NMR Structures of Apo L. casei Dihydrofolate Reductase and Its Complexes with Trimethoprim and NADPH: Contributions to Positive Cooperative Binding from Ligand-Induced Refolding, Conformational Changes, and Interligand Hydrophobic Interactions

bS Supporting Information The enzyme dihydrofolate reductase (DHFR; 5,6,7,8-tetra-hydrofolate:NADPH oxidoreductase, EC 1.5.1.3) catalyzes the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydro-folate (THF) using NADPH as coenzyme.1 Since THF and its metabolites are precursors of purine and pyrimidine bases, the normal functioning of this enzyme is essential for proliferating cells. This makes DHFR an excellent target for antifolate drugs such as methotrexate (anticancer), pyrimethamine (antimalarial), and trimethoprim (antibacterial). Such agents act by inhibiting the enzyme in parasitic or malignant cells.1,2 The cooperative binding of ligands to DHFR plays an important role not only in the enzyme catalytic cycle (negative cooperativity in THF/ NADPH binding)3 but also in enzyme inhibition (positive cooperativity in antifolate/NADPH binding).4 The effects of positive cooperative binding in controlling enzyme inhibition ar

The QTL within the H2 Complex Involved in the Control of Tuberculosis Infection in Mice Is the Classical Class II H2-Ab1 Gene.

The level of susceptibility to tuberculosis (TB) infection depends upon allelic variations in numerous interacting genes. In our mouse model system, the whole-genome quantitative trait loci (QTLs) scan revealed three QTLs involved in TB control on chromosomes 3, 9, and in the vicinity of the H2 complex on chromosome 17. For the present study, we have established a panel of new congenic, MHC-recombinant mouse strains bearing differential small segments of chromosome 17 transferred from the TB-susceptible I/St (H2j) strain onto the genetic background of TB-resistant C57BL/6 (B6) mice (H2b). This allowed narrowing the QTL interval to 17Ch: 33, 77-34, 34 Mb, containing 36 protein-encoding genes. Cloning and sequencing of the H2j allelic variants of these genes demonstrated profound polymorphic variations compare to the H2b haplotype. In two recombinant strains, B6.I-249.1.15.100 and B6.I-249.1.15.139, recombination breakpoints occurred in different sites of the H2-Aβ 1 gene (beta-chain of the Class II heterodimer H2-A), providing polymorphic variations in the domain β1 of the Aβ-chain. These variations were sufficient to produce different TB-relevant phenotypes: the more susceptible B6.I-249.1.15.100 strain demonstrated shorter survival time, more rapid body weight loss, higher mycobacterial loads in the lungs and more severe lung histopathology compared to the more resistant B6.I-249.1.15.139 strain. CD4+ T cells recognized mycobacterial antigens exclusively in the context of the H2-A Class II molecule, and the level of IFN-γ-producing CD4+ T cells in the lungs was significantly higher in the resistant strain. Thus, we directly demonstrated for the first time that the classical H2- Ab1 Class II gene is involved in TB control. Molecular modeling of the H2-Aj product predicts that amino acid (AA) substitutions in the Aβ-chain modify the motif of the peptide-MHC binding groove. Moreover, unique AA substitutions in both α- and β-chains of the H2-Aj molecule might affect its interactions with the T-cell receptor (TCR)

Survival curves of mice infected with <i>M</i>. <i>tuberculosis</i>.

<p>Survival of parental B6 and I/St and recombinant congenic mice (males) following aerosol challenge with ~500 CFU of <i>M</i>. <i>tuberculosis</i> H37Rv. Recombinant strains B6.I-9.3.19.8, B6.I-9.5.7, B6.I-9.5, B6.I-9.3, B6.I-249.1.15.46 and B6.I-249.1.15 all displayed similar (P > 0.1) intermediate mean survival time (MST) compared to hyper-susceptible I/St and relatively resistant B6 mice (<i>P</i> < 0.0001, log-rank test), which reflects the input of the intra-<i>H2</i> QTL in susceptibility. All recombinant strains were tested in 3–10 independent experiments (total N = 20–70 animals). Summary of 3–5 experiments is displayed (Kaplan-Meier survival analysis).</p

Possession of the <i>H2-Ab1</i>^j-like alleles results in a more severe TB infectious course.

<p><b>A–</b>Representative tuberculous lung lesions at day 35 post-infection. Hematoxylin and eosin staining, magnification x100. Arrows show granulomatous structures. <b>B</b>–TNF-α and IL-6 production in infected lungs at day 60 post-challenge. Whole-lung homogenates from individual mice (3 per group) were assessed in the ELISA format. The results are expressed as mean ± SD from two independent experiments (total N = 6), <i>P</i> < 0.05 for B6.I-100 and B6.I-139, ANOVA. <b>C–</b>The number of lung IFN-γ-producing CD4+ T cells was assessed by intracellular staining for IFN-γ at 35 days post-challenge. After culturing with mycobacterial sonicate, the lung cell population gated for CD3 expression was analyzed as displayed. Results of one of two similar experiments (total N = 6) are shown, with statistics for 3 individual mice per group provided in quadrants. In controls (cells from normal mice with mycobacterial sonicate, or cells from infected mice without antigen in culture) the per cent of IFN-γ-producing lung CD4+ T cells never exceed 0.1. <i>P</i> < 0.05 for B6.I-100 and B6.I-139, ANOVA.</p

Allelic polymorphisms in the coding parts of the protein-encoding genes^*.

<p>*The <i>H2</i>^j protein-encoding genes annotated for the region 34, 773331–34, 341959 were cloned and sequenced as described in Materials and Methods. Only genes with missense and non-synonymous mutations are included. Positions of (B6 → I/St) AA substitutions in the single letter code and deletions (Del) are displayed. Amino acid substitutions not reported previously (according to the Ensembl genetic variation data) are given in bold.</p><p>Allelic polymorphisms in the coding parts of the protein-encoding genes^{<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005672#t001fn001" target="_blank">*</a>}.</p

The differences in H2-Ab1 AA sequences between B6.I-100 and B6.I-139 mice.

<p>Protein structure alignment of H2-Ab1molecules. Gene annotations are from UniProt Domain structure <a href="http://www.uniprot.org/" target="_blank">http://www.uniprot.org</a>: 1–27 –signal peptide; 28–122 - β1 polymorphic domain; 123–216 - β2 conservative domain; 217–226 –connecting peptide (CP); 227–247 –transmembrane domain (TM); 248–265 –cytoplasmic domain (CD). H2^{b –}H2^j AA substitutions are highlighted.</p

Genotypes and TB phenotypes of new recombinant mouse strains B6.I-100 and B6.I-139.

<p><b>A–</b>The genome chart for B6.I-100 and B6.I-139 recombinant strains. Chromosome segments transferred from I/St onto B6 genetic background are shown in grey. <b>B–</b>survival curves of B6.I-100, B6.I-139 and parental strains mice after aerosol challenge with ~500 CFU of <i>M</i>. <i>tuberculosis</i>. MST ± SEM (days): B6 = 249 ± 10; I/St = 63 ± 11; B6.I-100 = 152 ± 13; B6.I-139 = 233 ± 14; B6.I-249.1.15.46 = 153 ± 11. Recombinant strains were tested in 3–5 independent experiments (total N = 20–40 males). Summary of 3 experiments is displayed (Kaplan-Meier survival analysis). <b>C–</b>CFU counts in infected lungs at days 28 and 70 post-challenge (4 mice per group, **<i>P</i> < 0.05, ANOVA). The representative results of one out of 2 independent experiments are present. <b>D–</b>genes and their positions in the D17Mit21 – <i>H2-Ea</i> interval according to Ensembl. Red borders–location of the candidate gene.</p