Rob Meijers

Crystal structure of the human CD4 N-terminal two-domain fragment complexed to a class II MHC molecule

The structural basis of the interaction between the CD4 coreceptor and a class II major histocompatibility complex (MHC) is described. The crystal structure of a complex containing the human CD4 N-terminal two-domain fragment and the murine I-Ak class II MHC molecule with associated peptide (pMHCII) shows that only the “top corner” of the CD4 molecule directly contacts pMHCII. The CD4 Phe-43 side chain extends into a hydrophobic concavity formed by MHC residues from both α2 and β2 domains. A ternary model of the CD4-pMHCII-T-cell receptor (TCR) reveals that the complex appears V-shaped with the membrane-proximal pMHCII at the apex. This configuration excludes a direct TCR–CD4 interaction and suggests how TCR and CD4 signaling is coordinated around the antigenic pMHCII complex. Human CD4 binds to HIV gp120 in a manner strikingly similar to the way in which CD4 interacts with pMHCII. Additional contacts between gp120 and CD4 give the CD4–gp120 complex a greater affinity. Thus, ligation of the viral envelope glycoprotein to CD4 occludes the pMHCII-binding site on CD4, contributing to immunodeficiency.

Structural basis of Dscam isoform specificity.

The Dscam gene gives rise to thousands of diverse cell surface receptors thought to provide homophilic and heterophilic recognition specificity for neuronal wiring and immune responses. Mutually exclusive splicing allows for the generation of sequence variability in three immunoglobulin ecto-domains, D2, D3 and D7. We report X-ray structures of the amino-terminal four immunoglobulin domains (D1–D4) of two distinct Dscam isoforms. The structures reveal a horseshoe configuration, with variable residues of D2 and D3 constituting two independent surface epitopes on either side of the receptor. Both isoforms engage in homo-dimerization coupling variable domain D2 with D2, and D3 with D3. These interactions involve symmetric, antiparallel pairing of identical peptide segments from epitopeu2009I that are unique to each isoform. Structure-guided mutagenesis and swapping of peptide segments confirm that epitopeu2009I, but not epitopeu2009II, confers homophilic binding specificity of full-length Dscam receptors. Phylogenetic analysis shows strong selection of matching peptide sequences only for epitopeu2009I. We propose that peptide complementarity of variable residues in epitopeu2009I of Dscam is essential for homophilic binding specificity.

Crystal structure of murine sCEACAM1a[1,4]: a coronavirus receptor in the CEA family

CEACAM1 is a member of the carcinoembryonic antigen (CEA) family. Isoforms of murine CEACAM1 serve as receptors for mouse hepatitis virus (MHV), a murine coronavirus. Here we report the crystal structure of soluble murine sCEACAM1a[1,4], which is composed of two Ig‐like domains and has MHV neutralizing activity. Its N‐terminal domain has a uniquely folded CC′ loop that encompasses key virus‐binding residues. This is the first atomic structure of any member of the CEA family, and provides a prototypic architecture for functional exploration of CEA family members. We discuss the structural basis of virus receptor activities of murine CEACAM1 proteins, binding of Neisseria to human CEACAM1, and other homophilic and heterophilic interactions of CEA family members.

Discovery of CD8+ T cell epitopes in Chlamydia trachomatis infection through use of caged class I MHC tetramers

Class I MHC tetramers allow direct phenotypic identification of CD8+ T cell populations, but their production remains laborious. A peptide exchange strategy that employs class I MHC products loaded with conditional ligands (caged MHC molecules) provides a fast and straightforward method to obtain diverse arrays of class I MHC tetramers and facilitates CD8+ T cell epitope discovery. Here, we describe the development of photocleavable analogs of the FAPGNYPAL (SV9) epitope that bind H-2Kb and H-2Db with full retention of their structural and functional integrity. We ranked all possible H-2Kb octameric and H-2Db nonameric epitopes that span the genome of Chlamydia trachomatis and prepared MHC tetramers from ≈2,000 of the highest scoring peptides by replacement of the SV9 analog with the peptide of choice. The resulting 2,000-member class I MHC tetramer array allowed the discovery of two variants of an epitope derived from polymorphic membrane protein I (PmpI) and an assessment of the kinetics of emergence and the effector function of the corresponding CD8+ T cells.

Structural Elucidation of the Bispecificity of a Domains as a Basis for Activating Non-Natural Amino Acids.

Many biologically active peptide secondary metabolites of bacteria are produced by modular enzyme complexes, the non-ribosomal peptide synthetases. Substrate selection occurs through an adenylation (A) domain, which activates the cognate amino acid with high fidelity. The recently discovered Au2005domain of an Anabaenopeptin synthetase from Planktothrix agardhii (ApnA A1) is capable of activating two chemically distinct amino acids (Arg and Tyr). Crystal structures of the Au2005domain reveal how both substrates fit into to binding pocket of the enzyme. Analysis of the binding pocket led to the identification of three residues that are critical for substrate recognition. Systematic mutagenesis of these residues created Au2005domains that were monospecific, or changed the substrate specificity to tryptophan. The non-natural amino acid 4-azidophenylalanine is also efficiently activated by a mutant Au2005domain, thus enabling the production of diversified non-ribosomal peptides for bioorthogonal labeling.

Bioorthogonal Cleavage and Exchange of Major Histocompatibility Complex Ligands by Employing Azobenzene-Containing Peptides.

Bioorthogonal cleavable linkers are attractive building blocks for compounds that can be manipulated to study biological and cellular processes. Sodium dithionite sensitive azobenzene-containing (Abc) peptides were applied for the temporary stabilization of recombinant MHC complexes, which can then be employed to generate libraries of MHC tetramers after exchange with a novel epitope. This technology represents an important tool for high-throughput studies of disease-specific Tu2005cell responses.