Crystal Structure of the Mp1p Ligand Binding Domain 2 Reveals Its Function as a Fatty Acid-binding Protein

Penicillium marneffei is a dimorphic, pathogenic fungus in Southeast Asia that mostly afflicts immunocompromised individuals. As the only dimorphic member of the genus, it goes through a phase transition from a mold to yeast form, which is believed to be a requisite for its pathogenicity. Mp1p, a cell wall antigenic mannoprotein existing widely in yeast, hyphae, and conidia of the fungus, plays a vital role in host immune response during infection. To understand the function of Mp1p, we have determined the x-ray crystal structure of its ligand binding domain 2 (LBD2) to 1.3 Å. The structure reveals a dimer between the two molecules. The dimer interface forms a ligand binding cavity, in which electron density was observed for a palmitic acid molecule interacting with LBD2 indirectly through hydrogen bonding networks via two structural water molecules. Isothermal titration calorimetry experiments measured the ligand binding affinity (K_d) of Mp1p at the micromolar level. Mutations of ligand-binding residues, namely S313A and S332A, resulted in a 9-fold suppression of ligand binding affinity. Analytical ultracentrifugation assays demonstrated that both LBD2 and Mp1p are mostly monomeric in vitro, no matter with or without ligand, and our dimeric crystal structure of LBD2 might be the result of crystal packing. Based on the conformation of the ligand-binding pocket in the dimer structure, a model for the closed, monomeric form of LBD2 is proposed. Further structural analysis indicated the biological importance of fatty acid binding of Mp1p for the survival and pathogenicity of the conditional pathogen.     

Lipoprotein succession in Borrelia burgdorferi: similar but distinct roles for OspC and VlsE at different stages of mammalian infection

Borrelia burgdorferi alternates between ticks and mammals, requiring variable gene expression and protein production to adapt to these diverse niches. These adaptations include shifting among the major outer surface lipoproteins OspA, OspC, and VlsE at different stages of the infectious cycle. We hypothesize that these proteins carry out a basic but essential function, and that OspC and VlsE fulfil this requirement during early and persistent stages of mammalian infection respectively. Previous work by other investigators suggested that several B. burgdorferi lipoproteins, including OspA and VlsE, could substitute for OspC at the initial stage of mouse infection, when OspC is transiently but absolutely required. In this study, we assessed whether vlsE and ospA could restore infectivity to an ospC mutant, and found that neither gene product effectively compensated for the absence of OspC during early infection. In contrast, we determined that OspC production was required by B. burgdorferi throughout SCID mouse infection if the vlsE gene were absent. Together, these results indicate that OspC can substitute for VlsE when antigenic variation is unnecessary, but that these two abundant lipoproteins are optimized for their related but specific roles during early and persistent mammalian infection by B. burgdorferi.     

Crystal structures of the extracellular domains of the CrRLK1L receptor‐like kinases ANXUR1 and ANXUR2

Catharanthus roseus Receptor‐Like Kinase 1‐like (CrRLK1L) proteins contain two tandem malectin‐like modules in their extracellular domains (ECDs) and function in diverse signaling pathways in plants. Malectin is a carbohydrate‐binding protein in animals and recognizes a number of diglucosides; however, it remains unclear how the two malectin‐like domains in the CrRLK1L proteins sense the ligand molecule. In this study, we reveal the crystal structures of the ECDs of ANXUR1 and ANXUR2, two CrRLK1L members in Arabidopsis thaliana that have critical functions in controlling pollen tube rupture during the fertilization process. We show that the two malectin‐like domains in these proteins pack together to form a rigid architecture. Unlike animal malectin, these malectin‐like domains lack residues involved in binding to the diglucosides, suggesting that they have a distinct ligand‐binding mechanism. A cleft is observed between the two malectin‐like domains, which might function as a potential ligand‐binding pocket.     

The ligand‐binding domain of a chemoreceptor from Comamonas testosteroni has a previously unknown homotrimeric structure

Transmembrane chemoreceptors are widely present in Bacteria and Archaea. They play a critical role in sensing various signals outside and transmitting to the cell interior. Here, we report the structure of the periplasmic ligand‐binding domain (LBD) of the transmembrane chemoreceptor MCP2201, which governs chemotaxis to citrate and other organic compounds in Comamonas testosteroni. The apo‐form LBD crystal revealed a typical four‐helix bundle homodimer, similar to previously well‐studied chemoreceptors such as Tar and Tsr of Escherichia coli. However, the citrate‐bound LBD revealed a four‐helix bundle homotrimer that had not been observed in bacterial chemoreceptor LBDs. This homotrimer was further confirmed with size‐exclusion chromatography, analytical ultracentrifugation and cross‐linking experiments. The physiological importance of the homotrimer for chemotaxis was demonstrated with site‐directed mutations of key amino acid residues in C. testosteroni mutants.     

Identification of B‐cell epitopes of Borrelia burgdorferi outer surface protein C by screening a phage‐displayed gene fragment library

Outer surface protein C (OspC) of Borrelia stimulates remarkable immune responses during early infection and is therefore currently considered a leading diagnostic and vaccine candidate. The sensitivity and specificity of serological tests based on whole protein OspC for diagnosis of Lyme disease are still unsatisfactory. Minimal B‐cell epitopes are key in the development of reliable immunodiagnostic tools. Using OspC fragments displayed on phage particles (phage library) and anti‐OspC antibodies isolated from sera of naturally infected patients, six OspC epitopes capable of distinguishing between LD patient and healthy control sera were identified. Three of these epitopes are located at the N‐terminus (OspC E1 aa19–27, OspC E2 aa38–53, OspC E3 aa62–66) and three at the C‐terminal end (OspC E4 aa155–163, OspC E5 aa184–190 and OspC E6 aa201–207). OspC E1, E4 and E6 were highly conserved among LD related Borreliae. To our knowledge, epitopes OspC E2, E3 and E5 were identified for the first time in this study. Minimal B‐cell epitopes may provide fundamental data for the development of multi‐epitope‐based diagnostic tools for Lyme disease.     

A call to order at the spirochaetal host–pathogen interface

As the Lyme disease spirochaete Borrelia burgdorferi shuttles back and forth between arthropod vector and vertebrate host, it encounters vastly different and hostile environments. Major mechanisms contributing to the success of this pathogen throughout this complex transmission cycle are phase and antigenic variation of abundant and serotype‐defining surface lipoproteins. These peripherally membrane‐anchored virulence factors mediate niche‐specific interactions with vector/host factors and protect the spirochaete from the perils of the mammalian immune response. In this issue of Molecular Microbiology, Tilly, Bestor and Rosa redefine the roles of two lipoproteins, OspC and VlsE, during mammalian infection. Using a variety of promoter fusions in combination with a sensitive in vivo ‘use it or lose it’ gene complementation assay, the authors demonstrate that proper sequential expression of OspC followed by VlsE indeed matters. A previously suggested general functional redundancy between these and other lipoproteins is shown to be limited and dependent on an immunodeficient experimental setting that is arguably of diminished ecological relevance. These data reinforce the notion that OspC plays a unique role during initial infection while the antigenically variant VlsE proteins allow for persistence in the mammalian host.     

Abrogation of ospAB constitutively activates the Rrp2‐RpoN‐RpoS pathway (sigmaN‐sigmaS cascade) in Borrelia burgdorferi

Molecular mechanisms underlying the reciprocal regulation of the two major surface lipoproteins and virulence factors of Borrelia burgdorferi, OspA and OspC, are not fully understood. Herein, we report that inactivation of the ospAB operon resulted in overproduction of OspC and many other lipoproteins via the constitutive activation of the Rrp2‐RpoN‐RpoS pathway. Complementing the ospAB mutant with a wild‐type copy of ospA, but not an ospA variant that lacks the lipoprotein signal sequence, restored normal regulation of the Rrp2‐RpoN‐RpoS pathway; these results indicate that the phenotype was not caused by spurious mutations. Interestingly, while most of the ospAB mutant clones displayed a constitutive ospC expression phenotype, some ospAB mutant clones showed little or no ospC expression. Further analyses revealed that this OspC‐negative phenotype was independent of abrogation of ospAB. While activation of the Rrp2‐RpoN‐RpoS pathway was recently shown to downregulate ospA, our findings suggest that reduction of OspA can also activate this pathway. We postulate that the activation of the Rrp2‐RpoN‐RpoS pathway and downregulation of OspA form a positive feedback loop that allows spirochaetes to produce and maintain a constant high level of OspC and other lipoproteins during tick feeding, a strategy that is critical for spirochaetal transmission and mammalian infection.     

Insight into the remarkable affinity and selectivity of the aminobenzosuberone scaffold for the M1 aminopeptidases family based on structure analysis

Aminopeptidases are ubiquitous hydrolases that cleave the N‐terminal residues of proteins and oligopeptides. They are broadly distributed throughout all kingdoms of life and have been implicated in a wide variety of physiological processes, including viral infection, parasite metabolism, protein processing, regulation of peptide hormones, and cancer cell proliferation. Members of the M1 family, also termed gluzincins, are defined by two highly conserved motifs in the catalytic domain: a zinc‐binding motif, HEXXH‐(X18)‐E; and an exopeptidase motif, GXMEN. We report the high‐resolution X‐ray structures of E. coli aminopeptidase N (PepN) in complex with three aminobenzosuberone scaffolds that display various Ki values (50, 0.33, and 0.034 µM) and provide a compelling view of the outstanding selectivity of these chemical entities for the M1 aminopeptidases. This series of inhibitors interacts as transition state mimics with highly conserved residues of the catalytic machinery and substrate recognition sites. Structural comparisons and model‐building studies allowed a deep interpretation of the SAR observed for bacterial, as well as mammalian enzymes. Proteins 2017; 85:1413–1421. © 2017 Wiley Periodicals, Inc.     

Two Distinct Determinants of Ligand Specificity in T1R1/T1R3 (the Umami Taste Receptor)

Umami taste perception in mammals is mediated by a heteromeric complex of two G-protein-coupled receptors, T1R1 and T1R3. T1R1/T1R3 exhibits species-dependent differences in ligand specificity; human T1R1/T1R3 specifically responds to l-Glu, whereas mouse T1R1/T1R3 responds more strongly to other l-amino acids than to l-Glu. The mechanism underlying this species difference remains unknown. In this study we analyzed chimeric human-mouse receptors and point mutants of T1R1/T1R3 and identified 12 key residues that modulate amino acid recognition in the human- and mouse-type responses in the extracellular Venus flytrap domain of T1R1. Molecular modeling revealed that the residues critical for human-type acidic amino acid recognition were located at the orthosteric ligand binding site. In contrast, all of the key residues for the mouse-type broad response were located at regions outside of both the orthosteric ligand binding site and the allosteric binding site for inosine-5′-monophosphate (IMP), a known natural umami taste enhancer. Site-directed mutagenesis demonstrated that the newly identified key residues for the mouse-type responses modulated receptor activity in a manner distinct from that of the allosteric modulation via IMP. Analyses of multiple point mutants suggested that the combination of two distinct determinants, amino acid selectivity at the orthosteric site and receptor activity modulation at the non-orthosteric sites, may mediate the ligand specificity of T1R1/T1R3. This hypothesis was supported by the results of studies using nonhuman primate T1R1 receptors. A complex molecular mechanism involving changes in the properties of both the orthosteric and non-orthosteric sites of T1R1 underlies the determination of ligand specificity in mammalian T1R1/T1R3.     

A high‐throughput screen for ligand binding reveals the specificities of three amino acid chemoreceptors from Pseudomonas syringae pv. actinidiae

Chemoreceptors play a central role in chemotaxis, allowing bacteria to detect chemical gradients and bias their swimming behavior in order to navigate toward favorable environments. The genome of the kiwifruit pathogen, Pseudomonas syringae pv. actinidiae (Psa) strain NZ‐V13 encodes 43 predicted chemoreceptors, none of which has been characterized. We developed a high‐throughput fluorescence‐based thermal shift assay for identifying the signal molecules that are recognized by a given chemoreceptor ligand binding domain (LBD). Using this assay, we characterized the ligand binding profiles of three Psa homologs of the P. aeruginosa PAO1 amino acid chemoreceptors PctA, PctB and PctC. Each recombinant LBD was screened against 95 potential ligands. The three Psa homologs, named pscA, pscB and pscC (P s a c hemoreceptors A , B and C ) bound 3, 10 and 3 amino acids respectively. In each case, their binding profiles were distinct from their P. aeruginosa PAO1 homologs. Notably, Psa PscA‐LBD only bound the acidic amino acids l ‐aspartate, d ‐aspartate and l ‐glutamate, whereas P. aeruginosa PctA‐LBD binds all of the l ‐proteinogenic amino acids except for l ‐aspartate and l ‐glutamate. A combination of homology modeling, site‐directed mutagenesis and functional screening identified a single amino acid residue in the Psa PscA‐LBD (Ala146) that is critically important for determining its narrow specificity.     

The BosR regulatory protein of Borrelia burgdorferi interfaces with the RpoS regulatory pathway and modulates both the oxidative stress response and pathogenic properties of the Lyme disease spirochete

Borrelia burgdorferi, the Lyme disease spirochete, adapts as it moves between the arthropod and mammalian hosts that it infects. We hypothesize that BosR serves as a global regulator in B. burgdorferi to modulate the oxidative stress response and adapt to mammalian hosts. To test this hypothesis, a bosR mutant in a low‐passage B. burgdorferi isolate was constructed. The resulting bosR::kan^R strain was altered when grown microaerobically or anaerobically suggesting that BosR is required for optimal replication under both growth conditions. The absence of BosR increased the sensitivity of B. burgdorferi to hydrogen peroxide and reduced the synthesis of Cdr and NapA, proteins important for cellular redox balance and the oxidative stress response, respectively, suggesting an important role for BosR in borrelial oxidative homeostasis. For the bosR mutant, the production of RpoS was abrogated and resulted in the loss of OspC and DbpA, suggesting that BosR interfaces with the Rrp2–RpoN–RpoS regulatory cascade. Consistent with the linkage to RpoS, cells lacking bosR were non‐infectious in the mouse model of infection. These results indicate that BosR is required for resistance to oxidative stressors and provides a regulatory response that is necessary for B. burgdorferi pathogenesis.     

Crystal structure of zebrafish complement 1qA globular domain

C1q contains three globular domains (C1qgD) that are the key functional component of the classical complement system. C1qgD can interact with important immune molecules, including IgG and C‐reactive protein (CRP) to form defense systems to protect animals. Here, the first non‐mammalian structure, zebrafish C1qA globular domain (Dare‐C1qAgD) was solved. Although the overall architecture of Dare‐C1qAgD is similar to human C1qA, residues involved in C1qBgD, C1qCgD, and CRP binding are somewhat different while residues involved in IgG binding are not present in zebrafish. The structure gives insight into how human and fish C1qA evolved from an ancestral protein.     

Pseudomonas signal molecule 3-oxo-C12-homoserine lactone interferes with binding of rosiglitazone to human PPARγ

Peroxisome proliferator activated receptor (PPARγ) has been suggested as a target for anti-inflammatory therapy in chronic lung disease, including infection with Pseudomonas aeruginosa. However, the P. aeruginosa signal molecule N-(3-oxo-dodecanoyl)-l-homoserine lactone (3-oxo-C12-HSL) has been reported to inhibit function of PPARs in mammalian cells. This suggests that binding of 3-oxo-C12-HSL to PPARs could increase inflammation during P. aeruginosa infection, particularly if it could compete for binding with other PPAR ligands. We investigated the ability of 3-oxo-C12-HSL to bind to a PPARγ ligand binding domain (LBD) construct, and to compete for binding with the highly active synthetic PPARγ agonist rosiglitazone. We demonstrate that 3-oxo-C12-HSL binds effectively to the PPARγ ligand binding domain, and that concentrations of 3-oxo-C12-HSL as low as 1 nM can effectively interfere with the binding of rosiglitazone to the PPARγ ligand binding domain. Because 3-oxo-C12 HSL has been demonstrated in lungs during P. aeruginosa infection, blockade of PPARγ-dependent signaling by 3-oxo-C12-HSL produced by the infecting P. aeruginosa could exacerbate infection-associated inflammation, and potentially impair the action of PPAR-activating therapy. Thus the proposed use of PPARγ agonists as anti-inflammatory therapy in lung P. aeruginosa infection may depend on their ability to counteract the effects of 3-oxo-C12-HSL.     

Fsr1, a striatin homologue,forms an endomembrane‐associated complex that regulates virulence in the maize pathogen Fusarium verticillioides

Fsr1, a homologue of mammalian striatin, containing multiple protein‐binding domains and a coiled‐coil (CC) domain, is critical for Fusarium verticillioides virulence. In mammals, striatin interacts with multiple proteins to form a STRIPAK (striatin‐interacting phosphatase and kinase) complex that regulates a variety of developmental processes and cellular mechanisms. In this study, we identified the homologue of a key mammalian STRIPAK component STRIP1/2 (striatin‐interacting proteins 1 and 2) in F. verticillioides, FvStp1, which interacts with Fsr1 in vivo. Gene deletion analysis indicates that FvStp1 is critical for F. verticillioides stalk rot virulence. In addition, we identified three proteins, designated FvCyp1, FvScp1 and FvSel1, which interact with the Fsr1 CC domain via a yeast two‐hybrid screen. Importantly, FvCyp1, FvScp1 and FvSel1 co‐localize to endomembrane structures, each having a preferred localization in the cell, and they are all required for F. verticillioides stalk rot virulence. Moreover, these proteins are necessary for the correct localization of Fsr1 to the endoplasmic reticulum (ER) and nuclear envelope. Thus, we identified several novel components in the STRIPAK complex that regulates F. verticillioides virulence, and propose that the correct organization and localization of Fsr1 are critical for STRIPAK complex function.     

Epitope mapping of Borrelia burgdorferi OspC protein in homodimeric fold

In current work, we used recombinant OspC protein derived from B. afzelii strain BRZ31 in the native homodimeric fold for mice immunization and following selection process to produce three mouse monoclonal antibodies able to bind to variable parts of up to five different OspC proteins. Applying the combination of mass spectrometry assisted epitope mapping and affinity based theoretical prediction we have localized regions responsible for antigen‐antibody interactions and approximate epitopes' amino acid composition. Two mAbs (3F4 and 2A9) binds to linear epitopes located in previously described immunogenic regions in the exposed part of OspC protein. The third mAb (2D1) recognises highly conserved discontinuous epitope close to the ligand binding domain 1.