Multidrug-resistant (MDR) gram-negative bacteria have increased the prevalence of fatal sepsis in modern times. Colistin is a cationic antimicrobial peptide (CAMP) antibiotic that permeabilizes the bacterial outer membrane (OM) and has been used to treat these infections. The OM outer leaflet is comprised of endotoxin containing lipid A, which can be modified to increase resistance to CAMPs and prevent clearance by the innate immune response. One type of lipid A modification involves the addition of phosphoethanolamine to the 1 and 4′ headgroup positions by phosphoethanolamine transferases. Previous structural work on a truncated form of this enzyme suggested that the full-length protein was required for correct lipid substrate binding and catalysis. We now report the crystal structure of a full-length lipid A phosphoethanolamine transferase from Neisseria meningitidis, determined to 2.75-Å resolution. The structure reveals a previously uncharacterized helical membrane domain and a periplasmic facing soluble domain. The domains are linked by a helix that runs along the membrane surface interacting with the phospholipid head groups. Two helices located in a periplasmic loop between two transmembrane helices contain conserved charged residues and are implicated in substrate binding. Intrinsic fluorescence, limited proteolysis, and molecular dynamics studies suggest the protein may sample different conformational states to enable the binding of two very different-sized lipid substrates. These results provide insights into the mechanism of endotoxin modification and will aid a structure-guided rational drug design approach to treating multidrug-resistant bacterial infections.lipid modification | multidrug resistance | molecular dynamics | Neisseria | membrane protein structure

Anthranilate phosphoribosyltransferase (AnPRT, EC 2.4.2.18) is a homodimeric enzyme that catalyzes the reaction between 5'-phosphoribosyl 1'-pyrophosphate (PRPP) and anthranilate, as part of the tryptophan biosynthesis pathway. Here we present the results of the first chemical screen for inhibitors against Mycobacterium tuberculosis AnPRT (Mtb-AnPRT), along with crystal structures of Mtb-AnPRT in complex with PRPP and several inhibitors. Previous work revealed that PRPP is bound at the base of a deep cleft in Mtb-AnPRT and predicted two anthranilate binding sites along the tunnel leading to the PRPP binding site. Unexpectedly, the inhibitors presented here almost exclusively bound at the entrance of the tunnel, in the presumed noncatalytic anthranilate binding site, previously hypothesized to have a role in substrate capture. The potencies of the inhibitors were measured, yielding Ki values of 1.5-119 μM, with the strongest inhibition displayed by a bianthranilate compound that makes hydrogen bond and salt bridge contacts with Mtb-AnPRT via its carboxyl groups. Our results reveal how the substrate capture mechanism of AnPRT can be exploited to inhibit the enzyme's activity and provide a scaffold for the design of improved Mtb-AnPRT inhibitors that may ultimately form the basis of new antituberculosis drugs with a novel mode of action.

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Fragile X syndrome, the most common form of inherited mental retardation, is the result of an unstable expansion of a CGG trinucleotide repeat in the 5' UTR of the fragile X mental retardation-1 (FMR1) gene. The abnormal hypermethylation of the expanded CGG repeats causes the transcriptional silencing of the FMR1 gene and, consequently, the loss of the fragile X mental retardation protein (FMRP). FMRP is an RNA binding protein that binds to G quartet forming RNA using its RGG box motif. In this study we have performed a thermodynamic analysis of the interactions between the FMRP RGG box domain and Sc1, an RNA molecule which had been previously shown to be bound with high affinity by both the full-length FMRP and by its RGG box domain. We have determined that the association between the FMRP RGG box and Sc1 RNA is dominated by hydrophobic and hydrogen bond interactions, with minor contributions from electrostatic interactions, and that the FMRP RGG box binding increases the stability of the G quartet RNA structure significantly. Interestingly, we found that the G quartet recognition is necessary but not sufficient for the FMRP RGG box binding to this RNA target, indicating that additional interactions of the peptide, possibly with the stem and/or stem-G quartet junction region, are required. Our results also indicate that the G quartet RNA recognition is not a general feature of the RGG box motif but rather carries some sequence, protein and/or RNA, specificity.

AnPRT (anthranilate phosphoribosyltransferase), required for the biosynthesis of tryptophan, is essential for the virulence of Mycobacterium tuberculosis (Mtb). AnPRT catalyses the Mg2+-dependent transfer of a phosphoribosyl group from PRPP (5'-phosphoribosyl-1'-pyrophosphate) to anthranilate to form PRA (5'-phosphoribosyl anthranilate). Mtb-AnPRT was shown to catalyse a sequential reaction and significant substrate inhibition by anthranilate was observed. Antimycobacterial fluoroanthranilates and methyl-substituted analogues were shown to act as alternative substrates for Mtb-AnPRT, producing the corresponding substituted PRA products. Structures of the enzyme complexed with anthranilate analogues reveal two distinct binding sites for anthranilate. One site is located over 8 Å (1 Å=0.1 nm) from PRPP at the entrance to a tunnel leading to the active site, whereas in the second, inner, site anthranilate is adjacent to PRPP, in a catalytically relevant position. Soaking the analogues for variable periods of time provides evidence for anthranilate located at transient positions during transfer from the outer site to the inner catalytic site. PRPP and Mg2+ binding have been shown to be associated with the rearrangement of two flexible loops, which is required to complete the inner anthranilate-binding site. It is proposed that anthranilate first binds to the outer site, providing an unusual mechanism for substrate capture and efficient transfer to the catalytic site following the binding of PRPP.

The three-dimensional structure of the enzyme dihydrodipicolinate synthase (KEGG entry Rv2753c, EC 4.2.1.52) from Mycobacterium tuberculosis (Mtb-DHDPS) was determined and refined at 2.28 A (1 A=0.1 nm) resolution. The asymmetric unit of the crystal contains two tetramers, each of which we propose to be the functional enzyme unit. This is supported by analytical ultracentrifugation studies, which show the enzyme to be tetrameric in solution. The structure of each subunit consists of an N-terminal (beta/alpha)(8)-barrel followed by a C-terminal alpha-helical domain. The active site comprises residues from two adjacent subunits, across an interface, and is located at the C-terminal side of the (beta/alpha)(8)-barrel domain. A comparison with the other known DHDPS structures shows that the overall architecture of the active site is largely conserved, albeit the proton relay motif comprising Tyr(143), Thr(54) and Tyr(117) appears to be disrupted. The kinetic parameters of the enzyme are reported: K(M)(ASA)=0.43+/-0.02 mM, K(M)(pyruvate)=0.17+/-0.01 mM and V(max)=4.42+/-0.08 micromol x s(-1) x mg(-1). Interestingly, the V(max) of Mtb-DHDPS is 6-fold higher than the corresponding value for Escherichia coli DHDPS, and the enzyme is insensitive to feedback inhibition by (S)-lysine. This can be explained by the three-dimensional structure, which shows that the (S)-lysine-binding site is not conserved in Mtb-DHDPS, when compared with DHDPS enzymes that are known to be inhibited by (S)-lysine. A selection of metabolites from the aspartate family of amino acids do not inhibit this enzyme. A comprehensive understanding of the structure and function of this important enzyme from the (S)-lysine biosynthesis pathway may provide the key for the design of new antibiotics to combat tuberculosis.

The emergence of extensively drug-resistant strains of Mycobacterium tuberculosis (Mtb) highlights the need for new therapeutics to treat tuberculosis. We are attempting to fast-track a targeted approach to drug design by generating analogues of a validated hit from molecular library screening that shares its chemical scaffold with a current therapeutic, the anti-arthritic drug Lobenzarit (LBZ). Our target, anthranilate phosphoribosyltransferase (AnPRT), is an enzyme from the tryptophan biosynthetic pathway in Mtb. A bifurcated hydrogen bond was found to be a key feature of the LBZ-like chemical scaffold and critical for enzyme inhibition. We have determined crystal structures of compounds in complex with the enzyme that indicate that the bifurcated hydrogen bond assists in orientating compounds in the correct conformation to interact with key residues in the substrate-binding tunnel of Mtb-AnPRT. Characterising the inhibitory potency of the hit and its analogues in different ways proved useful, due to the multiple substrates and substrate binding sites of this enzyme. Binding in a site other than the catalytic site was found to be associated with partial inhibition. An analogue, 2-(2-5-methylcarboxyphenylamino)-3-methylbenzoic acid, that bound at the catalytic site and caused complete, rather than partial, inhibition of enzyme activity was found. Therefore, we designed and synthesised an extended version of the scaffold on the basis of this observation. The resultant compound, 2,6-bis-(2-carboxyphenylamino)benzoate, is a 40-fold more potent inhibitor of the enzyme than the original hit and provides direction for further structure-based drug design.

Pentameric ligand-gated ion channels (pLGICs) or Cys-loop receptors are involved in fast synaptic signaling in the nervous system. Allosteric modulators bind to sites that are remote from the neurotransmitter binding site, but modify coupling of ligand binding to channel opening. In this study, we developed nanobodies (single domain antibodies), which are functionally active as allosteric modulators, and solved co-crystal structures of the prokaryote (Erwinia) channel ELIC bound either to a positive or a negative allosteric modulator. The allosteric nanobody binding sites partially overlap with those of small molecule modulators, including a vestibule binding site that is not accessible in some pLGICs. Using mutagenesis, we extrapolate the functional importance of the vestibule binding site to the human 5-HT3 receptor, suggesting a common mechanism of modulation in this protein and ELIC. Thus we identify key elements of allosteric binding sites, and extend drug design possibilities in pLGICs with an accessible vestibule site.