Dominique P. Frueh

A nuclear receptor-like pathway regulating multidrug resistance in fungi

Multidrug resistance (MDR) is a serious complication during treatment of opportunistic fungal infections that frequently afflict immunocompromised individuals, such as transplant recipients and cancer patients undergoing cytotoxic chemotherapy. Improved knowledge of the molecular pathways controlling MDR in pathogenic fungi should facilitate the development of novel therapies to combat these intransigent infections. MDR is often caused by upregulation of drug efflux pumps by members of the fungal zinc-cluster transcription-factor family (for example Pdr1p orthologues). However, the molecular mechanisms are poorly understood. Here we show that Pdr1p family members in Saccharomyces cerevisiae and the human pathogen Candida glabrata directly bind to structurally diverse drugs and xenobiotics, resulting in stimulated expression of drug efflux pumps and induction of MDR. Notably, this is mechanistically similar to regulation of MDR in vertebrates by the PXR nuclear receptor, revealing an unexpected functional analogy of fungal and metazoan regulators of MDR. We have also uncovered a critical and specific role of the Gal11p/MED15 subunit of the Mediator co-activator and its activator-targeted KIX domain in antifungal/xenobiotic-dependent regulation of MDR. This detailed mechanistic understanding of a fungal nuclear receptor-like gene regulatory pathway provides novel therapeutic targets for the treatment of multidrug-resistant fungal infections.

Dynamic thiolation–thioesterase structure of a non-ribosomal peptide synthetase

Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) produce numerous secondary metabolites with various therapeutic/antibiotic properties. Like fatty acid synthases (FAS), these enzymes are organized in modular assembly lines in which each module, made of conserved domains, incorporates a given monomer unit into the growing chain. Knowledge about domain or module interactions may enable reengineering of this assembly line enzymatic organization and open avenues for the design of new bioactive compounds with improved therapeutic properties. So far, little structural information has been available on how the domains interact and communicate. This may be because of inherent interdomain mobility hindering crystallization, or because crystallized molecules may not represent the active domain orientations. In solution, the large size and internal dynamics of multidomain fragments (>35 kilodaltons) make structure determination by nuclear magnetic resonance a challenge and require advanced technologies. Here we present the solution structure of the apo-thiolation–thioesterase (T–TE) di-domain fragment of the Escherichia coli enterobactin synthetase EntF NRPS subunit. In the holoenzyme, the T domain carries the growing chain tethered to a 4′-phosphopantetheine whereas the TE domain catalyses hydrolysis and cyclization of the iron chelator enterobactin. The T–TE di-domain forms a compact but dynamic structure with a well-defined domain interface; the two active sites are at a suitable distance for substrate transfer from T to TE. We observe extensive interdomain and intradomain motions for well-defined regions and show that these are modulated by interactions with proteins that participate in the biosynthesis. The T–TE interaction described here provides a model for NRPS, PKS and FAS function in general as T–TE-like di-domains typically catalyse the last step in numerous assembly-line chain-termination machineries.

In situ observation of protein phosphorylation by high-resolution NMR spectroscopy

Although the biological significance of protein phosphorylation in cellular signaling is widely appreciated, methods to directly detect these post-translational modifications in situ are lacking. Here we introduce the application of high-resolution NMR spectroscopy for observing de novo protein phosphorylation in vitro and in Xenopus laevis egg extracts and whole live oocyte cells. We found that the stepwise modification of adjacent casein kinase 2 (CK2) substrate sites within the viral SV40 large T antigen regulatory region proceeded in a defined order and through intermediate substrate release. This kinase mechanism contrasts with a more intuitive mode of CK2 action in which the kinase would remain substrate bound to perform both modification reactions without intermediate substrate release. For cellular signaling pathways, the transient availability of partially modified CK2 substrates could exert important switch-like regulatory functions.

Structural basis for the selectivity of the external thioesterase of the surfactin synthetase

Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) found in bacteria, fungi and plants use two different types of thioesterases for the production of highly active biological compounds. Type I thioesterases (TEI) catalyse the release step from the assembly line of the final product where it is transported from one reaction centre to the next as a thioester linked to a 4′-phosphopantetheine (4′-PP) cofactor that is covalently attached to thiolation (T) domains. The second enzyme involved in the synthesis of these secondary metabolites, the type II thioesterase (TEII), is a crucial repair enzyme for the regeneration of functional 4′-PP cofactors of holo-T domains of NRPS and PKS systems. Mispriming of 4′-PP cofactors by acetyl- and short-chain acyl-residues interrupts the biosynthetic system. This repair reaction is very important, because roughly 80% of CoA, the precursor of the 4′-PP cofactor, is acetylated in bacteria. Here we report the three-dimensional structure of a type II thioesterase from Bacillus subtilis free and in complex with a T domain. Comparison with structures of TEI enzymes shows the basis for substrate selectivity and the different modes of interaction of TEII and TEI enzymes with T domains. Furthermore, we show that the TEII enzyme exists in several conformations of which only one is selected on interaction with its native substrate, a modified holo-T domain.

FM reconstruction of non-uniformly sampled protein NMR data at higher dimensions and optimization by distillation

Non-uniform sampling (NUS) enables recording of multidimensional NMR data at resolutions matching the resolving power of modern instruments without using excessive measuring time. However, in order to obtain satisfying results, efficient reconstruction methods are needed. Here we describe an optimized version of the Forward Maximum entropy (FM) reconstruction method, which can reconstruct up to three indirect dimensions. For complex datasets, such as NOESY spectra, the performance of the procedure is enhanced by a distillation procedure that reduces artifacts stemming from intense peaks.

Internal motions in proteins and interference effects in nuclear magnetic resonance

These Ecole polytechnique federale de Lausanne EPFL, n° 2543 (2002)Section de chimieFaculte des sciences de baseInstitut des sciences et ingenierie chimiquesJury: Jonathan Boyd, Hubert Girault, Stephan Grzesiek, Andre Merbach Public defense: 2002-3-6 Reference doi:10.5075/epfl-thesis-2543Print copy in library catalog Record created on 2005-03-16, modified on 2016-08-08

NMR Methods for Studying Protein–Protein Interactions Involved in Translation Initiation

Translation in the cell is carried out by complex molecular machinery involving a dynamic network of protein-protein and protein-RNA interactions. Along the multiple steps of the translation pathway, individual interactions are constantly formed, remodeled, and broken, which presents special challenges when studying this sophisticated system. NMR is a still actively developing technology that has recently been used to solve the structures of several translation factors. However, NMR also has a number of other unique capabilities, of which the broader scientific community may not always be aware. In particular, when studying macromolecular interactions, NMR can be used for a wide range of tasks from testing unambiguously whether two molecules interact to solving the structure of the complex. NMR can also provide insights into the dynamics of the molecules, their folding/unfolding, as well as the effects of interactions with binding partners on these processes. In this chapter, we have tried to summarize, in a popular format, the various types of information about macromolecular interactions that can be obtained with NMR. Special attention is given to areas where the use of NMR provides unique information that is difficult to obtain with other approaches. Our intent was to help the general scientific audience become more familiar with the power of NMR, the current status of the technological limitations of individual NMR methods, as well as the numerous applications, in particular for studying protein-protein interactions in translation.

Effects of Redox Potential and Ca2+ on the Inositol 1,4,5-Trisphosphate Receptor L3-1 Loop Region IMPLICATIONS FOR RECEPTOR REGULATION

Inositol 1,4,5-trisphosphate receptor (IP3R) is a major intracellular Ca2+ channel, modulated by many factors in the cytosolic and lumenal compartments. Compared with cytosolic control, lumenal-side regulation has been much less studied, and some of its mechanistic aspects have been controversial. Of particular interest with regard to lumenal regulation are whether it involves direct interactions between IP3R and the regulators, and whether it involves conformational changes of the lumenal regions of IP3R. To understand these lumenal-side regulation mechanisms, we studied the effects of two important lumenal regulatory factors, the redox potential and Ca2+, on the L3-1 lumenal loop region of IP3R. The redox potential exerted direct and significant effects on the conformation of the loop region. By sharp contrast, Ca2+ showed little effect on the L3-1 conformation, suggesting that the regulation of Ca2+ is indirect or involves other receptor regions. GSH/oxidized glutathione-mediated oxidation introduced a unique intramolecular disulfide bond between Cys34 and Cys42. A variety of NMR experiments revealed that oxidation also induces localized helical characteristics in the Cys34-Cys42 region. Dynamics studies also showed reduced motions in the region upon oxidation, consistent with the conformational changes. The results raise the interesting possibility that Cys34 and Cys42 may act together as a reduction sensor, and that Cys65 may function as an oxidation sensor. Overall, our studies suggest that the redox potential and Ca2+ can regulate IP3R through totally different mechanisms: Ca2+ by the indirect effect and the redox potential by direct action causing conformational changes.

Triple quantum decoherence under multiple refocusing: slow correlated chemical shift modulations of C' and N nuclei in proteins.

A new experiment allows the identification of residues that feature slow conformational exchange in macromolecules. Rotations about dihedral angles that are slower than the global correlation time τc cause a modulation of the isotropic chemical shifts of the nuclei. If these fluctuations are correlated they induce a differential line broadening between three-spin single-quantum and triple-quantum coherences involving three nuclei such as the carbonyl C′, the neighbouring amide nitrogen N and the amide proton HN belonging to a pair of consecutive amino acids. A cross-correlated relaxation rate RCS/CSC′N can be determined that corresponds to the sum of the isotropic and anisotropic contributions to the chemical shift modulations of the carbonyl carbon and nitrogen nuclei. Only the isotropic contributions depend on the pulse repetition rate of a multiple-refocusing sequence. An attenuation of the relaxation rate with increasing pulse repetition rate can therefore be attributed to slow motions. The asparagine N25 residue of ubiquitin, located in the first α-helix, is shown to feature significant slow conformational exchange.

Practical aspects of NMR signal assignment in larger and challenging proteins

NMR has matured into a technique routinely employed for studying proteins in near physiological conditions. However, applications to larger proteins are impeded by the complexity of the various correlation maps necessary to assign NMR signals. This article reviews the data analysis techniques traditionally employed for resonance assignment and describes alternative protocols necessary for overcoming challenges in large protein spectra. In particular, simultaneous analysis of multiple spectra may help overcome ambiguities or may reveal correlations in an indirect manner. Similarly, visualization of orthogonal planes in a multidimensional spectrum can provide alternative assignment procedures. We describe examples of such strategies for assignment of backbone, methyl, and nOe resonances. We describe experimental aspects of data acquisition for the related experiments and provide guidelines for preliminary studies. Focus is placed on large folded monomeric proteins and examples are provided for 37, 48, 53, and 81 kDa proteins.