Immunoglobulin and T-cell receptor (TCR) molecules are central to the adaptive immune system. Sequence conservation, similarities in domain structure, and usage of similar recombination signal sequences and recombination machinery indicate that there was probably a time during evolution when an ancestral receptor diverged to the modern-day immunoglobulin and TCR. Other molecules that undergo rearrangement have not been described in vertebrates, nor have intermediates been identified that have features of both these gene families. We report here the isolation of a new member of the immunoglobulin superfamily from the nurse shark, Ginglymostoma cirratum, which contains one variable and five constant domains and is found as a dimer in serum.

Compared with the MHC of typical mammals, the chicken MHC is smaller and simpler, with only two class I genes found in the B12 haplotype. We make five points to show that there is a singledominantly expressed class I molecule that can have a strong effect on MHC function. First, we find only one cDNA for two MHC haplotypes (B14 and B15) and cDNAs corresponding to two genes for the other six (B2, B4, B6, B12, B19, and B21). Second, we find, for the B4, B12, and B15 haplotypes, that one cDNA is at least 10-fold more abundant than the other. Third, we use 2D gel electrophoresis of class I molecules from pulse-labeled cells to show that there is only one heavy chain spot for the B4 and B15 haplotypes, and one major spot for the B12 haplotype. Fourth, we determine the peptide motifs for B4, B12, and B15 cells in detail, including pool sequences and individual peptides, and show that the motifs are consistent with the peptides binding to models of the class I molecule encoded by the abundant cDNA. Finally, having shown for three haplotypes that there is a single dominantly expressed class I molecule at the level of RNA, protein, and antigenic peptide, we show that the motifs can explain the striking MHC-determined resistance and susceptibility to Rous sarcoma virus. These results are consistent with the concept of a ''minimal essential MHC'' for chickens, in strong contrast to typical mammals.antigen presentation ͉ avian ͉ essential ͉ evolution ͉ minimal

Little is known about the structure of major histocompatibility complex (MHC) molecules outside of mammals. Only one class I molecule in the chicken MHC is highly expressed, leading to strong genetic associations with infectious pathogens. Here, we report two structures of the MHC class I molecule BF2*2101 from the B21 haplotype, which is known to confer resistance to Marek's disease caused by an oncogenic herpesvirus. The binding groove has an unusually large central cavity, which confers substantial conformational flexibility to the crucial residue Arg9, allowing remodeling of key peptide-binding sites. The coupled variation of anchor residues from the peptide, utilizing a charge-transfer system unprecedented in MHC molecules, allows peptides with conspicuously different sequences to be bound. This promiscuous binding extends our understanding of ways in which MHC class I molecules can present peptides to the immune system and might explain the resistance of the B21 haplotype to Marek's disease.

We recently have identified an antigen receptor in sharks called NAR (new or nurse shark antigen receptor) that is secreted by splenocytes but does not associate with Ig light (L) chains. The NAR variable (V) region undergoes high levels of somatic mutation and is equally divergent from both Ig and T cell receptors (TCR). Here we show by electron microscopy that NAR V regions, unlike those of conventional Ig and TCR, do not form dimers but rather are independent, f lexible domains. This unusual feature is analogous to bona fide camelid IgG in which modifications of Ig heavy chain V (V H ) sequences prevent dimer formation with L chains. NAR also displays a uniquely f lexible constant (C) region. Sequence analysis and modeling show that there are only two types of expressed NAR genes, each having different combinations of noncanonical cysteine (Cys) residues in the V domains that likely form disulfide bonds to stabilize the single antigen-recognition unit. In one NAR class, rearrangement events result in mature genes encoding an even number of Cys (two or four) in complementarity-determining region 3 (CDR3), which is analogous to Cys codon expression in an unusual human diversity (D) segment family. The NAR CDR3 Cys generally are encoded by preferred reading frames of rearranging D segments, providing a clear design for use of preferred reading frame in antigen receptor D regions. These unusual characteristics shared by NAR and unconventional mammalian Ig are most likely the result of convergent evolution at the molecular level.At the heart of the adaptive immune system are the antigen receptors, Ig and T cell receptor (TCR), that are generated in anticipation of recognition of pathogens (1). The typical antigen receptor is composed of two polypeptide chains [heavy (H) and light (L) for Igs and ␣ and ␤ or ␥ and ␦ for TCRs]. Each chain, in turn, is composed of a single, variable (V) domain at the N-terminal end followed by one to seven constant (C) domains. C domains define the effector functions characteristic of a given class of Ig whereas V domains each display a unique sequence and structure defining antigen specificity. Igs can be subdivided further into Fab and Fc fragments, responsible for antigen binding and for effector function, respectively. Ig and TCR V regions are encoded by a mosaic of genes ligated together somatically during lymphocyte ontogeny (2). Specifically, single V and J elements are joined together at the DNA level for Ig L chain or TCR ␣ and ␥ V regions. In Ig H chains and TCR ␤ and ␦ chains, one or, occasionally, two D elements are joined between the V and J segments. Together, the V, (D), and J elements encode framework (FR, responsible for protein folding and structure) and complementarity-determining regions (CDR, responsible for antigen interactions) within the V domains.The evolutionary origin of antigen receptors is unknown, but the first indication of their emergence phylogenetically is in cartilaginous fish (sharks, skates, and rays), where at least three types of Ig (3-9)...

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In recent years, short coiled coils have been used for applications ranging from biomaterial to medical sciences. For many of these applications knowledge of the factors that control the topology of the engineered protein systems is essential. Here, we demonstrate that trimerization of short coiled coils is determined by a distinct structural motif that encompasses specific networks of surface salt bridges and optimal hydrophobic packing interactions. The motif is conserved among intracellular, extracellular, viral, and synthetic proteins and defines a universal molecular determinant for trimer formation of short coiled coils. In addition to being of particular interest for the biotechnological production of candidate therapeutic proteins, these findings may be of interest for viral drug development strategies.protein engineering ͉ sequence-to-structure rules ͉ protein-protein interaction ͉ salt bridges ͉ x-ray crystallography T he potential of short ␣-helical coiled coils for protein engineering, biotechnological, biomaterial, basic research, and medical applications has recently been recognized (1-12). The wide range of applications underscores the need for topological control of coiled-coil peptides. Although seemingly simple, coiled coils can form a variety of different assemblies ranging from dimers to pentamers (13,14). Furthermore, coiled coils can form homomers or heteromers with their chains arranged in a parallel or antiparallel fashion. To understand how side-chainside-chain interactions determine a particular structure, it is important to discriminate the factors responsible for specific interhelical interactions from those that are common to all coiled coils such as the heptad repeat.It is generally acknowledged that the detailed packing geometry of hydrophobic core residues correlates with the oligomerization state (15,16). In dimers the side chains of the residues at a and d positions pack in a manner termed parallel and perpendicular, respectively. In the four-stranded state the dimer mode is reversed. Trimeric coiled coils are characterized by an intermediate geometry of these residues, termed acute. Previous studies found that these oligomerization states are determined in particular by the distribution of isoleucine and leucine residues that prefer the parallel and acute, and the acute and perpendicular geometries, respectively (15, 16).Buried polar residues in hydrophobic interfaces also play an important role in determining the number and orientation of strands in coiled coils. Many two-stranded leucine zippers of transcription regulators contain at least one conserved polar residue. Frequently an asparagine or a lysine residue occupies heptad position a toward the center of the sequence, suggesting a common mechanism for determining dimer specificity in these molecules (17). Polar residues like glutamine and threonine at positions a and d, respectively, can favor trimer formation (18,19). Interhelical interactions between side chains of residues at the e and g positions as well as the packi...

In most vertebrate embryos and neonates studied to date unique antigen receptors (antibodies and T cell receptors) are expressed that possess a limited immune repertoire. We have isolated a subclass of IgM, IgM 1gj, from the nurse shark Ginglymostoma cirratum that is preferentially expressed in neonates. The variable (V) region gene encoding the heavy (H) chain underwent V-D-J rearrangement in germ cells (''germline-joined''). Such H chain V genes were discovered over 10 years ago in sharks but until now were not shown to be expressed at appreciable levels; we find expression of H 1gj in primary and secondary lymphoid tissues early in life, but in adults only in primary lymphoid tissue, which is identified in this work as the epigonal organ. H 1gj chain associates covalently with light (L) chains and is most similar in sequence to IgM H chains, but like mammalian IgG has three rather than the four IgM constant domains; deletion of the ancestral IgM C2 domain thus defines both IgG and IgM 1gj. Because sharks are the members of the oldest vertebrate class known to possess antibodies, unique or specialized antibodies expressed early in ontogeny in sharks and other vertebrates were likely present at the inception of the adaptive immune system.

In mammals, there are MHC class II molecules with distinctive sequence features, such as the classical isotypes DR, DQ and DP. These particular isotypes have not been reported in non-mammalian vertebrates. We have isolated the class II (B-L) alpha chain from outbred chickens as the basis for the cloning and sequencing of the cDNA. We found only one class II alpha chain transcript, which bears the major features of a classical class II alpha sequence, including the critical peptide-binding residues. The chicken sequence is more similar to human DR than to the DQ, DP, DO or DM isotypes, most significantly in the peptide-binding alpha(1) domain. The cDNA and genomic DNA sequences from chickens of diverse origins show few alleles, which differ in only four nucleotides and one amino acid. In contrast, significant restriction fragment length polymorphism is detected by Southern blot analysis of genomic DNA, suggesting considerable diversity around the gene. Analysis of a large back-cross family indicates that the class II alpha chain locus ( B-LA) is located roughly 5.6 cM from the MHC locus, which encodes the classical class II beta chains. Thus the chicken class II alpha chain gene is like the mammalian DR and E isotypes in three properties: the presence of the critical peptide-binding residues, the low level of polymorphism and sequence diversity, and the recombinational separation from the class II beta chain genes. These results indicate that the sequence features of this lineage are both functionally important and at least 300 million years old.