CD2 is a 50-60 KDa glycoprotein comprising of two extracellular immunoglobulin superfamily (IgSF) domains and an intracellular C-terminal domain linked by a putative transmembrane helix. The structure of the CD2 extracellular portion (Fig. 1.2), the ectodomain, has been determined by X-ray crystallography (Bodian et aL, 1994; Jones et aL, 1992), while the solution structures of N-terminal domain of CD2 (CD2d 1 ) has been determined by NMR spectroscopy (Driscoll et aL,
1991; Withka et aL, 1993). The N-terminal domain of CD2 (CD2dl), the focus of this investigation, was demonstrated to be solely responsible for the adhesion function CD2 (Hahn et aL, 1992a; Peterson & Seed, 1987; Sayre et aL, 1989).
Domain 2
Domain 1
Domain 1
Domain 2
Fig. 1.2 Two orthogonal views o f the crystal structure o f the 2-domain extracellular portion o f rat CD2 (PDB ID: IHNG) in a ‘head-to-head’ dimeric orientation. Each monomer is indicated by a different colour.
CD2 ectodomain - From the structures of CD2 as determined by X-ray crystallography and NMR spectroscopy, both extracellular domains are shown to be of IgSF fold - the N-terminal domain CD2dl is of V-set fold while the membrane- proximal domain is of C2-set. The V-type fold of domain 1 (CD2dl) consists of 9 stranded (3-sheet, with five antiparallel strand GFCC’C” forming one sheet and four antiparallel strands forming the ABED sheet. The A strand, however, can also be considered to be shared by both sheets. The intrachain disulphide bond that normally holds together the two sheets and stabilises the structure in V-set immunoglobulin is absent in CD2dl. Two cysteines, however, can be inserted at the predicted positions (118 and V78) to form a disulphide bond (Gray et aL, 1993). There are differences in CD2 to the standard V-type fold in its loop configuration: its DE loop is very short, while the EC loop is longer. The ^-strands B, D and E are also truncated (Jones et al., 1992), with the short B strand being due to the presence of Pro 19 which result in a kink at the beginning of the BC loop.
Fig. 1.3 Ribbon diagram o f the N-terminal domain o f CD2.
The C2 fold of the second domain is consisted of 7 stranded (3-sheet. This second domain of CD2 may be involved in CD2 function by interacting with adjacent molecules. The domain 1 and 2 are linked by a highly conserved flexible linker region which allow up to 20° orientational freedom for domain 1 relative to domain
2. This may be important in the binding interaction by allowing the CD2dl to be orientated such that the GFCC’C” face form a platform nearly parallel to the cell surface. The linker may therefore enhance binding by maximising exposure. The stalk linking the extracellular portion to the membrane, for unknown reason, is also highly conserved.
There are four Asn-linked glycans in rat CD2, with three on the N-terminal domain CD2dl (Jones et aL, 1992; Williams & Barclay, 1988). The adhesion surface, however, is free from glycosylation site and the binding of rat CD2 to CD48 is independent of glycosylation (Davis et aL, 1995a; van der Merwe et aL, 1993a; van der Merwe et aL, 1993b). It was previously thought that the glycosylation found on human CD2dl may contribute to ligand binding (Recny et aL, 1992), however, structural studies have shown that this N-linked glycan is located opposite to the ligand binding site and therefore not directly involved in binding (Wyss et aL, 1995). It was also suggested that the N-glycan is crucial for stabilising the protein fold and counterbalancing an unfavourable clustering of positive charges (Wyss et aL, 1995), however, this interpretation is disputed (Davis & van der Merwe, 1996). The N- glycan attached to the membrane proximal domain was proposed to have a function in maintaining the upright position of the extracellular portion of CD2, orientating the binding surface of CD2 in such a manner as to promote fran^-interaction of CD2 with CD48 instead of cw-interaction (Dustin et aL, 1996). The glycans may also prevent non-specific protein-protein interaction during receptor clustering, protect against proteases and play a general role in controlling the assembly and stabilisation of the complexes in the immunological synapse (Rudd et aL, 1999).
Cytoplasmic domain - While the N-terminal domain is responsible for adhesion, the cytoplasmic domain is responsible for attachment to cytoskeleton and signalling. The cytoplasmic tail contains 116 amino acids and is largely unstructured. It is highly basic ( p i- 12), contains large number of prolines (-20%), and possesses the most highly conserved sequences among all CD2 homologues (Fig. 1.4) (Clayton et aL, 1987; Tavemor et aL, 1994). The cytoplasmic tail contains several functional regions which may be important in mediating signal triggered by extracellular stimulus (He et aL, 1988; Kivens et aL, 1998; Moingeon et aL, 1989b). As mentioned earlier, the cytoplasmic domain may be bound by several adaptor proteins;
for example, the KGPPLPRPRV sequence at the C-terminus can be bound by the SH3 domains of CD2BP1 and CD2AP (Dustin et aL, 1998; Li et aL, 1998). The CD2AP is associated with CD2-triggered cytoskeletal rearrangements. The CD2BP1 can recruit cytosolic protein tyrosine phosphatase (PTP)-PEST to the cytoplasmic tail and is associated with regulation of adhesion by CD2 and integrins (Li et aL, 1998), it may also induce clustering of CD2. The cytoplasmic domain of human CD2 also contains two PPPGHR segments that are bound by CD2BP2 proline-binding modules, the glycine-tyrosine-phenylalanine (GYF) domain which regulates protein- protein interaction (Freund et aL, 1999; Nishizawa et aL, 1998). CD2BP2 is involved in IL-2 production and Ca^"^ flux. The C-terminus may also become associated with the SH3 domain of p561ck and p59^" (Gassmann et aL, 1994; Lin et aL, 1998). There is, however, no tyrosine present that can mediate interaction with SH2 domain-containing signalling elements. The C-terminal asparagine appeared to be important for the upregulation of the CD2 avidity as substitution of this residue to alanine or aspartic acid completely abrogates CD2 capacity for upregulation (Hahn et aL, 1992b).