Structure-Function Analysis of the Acyl Carrier Protein Synthase (AcpS) from Mycobacterium tuberculosis

We have solved the crystal structure of the acyl carrier protein synthase (AcpS) from Mycobacterium tuberculosis (Mtb) at 1.95 Å resolution. AcpS, a 4-phosphopantetheinyl transferase, activates two distinct acyl carrier proteins (ACPs) that are present in fatty acid synthase (FAS) systems FAS-I and FAS-II, the ACP-I domain and the mycobacterial ACP-II protein (ACPM), respectively. Mtb, the causal agent of tuberculosis (TB), and all other members of the Corynebacterineae family are unique in possessing both FAS systems to produce and to elongate fatty acids to mycolic acids, the hallmark of mycobacterial cell wall. Various steps in this process are prime targets for first-line anti-TB agents. A comparison of the Mtb AcpS structure determined here with those of other AcpS proteins revealed unique structural features in Mtb AcpS, namely, the presence of an elongated helix followed by a flexible loop and a moderately electronegative surface unlike the positive surface common to other AcpSs. A structure-based sequence comparison between AcpS and its ACP substrates from various species demonstrated that the proteins of the Corynebacterineae family display high sequence conservation, forming a segregated subgroup of AcpS and ACPs. Analysis of the putative interactions between AcpS and ACPM from Mtb, based on a comparison with the complex structure from Bacillus subtilis, showed that the Mtb AcpS and ACPM lack the electrostatic complementarity observed in B. subtilis. Taken together, the common characteristic of the Corynebacterineae family is likely reflected in the participation of different residues and interactions used for binding the Mtb AcpS to ACP-I and ACPM. The distinct features and essentiality of AcpS, as well as the mode of interaction with ACPM and ACP-I in Mtb, could be exploited for the design of AcpS inhibitors, which, similarly to other inhibitors of fatty acid synthesis, are expected to be effective anti-TB-specific drugs.     

An alternative mechanism for the catalysis of peptide bond formation by L/F transferase: substrate binding and orientation

Eubacterial leucyl/phenylalanyl tRNA protein transferase (L/F transferase) catalyzes the transfer of a leucine or a phenylalanine from an aminoacyl-tRNA to the N-terminus of a protein substrate. This N-terminal addition of an amino acid is analogous to that of peptide synthesis by ribosomes. A previously proposed catalytic mechanism for Escherichia coli L/F transferase identified the conserved aspartate 186 (D186) and glutamine 188 (Q188) as key catalytic residues. We have reassessed the role of D186 and Q188 by investigating the enzymatic reactions and kinetics of enzymes possessing mutations to these active-site residues. Additionally three other amino acids proposed to be involved in aminoacyl-tRNA substrate binding are investigated for comparison. By quantitatively measuring product formation using a quantitative matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based assay, our results clearly demonstrate that, despite significant reduction in enzymatic activity as a result of different point mutations introduced into the active site of L/F transferase, the formation of product is still observed upon extended incubations. Our kinetic data and existing X-ray crystal structures result in a proposal that the critical roles of D186 and Q188, like the other amino acids in the active site, are for substrate binding and orientation and do not directly participate in the chemistry of peptide bond formation. Overall, we propose that L/F transferase does not directly participate in the chemistry of peptide bond formation but catalyzes the reaction by binding and orientating the substrates for reaction in an analogous mechanism that has been described for ribosomes.     

Ligand-Induced Structural Transitions, Mutational Analysis, and ‘Open’ Quaternary Structure of the M. tuberculosis Feast/Famine Regulatory Protein (Rv3291c)

Rv3291c is a member of the feast/famine regulatory protein family that is known to form stable protein-DNA complexes. We report a specific oligomeric transition between hexadecameric and octameric/lower-order oligomers in the presence of Phe that supports an effector-mediated model for the disassembly of a nucleosome-like particle. We had generated two mutants, Gly102Thr and Glu104Ala, which are part of the essential 100-106 effector-binding loop. The Gly102Thr mutant adopts an unusual ‘open’ quaternary structure and offers interesting functional insights co-related to the binding of an effector. This is similar to the previously reported Escherichia coli Lrp co-crystallized in the presence of DNA where the interactions of the substrate with the N-terminal DNA binding domain presumably lead to symmetry deviations to the oligomeric association. The present structure represents a direct evidence to support that changes made to the effector-binding domain at the C-terminus also result in a functionally relevant quaternary structural change. Conversely, the Glu104Ala mutant retains the closed quaternary association observed in the native protein and reveals nonsymmetrical interaction effects in the two subunits of the dimer. We also report that the native protein unexpectedly binds Lys but does not recognize Arg and offer a structural explanation for it. Error-scaled difference distance matrix analysis suggests that the protein has a relatively flexible core that is presumably needed to mediate the structural changes necessary for the protein's regulatory functions.     

Downhill versus Barrier-Limited Folding of BBL 1: Energetic and Structural Perturbation Effects upon Protonation of a Histidine of Unusually Low pKa

A dispersion of melting temperatures at pH 5.3 for individual residues of the BBL protein domain has been adduced as evidence for barrier-free downhill folding. Other members of the peripheral subunit domain family fold cooperatively at pH 7. To search for possible causes of anomalies in BBL's denaturation behavior, we measured the pH titration of individual residues by heteronuclear NMR. At 298 K, the pK_a of His142 was close to that of free histidine at 6.47 ± 0.04, while that of the more buried His166 was highly perturbed at 5.39 ± 0.02. Protonation of His166 is thus energetically unfavorable and destabilizes the protein by ∼ 1.5 kcal/mol. Changes in C^α secondary shifts at pH 5.3 showed a decrease in helicity of the C-terminus of helix 2, where His166 is located, which was accompanied by a measured decrease of 1.1 ± 0.2 kcal/mol in stability from pH 7 to 5.3. Protonation of His166 perturbs, therefore, the structure of BBL. Only ∼ 1% of the structurally perturbed state will be present at the biologically relevant pH 7.6. Experiments at pH 5.3 report on a near-equal mixture of the two different native states. Further, at this pH, small changes of pH and pK_a induced by changes in temperature will have near-maximal effects on pH-dependent conformational equilibria and on propagation of experimental error. Accordingly, conventional barrier-limited folding predicts some dispersion of measured thermal unfolding curves of individual residues at pH 5.3.     

Carbohydrate and Domain Architecture of an Immature Antibody Glycoform Exhibiting Enhanced Effector Functions

Antibodies contain a conserved glycosylation site that has emerged as a target for the modulation of antibody effector functions. The crystal structure of a biosynthetic intermediate of human IgG1, bearing immature oligomannose-type glycans and reported to display increased antibody-dependent cellular cytotoxicity, demonstrates that glycan engineering can bias the Fc to an open conformation primed for receptor binding.     

Oxidative folding and structural analyses of a Kunitz-related inhibitor and its disulfide intermediates: functional implications

Tick-derived protease inhibitor (TdPI) is a tight-binding Kunitz-related inhibitor of human tryptase β with a unique structure and disulfide-bond pattern. Here we analyzed its oxidative folding and reductive unfolding by chromatographic and disulfide analyses of acid-trapped intermediates. TdPI folds through a stepwise generation of heterogeneous populations of one-disulfide, two-disulfide, and three-disulfide intermediates, with a major accumulation of the nonnative three-disulfide species IIIa. The rate-limiting step of the process is disulfide reshuffling within the three-disulfide population towards a productive intermediate that oxidizes directly into the native four-disulfide protein. TdPI unfolds through a major accumulation of the native three-disulfide species IIIb and the subsequent formation of two-disulfide and one-disulfide intermediates. NMR characterization of the acid-trapped and further isolated IIIa intermediate revealed a highly disordered conformation that is maintained by the presence of the disulfide bonds. Conversely, the NMR structure of IIIb showed a native-like conformation, with three native disulfide bonds and increased flexibility only around the two free cysteines, thus providing a molecular basis for its role as a productive intermediate. Comparison of TdPI with a shortened variant lacking the flexible prehead and posthead segments revealed that these regions do not contribute to the protein conformational stability or the inhibition of trypsin but are important for both the initial steps of the folding reaction and the inhibition of tryptase β. Taken together, the results provide insights into the mechanism of oxidative folding of Kunitz inhibitors and pave the way for the design of TdPI variants with improved properties for biomedical applications.     

Chloramphenicol Biosynthesis: The Structure of CmlS, a Flavin-Dependent Halogenase Showing a Covalent Flavin-Aspartate Bond

Chloramphenicol is a halogenated natural product bearing an unusual dichloroacetyl moiety that is critical for its antibiotic activity. The operon for chloramphenicol biosynthesis in Streptomyces venezuelae encodes the chloramphenicol halogenase CmlS, which belongs to the large and diverse family of flavin-dependent halogenases (FDH’s). CmlS was previously shown to be essential for the formation of the dichloroacetyl group. Here we report the X-ray crystal structure of CmlS determined at 2.2 Å resolution, revealing a flavin monooxygenase domain shared by all FDHs, but also a unique ‘winged-helix’ C-terminal domain that creates a T-shaped tunnel leading to the halogenation active site. Intriguingly, the C-terminal tail of this domain blocks access to the halogenation active site, suggesting a structurally dynamic role during catalysis. The halogenation active site is notably nonpolar and shares nearly identical residues with Chondromyces crocatus tyrosyl halogenase (CndH), including the conserved Lys (K71) that forms the reactive chloramine intermediate. The exception is Y350, which could be used to stabilize enolate formation during substrate halogenation. The strictly conserved residue E44, located near the isoalloxazine ring of the bound flavin adenine dinucleotide (FAD) cofactor, is optimally positioned to function as a remote general acid, through a water-mediated proton relay, which could accelerate the reaction of the chloramine intermediate during substrate halogenation, or the oxidation of chloride by the FAD(C4α)-OOH intermediate. Strikingly, the 8α carbon of the FAD cofactor is observed to be covalently attached to D277 of CmlS, a residue that is highly conserved in the FDH family. In addition to representing a new type of flavin modification, this has intriguing implications for the mechanism of FDHs. Based on the crystal structure and in analogy to known halogenases, we propose a reaction mechanism for CmlS.     

Structure and Function of the Oxidoreductase DsbA1 from Neisseria meningitidis

Neisseria meningitidis encodes three DsbA oxidoreductases (NmDsbA1-NmDsbA3) that are vital for the oxidative folding of many membrane and secreted proteins, and these three enzymes are considered to exhibit different substrate specificities. This has led to the suggestion that each N. meningitidis DsbA (NmDsbA) may play a specialized role in different stages of pathogenesis; however, the molecular and structural bases of the different roles of NmDsbAs are unclear. With the aim of determining the molecular basis for substrate specificity and how this correlates to pathogenesis, we undertook a biochemical and structural characterization of the three NmDsbAs. We report the 2.0-Å-resolution crystal structure of the oxidized form of NmDsbA1, which adopted a canonical DsbA fold similar to that observed in the structures of NmDsbA3 and Escherichia coli DsbA (EcDsbA). Structural comparisons revealed variations around the active site and candidate peptide-binding region. Additionally, we demonstrate that all three NmDsbAs are strong oxidases with similar redox potentials; however, they differ from EcDsbA in their ability to be reoxidized by E. coli DsbB. Collectively, our studies suggest that the small structural differences between the NmDsbA enzymes and EcDsbA are functionally significant and are the likely determinants of substrate specificity.     

Physiological sites of deamidation and methyl esterification in sensory transducers of Halobacterium salinarum

In Halobacterium salinarum, up to 18 sensory transducers (Htrs) relay environmental stimuli to an intracellular signaling system to induce tactic responses. As known from the extensively studied enterobacterial system, sensory adaptation to persisting stimulus intensities involves reversible methylation of certain transducer glutamate residues, some of which originate from glutamine residues by deamidation. This study analyzes the in vivo deamidation and methylation of membrane-bound Htrs under physiological conditions. Electrospray ionization tandem mass spectrometry of chromatographically separated proteolytic peptides identified 19 methylation sites in 10 of the 12 predicted membrane-spanning Htrs. Matrix-assisted laser desorption/ionization mass spectrometry additionally detected three sites in two soluble Htrs. Sensory transducers contain a cytoplasmic coiled-coil region, composed of hydrophobic heptads, seven-residue repeats in which the first and the fourth residues are mostly hydrophobic. All identified Htr methylations occurred at glutamate residues at the second and/or third position of such heptads. In addition to singly methylated pairs of glutamate and/or glutamine residues, we identified singly methylated aspartate-glutamate and alanine-glutamate pairs and doubly methylated glutamate pairs. The largest methylatable regions detected in Htrs comprise six heptads along the coiled coil. One methylated glutamate residue was detected outside of such a region, in the signaling region of Htr14. Our analysis produced evidence supporting the predicted methyltransferase and methylesterase activities of halobacterial CheR and CheB, respectively. It furthermore demonstrated that CheB is required for Htr deamidations, at least at a specific glutamine-glutamate pair in Htr2 and a specific aspartate-glutamine pair in Htr4. Compared to previously reported methods, the described approach significantly facilitates the identification of physiological transducer modification sites.     

The Crystal Structure of the [NiFe] Hydrogenase from the Photosynthetic Bacterium Allochromatium vinosum: Characterization of the Oxidized Enzyme (Ni-A State)

The crystal structure of the membrane-associated [NiFe] hydrogenase from Allochromatium vinosum has been determined to 2.1 Å resolution. Electron paramagnetic resonance (EPR) and Fourier transform infrared spectroscopy on dissolved crystals showed that it is present in the Ni-A state (> 90%). The structure of the A. vinosum [NiFe] hydrogenase shows significant similarities with [NiFe] hydrogenase structures derived from Desulfovibrio species. The amino acid sequence identity is ∼ 50%. The bimetallic [NiFe] active site is located in the large subunit of the heterodimer and possesses three diatomic non-protein ligands coordinated to the Fe (two CN⁻ , one CO). Ni is bound to the protein backbone via four cysteine thiolates; two of them also bridge the two metals. One of the bridging cysteines (Cys64) exhibits a modified thiolate in part of the sample. A mono-oxo bridging ligand was assigned between the metal ions of the catalytic center. This is in contrast to a proposal for Desulfovibrio sp. hydrogenases that show a di-oxo species in this position for the Ni-A state. The additional metal site located in the large subunit appears to be a Mg²⁺ ion. Three iron-sulfur clusters were found in the small subunit that forms the electron transfer chain connecting the catalytic site with the molecular surface. The calculated anomalous Fourier map indicates a distorted proximal iron-sulfur cluster in part of the crystals. This altered proximal cluster is supposed to be paramagnetic and is exchange coupled to the Ni³⁺ ion and the medial [Fe₃S₄]⁺ cluster that are both EPR active (S = 1/2 species). This finding of a modified proximal cluster in the [NiFe] hydrogenase might explain the observation of split EPR signals that are occasionally detected in the oxidized state of membrane-bound [NiFe] hydrogenases as from A. vinosum.     

Levansucrases from Pseudomonas syringae pv. tomato and P. chlororaphis subsp. aurantiaca: substrate specificity, polymerizing properties and usage of different acceptors for fructosylation

Levansucrases of Pseudomonas syringae pv. tomato DC3000 (Lsc3) and Pseudomonas chlororaphis subsp. aurantiaca (also Pseudomonas aurantiaca) (LscA) have 73% identity of protein sequences, similar substrate specificity and kinetic properties. Both enzymes produce levan and fructooligosaccharides (FOS) of varied length from sucrose, raffinose and sugar beet molasses. A novel high-throughput chip-based nanoelectrospray mass spectrometric method was applied to screen alternative fructosyl acceptors for levansucrases. Lsc3 and LscA could both transfructosylate d-xylose, d-fucose, l- and d-arabinose, d-ribose, d-sorbitol, xylitol, xylobiose, d-mannitol, d-galacturonic acid and methyl-α-d-glucopyranoside and heterooligofructans with degree of polymerization up to 5 were detected. The ability of d-sorbitol, xylobiose, d-galacturonic acid, d-mannitol, xylitol and methyl-α-d-glucopyranoside to serve as fructosyl acceptors for levansucrases is shown for the first time. Expectedly, site-directed mutagenesis of His321 in Lsc3 to Arg, Lys, Leu and Ser resulted in proteins with decreased catalytic activity, affinity for sucrose and polymerizing ability. Random mutagenesis yielded a Lsc3 mutant Thr302Pro with reduced synthesis of levan and long-chain FOS. Thr302 is located in conserved DQTERP region of levansucrases adjacent to predicted acid-base catalyst Glu303. Thr302 and His321 are predicted to belong to +1 subsite of the substrate binding region of Lsc3.     

10-Å CryoEM Structure and Molecular Model of the Myriapod (Scutigera) 6 × 6mer Hemocyanin: Understanding a Giant Oxygen Transport Protein

Oxygen transport in Myriapoda is maintained by a unique 6 × 6mer hemocyanin, that is, 36 subunits arranged as six hexamers (1 × 6mers). In the sluggish diplopod Spirostreptus, the 1 × 6mers seem to operate as almost or fully independent allosteric units (h ∼ 1.3; P₅₀ ∼ 5 torr), whereas in the swift centipede Scutigera, they intensively cooperate allosterically (h ∼ 10; P₅₀ ∼ 50 torr). Here, we show the chemomechanical basis of this differential behavior as deduced from hybrid 6 × 6mer structures, obtained by single-particle cryo-electron microscopy of the Scutigera 6 × 6mer (10.0 Å resolution according to the 0.5 criterion) and docking of homology-modeled subunits from Scutigera and two diplopods, Spirostreptus and Polydesmus. The Scutigera 6 × 6mer hemocyanin is a trigonal antiprism assembled from six smaller trigonal antiprisms (1 × 6mers), thereby exhibiting D3 point group symmetry. It can be described as two staggered 3 × 6mers or three oblique 2 × 6mers. Topologically, the 6 × 6mer is subdivided into six subunit zones, thereby exhibiting a mantle (24 subunits) and a core (12 subunits). The six hexamers are linked by 21 bridges, subdivided into five types: two within each 3 × 6mer and three between both 3 × 6mers. The molecular models of the 6 × 6mer reveal intriguing amino acid appositions at these inter-hexamer interfaces. Besides opportunities for salt bridges, we found pairs of carboxylate residues for possible bridging via a Ca²⁺ or Mg²⁺ ion. Moreover, we detected histidine clusters, notably in Scutigera, allowing us to advance hypotheses as to how the hexamers are allosterically coupled in centipede hemocyanin and why they act more independently in diplopod hemocyanin.     

Characterization of Plasmodium falciparum 6-Phosphogluconate Dehydrogenase as an Antimalarial Drug Target

The enzyme 6-phosphogluconate dehydrogenase (6PGD) of the malaria parasite Plasmodium falciparum catalyzes the third step of the pentose phosphate pathway converting 6-phosphogluconate (6PG) to ribulose 5-phosphate. The NADPH produced by 6PGD is crucial for antioxidant defense and redox regulation, and ribose 5-phosphate is essential for DNA and RNA synthesis in the rapidly growing parasite. Thus, 6PGD represents an attractive antimalarial drug target. In this study, we present the X-ray structures of Pf6PGD in native form as well as in complex with 6PG or nicotinamide adenine dinucleotide phosphate (NADP⁺) at resolutions of 2.8, 1.9, and 2.9?Å, respectively. The overall structure of the protein is similar to structures of 6PGDs from other species; however, a flexible loop close to the active site rearranges upon binding of 6PG and likely regulates the conformation of the cofactor NADP⁺. Upon binding of 6PG, the active site loop adopts a closed conformation. In the absence of 6PG, the loop opens and NADP⁺ is bound in a waiting position, indicating that the cofactor and 6PG bind independently from each other. This sequential binding mechanism was supported by kinetic studies on the homodimeric wild-type Pf6PGD. Furthermore, the function of the Plasmodium-specific residue W104L mutant was characterized by site-directed mutagenesis. Notably, the activity of Pf6PGD was found to be post-translationally redox regulated via S-nitrosylation, and screening the Medicines for Malaria Venture Malaria Box identified several compounds with IC₅₀s in the low micromolar range. Together with the three-dimensional structure of the protein, this is a promising starting point for further drug discovery approaches.     

Chemoenzymatic Synthesis, Inhibition Studies, and X-ray Crystallographic Analysis of the Phosphono Analog of UDP-Galp as an Inhibitor and Mechanistic Probe for UDP-Galactopyranose Mutase

UDP (uridine diphosphate) galactopyranose mutase (UGM) is involved in the cell wall biosynthesis of many pathogenic microorganisms. UGM catalyzes the reversible conversion of UDP-α-d-galactopyranose into UDP-α-d-galactofuranose, with the latter being the precursor of galactofuranose (Galf) residues in cell walls. Glycoconjugates of Galf are essential components in the cell wall of various pathogenic bacteria, including Mycobacterium tuberculosis, the causative agent of tuberculosis. The absence of Galf in humans and its bacterial requirement make UGM a potential target for developing novel antibacterial agents. In this article, we report the synthesis, inhibitory activity, and X-ray crystallographic studies of UDP-phosphono-galactopyranose, a nonhydrolyzable C-glycosidic phosphonate. This is the first report on the synthesis of a phosphonate analog of UDP-α-d-galactopyranose by a chemoenzymatic phosphoryl coupling method. The phosphonate was evaluated against three bacterial UGMs and showed only moderate inhibition. We determined the crystal structure of the phosphonate analog bound to Deinococcus radiodurans UGM at 2.6 Å resolution. The phosphonate analog is bound in a novel conformation not observed in UGM-substrate complex structures or in other enzyme-sugar nucleotide phosphonate complexes. This complex structure provides a structural basis for the observed micromolar inhibition towards UGM. Steric clashes, loss of electrostatic stabilization between an active-site arginine (Arg305) and the phosphonate analog, and a 180° flip of the hexose moiety account for the differences in the binding orientations of the isosteric phosphonate analog and the physiological substrate. This provides new insight into the ability of a sugar-nucleotide-binding enzyme to orient a substrate analog in an unexpected geometry and should be taken into consideration in designing such enzyme inhibitors.