Reinhard Wimmer

NMR Structure and Metal Interactions of the CopZ Copper Chaperone

A recently discovered family of proteins that function as copper chaperones route copper to proteins that either require it for their function or are involved in its transport. InEnterococcus hirae the copper chaperone function is performed by the 8-kDa protein CopZ. This paper describes the NMR structure of apo-CopZ, obtained using uniformly15N-labeled CopZ overexpressed in Escherichia coli and NMR studies of the impact of Cu(I) binding on the CopZ structure. The protein has a βαββαβ fold, where the four β-strands form an antiparallel twisted β-sheet, and the two helices are located on the same side of the β-sheet. A sequence motif GMXCXXC in the loop between the first β-strand and the first α-helix contains the primary ligands, which bind copper(I). Binding of copper(I) caused major structural changes in this molecular region, as manifested by the fact that most NMR signals of the loop and the N-terminal part of the first helix were broadened beyond detection. This effect was strictly localized, because the remainder of the apo-CopZ structure was maintained after addition of Cu(I). NMR relaxation data showed a decreased correlation time of overall molecular tumbling for Cu(I)-CopZ when compared withapo-CopZ, indicating aggregation of Cu(I)-CopZ. The structure of CopZ is the first three-dimensional structure of a cupro-protein for which the metal ion is an exchangeable substrate rather than an integral part of the structure. Implications of the present structural work for the in vivo function of CopZ are discussed, whereby it is of special interest that the distribution of charged residues on the CopZ surface is highly uneven and suggests preferred recognition sites for other proteins that might be involved in copper transfer.

A metabolic model for members of the genus Tetrasphaera involved in enhanced biological phosphorus removal

Members of the genus Tetrasphaera are considered to be putative polyphosphate accumulating organisms (PAOs) in enhanced biological phosphorus removal (EBPR) from wastewater. Although abundant in Danish full-scale wastewater EBPR plants, how similar their ecophysiology is to ‘Candidatus Accumulibacter phosphatis’ is unclear, although they may occupy different ecological niches in EBPR communities. The genomes of four Tetrasphaera isolates (T. australiensis, T. japonica, T. elongata and T. jenkinsii) were sequenced and annotated, and the data used to construct metabolic models. These models incorporate central aspects of carbon and phosphorus metabolism critical to understanding their behavior under the alternating anaerobic/aerobic conditions encountered in EBPR systems. Key features of these metabolic pathways were investigated in pure cultures, although poor growth limited their analyses to T. japonica and T. elongata. Based on the models, we propose that under anaerobic conditions the Tetrasphaera-related PAOs take up glucose and ferment this to succinate and other components. They also synthesize glycogen as a storage polymer, using energy generated from the degradation of stored polyphosphate and substrate fermentation. During the aerobic phase, the stored glycogen is catabolized to provide energy for growth and to replenish the intracellular polyphosphate reserves needed for subsequent anaerobic metabolism. They are also able to denitrify. This physiology is markedly different to that displayed by ‘Candidatus Accumulibacter phosphatis’, and reveals Tetrasphaera populations to be unusual and physiologically versatile PAOs carrying out denitrification, fermentation and polyphosphate accumulation.

Structural basis for cyclodextrins' suppression of human growth hormone aggregation

Many therapeutic proteins require storage at room temperature for extended periods of time. This can lead to aggregation and loss of function. Cyclodextrins (CDs) have been shown to function as aggregation suppressors for a wide range of proteins. Their potency is often ascribed to their affinity for aromatic amino acids, whose surface exposure would otherwise lead to protein association. However, no detailed structural studies are available. Here we investigate the interactions between human growth hormone (hGH) and different CDs at low pH. Although hGH aggregates readily at pH 2.5 in 1 M NaCl to form amorphous aggregates, the presence of 25 to 50 mM of various β‐CD derivatives is sufficient to completely avoid this. α‐ and γ‐CD are considerably less effective. Stopped‐flow data on the aggregation reaction in the presence of β‐CD are analyzed according to a minimalist association model to yield an apparent hGH‐β‐CD dissociation constant of ∼6 mM. This value is very similar to that obtained by simple fluorescence‐based titration of hGH with β‐CD. Nuclear magnetic resonance studies indicate that β‐CD leads to a more unfolded conformation of hGH at low pH and predominantly binds to the aromatic side‐chains. This indicates that aromatic amino acids are important components of regions of residual structure that may form nuclei for aggregation.

Production of novel fusarielins by ectopic activation of the polyketide synthase 9 cluster in Fusarium graminearum.

Like many other filamentous fungi, Fusarium graminearum has the genetic potential to produce a vast array of unknown secondary metabolites. A promising approach to determine the nature of these is to activate silent secondary metabolite gene clusters through constitutive expression of cluster specific transcription factors. We have developed a system in which an expression cassette containing the transcription factor from the targeted PKS cluster disrupts the production of the red mycelium pigment aurofusarin. This aids with identification of mutants as they appear as white colonies and metabolite analyses where aurofusarin and its intermediates are normally among the most abundant compounds. The system was used for constitutive expression of the local transcription factor from the PKS9 cluster (renamed FSL) leading to production of three novel fusarielins, F, G and H. This group of compounds has not previously been reported from F. graminearum or linked to a biosynthetic gene in any fungal species. The toxicity of the three novel fusarielins was examined against colorectal cancer cell lines where fusarielin H was more potent than fusarielin F and G.

Synthesis of sucrose laurate using a new alkaline protease

Abstract Sucrose laurate esters were synthesized from sucrose and vinyl laurate in organic solvents using an alkaline protease from a new alkalophilic strain, Bacillus pseudofirmus AL-89. Maximum synthetic activity was observed in the presence of 7.5% v/v water and in the pH range of 7–10. With protease AL-89 esterification occurred predominantly at the 2- O -position while subtilisin A-catalyzed monoester formation predominantly at the 1′- O position. In the absence of enzyme, buffer salts catalyzed non-specific reactions, resulting in the formation of a number of esters. Non-specific catalysis was also observed upon inhibition of the enzyme using a serine protease inhibitor or upon deactivation of the enzyme at pH above 10.

The Lantibiotic NAI-107 Binds to Bactoprenol-bound Cell Wall Precursors and Impairs Membrane Functions

Background: NAI-107 is a potent lantibiotic with an unknown mode of action. Results: NAI-107 targets bactoprenol-bound cell envelope precursors, e.g. lipid II, and in addition affects the bacterial membrane. Conclusion: Cell wall biosynthesis is blocked by sequestration of lipid II and functional disorganization of the cell wall machinery. Significance: The dual mechanism of action may explain the potency of NAI-107 and related lantibiotics. The lantibiotic NAI-107 is active against Gram-positive bacteria including vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus. To identify the molecular basis of its potency, we studied the mode of action in a series of whole cell and in vitro assays and analyzed structural features by nuclear magnetic resonance (NMR). The lantibiotic efficiently interfered with late stages of cell wall biosynthesis and induced accumulation of the soluble peptidoglycan precursor UDP-N-acetylmuramic acid-pentapeptide (UDP-MurNAc-pentapeptide) in the cytoplasm. Using membrane preparations and a complete cascade of purified, recombinant late stage peptidoglycan biosynthetic enzymes (MraY, MurG, FemX, PBP2) and their respective purified substrates, we showed that NAI-107 forms complexes with bactoprenol-pyrophosphate-coupled precursors of the bacterial cell wall. Titration experiments indicate that first a 1:1 stoichiometric complex occurs, which then transforms into a 2:1 (peptide: lipid II) complex, when excess peptide is added. Furthermore, lipid II and related molecules obviously could not serve as anchor molecules for the formation of defined and stable nisin-like pores, however, slow membrane depolarization was observed after NAI-107 treatment, which could contribute to killing of the bacterial cell.

Eurocin, a New Fungal Defensin: STRUCTURE, LIPID BINDING, AND ITS MODE OF ACTION*

Background: Antimicrobial peptides are new antibiotics avoiding resistance problems. Results: Eurocin is a new antimicrobial peptide featuring a cysteine-stabilized αβ-fold. Eurocin binds the cell wall precursor lipid II but does not disrupt cell membranes. Conclusion: Eurocin acts by inhibiting cell wall synthesis. Its structure is typical for invertebrate defensins. Significance: Knowing the mode of action and structure is a prerequisite for pharmaceutical application of an antibiotic. Antimicrobial peptides are a new class of antibiotics that are promising for pharmaceutical applications because they have retained efficacy throughout evolution. One class of antimicrobial peptides are the defensins, which have been found in different species. Here we describe a new fungal defensin, eurocin. Eurocin acts against a range of Gram-positive human pathogens but not against Gram-negative bacteria. Eurocin consists of 42 amino acids, forming a cysteine-stabilized α/β-fold. The thermal denaturation data point shows the disulfide bridges being responsible for the stability of the fold. Eurocin does not form pores in cell membranes at physiologically relevant concentrations; it does, however, lead to limited leakage of a fluorophore from small unilamellar vesicles. Eurocin interacts with detergent micelles, and it inhibits the synthesis of cell walls by binding equimolarly to the cell wall precursor lipid II.

p25α is flexible but natively folded and binds tubulin with oligomeric stoichiometry

p25α is a 219‐residue proteinwhich stimulates aberrant tubulin polymerization and is implicated in a variety of other functions. The protein has unusual secondary structure involving significant amounts of random coil, and binding to microtubules is accompanied by a large structural change, suggesting a high degree of plasticity. p25α has been proposed to be natively unfolded, so that folding is coupled to interaction with its physiological partners. Here we show that recombinant human p25α is folded under physiological conditions, since it has a well structured and solvent‐sequestered aromatic environment and considerable chemical shift dispersion of amide and aliphatic protons. With increasing urea concentrations, p25α undergoes clear spectral changes suggesting significant loss of structure. p25α unfolds cooperatively in urea according to a simple two‐state transition with a stability in water of ∼5 kcal/mol. The protein behaves as a monomer and refolds with a transient on‐pathway folding intermediate. However, high sensitivity to proteolytic attack and abnormal gel filtration migration behavior suggests a relatively extended structure, possibly organized in distinct domains. A deletion mutant of p25α lacking residues 3–43 also unfolds cooperatively and with similar stability, suggesting that the N‐terminal region is largely unstructured. Both proteins undergo significant loss of structure when bound to monomeric tubulin. The stoichiometry of binding is estimated to be 3–4 molecules of tubulin per p25α and is not significantly affected by the deletion of residues 3–43. In conclusion, we dismiss the proposal that p25α is natively unfolded, although the protein is relatively flexible. This flexibility may be linked to its tubulin‐binding properties.

Interactions of a fungal lytic polysaccharide monooxygenase with β-glucan substrates and cellobiose dehydrogenase

Significance Copper-dependent lytic polysaccharide monooxygenases (LPMOs) are key players in the enzymatic conversion of biomass. LPMOs catalyze oxidative cleavage of glycosidic bonds in a process involving molecular oxygen and an electron donor, such as cellobiose dehydrogenase (CDH). Using protein NMR and isothermal titration calorimetry we have studied the interactions between a fungal LPMO and three soluble substrates and CDH. The results reveal which areas on the LPMO surface interact with the varying substrates and unambiguously show that both the substrate and CDH bind to a region that is centered around the copper site. The data presented here suggest that electron transfer occurs before substrate binding, providing important new leads for understanding the reaction mechanism of LPMOs. Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze oxidative cleavage of glycosidic bonds using molecular oxygen and an external electron donor. We have used NMR and isothermal titration calorimetry (ITC) to study the interactions of a broad-specificity fungal LPMO, NcLPMO9C, with various substrates and with cellobiose dehydrogenase (CDH), a known natural supplier of electrons. The NMR studies revealed interactions with cellohexaose that center around the copper site. NMR studies with xyloglucans, i.e., branched β-glucans, showed an extended binding surface compared with cellohexaose, whereas ITC experiments showed slightly higher affinity and a different thermodynamic signature of binding. The ITC data also showed that although the copper ion alone hardly contributes to affinity, substrate binding is enhanced for metal-loaded enzymes that are supplied with cyanide, a mimic of O2−. Studies with CDH and its isolated heme b cytochrome domain unambiguously showed that the cytochrome domain of CDH interacts with the copper site of the LPMO and that substrate binding precludes interaction with CDH. Apart from providing insights into enzyme–substrate interactions in LPMOs, the present observations shed new light on possible mechanisms for electron supply during LPMO action.

CopY-like copper inducible repressors are putative 'winged helix' proteins

CopY of Enterococcus hirae is a well characterized copper-responsive repressor involved in copper homeostasis. In the absence of copper, it binds to the promoter. In high copper, the CopZ copper chaperone donates copper to CopY, thereby releasing it from the promoter and allowing transcription of the downstream copper homeostatic genes of the cop operon. We here show that the CopY-like repressors from E. hirae, Lactococcus lactis, and Streptococcus mutans have similar affinities not only for their native promoters, but also for heterologous cop promoters. CopZ of L. lactis accelerated the release of CopY from the promoter, suggesting that CopZ of L. lactis acts as copper chaperone, similar to CopZ in E. hirae. The consensus binding motif of the CopY-like repressors was shown to be TACAxxTGTA. The same binding motif is present in promoters controlled by BlaI of Bacillus licheniformis, MecI of Staphylococcus aureus and related repressors. BlaI and MecI have known structures and belong to the family of ‘winged helix’ proteins. In the N- terminal domain, they share significant sequence similarity with CopY of E. hirae. Moreover, they bind to the same TACAxxTGTA motif. NMR analysis of the N-terminal DNA binding domain of CopY of L. lactis showed that it contained the same α-helical content like the same regions of BlaI and MecI. These findings suggest that the DNA binding domains of CopY-like repressors are also of the ‘winged helix’ type.