Sequence, structure, function, immunity: structural genomics of costimulation

Abstract

Costimulatory receptors and ligands trigger the signaling pathways that are responsible for modulating the strength, course, and duration of an immune response. High-resolution structures have provided invaluable mechanistic insights by defining the chemical and physical features underlying costimulatory receptor:ligand specificity, affinity, oligomeric state, and valency. Furthermore, these structures revealed general architectural features that are important for the integration of these interactions and their associated signaling pathways into overall cellular physiology. Recent technological advances in structural biology promise unprecedented opportunities for furthering our understanding of the structural features and mechanisms that govern costimulation. In this review, we highlight unique insights that have been revealed by structures of costimulatory molecules from the immunoglobulin and tumor necrosis factor superfamilies and describe a vision for future structural and mechanistic analysis of costimulation. This vision includes simple strategies for the selection of candidate molecules for structure determination and highlights the critical role of structure in the design of mutant costimulatory molecules for the generation of in vivo structure-function correlations in a mammalian model system. This integrated 'atoms-to-animals' paradigm provides a comprehensive approach for defining atomic and molecular mechanisms.

Fig. 1. Structures and folds of immunological significance

(A) T-cell receptor (yellow and magenta):MHC-peptide (blue and orange with peptide in stick representation) complex from mouse, PDB Code 1G6R; (B) Human MICA (an MHC class-I-related molecule; magenta) in complex with NK cell receptor NKG2-D (a member of the lectin superfamily; yellow and green), PDB Code 1HYR; (C) Herpesvirus M3 decoy receptor (blue and green) in complex with the CC-chemokine MCP-1 (red and yellow), PDB Code 1ML0; and (D) human GPCR A2a adenosine receptor; this structure provides a model for the many GPCRs relevant to immunity, PDB Code 3EML.

Fig. 2. Biological and genomic strategies for identifying protein targets for structure determination

(A) Systems considerations. DcR3 (red), a soluble TNFR superfamily member, represents a hub protein, as it neutralizes three trimeric TNF ligands, FasL (yellow), TL1A (green) and LIGHT (blue). Arrows indicate functional interactions between the ligands and their cognate receptors. Cysteine rich domains of the receptors are represented as squares. (B) Sequence-based network graph highlighting the sequence similarities between genes in the Nectin family. Genes are illustrated as nodes (circles) and related sequences are connected by edges (i.e., lines). A dashed line depicts adjacent genes in the genome. The only member of this diverse family for which a structure has been determined (CADM3; PDB 1Z9M) is highlighted in black. Cytoscape (131) was used to layout the network. (C) Physically Linked Gene Families. The mapping of selected Ig superfamily (red) and all known TNF (blue) and TNFR (green) genes on the human karyotype highlights clusters of evolutionary related adjacent genes. Genes sharing a vertical line have immediately adjacent chromosomal positions.

Fig. 3. Unique sequence features reveal structural and functional specialization

As an example, multiple sequence alignments of the IgV domains of three members of the human TIM family (TIM1, TIM3, and TIM4) and three members of the human CD28 family (CD28, ICOS, and CTLA4) are displayed. Secondary structure calculated from representatives of each family is illustrated at the top and bottom of the alignment for the TIM and CD28 families. Highlighted in red are positions that are invariant in both families; for example the two cysteines that form the canonical disulfide bond between the B and the F strands (red circles). In blue are sequence positions that are conserved in one family but are absent in the other; these positions define family-specific sequence signatures, and suggest unique structural features that are directly relevant to functional specialization. ALSCRIPT (132) was used to format the multiple sequence alignment.

Fig. 4. Structure of the IgV domain and its diversification in costimulation

(A) Structure of an IgV domain. Strands of the front (green) and back (blue) sheets of the heavy-chain IgV domain from an Fv molecule (PDB Code 1A6W) are labeled according to convention and the conserved disulfide bond connecting strands B and F is colored in orange. The CDR loops connecting strands B and C (CDR1), strands C′ and C″ (CDR2) and strands F and G (CDR3) are highlighted in red. (B) Typical IgV interface formed by the front sheets. IgV dimer formed by heavy- and light-chain IgV domains of an Fv molecule (PDB Code 1A6W). (C) Structural and organizational variations of the Ig superfamily. Costimulatory receptors of the CD28 superfamily are predominantly disulfide-linked dimers of single IgV domains; SLAM family members contain both IgV and IgC domains; TIM family molecules contain one IgV domain attached to a highly glycosylated stalk region; nectin and nectin-like molecules consist of three Ig domains (one IgV and two IgC)s.

Fig. 5. Organizational and sequence features of the CD28:B7 families

(A) Domain organization of the CD28:B7 families. CD28, CTLA-4 and ICOS are immediately adjacent on the chromosome and form disulfide-linked homodimers; PD-1 is monomeric and not part of this cluster. CD28-type receptors consist of a single IgV domain, linked to stalk region and cytoplasmic tail containing tyrosine-based signaling motifs. (B) Organization of B7 ligands. The B7 ligands possess ectodomains composed of a membrane proximal IgC and a membrane distal IgV domain, linked to a stalk region and cytoplasmic tail. (C) Sequence alignment of the ectodomains of CD28 family receptors. Secondary structure is denoted on the basis of the CTLA-4 crystal structure; conserved residues are white with red background; residues with similar properties are labeled red; residues that contribute to the dimer interface of CTLA-4 are highlighted with black circles, those contributing to the CD28 dimer interface with red circles, grey circles annotate those positions that contribute to the dimer interface in both molecules. The conserved cysteine mediating covalent dimerization in CD28, CTLA-4 and ICOS is highlighted with a red asterisk; PD-1 has a Ser at this position. Residues that contribute to the proline-rich ligand-binding motif in CD28, CTLA-4 and ICOS are highlighted with green asterisks; PD-1 lacks this motif. Numbering corresponds to the human CTLA-4.

Fig. 6. CD28 and CTLA-4 share a similar binding mode for the B7 molecules

(A) Superposition of the CD28 monomer (PDB Code 1YJD) and the CTLA-4:B7-1 (PDB Code 1I8L) complex. The tip of the FG loop contains the MYPPPY motif that is crucial for binding. (B) Detailed view of the MYPPPY motif. The consensus ligand-binding sequence shared in CD28 and CTLA-4 makes contacts with residues on the front face (C, F, and G strands) of B7-1. Residues in the MYPPPY motif are numbered according to human CTLA-4.

Fig. 7. CTLA-4 and CD28 have different valencies for the B7 ligands

(A) Structure of the CTLA-4 dimer. This structure (PDB Code 1I8L) reveals a side-to-side dimer in which the interface is formed by the bases of the A and G strands, placing the FG ligand-binding loop distal from the dimer interface. This relative orientation of the monomers allows for a bivalent ligand binding. (B) Structure of the CD28 dimer. This structure (PDB Code 1YJD) shows a different dimer interface, which results in a more compact dimer and a different placement of the FG ligand-binding loops. (C) Crystal structure of bivalent CTLA-4 dimers binding to bivalent B7-1 dimers. These properties result in a periodic network that may have important functional consequences (PDB Code 1I8L). (D) The CD28 dimer is monovalent. A model of the putative CD28:B7-1 complex was constructed by superimposing a single CTLA-4:B7-1 complex on each of the two molecules in the CD28 dimer. This model is incompatible with the simultaneous binding of two distinct B7 molecules to the CD28 dimer due to unfavorable steric interactions and supports monovalency.

Fig. 8. ‘Crystallographic view’ of the immunological synapse

Composite model of the MHC:TCR complex and costimulatory receptor:ligand complexes in the central region of the immunological synapse. The MHC:TCR (PDB Code 1G6R), PD-1:PD-L1 (PDB Code 3BIK), PD-1:PD-L2 (PDB Code 3BP5) and CTLA-4:B7-1 (PDB Code 1I8L) complexes are based on existing crystal structures; the model of the CD28:B7-1 complex was generated as described in Fig. 7; the generation of the model of the full length CD2:CD58 complex was described previously (97). The approximate dimensions (i.e., lengths) of the complexes are shown, as well as the number of residues connecting the structured Ig domains to the membrane. Also noted is the ∼140 Å distance that characterizes the separation between the plasma membranes in the immunological synapse.

Fig. 9. Comparison of the PD-1:PD-L and CTLA-4:B7 complexes

(A) PD-L1 and PD-L2 form similar complexes with PD-1. Superposition of the PD-1:PD-L1 and PD-1:PD-L2 complexes shows very similar overall structure features. (B) Different organization of the PD-1:PD-L and CTLA-4:B7 complexes. Superposition of the PD-1:PD-L2 and CTLA-4:B7-1 complexes reveal different overall organizations. The PD-1:PD-L complexes are more compact, spanning an end-to-end distance of ∼76 Å, compared to ∼100Å in the CTLA-4:B7 complexes. Longer stalk regions in PD-1 and PD-Ls presumably compensate for this difference, allowing for all of these complexes to be recruited to the central zone of the immunological synapse.

Fig. 10. Overall organization of TIM family receptors

The TIM family receptors are encoded by tightly clustered genes (see Fig. 2). All TIM receptors possess an IgV domain and a variable length mucin domain that can be highly highly O-glycosylated, followed by stalk region, transmembrane region, and cytoplasmic tail with tyrosine-based signaling motif, except for TIM-4.

Fig. 11. The TIM family IgV Domains are unique in the Ig superfamily

(A) Comparison of a typical IgV domain (PD-1; PDB Code 1NPU) and the TIM-3 (PDB Code 2OYP) IgV domain. The strands are labeled, as are the CC′ and FG loops; the C and C′ strands and the CC′ loop are displayed in red. Residues in TIM-3 CC′-FG cleft are highlighted in green stick representation. (B) Structures of TIM-1 (PDB Code 2OR8), TIM-2 (PDB Code 2OR7), and TIM-4 (PDB Code 3BIB). The C and C′ strands and the CC′ loop are colored as red. These structures highlight the unique structural variation present in all TIM family.

Fig. 12. Structure of TIM-4:phosphatidylserine (PS) complex

(A) Overall arrangement of the TIM-4:PS structure. TIM-4 exploits the CC′-FG cleft to bind PS in a metal-dependent fashion (purple sphere) involving one water molecule (red sphere) (PDB Code 3BIB). The C and C′ strands and the CC′ loop are highlighted in red. (B) Detailed view of the PS binding site. The TIM-4:PS binding site, including the CC′-FG cleft, metal atom, PS and water, in the same orientation as panel A. The metal atom (purple sphere) is coordinated to side chains of N121, D122, main chain of V116 and G118, PS and water. The PS head group forms hydrogen bonds with side chains of S62 and K63. (C) The multiple sequence alignment of CC′ and FG loop in human and mouse TIM family members. Residues with greater than 50% conservation are colored red; invariant residues are in bold white with red background. The secondary structure of the TIM-4 IgV domain is shown above the alignment.

Fig. 13. Domain organization of the CD2/SLAM family receptors

Seven of these genes are immediately proximal to one another; two others are nearby; CD2 and CD58 form a second cluster on the same chromosome (see Fig. 2). All CD2/SLAM family members are composed of a membrane distal IgV domain and a membrane proximal IgC2 domain; Ly-9 is the sole exception with tandem repeats of IgV-IgC2 motif. Tyrosine-based signaling motifs are highlighted. The GPI-linkages in CD48 and CD58 are denoted as arrows. The homophilic receptors and the heterophilic receptors are labeled in red and blue, respectively. Receptors with unknown ligand are denoted in green.

Fig. 14. Specificity of homophilic and heterophilic interactions within CD2/SLAM family

The left panels show ribbon representations of the structures of the interacting IgV domains of NTB-A (PDB Code 2IF7) and CD84 (PDB Code 2PKD) homophilic dimers and the CD2:CD58 (PDB Code 1QA9) and 2B4:CD48 heterophilic dimers (2PTT). The right panels show surface representations of the homophilic or heterophilic dimer interfaces; the two molecules are each rotated 90° in opposite directions about a vertical axis to expose the dimer interface. The residues involved forming hydrogen bonds and hydrophobic interactions across the dimer interface are colored as green and yellow, respectively. Positively and negatively charged residues involved in ionic interactions across the dimer interface are colored as blue and red, respectively. Each pair of molecules presents a distinct set of surface features that underlie binding specificity.

Fig. 15. Structure and organization of the NTB-A homophilic dimer

The crystal structure of full length NTB-A shows a homophilic dimer formed by the interaction of the two front sheets of the IgV domains (cyan and green); the IgV and IgC2 domains are labeled (PDB Code 2IF7). The end-to-end distance of NTB-A homophilic dimer is ∼100 Å and this organization is consistent with the engagement of monomers from two interacting cell surfaces.

Fig. 16. Structure and assembly of a classical TNF trimer

(A) TNF-β monomer. Ribbon diagram of human TNF-β monomer (PDB Code 1TNR). The ten anti-parallel β-strands are labeled. (B) Conventional TNF trimer. Ribbon diagram of human TNF-β trimer shows typical compact architecture of conventional THDs. (C) Conventional TNF:TNFR complex (PDB Code 1TNR). Human TNF-β trimer (shown in surface representation) engages its receptor (blue ribbon) to yield a complex with 3:3 receptor:ligand stoichiometry and results in a separation of ∼35 Å between individual receptor molecules.

Fig. 17. Atypical TNF trimers

(A) and (B) Ribbon diagrams of human GITRL (A: PDB Code 2Q1M) and human OX40L (B: PDB Code 2HEV) from the divergent family of the TNF superfamily exhibiting an atypical expanded trimeric assemblies. (C) and (D) Comparison of a conventional TNF:TNFR complex (C: PDB Code 1TNR) and the atypical human OX40L:OX40 complex (D: PDB Code 2HEV), highlighting the differences in overall organization, including the distinct placement of the receptor C-termini.

Fig. 18. Dynamic self-assembly of human GITRL

(A) Schematic of the reversible monomer-trimer equilibrium of human GITRL. As only the trimeric GITRL is competent bind receptor, this self-association behavior imposes an energetic penalty that results in modest apparent receptor binding affinity. (B) Ribbon diagram of the high affinity coiled-coil construct of human GITRL trimer that does not exhibit measurable dissociation (PDB Code 2R32).

Fig. 19. Novel dimeric organization of mouse GITRL

(A) Mouse GITRL dimmer. This structure exhibits a novel dimer that involves domain swapping of the C-termini (PDB Code 2QDN). (B) Detailed view of the C-terminal domain swap. (C) GITRL amino acid sequence alignment. Residues involved in hydrogen bond interactions at the murine dimer interface are denoted with red asterisks. Residues involved in the domain-swap interactions are underlined in red. Residues forming contacts at the subunit interfaces of human GITRL trimer are marked with black asterisks on the top of the alignment.