Peptidoglycan glycosyltransferase-ligand binding assay based on tryptophan fluorescence quenching.

Peptidoglycan glycosyltransferases (GTase) of family 51 are essential enzymes for the synthesis of the glycan chains of the bacterial cell wall. They are considered potential antibacterial target, but discovery of inhibitors was hampered so far by the lack of efficient and affordable screening assay. Here we used Staphylococcus aureus MtgA to introduce a single tryptophan reporter residue in selected positions flanking the substrates binding cavity of the protein. We selected a mutant (Y181W) that shows strong fluorescence quenching in the presence of moenomycin A and two lipid II analogs inhibitors. The assay provides a simple method to study GTase-ligand interactions and can be used as primary high throughput screening of GTase inhibitors without the need for lipid II substrate or reporter ligands.

The Role of Active Site Flexible Loops in Catalysis and of Zinc in Conformational Stability of Bacillus cereus 569/H/9 ß-Lactamase.

Metallo-ß-lactamases catalyze the hydrolysis of most ß-lactam antibiotics and hence represent a major clinical concern. The development of inhibitors for these enzymes is complicated by the diversity and flexibility of their substrate-binding sites, motivating research into their structure and function. In this study, we examined the conformational properties of the Bacillus cereus ß-lactamase II in the presence of chemical denaturants using a variety of biochemical and biophysical techniques. The apoenzyme was found to unfold cooperatively, with a Gibbs free energy of stabilization (ΔG(0)) of 32 ± 2 kJ·mol(-1) For holoBcII, a first non-cooperative transition leads to multiple interconverting native-like states, in which both zinc atoms remain bound in an apparently unaltered active site, and the protein displays a well organized compact hydrophobic core with structural changes confined to the enzyme surface, but with no catalytic activity. Two-dimensional NMR data revealed that the loss of activity occurs concomitantly with perturbations in two loops that border the enzyme active site. A second cooperative transition, corresponding to global unfolding, is observed at higher denaturant concentrations, with ΔG(0) value of 65 ± 1.4 kJ·mol(-1) These combined data highlight the importance of the two zinc ions in maintaining structure as well as a relatively well defined conformation for both active site loops to maintain enzymatic activity.

Accessibility changes within diphtheria toxin T domain upon membrane penetration probed by hydrogen exchange and mass spectrometry.

The translocation domain of diphtheria toxin inserts in membrane and becomes functional when the pH inside endosomes is acid. At that stage, the domain is in a partially folded state; this prevents the use of high-resolution methods for the characterization of its functional structure. On that purpose, we report here the use of hydrogen/deuterium exchange experiments coupled to mass spectrometry. The conformation changes during the different steps of insertion into lipid bilayer are monitored with a resolution of few residues. Three parts of the translocation domain can be distinguished. With a high protection against exchange, the C-terminal hydrophobic helical hairpin is embedded in the membrane. Despite a lower protection, a significant effect in the presence of lipid vesicles shows that the N-terminal part is in interaction with the membrane interface. The sensitivity to the ionic strength indicates that electrostatic interactions are important for the binding. The middle part of the domain has an intermediate protection; this suggests that this part of the domain can be embedded within the membrane but remains quite dynamic. These results provide unprecedented insight into the structure reorganization of the protein to go from a soluble state to a membrane-inserted one.

Translocators YopB and YopD from Yersinia enterocolitica form a multimeric integral membrane complex in eukaryotic cell membranes.

The type III secretion systems are contact-activated secretion systems that allow bacteria to inject effector proteins across eukaryotic cell membranes. The secretion apparatus, called injectisome or needle complex, includes a needle that terminates with a tip structure. The injectisome exports its own distal components, like the needle subunit and the needle tip. Upon contact, it exports two hydrophobic proteins called translocators (YopB and YopD in Yersinia enterocolitica) and the effectors. The translocators, assisted by the needle tip, form a pore in the target cell membrane, but the structure of this pore remains elusive. Here, we purified the membranes from infected sheep erythrocytes, and we show that they contain integrated and not simply adherent YopB and YopD. In blue native PAGE, these proteins appeared as a multimeric 500- to 700-kDa complex. This heteropolymeric YopBD complex could be copurified after solubilization in 0.5% dodecyl maltoside but not visualized in the electron microscope. We speculate that this complex may not be stable and rigid but only transient.

Accessibility changes within diphtheria toxin T domain when in the functional molten globule state, as determined using hydrogen/deuterium exchange measurements.

The translocation domain (T domain) of diphtheria toxin adopts a partially folded state, the so-called molten globule state, to become functional at acidic pH. We compared, using hydrogen/deuterium exchange experiments associated with MS, the structures of the T domain in its soluble folded state at neutral pH and in its functional molten globule state at acidic pH. In the native state, the alpha-helices TH5 and TH8 are identified as the core of the domain. Based on the high-resolution structure of the T domain, we propose that TH8 is highly protected because it is buried within the native structure. According to the same structure, TH5 is partly accessible at the surface of the T domain. We propose that its high protection is caused by the formation of dimers. Within the molten globule state, high protection is still observed within the helical hairpin TH8-TH9, which is responsible for the insertion of the T domain into the membrane. In the absence of the lipid bilayer, this hydrophobic part of the domain self-assembles, leading to the formation of oligomers. Overall, hydrogen/deuterium-exchange measurements allow the analysis of interaction contacts within small oligomers made of partially folded proteins. Such information, together with crystal structure data, are particularly valuable for using to analyze the self-assembly of proteins.

Membrane Interaction of botulinum neurotoxin A translocation (T) domain. The belt region is a regulatory loop for membrane interaction.

The translocation of the catalytic domain through the membrane of the endosome to the cell cytoplasm is a key step of intoxication by botulinum neurotoxin (BoNT). This step is mediated by the translocation (T) domain upon endosome acidification, although the mechanism of interaction of the T domain with the membrane is still poorly understood. Using physicochemical approaches and spectroscopic methods, we studied the interaction of the BoNT/A T domain with the membrane as a function of pH. We found that the interaction with membranes does not involve major secondary or tertiary structural changes, as reported for other toxins like diphtheria toxin. The T domain becomes insoluble around its pI value and then penetrates into the membrane. At that stage, the T domain becomes able to permeabilize lipid vesicles. This occurs for pH values lower than 5.5, in agreement with the pH encountered by the toxin within endosomes. Electrostatic interactions are also important for the process. The role of the so-called belt region was investigated with four variant proteins presenting different lengths of the N-extremity of the T domain. We observed that this part of the T domain, which contains numerous negatively charged residues, limits the protein-membrane interaction. Indeed, interaction with the membrane of the protein deleted of this extremity takes place for higher pH values than for the entire T domain. Overall, the data suggest that acidification eliminates repulsive electrostatic interactions between the T domain and the membrane, allowing its penetration into the membrane without triggering detectable structural changes.

Defining the interacting regions between apomyoglobin and lipid membrane by hydrogen/deuterium exchange coupled to mass spectrometry.

Sperm whale myoglobin can be considered as the model protein of the globin family. The pH-dependence of the interactions of apomyoglobin with lipid bilayers shares some similarities with the behavior of pore-forming domains of bacterial toxins belonging also to the globin family. Two different states of apomyoglobin bound to a lipid bilayer have been characterized by using hydrogen/deuterium exchange experiments and mass spectrometry. When bound to the membrane at pH 5.5, apomyoglobin remains mostly native-like and interacts through alpha-helix A. At pH 4, the binding is related to the stabilization of a partially folded state. In that case, alpha-helices A and G are involved in the interaction. At this pH, alpha-helix G, which is the most hydrophobic region of apomyoglobin, is available for interaction with the lipid bilayer because of the loss of the tertiary structure. Our results show the feasibility of such experiments and their potential for the characterization of various membrane-bound states of amphitropic proteins such as pore-forming domains of bacterial toxins. This is not possible with other high-resolution methods, because these proteins are usually in partially folded states when interacting with membranes.

Interactions of apomyoglobin with membranes: mechanisms and effects on heme uptake.

The last step of the folding reaction of myoglobin is the incorporation of a prosthetic group. In cells, myoglobin is soluble, while heme resides in the mitochondrial membrane. We report here an exhaustive study of the interactions of apomyoglobin with lipid vesicles. We show that apomyoglobin interacts with large unilamellar vesicles under acidic conditions, and that this requires the presence of negatively charged phospholipids. The pH dependence of apomyoglobin interactions with membranes is a two-step process, and involves a partially folded state stabilized at acidic pH. An evident role for the interaction of apomyoglobin with lipid bilayers would be to facilitate the uptake of heme from the outer mitochondrial membrane. However, heme binding to apomyoglobin is observed at neutral pH when the protein remains in solution, and slows down as the pH becomes more favorable to membrane interactions. The effective incorporation of soluble heme into apomyoglobin at neutral pH suggests that the interaction of apomyoglobin with membranes is not necessary for the heme uptake from the lipid bilayer. In vivo, however, the ability of apomyoglobin to interact with membrane may facilitate its localization in the vicinity of the mitochondrial membranes, and so may increase the yield of heme uptake. Moreover, the behavior of apomyoglobin in the presence of membranes shows striking similarities with that of other proteins with a globin fold. This suggests that the globin fold is well adapted for soluble proteins whose functions require interactions with membranes.

Behavior of the N-terminal helices of the diphtheria toxin T domain during the successive steps of membrane interaction.

During intoxication of a cell, the translocation (T) domain of the diphtheria toxin helps the passage of the catalytic domain across the membrane of the endosome into the cytoplasm. We have investigated the behavior of the N-terminal region of the T domain during the successive steps of its interaction with membranes at acidic pH using tryptophan fluorescence, its quenching by brominated lipids, and trypsin digestion. The change in the environment of this region was monitored using mutant W281F carrying a single native tryptophan at position 206 at the tip of helix TH1. The intrinsic propensity to interact with the membrane of each helix of the N-terminus of the T domain, TH1, TH2, TH3, and TH4, was also studied using synthetic peptides. We showed the N-terminal region of the T domain was not involved in the binding of the domain to the membrane, which occurred at pH 6 mainly through hydrophobic effects. At that stage of the interaction, the N-terminal region remained strongly solvated. Further acidification eliminated repulsive electrostatic interactions between this region and the membrane, allowing its penetration into the membrane by attractive electrostatic interactions and hydrophobic effects. The peptide study indicated the nature of forces contributing to membrane penetration. Overall, the data suggested that the acidic pH found in the endosome not only triggers the formation of the molten globule state of the T domain required for membrane interaction but also governs a progressive penetration of the N-terminal part of the T domain in the membrane. We propose that these physicochemical properties are necessary for the translocation of the catalytic domain.