Ada Prochnicka-Chalufour

Implications of a Fab-like structure for the T-cell receptor.

The antigen-specific receptor of T lymphocytes (TCR) and the Fab moiety of immunoglobulins are expected to fold into similar three-dimensional structures because of their identical protein domain organization, the conservation of key residues and their overall sequence homology. However, T cells mostly appear to recognize short peptide antigens bound to MHC class I or class II presenting molecules. A complete model of the human leucocyte antigen molecule (HLA-A2) reconstructed from the alpha-carbon coordinates was used to investigate the putative organization of a TCR/peptide/HLA-A2 complex. In this article, Jean-Michel Claverie and co-workers show that the respective geometries of a Fab-like TCR structure and of the HLA-A2 antigen binding site suggest a model where the third variable regions of both chains of the TCR mainly interact with the peptide antigen, while the first and/or second less variable regions are in position for making contact with residues pointing up from the alpha 1 and alpha 2 helical regions of the HLA-A2 molecule.

Novel STAT1 alleles in otherwise healthy patients with mycobacterial disease.

The transcription factor signal transducer and activator of transcription-1 (STAT1) plays a key role in immunity against mycobacterial and viral infections. Here, we characterize three human STAT1 germline alleles from otherwise healthy patients with mycobacterial disease. The previously reported L706S, like the novel Q463H and E320Q alleles, are intrinsically deleterious for both interferon gamma (IFNG)–induced gamma-activating factor–mediated immunity and interferon alpha (IFNA)–induced interferon-stimulated genes factor 3–mediated immunity, as shown in STAT1-deficient cells transfected with the corresponding alleles. Their phenotypic effects are however mediated by different molecular mechanisms, L706S affecting STAT1 phosphorylation and Q463H and E320Q affecting STAT1 DNA-binding activity. Heterozygous patients display specifically impaired IFNG-induced gamma-activating factor–mediated immunity, resulting in susceptibility to mycobacteria. Indeed, IFNA-induced interferon-stimulated genes factor 3–mediated immunity is not affected, and these patients are not particularly susceptible to viral disease, unlike patients homozygous for other, equally deleterious STAT1 mutations recessive for both phenotypes. The three STAT1 alleles are therefore dominant for IFNG-mediated antimycobacterial immunity but recessive for IFNA-mediated antiviral immunity at the cellular and clinical levels. These STAT1 alleles define two forms of dominant STAT1 deficiency, depending on whether the mutations impair STAT1 phosphorylation or DNA binding.

Novel mutations in the RFXANK gene: RFX complex containing in-vitro-generated RFXANK mutant binds the promoter without transactivating MHC II

MHC class II deficiency is a combined immunodeficiency caused by defects in the four regulatory factors, CIITA, RFXANK, RFX5 and RFXAP, that control MHC II expression at the transcriptional level. The RFXANK gene encodes one subunit of the heterotrimeric RFX complex that is involved in the assembly of several transcription factors on MHC II promoters. Seven different RFXANK mutations have previously been reported in 26 unrelated patients. The most frequent mutation, a 26-bp deletion (752delG-25), has been identified in 21 patients. The other mutations are all nonsense or splice-site mutations, leading to proteins lacking all or part of the RFXANK ankyrin repeat region. We report two novel missense mutations, D121V and R212X, resulting in loss of function of the gene. We investigated the in vivo effects of these mutations and of three other point mutations on the expression of the RFXANK RNA and protein. The number of RFXANK transcripts was severely reduced in all patients except one. The RFXANK protein was barely detected in two cases. In addition, guided by a structural model of RFXANK, we investigated experimental mutants of the C-terminal tyrosine 224. Substitution Y224A, but not Y224F, led to the loss of function of RFXANK. Two null mutants, D121V and Y224A, were tested in protein interaction and DNA binding assays. The D121V mutant was unable to form the RFX complex, indicating that D121 is required for RFXAP binding. The Y224A mutant formed an RFX complex that bound normally to the MHC II promoter, but did not lead to MHC class II expression, whereas Y224F RFXANK retained the wild-type function. This indicates that an aromatic ring, but not the phenyl chain of tyrosine, is necessary at position 224 for normal RFXANK function. Studies on the Y224A mutant suggest that, in addition to the RFX subunits and CIITA, another protein is essential for MHC class II expression. This protein appears to interact with the fourth ankyrin repeat of RFXANK.

Extensive structural homology between H-2 K/D/L antigens and non-polymorphic class I Qa, Tla and “37” molecules suggests they may act as peptide carriers

Key structural features of H-2 K/D/L and HLA A/B/C class I molecules were identified by analysing the available sequences with reference to the 3-D structure of HLA-A2. Most of them were found to be conserved in a panel of 6 Qa and 4 Tla sequences. This finding, in addition to the high overall sequence similarity between polymorphic and non-polymorphic class I molecules strongly suggests a possible role for the latter in peptide binding, transport and/or presentation.

Structure and Dynamics of the Anticodon Arm Binding Domain of Bacillus stearothermophilus Tyrosyl-tRNA Synthetase

The structure of a recombinant protein, TyrRS(delta4), corresponding to the anticodon arm binding domain of Bacillus stearothermophilus tyrosyl-tRNA synthetase, has been solved, and its dynamics have been studied by nuclear magnetic resonance (NMR). It is the first structure described for such a domain of a tyrosyl-tRNA synthetase. It consists of a five-stranded beta sheet, packed against two alpha helices on one side and one alpha helix on the other side. A large part of the domain is structurally similar to other functionally unrelated RNA binding proteins. The basic residues known to be essential for tRNA binding and charging are exposed to the solvent on the same face of the molecule. The structure of TyrRS(delta4), together with previous mutagenesis data, allows one to delineate the region of interaction with tRNATyr.

The Structure of HasB Reveals a New Class of TonB Protein Fold

TonB is a key protein in active transport of essential nutrients like vitamin B12 and metal sources through the outer membrane transporters of Gram-negative bacteria. This inner membrane protein spans the periplasm, contacts the outer membrane receptor by its periplasmic domain and transduces energy from the cytoplasmic membrane pmf to the receptor allowing nutrient internalization. Whereas generally a single TonB protein allows the acquisition of several nutrients through their cognate receptor, in some species one particular TonB is dedicated to a specific system. Despite a considerable amount of data available, the molecular mechanism of TonB-dependent active transport is still poorly understood. In this work, we present a structural study of a TonB-like protein, HasB dedicated to the HasR receptor. HasR acquires heme either free or via an extracellular heme transporter, the hemophore HasA. Heme is used as an iron source by bacteria. We have solved the structure of the HasB periplasmic domain of Serratia marcescens and describe its interaction with a critical region of HasR. Some important differences are observed between HasB and TonB structures. The HasB fold reveals a new structural class of TonB-like proteins. Furthermore, we have identified the structural features that explain the functional specificity of HasB. These results give a new insight into the molecular mechanism of nutrient active transport through the bacterial outer membrane and present the first detailed structural study of a specific TonB-like protein and its interaction with the receptor.

1H NMR structural analysis of novel potassium blocking toxins using a nano-NMR probe

A new class of toxin acting on potassium channels and cross-linked by four disulfide bridges instead of three has been recently described. Two peptides, Pi1 and Pi7, purified from the venom of the scorpion Pandinus imperator belong to this new class. Structural features of one of these new toxins. Pi1, have been investigated by proton nuclear magnetic resonance using a new technology that allows to work with very small amount of compound, in the nanomole range. It is shown that it is possible to collect high quality data set in terms of resolution, lineshape and sensitivity with nanomolar amount of compound using this technology. Preliminary results on Pi7 are also presented. The approach described here is quite attractive for the study of natural compounds such as toxins often available at low amounts.

Solution structure of Cn5, a crustacean toxin found in the venom of the scorpions Centruroides noxius and Centruroides suffusus suffusus

The crustacean toxin Cn5 from Centruroides noxius Hoffmann and peptide Css39.8 from Centruroides suffusus suffusus scorpion venoms are identical peptides, as confirmed by amino acid sequence of purified toxins and by DNA sequencing of the two respective cloned genes. Therefore in this communication they will be simply named Cn5. Cn5 is a 66 amino acid long peptide with four disulfide bridges, formed between pairs of cysteines: C1-C8, C2-C5, C3-C6, and C4-C7 (the numbers indicate the relative positions of the cysteine residues in the primary structure). This peptide is non-toxic to mammals but deadly to arthropods (LD(50) 28.5 mg/g body weight of crayfish). Its three-dimensional structure was determined by NMR using a total of 965 meaningful distance constraints derived from the volume integration of the 2D NOESY spectra. The Cn5 structure displays a mixed alpha/beta fold stabilized by four disulfide bridges, with a kink induced by a cis-proline in its C-terminal part. Cn5 electrostatic surface is compared to that of Cn2 toxin toxic to mammals. The local differences produced by additional or substituted residues that would influence toxin selectivity towards mammalian or crustacean Na(+) channels are discussed.

Structural basis of the signalling through a bacterial membrane receptor HasR deciphered by an integrative approach

In bacteria, some scarce nutrients are sensed, bound and internalized by their specific transporter. In the present study, using an integrative structural approach, we study HasR, a bacterial haem transporter in both its free and its loaded forms.

Interaction of a Partially Disordered Antisigma Factor with Its Partner, the Signaling Domain of the TonB-Dependent Transporter HasR

Bacteria use diverse signaling pathways to control gene expression in response to external stimuli. In Gram-negative bacteria, the binding of a nutrient is sensed by an outer membrane transporter. This signal is then transmitted to an antisigma factor and subsequently to the cytoplasm where an ECF sigma factor induces expression of genes related to the acquisition of this nutrient. The molecular interactions involved in this transmembrane signaling are poorly understood and structural data on this family of antisigma factor are rare. Here, we present the first structural study of the periplasmic domain of an antisigma factor and its interaction with the transporter. The study concerns the signaling in the heme acquisition system (Has) of Serratia marcescens. Our data support unprecedented partially disordered periplasmic domain of an anti-sigma factor HasS in contact with a membrane-mimicking environment. We solved the 3D structure of the signaling domain of HasR transporter and identified the residues at the HasS−HasR interface. Their conservation in several bacteria suggests wider significance of the proposed model for the understanding of bacterial transmembrane signaling.