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.

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Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) produce numerous secondary metabolites with various therapeutic/antibiotic properties 1 . 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 2 . 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.The Escherichia coli enterobactin synthetase (Ent) synthesizes the siderophore enterobactin, a virulence factor used by E. coli to infect iron-limited microenvironments of vertebrate hosts. Enterobactin is obtained by three iterative condensations of dihydroxybenzoate (DHB) with serine, followed by macrocyclizing release of the macrolactone that can form high-affinity hexadentate complexes with ferric iron 3,4 (Fig. 1). Ent is a two-module (EntB/EntE and EntF), three-protein assembly line that combines the features of both a type I NRPS, in which all domains interact in cis in a single protein chain (EntF), and a type II NRPS, in which the (Fig. 1a). The EntE and EntF adenylation (A) domains select the appropriate substrates (DHB or serine) and activate them as acyl-adenosinemonophosphates. These are then loaded on the 4′-PP arms attached to Ser 245 of the holo-EntB aryl carrier protein (ArCP) and to Ser 48 of holo-EntF T domains, respectively (Fig. 1b). The EntF condensation (C) domain catalyses the DHB-Ser amide-bond formation (Fig. 1c). The DHB-Ser chains are next transferre...

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.

Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) found in bacteria, fungi and plants utilize two different types of thioesterases for the production of highly active biological compounds1 , 2. Type I thioesterases (TEI) catalyze the release step from the assembly line3 of the final product where it is transported from one reaction center to the next as a thioester linked to a 4′-phosphopantetheine cofactor (4′-PP) that is covalently attached to thiolation (T) domains4 -9. 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 systems11 -13. Mispriming of 4′-PP cofactors by acetyl-and short chain acylresidues interrupts the biosynthetic system. This repair reaction is very important, since roughly 80% of coenzyme A (CoA), the precursor of the 4′-phosphopantetheine cofactor, is acetylated in bacteria14. Here we report the first three-dimensional structure of a type II thioesterase free and in complex with a T domain. Comparison with structures of TEI enzymes3 , 15 shows the basis for substrate selectivity and the different modes of interaction of TEII and TEI enzymes with T domains. In addition, we show that the TEII enzyme exists in several conformations of which only one is selected upon interaction with its native substrate, a modified holo-T domain. The transport of the growing chain between individual modules is achieved by small ~80 amino acid long T domains that interact with the aminoacyl-forming activation A-domain and both the up-and downstream peptide-bond-forming condensation C-domains 4-10 . In addition, some of the T domains of the Surfactin-synthetase have to interact with epimerization domains, located at the C-terminus of subunits SrfA-A and SrfA-B, or with the covalently linked type I thioesterase 3 . The type I thioesterase catalyzes the macrolactone formation between Leu (7) and the β-hydroxy fatty acid to release the mature surfactin. Modifications blocking the reactive thiol group of the 4′-PP cofactor attached to any T domain can occur with small molecules present in the cell (acetylation, succinylation and modification with fatty acids) and pose a significant challenge for the organism to keep the assembly line running.The surveillance and repair tasks for the surfactin assembly line are carried out by the standalone surfactin type II thioesterase (SrfTEII) 11-13. The importance of this enzyme has been demonstrated by genetic deletions that reduced the production of surfactin by 84%17. Due to the large variety of acylation modifications and the fact that the SrfTEII has to be able to interact with all seven T domains of the entire assembly line, this TEII has to be -in contrast to the type I thioesterase at the end of the last module -rather non-specific. At the same time, premature cleavage of the correct growing peptide chain has to be avoided. In addition to this repair function, the SrfTEII mi...

Nonribosomal peptide synthetases (NRPSs) are microbial enzymes that produce a wealth of important natural products by condensing substrates in an assembly line manner. The proper sequence of substrates is obtained by tethering them to phosphopantetheinyl arms of holo carrier proteins (CPs) via a thioester bond. CPs in holo and substrate-loaded forms visit NRPS catalytic domains in a series of transient interactions. A lack of structural information on substrate-loaded carrier proteins has hindered our understanding of NRPS synthesis. Here, we present the first structure of an NRPS aryl carrier protein loaded with its substrate via a native thioester bond, together with the structure of its holo form. We also present the first quantification of NRPS CP backbone dynamics. Our results indicate that prosthetic moieties in both holo and loaded forms are in contact with the protein core, but they also sample states in which they are disordered and extend in solution. We observe that substrate loading induces a large conformational change in the phosphopantetheinyl arm, thereby modulating surfaces accessible for binding to other domains. Our results are discussed in the context of NRPS domain interactions.

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.

The initial step of protein NMR resonance assignments typically identifies the sequence positions of 1H-15N HSQC cross-peaks. This is usually achieved by tediously comparing strips of multiple triple-resonance experiments. More conveniently, this could be obtained directly with hNcaNH and hNcocaNH-type experiments. However, in large proteins and at very high fields, rapid transverse relaxation severely limits the sensitivity of these experiments, and the limited spectral resolution obtainable in conventionally recorded experiments leaves many assignments ambiguous. We have developed alternative hNcaNH experiments that overcome most of these limitations. The TROSY technique was implemented for semiconstant time evolutions in both indirect dimensions, which results in remarkable sensitivity and resolution enhancements. Non-uniform sampling in both indirect dimensions combined with Maximum Entropy (MaxEnt) reconstruction enables such dramatic resolution enhancement while maintaining short measuring times. Experiments are presented that provide either bidirectional or unidirectional connectivities. The experiments do not involve carbonyl coherences and thus do not suffer from fast chemical shift anisotropy-mediated relaxation otherwise encountered at very high fields. The method was applied to a 300 microM sample of a 37 kDa fragment of the E. coli enterobactin synthetase module EntF, for which high-resolution spectra with an excellent signal-to-noise ratio were obtained within 4 days each.