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The T cell antigen receptor (TCR) mediates recognition of peptide antigens bound in the groove of major histocompatibility complex (MHC) molecules. This dual recognition is mediated by the complementarity-determining residue (CDR) loops of the alpha and beta chains of a single TCR which contact exposed residues of the peptide antigen and amino acids along the MHC alpha helices. The recent description of T cells that recognize hydrophobic microbial lipid antigens has challenged immunologists to explain, in molecular terms, the nature of this interaction. Structural studies on the murine CD1d1 molecule revealed an electrostatically neutral putative antigen-binding groove beneath the CD1 alpha helices. Here, we demonstrate that alpha/beta TCRs, when transferred into TCR-deficient recipient cells, confer specificity for both the foreign lipid antigen and CD1 isoform. Sequence analysis of a panel of CD1-restricted, lipid-specific TCRs reveals the incorporation of template-independent N nucleotides that encode diverse sequences and frequent charged basic residues at the V(D)J junctions. These sequences permit a model for recognition in which the TCR CDR3 loops containing charged residues project between the CD1 alpha helices, contacting the lipid antigen hydrophilic head moieties as well as adjacent CD1 residues in a manner that explains antigen specificity and CD1 restriction.

Conformational changes associated with the shift of trypanosomal nucleoside N-ribohydrolases from unliganded to transition state were investigated. Tightly bound calcium ions as a catalytic site ligand were identified after the nonspecific nucleoside hydrolase from Crithidia fasciculata was cocrystallized with p-aminophenyliminoribitol. Results showed that the complex with the transition-state inhibitor is characterized by large protein conformational changes to form a hydrophobic leaving group site (2) C3'-exo geometry for the inhibitor.

The T cell receptor (TCR) inherently has dual specificity. T cells must recognize self-antigens in the thymus during maturation and then discriminate between foreign pathogens in the periphery. A molecular basis for this cross-reactivity is elucidated by the crystal structure of the alloreactive 2C TCR bound to self peptide -- major histocompatibility complex (pMHC) antigen H-[2K.sup.b]-dEV8 refined against anisotropic 3.0 angstrom resolution x-ray data. The interface between peptide and TCR exhibits extremely poor shape complementarity, and the TCR Β chain complementarity-determining region 3 (CDR3) has minimal interaction with the dEV8 peptide. Large conformational changes in three of the TCR CDR loops are induced upon binding, providing a mechanism of structural plasticity to accommodate a variety of different peptide antigens. Extensive TCR interaction with the pMHC [Alpha] helices suggests a generalized orientation that is mediated by the [V.sub.[Alpha]] domain of the TCR and rationalizes how TCRs can effectively 'scan' different peptides bound within a large, low-affinity MHC structural framework for those that provide the slight additional kinetic stabilization required for signaling., The phenomenon of MHC restriction is the basis of the cell-mediated immune response to foreign pathogens(1). The central molecular event governing this process is the engagement of the clonotypic [Alpha]Β [...]

Cellular immunity is mediated by the interaction of an [Alpha][Beta] T cell receptor (TCR) with a peptide presented within the context era major histocompatibility complex (MHC) molecule. Allereactive T cells have [Alpha][Beta] TCRs that can recognize both self- and foreign peptide - MHC (pMHC) complexes, implying that the TCR has significant complementarity with different pMHC. To characterize the molecular basis for alloreactive TCR recognition of pMHC, we have produced a soluble, recombinant form of an allereactive [Alpha][Beta] T cell receptor in Drosophila melanogaster cells. This recombinant TCR, 2C, is expressed as a correctly paired [Alpha][Beta] heterodimer, with the chains covalently connected via a disulfide bond in the C-terminal region. The native conformation of the 2C TCR was probed by surface plasmon resonance (SPR) analysis by using conformation-specific monoclonal antibodies, as well as syngeneic and allogeneic pMHC ligands. The 2C interaction with H-2[K.sub.b]-dEV8, H-2[K.sup.bm3]-dEV8, H-2[K.sup.b]-SIYR and H-2[L.sup.d]-p2Ca spans a range of affinities from [K.sub.d] = [10.sup.-4] to [10.sup.-6]M for the syngeneic (H-2[K.sup.b]) and allogeneic (H-2[K.sup.bm3], H-2[L.sup.d]) ligands. In general, the syngeneic ligands bind with weaker affinities than the allegeneic ligands, consistent with current threshold models of thymic selection and T cell activation. Crystallization of the 2C TCR required proteolytic trimming of the C-terminal residues of the [Alpha] and [Beta] chains. X-ray quality crystals of complexes or 2C with H-2[K.sup.b]-dEV8, H-2[K.sup.bm3]-dEV8 and H-2[K.sup.b]-SIYR have been grown.

The central event in the cellular immune response to invading microorganisms is the specific recognition of foreign peptides bound to major histocompatibility complex (MHC) molecules by the αβ T cell receptor (TCR). The x-ray structure of the complete extracellular fragment of a glycosylated αβ TCR was determined at 2.5 angstroms, and its orientation bound to a class I MHC-peptide (pMHC) complex was elucidated from crystals of the TCR-pMHC complex. The TCR resembles an antibody in the variable Vα and Vβ domains but deviates in the constant Cα domain and in the interdomain pairing of Cα with Cβ. Four of seven possible asparagine-linked glycosylation sites have ordered carbohydrate moieties, one of which lies in the Cα-Cβ interface. The TCR combining site is relatively flat except for a deep hydrophobic cavity between the hypervariable CDR3s (complementarily-determining regions) of the α and β chains. The 2C TCR covers the class I MHC H-[2K.sup.b] binding groove so that the [V.sub.α] CDRs 1 and 2 are positioned over the amino-terminal region of the bound dEV8 peptide, the Vβ chain CDRs 1 and 2 are over the carboxyl-terminal region of the peptide, and the Vα and Vβ CDR3s straddle the peptide between the helices around the central position of the peptide., T lymphocytes respond to a wide variety of foreign antigens that are presented as peptides in the context of major histocompatibility molecules (MHC) (1). Specific recognition of peptide-MHC (pMHC) complexes [...]

The three-dimensional structure of inosine-uridine nucleoside N-ribohydrolase from Crithidia fasciculata is resolved using X-ray crystallography. The protein comprises an eight-stranded beta-sheet and six alpha-helices. The catalytic site is situated at the C-terminal end of beta-strands beta-1 and beta-4. The part of the catalytic site envisaged to be involved in purine salvage is hydrophobic and contains a single ionizable histidine. The substrate binding site has a novel architecture.

The enzyme inosine-uridine (IU) nucleoside hydrolase in the protozoa Crithidia fasciculata requires histidine at position 241 for effective hydrolysis of inosine but not for nitrophenyl riboside. The enzyme is cloned using polymerase chain reaction and purified after expressing the cDNA in Escherichia coli. The amino acid sequence of IU-nucleoside hydrolase has no resemblance to that of any known protein except the N-terminal sequence which resembles a cDNA clone of Leishmania major. X-ray crystal structure of the enzyme is discussed.

High-resolution crystal structures of catalytically active soybean beta-amylase (SBA), unliganded and complexed with maltose and maltal reveal the carboxyl groups of two glutamic acid residues as the catalytic sites in SBA. Ligand binding has no influence on the spatial relationship between the two glutamic residues, Glu 186 and Glu 380. The mobile hinged loop 3 segment in SBA is similar to the hinged loop associated with triosephosphate isomerase.

Abstract The characteristics of intra-host human immunodeficiency virus type 1 (HIV-1) env evolution were evaluated in untreated HIV-1-infected subjects with different patterns of disease progression, including 2 normal progressor [NP], and 5 Long term non-progressor [LTNP] patients. High-resolution phylogenetic analysis of the C2-C5 env gene sequences of the replicating HIV-1 was performed in sequential samples collected over a 3–5 year period; overall, 301 HIV-1 genomic RNA sequences were amplified from plasma samples, cloned, sequenced and analyzed. Firstly, the evolutionary rate was calculated separately in the 3 codon positions. In all LTNPs, the 3rd codon mutation rate was equal or even lower than that observed at the 1st and 2nd positions (p = 0.016), thus suggesting strong ongoing positive selection. A Bayesian approach and a maximum-likelihood (ML) method were used to estimate the rate of virus evolution within each subject and to detect positively selected sites respectively. A great number of N-linked glycosylation sites under positive selection were identified in both NP and LTNP subjects. Viral sequences from 4 of the 5 LTNPs showed extensive positive selective pressure on the CD4-binding site (CD4bs). In addition, localized pressure in the area of the IgG-b12 epitope, a broad neutralizing human monoclonal antibody targeting the CD4bs, was documented in one LTNP subject, using a graphic colour grade 3-dimensional visualization. Overall, the data shown here documenting high selective pressure on the HIV-1 CD4bs of a group of LTNP subjects offers important insights for planning novel strategies for the immune control of HIV-1 infection.

Abstract Background MHC class I-peptide tetramers are currently utilised to characterize CD8+ T cell responses at single cell level. The generation and use of MHC class II tetramers to study antigen-specific CD4+ T cells appears less straightforward. Most MHC class II tetramers are produced with a homogeneously built-in peptide, reducing greatly their flexibility of use. We attempted the generation of "empty" functional HLA-DR*1101 tetramers, receptive for loading with synthetic peptides by incubation. No such reagent is in fact available for this HLA-DR allele, one of the most frequent in the Caucasian population. Results We compared soluble MHC class II-immunoglobulin fusion proteins (HLA-DR*1101-Ig) with soluble MHC class II protein fused with an optimised Bir site for enzymatic biotynilation (HLA-DR*1101-Bir), both produced in insect cells. The molecules were multimerised by binding fluorochrome-protein A or fluorochrome-streptavidin, respectively. We find that HLA-DR*1101-Bir molecules are superior to the HLA-DR*1101-Ig ones both in biochemical and functional terms. HLA-DR*1101-Bir molecules can be pulsed with at least three different promiscuous peptide epitopes, derived from Tetanus Toxoid, influenza HA and the tumour associated antigen MAGE-3 respectively, to stain specific CD4+ T cells. Both staining temperature and activation state of CD4+ T cells are critical for the binding of peptide-pulsed HLA-DR*1101-Bir to the cognate TCR. Conclusion It is therefore possible to generate a soluble recombinant HLA-DR*1101 backbone that is receptive for loading with different peptides to stain specific CD4+ T cells. As shown for other HLA-DR alleles, we confirm that not all the strategies to produce soluble HLA-DR*1101 multimers are equivalent.

A novel in silico approach based on the combined use of essential dynamics sampling and nonequilibrium free-energy calculations is introduced to obtain quantitative data on conformational energy landscapes. The proposed approach provides first evidence for a rate-limiting character of the conformational transition in this enzyme, and an unexpected role of the protonation state of a single residue in regulating substrate binding and product release.

The first functional and structural characterization of the inosine-guanosine-specific nucleoside hydrolase (NH) from Trypanosoma brucei brucei is reported. The study identified nanomolar iminoribitol-based inhibitors which could be important lead compounds for the development of novel therapeutic strategies against trypanosomal diseases.

Cryotrapping and X-ray crystallography were used to characterize the binding interactions between the slowly hydrolyzed substrate inosine and the Escherichia coli pyrimidine-specific nucloeside hydrolase (NH) YeiK. The results provided direct evidence for the distinct subsets of amino acid residues which are involved in the hydrolysis of purine or pyrimidine nucleosides in group I NHs.

An alternative set of torsional parameters of pyrimidinic bases to be used in conjunction with the AMBER force field which could provide an accurate representation of the flexibility of the nitrogenous base is nucleotides is presented.

The functional characterization of the Escherichia coli YbeK (RihA) protein as a pyrimidine nucleoside-preferring nucleoside hydrolases (NH), and its first crystal structure to 1.8 Angstrom resolution is presented. The enzyme active site by alpha or beta anomer of ribose and provides the first structural description of the binding of the NH reaction product, while the amino acid residues involved in ribosyl binding are conserved in pyrimidine specific NHs.

The structural basis for the T cell response to glycolipid antigens (Ags) remains poorly understood. T lymphocytes autoreactive for mouse CD1 (mCD1.1) or reactive for the glycosphingolipid [Alpha]-galactosylceramide ([Alpha]-GalCer) presented by mCD1.1 have been described previously. In this paper it is shown that mutations at the top of the [Alpha] helices and in the bottom of the Ag-binding groove can disrupt both mCD1.1 autoreactivity and [Alpha]-GalCer recognition. The locations of the positions that affect T cell responses indicate that recognition of mCD1.1 is not likely to be unconventional or superantigen-like. Furthermore, the effects of the bottom of the pocket mutation suggest that the autoreactive response could require an autologous ligand, and they indicate that [Alpha]-GalCer binds to the groove of mCD1.1, most likely with the shorter 18-carbon hydrophobic chain in the A' pocket. Natural killer T cell hybridomas with identical T cell antigen receptor (TCR) [Alpha] chains and different [Beta] chains respond differently to [Alpha]-GalCer presented by mCD1.1 mutants. This finding indicates a role for TCR [Beta] in defining natural killer T cell specificity, despite the more restricted diversity of the [Alpha] chains in these cells. Overall, the data are consistent with a mode of lipoglycan recognition similar to that proposed for glycopeptides, in which the TCR [Alpha] and [Beta] chains survey a surface composed of both mCD1.1 and the carbohydrate portion of [Alpha]-GalCer.

Murine T10 and T22 are highly related nonclassical major histocompatibility complex (MHC) class lb proteins that bind to certain [Gamma]Δ T cell receptors (TCRs) in the absence of other components. The crystal structure of [T22.sup.b] at 3.1 angstroms reveals similarities to MHC class I molecules, but one side of the normal peptide-binding groove is severely truncated, which allows direct access to the Β-sheet floor. Potential [Gamma]Δ TCR-binding sites can be inferred from functional mapping of T10 and T22 point mutants and allelic variants. Thus, T22 represents an unusual variant of the MHC-like fold and indicates that [Gamma]Δ and [Alpha]Β TCRs interact differently with their respective MHC ligands., Classical MHC class I (class Ia) molecules participate in immune responses by presenting peptide antigens to cytolytic [Alpha]Β T cells (1). Many nonclassical MHC class I (class Ib) molecules have [...]