A. Louise Creagh

Characterization and affinity applications of cellulose-binding domains

Cellulose-binding domains (CBDs) are discrete protein modules found in a large number of carbohydrolases and a few nonhydrolytic proteins. To date, almost 200 sequences can be classified in 13 different families with distinctly different properties. CBDs vary in size from 4 to 20 kDa and occur at different positions within the polypeptides; N-terminal, C-terminal and internal. They have a moderately high and specific affinity for insoluble or soluble cellulosics with dissociation constants in the low micromolar range. Some CBDs bind irreversibly to cellulose and can be used for applications involving immobilization, others bind reversibly and are more useful for separations and purifications. Dependent on the CBD used, desorption from the matrix can be promoted under various different conditions including denaturants (urea, high pH), water, or specific competitive ligands (e.g. cellobiose). Family I and IV CBDs bind reversibly to cellulose in contrast to family II and III CBDs which are in general, irreversibly bound. The binding of family II CBDs (CBD(Cex)) to crystalline cellulose is characterized by a large favourable increase in entropy indicating that dehydration of the sorbent and the protein are the major driving forces for binding. In contrast, binding of family IV CBDs (CBD(N1)) to amorphous or soluble cellulosics is driven by a favourable change in enthalpy which is partially offset by an unfavourable entropy change. Hydrogen bond formation and van der Waals interactions are the main driving forces for binding. CBDs with affinity for crystalline cellulose are useful tags for classical column affinity chromatography. The affinity of CBD(N1) for soluble cellulosics makes it suitable for use in large-scale aqueous two-phase affinity partitioning systems.

A discrete genetic locus confers xyloglucan metabolism in select human gut Bacteroidetes

A well-balanced human diet includes a significant intake of non-starch polysaccharides, collectively termed ‘dietary fibre’, from the cell walls of diverse fruits and vegetables. Owing to the paucity of alimentary enzymes encoded by the human genome, our ability to derive energy from dietary fibre depends on the saccharification and fermentation of complex carbohydrates by the massive microbial community residing in our distal gut. The xyloglucans (XyGs) are a ubiquitous family of highly branched plant cell wall polysaccharides whose mechanism(s) of degradation in the human gut and consequent importance in nutrition have been unclear. Here we demonstrate that a single, complex gene locus in Bacteroides ovatus confers XyG catabolism in this common colonic symbiont. Through targeted gene disruption, biochemical analysis of all predicted glycoside hydrolases and carbohydrate-binding proteins, and three-dimensional structural determination of the vanguard endo-xyloglucanase, we reveal the molecular mechanisms through which XyGs are hydrolysed to component monosaccharides for further metabolism. We also observe that orthologous XyG utilization loci (XyGULs) serve as genetic markers of XyG catabolism in Bacteroidetes, that XyGULs are restricted to a limited number of phylogenetically diverse strains, and that XyGULs are ubiquitous in surveyed human metagenomes. Our findings reveal that the metabolism of even highly abundant components of dietary fibre may be mediated by niche species, which has immediate fundamental and practical implications for gut symbiont population ecology in the context of human diet, nutrition and health.

The crystal structure of MexR from Pseudomonas aeruginosa in complex with its antirepressor ArmR

The intrinsic antimicrobial resistance of the opportunistic human pathogen Pseudomonas aeruginosa is compounded in mutant strains that overexpress multidrug efflux pumps such as the prominent drug-proton antiporter, MexAB-OprM. The primary regulator of the mexAB-oprM operon is the MarR family repressor, MexR. An additional repressor, NalC, also regulates mexAB-oprM by controlling expression of ArmR, an antirepressor peptide that is hypothesized to prevent the binding of MexR to its cognate DNA operator via an allosteric protein–peptide interaction. To better understand how ArmR modulates MexR, we determined the MexR-binding region of ArmR as its C-terminal 25 residues and solved the crystal structure of MexR in a 2:1 complex with this ArmR fragment at 1.8 Å resolution. This structure reveals that the C-terminal residues of ArmR form a kinked α-helix, which occupies a pseudosymmetrical and largely hydrophobic binding cavity located at the centre of the MexR dimer. Although the ArmR-binding cavity partially overlaps with the small molecule effector-binding sites of other MarR family members, it possesses a larger and more complex binding surface to accommodate the greater size and specific physicochemical properties of a peptide effector. Comparison with the structure of apo-MexR reveals that ArmR stabilizes a dramatic conformational change that is incompatible with DNA-binding. Thus, this work defines the structural mechanism by which ArmR allosterically derepresses MexR-controlled gene expression in P. aeruginosa and reveals important insights into the regulation of multidrug resistance.

Structural Characterization of the Type-III Pilot-Secretin Complex from Shigella flexneri

Assembly of the type-III secretion apparatus, which translocates proteins through both membranes of Gram-negative bacterial pathogens into host cells, requires the formation of an integral outer-membrane secretin ring. Typically, a small lipidated pilot protein is necessary for the stabilization and localization of this ring. Using NMR spectroscopy, we demonstrate that the C-terminal residues 553-570 of the Shigella flexneri secretin MxiD encompass the minimal binding domain for its cognate pilot MxiM. Although unstructured in isolation, upon complex formation with MxiM, these residues fold into an amphipathic turn-helix motif that caps the elongated hydrophobic cavity of the cracked beta-barrel pilot. Along with a rearrangement of core aromatic residues, this prevents the binding of lipids within the cavity. The mutually exclusive association of lipids and MxiD with MxiM establishes a framework for understanding the role of a pilot in the outer-membrane insertion and multimerization of the secretin ring.

The Mannoprotein Cig1 supports iron acquisition from heme and virulence in the pathogenic fungus Cryptococcus neoformans.

Iron acquisition is critical for virulence of the human pathogenic fungus Cryptococcus neoformans. The cryptococcal transcript for the extracellular mannoprotein Cig1 is highly regulated by iron and abundant in iron-starved cells, suggesting a role in iron acquisition. Indeed, loss of Cig1 resulted in delayed growth on heme at physiological pH. Expression of CIG1 is regulated by the pH-responsive transcription factor Rim101, and loss of Rim101 also impaired growth on heme. A cig1Δ mutant was less susceptible than the wild-type strain to noniron metalloporphyrins, further indicating a role for Cig1 in heme uptake. Recombinant Cig1 exhibited the absorbance spectrum of a heme-binding protein upon heme titration, and Cig1 may therefore function as a hemophore at the cell surface. Cig1 contributed to virulence in a mouse model of cryptococcosis but only in a mutant that also lacked the high-affinity iron uptake system. Overall, Cig1-mediated heme uptake is a potential therapeutic target in C. neoformans.

Germline V-genes sculpt the binding site of a family of antibodies neutralizing human cytomegalovirus.

Immunoglobulin genes are generated somatically through specialized mechanisms resulting in a vast repertoire of antigen‐binding sites. Despite the stochastic nature of these processes, the V‐genes that encode most of the antigen‐combining site are under positive evolutionary selection, raising the possibility that V‐genes have been selected to encode key structural features of binding sites of protective antibodies against certain pathogens. Human, neutralizing antibodies to human cytomegalovirus that bind the AD‐2S1 epitope on its gB envelope protein repeatedly use a pair of well‐conserved, germline V‐genes IGHV3‐30 and IGKV3‐11. Here, we present crystallographic, kinetic and thermodynamic analyses of the binding site of such an antibody and that of its primary immunoglobulin ancestor. These show that these germline V‐genes encode key side chain contacts with the viral antigen and thereby dictate key structural features of the hypermutated, high‐affinity neutralizing antibody. V‐genes may thus encode an innate, protective immunological memory that targets vulnerable, invariant sites on multiple pathogens.

Affinity-based design of a synthetic universal reversal agent for heparin anticoagulants

A safe, synthetic anticoagulant-reversal agent based on a dendritic polymer is effective against all the clinical heparin anticoagulants and may be a treatment for bleeding in high-risk surgical procedures. One Drug to Rule Them All To clot or not to clot—that is the question. Prevention of blood clotting—anticoagulation—is preferred during surgical procedures or in blood vessels where clots can cause blockage. In fact, heparin-based anticoagulant drugs are used broadly for such purposes. But on the flip side, these anticoagulants are associated with bleeding risks that make close monitoring and neutralization necessary. Currently, only protamine has been clinically approved as an antidote to heparin-based anticoagulants; but the drug displays some adverse effects and is impotent against certain heparins and heparin-related medications. Now, Shenoi et al. describe a fully synthetic dendritic polymer–based universal heparin reversal agent (UHRA) that functions via multivalent presentation of branched cationic heparin binding groups (HBGs). The authors varied the agent’s scaffold, protective shell, and number and array of HBGs to develop an antidote that neutralized all clinically used heparin-related anticoagulants. The UHRA displayed safety and efficacy in animal models of heparin-induced bleeding. The new therapeutic may one day benefit patients in situations where the goal is to clot—such as in the treatment of excessive bleeding during anticoagulant therapy or high-risk surgery. Heparin-based anticoagulant drugs have been widely used for the prevention of blood clotting during surgical procedures and for the treatment of thromboembolic events. However, bleeding risks associated with these anticoagulants demand continuous monitoring and neutralization with suitable antidotes. Protamine, the only clinically approved antidote to heparin, has shown adverse effects and ineffectiveness against low–molecular weight heparins and fondaparinux, a heparin-related medication. Alternative approaches based on cationic molecules and recombinant proteins have several drawbacks including limited efficacy, toxicity, immunogenicity, and high cost. Thus, there is an unmet clinical need for safer, rapid, predictable, and cost-effective anticoagulant-reversal agents for all clinically used heparins. We report a design strategy for a fully synthetic dendritic polymer–based universal heparin reversal agent (UHRA) that makes use of multivalent presentation of branched cationic heparin binding groups (HBGs). Optimization of the UHRA design was aided by isothermal titration calorimetry studies, biocompatibility evaluation, and heparin neutralization analysis. By controlling the scaffold’s molecular weight, the nature of the protective shell, and the presentation of HBGs on the polymer scaffold, we arrived at lead UHRA molecules that completely neutralized the activity of all clinically used heparins. The optimized UHRA molecules demonstrated superior efficacy and safety profiles and mitigated heparin-induced bleeding in animal models. This new polymer therapeutic may benefit patients undergoing high-risk surgical procedures and has potential for the treatment of anticoagulant-related bleeding problems.

Topical delivery of interferon alpha by biphasic vesicles: evidence for a novel nanopathway across the stratum corneum.

Noninvasive delivery of macromolecules across intact skin is challenging but would allow for needle-free administration of many pharmaceuticals. Biphasic vesicles, a novel lipid-based topical delivery system, have been shown to deliver macromolecules into the skin. Investigation of the delivery mechanism of interferon alpha (IFN alpha), as a model protein, by biphasic vesicles could improve understanding of molecular transport through the stratum corneum and allow for the design of more effective delivery systems. The interaction of biphasic vesicles with human skin and isolated stratum corneum membrane was investigated by confocal microscopy, differential scanning calorimetry (DSC) and small- and wide-angle X-ray scattering (SAXS and WAXS). Confocal microscopy revealed that biphasic vesicles delivered IFN alpha intercellularly, to a depth of 70 microm, well below the stratum corneum and into the viable epidermis. DSC and SAXS/WAXS data suggest that the interaction of biphasic vesicles with SC lipids resulted in the formation of a three-dimensional cubic Pn3m polymorphic phase by the molecular rearrangement of intercellular lipids. This cubic phase could be an intercellular permeation nanopathway that may explain the increased delivery of IFN alpha by biphasic vesicles. Liposomes and submicrometer emulsion (the individual building blocks of biphasic vesicles) separately and methylcellulose gel, an alternative topical vehicle, did not induce a cubic phase and delivered low amounts of IFN alpha below the stratum corneum. Molecular modeling of the cubic Pn3m phase and lamellar-to-cubic phase transitions provides a plausible mechanism for transport of IFN alpha. It is hypothesized that induction of a Pn3m cubic phase in stratum corneum lipids could make dermal and transdermal delivery of other macromolecules also possible.

Calorimetric studies of the interaction between the insulin-enhancing drug candidate bis(maltolato)oxovanadium(IV) (BMOV) and human serum apo-transferrin

Bis(maltolato)oxovanadium(IV) (BMOV), and its ethylmaltol analog, bis(ethylmaltolato)oxovanadium(IV) (BEOV), are candidate insulin-enhancing agents for the treatment of type 2 diabetes mellitus; in mid-2008, BEOV advanced to phase II clinical testing. The interactions of BMOV and its inorganic congener, vanadyl sulfate (VOSO(4)), with human serum apo-transferrin (hTf) were investigated using differential scanning calorimetry (DSC). Addition of BMOV or VOSO(4) to apo-hTf resulted in an increase in thermal stability of both the C- and N-lobes of transferrin as a result of binding to either vanadyl compound. A series of DSC thermograms of hTf solutions containing different molar ratios of BMOV and VOSO(4) were used to determine binding constants; at 25 degrees C the binding constants of BMOV to the C- and N-lobes of apo-hTf were found to be 3 (+/-1)x10(5) and 1.8 (+/-0.7)x10(5)M(-1), respectively. The corresponding values for VOSO(4) were 1.7 (+/-0.3)x10(5) and 7 (+/-2)x10(4)M(-1). The results show that the vanadium species initially presented as either BMOV or VOSO(4) had similar affinities for human serum transferrin due to oxidation of solvated vanadyl(IV) prior to complexation to transferrin. Binding of metavanadate (VO(3)(-)) was confirmed by DSC and isothermal titration calorimetry (ITC) experiments of the interaction between sodium metavanadate (NaVO(3)) and hTf.

Ion exchange in hydroxyapatite with lanthanides.

Naturally occurring hydroxyapatite, Ca5(PO4)3(OH) (HAP), is the main inorganic component of bone matrix, with synthetic analogues finding applications in bioceramics and catalysis. An interesting and valuable property of both natural and synthetic HAP is the ability to undergo cationic and anionic substitution. The lanthanides are well-suited for substitution for the Ca(2+) sites within HAP, because of their similarities in ionic radii, donor atom requirements, and coordination geometries. We have used isothermal titration calorimetry (ITC) to investigate the thermodynamics of ion exchange in HAP with a representative series of lanthanide ions, La(3+), Sm(3+), Gd(3+), Ho(3+), Yb(3+) and Lu(3+), reporting the association constant (Ka), ion-exchange thermodynamic parameters (ΔH, ΔS, ΔG), and binding stoichiometry (n). We also probe the nature of the La(3+):HAP interaction by solid-state nuclear magnetic resonance ((31)P NMR), X-ray diffraction (XRD), and inductively coupled plasma-optical emission spectroscopy (ICP-OES), in support of the ITC results.