen- domains 1 and 2 need to bind in order to activate T cells. tal evidence for the role of these oligosaccharide chains in Mutations in the CC′ loop of domain 2 (so-called region 3) binding, but as there are so many of them they must influence affect mAb and ligand binding (Peterson and Seed, 1987). Now docking interactions. The one clear example of the influence ′ for ICAM-3, mutations in domain 2 (R127 in the C-C loop of glycans is in the binding of ICAM-1 to its β2 integrin and D166 within the F-G loop) reduce binding to LFA-1 receptor Mac-1 (CD11b/CD18; Diamond et al., 1991). N- (Holness et al., unpublished data). Interestingly, these are in linked glycans decorating domain 3 of ICAM-1 negatively equivalent regions to the key residues identified in domain 1. affect Mac-1 binding; removing them, by either enzymic However, it is clear that the N-terminal two domains in both cleavage or use of inhibitors of glycosylation, increased these cases do not contribute equally to the binding site. ICAM-1/Mac-1 adhesion. However, in other cases the glycans Domain 1 is dominant with respect to binding, with domain 2 may not play such a significant role, since they may not playing a less critical role. Mutation of the key residues in impinge on the direct interaction face. This appears to be true domain 1 prevents interaction with ligand, whilst a reduced for CD2 where the three-dimensional structure of the fully gly- level of binding is still retained after mutation of the important cosylated form of domain 1 of human CD2, determined by sites in domain 2. From this work, we speculate that binding nuclear magnetic resonance spectroscopy, places the oligosac- may be (at least) a two-step process: an initial weak/low- charide at the top of the connecting loops between the β- affinity binding or docking with domain 2, followed by a high- strands, at the perimeter of the CD58 ligand binding site affinity engagement of key residues in domain 1. (Withka et al., 1993). Clearly, more experimental studies on The interactions of CD4 with MHC class II, and CD8 with the influence of glycans on binding and structural models of MHC class I, offer a more complex mode of interaction their positions relative to binding surfaces are needed. where binding occurs over very extended surfaces that involve multiple domains. In addition, there may be multiple interactions with dimeric forms of the molecules, especially single CD4 molecules binding to dimers of class II molecules HOMOTYPIC CAMs AND INTERNAL ALIGNMENTS and αβ heterodimers of CD8 binding to single class I molecules. The crystal structures of class II revealed a The structural themes emerging from homotypic CAM inter- dimeric interaction between class II molecules, i.e. a dimer actions are distinct from the heterotypic mode. The binding of dimers (Brown et al., 1993). It was proposed that this sites seem to be located in internal domains and homotypic dimer could interact with two CD4 molecules simultane- interaction requires an inter-digitating alignment of anti- ously, and this agrees with the accumulated data from muta- parallel molecules (Fig. 2). genesis studies that were previously contradictory but can Definition of the binding site in NCAM, for so long the pro- now be reconciled. Mutagenesis screens had indicated that all totypic CAM, has lagged behind the leukocyte CAMs. The lateral faces (the ABE face and the CFG face) of CD4 seem extracellular domain of NCAM consists of five Ig domains and to bind to class II. If two class II molecules can bind together two type III fibronectin repeat modules. Recently, a region in as a dimer, a single CD4 can engage them via these two a C′ strand (KYSFNYDGSE) in a CDR2-like region in domain opposite faces (Fig. 1) (Moebius et al., 1993). This contrasts 3 has been shown to mediate homotypic binding (Rao et al., with the restricted binding site for HIV gp120, which 1992). It is not known how the two NCAM molecules need to involves only the CDR-3-like loop, on one face of CD4 be correctly aligned to enable this homotypic site to bind, i.e. (Peterson and Seed, 1988). whether they need to be aligned in a parallel or anti-parallel Recently, one group has proposed that CD4 can self- mode. An early model for how homotypic binding may occur dimerise. There is some evidence for the role of a CDR3 loop came from the work on gp80, the major adhesin expressed in in domain 1 acting as a low-affinity dimerisation inducer; a the late slug phase of the slime mould Dictyostelium discoid- peptide derived from CD4 domain 1 in a CDR3-like loop (D1 ium. gp80 is not thought to be a member of the IgSF, but a residues 87-98; EDQKEEVQLLVF) binds to CD4-Fc in an in short motif (YKLNVNDS) was defined as mediating vitro solid-phase adhesion system (Langedijk et al., 1993). homotypic adhesion (Kamboj et al., 1989). By aligning two However, there is little evidence for the existence of CD4 gp80 molecules in an anti-parallel manner, an ionic salt-bridge dimers at the cell surface, and although dimers were evident bond could form between the two oppositely charged ends of in CD4 crystals (Ryu et al., 1990; Wang et al., 1990; Brady this motif. This is an attractive model, though not formally et al., 1993), the area of contact in these dimers was deemed proven. to be insignificant. This contrasts with CD8, where there is Certain members of the family clear evidence for the existence of CD8 αβ heterodimers at (CEA), which contains over 30 closely related IgSF molecules the cell surface, and the CD8α crystals contained αα homod- (CD66 a,b,c,d,e), seem to bind in an anti-parallel mode imers that had extensive surface area in contact (Leahy et al., requiring a reciprocal interdigitating alignment of two 1992). molecules expressed on apposing cells (Zhou et al., 1993; and 2068 C. L. Holness and D. L. Simmons

located in domain 2 and the other in domain 6. Again, the inter- A NCAM AAAA action of homotypic CAMs may be a two-part process: an FNIII FNIII D5 D4 AAAA D3 D2 D1 initial alignment of apposed CAMs, most probably in an anti- AAAA parallel orientation, followed by docking of specific domains AAAA that mediate homotypic binding. Thus the overall pattern of homotypic IgSF interactions that has emerged from recent studies points to a more complex mode than for heterotypic adhesion, where dominant homotypic site domain(s) are located in membrane distal N-terminal positions and interact head to head. Homotypic IgSF CAMs display a CEA B range of binding modes from the simple head-to-head interac- D7 D6 D5 D4 D3 D2 D1 tions to more complex interdigitating interactions requiring correct alignments of the CAMs along their lengths. Growth factor receptor binding pockets The main focus of this Commentary has been IgSF members involved in cell adhesion or cell-cell recognition. However, the extracellular domains of many growth factor receptors (M- CSF1-R, PDGF-R, FGF-R) contain IgSF domains. The ligand C CD31 binding sites of some of these receptors have been analysed D6 D5 D4 D3 D2 D1 recently and reveal a common yet distinct binding pattern (Fig. 3). The M-CSF-1 receptor, whose extracellular domain consists of five Ig domains, binds its ligand in the third Ig domain (Wang et al., 1993). Similarly, the PDGF receptor (extracellular domain also consisting of five Ig domains; Heidaran et al., 1990) and some of the FGF receptor family bind their ligands via D3 (Yayon et al., 1992). All these ligands are small and the binding pocket is, therefore, likely to be D CD66 confined to a single domain. The ligand/Ig domain contact D4 D3 D2 D1 points have not been defined. Certain members of the FGF receptor family use a novel mechanism for altering ligand specificity, based on differential splicing of a 50 amino acid module into the C-terminal half of D3 (Yayon et al., 1992). So far, this remains unique to these FGF receptors and a similar mechanism for altering ligand specificity has not been found Fig. 2. Binding sites in homotypic CAMs. (A) NCAM; (B) CEA- in any of the adhesion molecule IgSF members. CEA anti-parallel double reciprocal binding; (C) CD31-CD31; (D) For many growth factors, a key event induced by ligand CD66-CD66 head-to-head interaction. binding is receptor dimerisation. The dimerisation of the extra- cellular domains is thought to lead to pairing of the cytoplas- Fig. 2). However, the precise domains mediating this binding mic tails, which then activate intrinsic tyrosine kinases or allow are not known, though internal domains must be involved. A association of intracellular kinases. Apart from the proposed model has been proposed for the structure of CEA where all model for class II dimers binding to CD4 (dimers?), this seven Ig domains are arranged as an extended linear rod (Bates mechanism for adhesion molecule has not et al., 1992). Anti-parallel alignment of two apposing CEA been studied. As recognition/adhesion events between adjacent molecules could then potentially involve interactions between or apposed cells lead to mass pairings of CAMs, it is possible all domains. However, an analysis of ‘shorter’ members of the that the cytoplasmic tails of these multimerised CAMs undergo CEA family, containing only four Ig domains, pointed to a conformational changes allowing association with signalling dominant role for the N-terminal domain, with merely an and cytoskeletal machinery. accessory role played by the other domains related to appro- priate presentation and accessibility of the binding site to the CONCLUSIONS apposed CAM (Cheung et al., 1993). This agrees with our own recent work on CD66a/biliary glycoprotein 1, which contains In spite of the very conserved patterns of domain folding and four Ig domains (Teixera et al., 1994; Watt et al., 1994). The domain organisation seen in members of the IgSF, there is a N-terminal domain contains the dominant binding site and the surprising diversity in their modes of interactions. Binding remaining domains merely act as a stalk to present this site sites can be localised to single domains or involve extended away from the cell surface glycocalyx. faces encompassing several domains. The sites can be located Another example of the ‘internal domain binding mode’ is in N-terminal membrane distal domains or internal and even provided by CD31, which is an abundantly expressed endo- membrane proximal domains. Molecules can interact via N- thelial homotypic CAM, whose extracellular region consists of terminal membrane distal domains in a head-to-head manner, six Ig domains. Present data support a similar anti-parallel or require full inter-digitation of many domains of the CAMs mode of interaction, with one part of the binding site being in an anti-parallel orientation on apposed cell surfaces.

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