Structures of the L27 Domain of Disc Large Homologue 1 Protein Illustrate a Self-Assembly Module

Disc large 1 (Dlg1) proteins, members of the MAGUK protein family, are linked to cell polarity via their participation in multiprotein assemblies. At their N-termini, Dlg1 proteins contain a L27 domain. Typically, the L27 domains participate in the formation of obligate hetero-oligomers with the L27 domains from their cognate partners. Among the MAGUKs, Dlg1 proteins exist as homo-oligomers, and the oligomerization is solely dependent on the L27 domain. Here we provide biochemical and structural evidence of homodimerization via the L27 domain of Dlg1 from <i>Drosophila melanogaster</i>. The structure reveals that the core of the dimer is formed by a distinctive six-helix assembly, involving all three conserved helices from each subunit (monomer). The homodimer interface is extended by the C-terminal tail of the L27 domain of Dlg1, which forms a two-stranded antiparallel β-sheet. The structure reconciles and provides a structural context for a large body of available mutational data. From our analyses, we conclude that the observed L27 homodimerization is most likely a feature unique to the Dlg1 orthologs within the MAGUK family

A Small Protein Associated with Fungal Energy Metabolism Affects the Virulence of <i>Cryptococcus neoformans</i> in Mammals

<div><p>The pathogenic yeast <i>Cryptococcus neoformans</i> causes cryptococcosis, a life-threatening fungal disease. <i>C</i>. <i>neoformans</i> has multiple virulence mechanisms that are non-host specific, induce damage and interfere with immune clearance. Microarray analysis of <i>C</i>. <i>neoformans</i> strains serially passaged in mice associated a small gene (CNAG_02591) with virulence. This gene, hereafter identified as <i>HVA1</i> (hypervirulence-associated protein 1), encodes a protein that has homologs of unknown function in plant and animal fungi, consistent with a conserved mechanism. Expression of <i>HVA1</i> was negatively correlated with virulence and was reduced <i>in vitro</i> and <i>in vivo</i> in both mouse- and <i>Galleria</i>-passaged strains of <i>C</i>. <i>neoformans</i>. Phenotypic analysis in <i>hva1</i>Δ and <i>hva1</i>Δ+<i>HVA1</i> strains revealed no significant differences in established virulence factors. Mice infected intravenously with the <i>hva1</i>Δ strain had higher fungal burden in the spleen and brain, but lower fungal burden in the lungs, and died faster than mice infected with H99W or the <i>hva1</i>Δ+<i>HVA1</i> strain. Metabolomics analysis demonstrated a general increase in all amino acids measured in the disrupted strain and a block in the TCA cycle at isocitrate dehydrogenase, possibly due to alterations in the nicotinamide cofactor pool. Macrophage fungal burden experiments recapitulated the mouse hypervirulent phenotype of the <i>hva1Δ</i> strain only in the presence of exogenous NADPH. The crystal structure of the Hva1 protein was solved, and a comparison of structurally similar proteins correlated with the metabolomics data and potential interactions with NADPH. We report a new gene that modulates virulence through a mechanism associated with changes in fungal metabolism.</p></div

<i>In vivo</i> gene expression levels of <i>HVA1</i> in the liver are correlated with virulence.

<p><i>In vivo</i> gene expression levels of <i>HVA1</i> were measured using qRT-PCR for six different mouse-passaged strains and plotted against the average time to death in 10 mice caused by each strain.</p

Microarray analysis comparing the <i>hva1Δ</i> and <i>hva1</i>Δ+<i>HVA1</i>strains. All genes shown were up-regulated in the <i>hva1</i>Δ+<i>HVA1</i> strain compared to the <i>hva1Δ</i> strain.

<p>Microarray analysis comparing the <i>hva1Δ</i> and <i>hva1</i>Δ+<i>HVA1</i>strains. All genes shown were up-regulated in the <i>hva1</i>Δ+<i>HVA1</i> strain compared to the <i>hva1Δ</i> strain.</p

Lung histology at day 7.

<p>Mice were infected with the pre-passage H99W (A), the <i>hva1Δ</i> and <i>hva1Δ+HVA1</i> strains (B and C, respectively). The <i>hva1Δ</i> strain showed lower fungal burden and dense areas compared to H99W and the <i>hva1Δ+HVA1</i> strain, which showed organized inflammation around <i>C</i>. <i>neoformans</i>.</p

Volcano plot comparing the <i>hva1Δ</i> and H99W strains.

<p>Metabolites that are elevated in the <i>hva1Δ</i> strain versus H99W are red, while those that are decreased in the <i>hva1Δ</i> strain versus H99W are blue. To be colored the fold change must be greater than 1.5 and p-value<0.05. These data are suggestive of a block in the in the production of 2-ketoglutarate in the <i>hva1Δ</i> strain compared to H99W.</p

The <i>hva1Δ</i> strain has a hypervirulent phenotype.

<p>Mouse fungal burden in the spleen (A), brain (B) and lungs (C) of mice infected intravenously with the pre-passage H99W, the <i>hva1Δ</i> and <i>hva1Δ+HVA1</i> strains. Six mice were infected for each group. Error bars depict standard deviation. D) Survival data for mice infected intravenously with the pre-passage H99W, the <i>hva1Δ</i> and <i>hva1Δ+HVA1</i> strains. Ten mice were infected for each group (only 9 mice reported for H99W).</p

Gene expression of <i>HVA1</i> from microarray and qRT-PCR analysis of two mouse-passaged and one <i>Galleria</i>-passaged <i>C</i>. <i>neoformans</i> strains.

<p>Gene expression of <i>HVA1</i> from microarray and qRT-PCR analysis of two mouse-passaged and one <i>Galleria</i>-passaged <i>C</i>. <i>neoformans</i> strains.</p

Crystal structure of Hva1.

<p>(A) The two monomers of the asymmetric unit are distinguished in cyan and magenta. Regions corresponding to β-strands and α-helix are shown as arrows and a coil, respectively. The β-strands that form the twisted sheet are labelled as β1–5 in the magenta model of this figure with β5 not represented in the arrow format to improve clarity. Overall, the architecture of Hva1 is very ordered and the two monomers are associated with each other via an extensive interface. (B) Connecting topology of the various secondary structural motifs of Hva1. The two monomers of the asymmetric unit are distinguished in cyan and magenta. Arrows represent β-strands which are labelled as indicated in (A). The regions that form the monomer-monomer interface are highlighted by dotted circles.</p

Comparison of <i>Alicyclobacillus acidocaldarius</i> <i>o</i>‑Succinylbenzoate Synthase to Its Promiscuous <i>N</i>‑Succinylamino Acid Racemase/<i>o</i>‑Succinylbenzoate Synthase Relatives

Studying the evolution of catalytically promiscuous enzymes like those from the <i>N</i>-succinylamino acid racemase/<i>o</i>-succinylbenzoate synthase (NSAR/OSBS) subfamily can reveal mechanisms by which new functions evolve. Some enzymes in this subfamily have only OSBS activity, while others catalyze OSBS and NSAR reactions. We characterized several NSAR/OSBS subfamily enzymes as a step toward determining the structural basis for evolving NSAR activity. Three enzymes were promiscuous, like most other characterized NSAR/OSBS subfamily enzymes. However, <i>Alicyclobacillus acidocaldarius</i> OSBS (AaOSBS) efficiently catalyzes OSBS activity but lacks detectable NSAR activity. Competitive inhibition and molecular modeling show that AaOSBS binds <i>N</i>-succinylphenylglycine with moderate affinity in a site that overlaps its normal substrate. On the basis of possible steric conflicts identified by molecular modeling and sequence conservation within the NSAR/OSBS subfamily, we identified one mutation, Y299I, that increased NSAR activity from undetectable to 1.2 × 10² M^–1 s^–1 without affecting OSBS activity. This mutation does not appear to affect binding affinity but instead affects <i>k</i>_cat, by reorienting the substrate or modifying conformational changes to allow both catalytic lysines to access the proton that is moved during the reaction. This is the first site known to affect reaction specificity in the NSAR/OSBS subfamily. However, this gain of activity was obliterated by a second mutation, M18F. Epistatic interference by M18F was unexpected because a phenylalanine at this position is important in another NSAR/OSBS enzyme. Together, modest NSAR activity of Y299I AaOSBS and epistasis between sites 18 and 299 indicate that additional sites influenced the evolution of NSAR reaction specificity in the NSAR/OSBS subfamily