Abstract The World Health Organization has identified antibiotic resistance as one of the three greatest threats to human health. The need for antibiotics is a pressing matter that requires immediate attention. Here, computer-aided drug design is used to develop a structurally unique antibiotic family targeting holo-acyl carrier protein synthase (AcpS). AcpS is a highly conserved enzyme essential for bacterial survival that catalyzes the first step in lipid synthesis. To the best of our knowledge, there are no current antibiotics targeting AcpS making this drug development program of high interest. We synthesize a library of > 700 novel compounds targeting AcpS, from which 33 inhibit bacterial growth in vitro at ≤ 2 μg/mL. We demonstrate that compounds from this class have stand-alone activity against a broad spectrum of Gram-positive organisms and synergize with colistin to enable coverage of Gram-negative species. We demonstrate efficacy against clinically relevant multi-drug resistant strains in vitro and in animal models of infection in vivo including a difficult-to-treat ischemic infection exemplified by diabetic foot ulcer infections in humans. This antibiotic family could form the basis for several multi-drug-resistant antimicrobial programs.

The protozoan parasite Plasmodium falciparum is responsible for the most lethal form of malaria and kills about one million people annually, most of whom are children in sub‐Saharan Africa. Fatty acid synthesis (FAS) is a validated antimalarial strategy: it occurs in a specialized parasite organelle (the apicoplast) and uses a dissociated type II FAS system similar to bacteria. Bioinformatics analysis indicates that P. falciparum encodes a single enzyme, holo‐ACP synthase (PfAcpS), responsible for activating acyl carrier protein (ACP) involved in FAS. PfAcpS consists of a signal/leader peptide, a large N‐terminal domain of unknown function, and a C‐terminal domain (PfAcpS‐C) that is highly homologous (30–50% similarity) to trimeric bacterial AcpS enzymes. We have expressed and isolated the soluble PfAcpS‐C domain and shown that it catalyzes the transfer of (acyl)phosphopantetheine to PfACP from coenzyme A or from its derivatives acetyl‐CoA and octanoyl CoA. PfAcpS exhibited much lower activity than E. coli AcpS and preferred higher concentrations of CoA derivatives. Several novel inhibitors of E. coli AcpS also blocked PfAcpS activity, although the degree of inhibition differed between the enzymes. The sequence similarity between PfAcpS‐C and bacterial AcpS enzymes suggests that drugs targeting the latter might also inhibit P. falciparum growth and pathogenesis. (Supported by NSERC)

As an essential protein in bacterial fatty acid synthesis, acyl carrier protein (ACP) is an attractive anti‐microbial drug target. The three‐dimensional structure of ACP is comprised of three main parallel α–helices, which create a hydrophobic binding pocket that encloses a fatty acyl chain when attached. ACP interacts with most partner enzymes through the acidic helix II, but the exact mechanism of interaction and acyl transfer to a partner enzyme is poorly understood. For acyl transfer to occur, we hypothesize that a conformational change is required. To examine this requirement, we have analyzed a cyclic version of Vibrio harveyi ACP created by split‐intein technology (Volkmann, et al. (2010) JBC 285: 8605) using molecular dynamics, mass spectrometry, tryptophan fluorescence, and other biophysical techniques. We have demonstrated that cyclic ACP is more thermodynamically stable than wild‐type linear ACP. Furthermore, we show that cyclic ACP is able to act as a substrate for holo‐ACP synthase, yielding a cyclic ACP capable of carrying fatty acids. (Supported by NSERC and an NSHRF Student Research Award)

Bacterial acyl carrier protein (ACP) is essential for the synthesis of fatty acids and serves as the major acyl donor for the formation of phospholipids and other lipid products. Acyl-ACP encloses attached fatty acyl groups in a hydrophobic pocket within a four-helix bundle, but must at least partially unfold to present the acyl chain to the active sites of its multiple enzyme partners. To further examine the constraints of ACP structure and function, we have constructed a cyclic version of Vibrio harveyi ACP, using split-intein technology to covalently join its closely apposed N and C termini. Cyclization stabilized ACP in a folded helical conformation as indicated by gel electrophoresis, circular dichroism, fluorescence, and mass spectrometry. Molecular dynamics simulations also indicated overall decreased polypeptide chain mobility in cyclic ACP, although no major conformational rearrangements over a 10-ns period were noted. In vivo complementation assays revealed that cyclic ACP can functionally replace the linear wild-type protein and support growth of an Escherichia coli ACP-null mutant strain. Cyclization of a folding-deficient ACP mutant (F50A) both restored its ability to adopt a folded conformation and enhanced complementation of growth. Our results thus suggest that ACP must be able to adopt a folded conformation for biological activity, and that its function does not require complete unfolding of the protein.

Bacterial acyl carrier protein (ACP) is a highly anionic, 9 kDa protein that functions as a cofactor protein in fatty acid biosynthesis. Escherichia coli ACP is folded at neutral pH and in the absence of divalent cations, while Vibrio harveyi ACP, which is very similar at 86% sequence identity, is unfolded under the same conditions. V. harveyi ACP adopts a folded conformation upon the addition of divalent cations such as Ca(2+) and Mg(2+) and a mutant, A75H, was previously identified that restores the folded conformation at pH 7 in the absence of divalent cations. In this study we sought to understand the unique folding behavior of V. harveyi ACP using NMR spectroscopy and biophysical methods. The NMR solution structure of V. harveyi ACP A75H displays the canonical ACP structure with four helices surrounding a hydrophobic core, with a narrow pocket closed off from the solvent to house the acyl chain. His-75, which is charged at neutral pH, participates in a stacking interaction with Tyr-71 in the far C-terminal end of helix IV. pH titrations and the electrostatic profile of ACP suggest that V. harveyi ACP is destabilized by anionic charge repulsion around helix II that can be partially neutralized by His-75 and is further reduced by divalent cation binding. This is supported by differential scanning calorimetry data which indicate that calcium binding further increases the melting temperature of V. harveyi ACP A75H by ∼20 °C. Divalent cation binding does not alter ACP dynamics on the ps-ns timescale as determined by (15)N NMR relaxation experiments, however, it clearly stabilizes the protein fold as observed by hydrogen-deuterium exchange studies. Finally, we demonstrate that the E. coli ACP H75A mutant is similarly unfolded as wild-type V. harveyi ACP, further stressing the importance of this particular residue for proper protein folding.

Insulin stimulates phosphorylation cascades, including phosphatidylinositol-3-kinase (PI3K), phosphatidylinositol-dependent kinase (PDK1), Akt, and protein kinase C (PKC). Myristoylated alanine-rich C-kinase substrate (MARCKS), a PKCβII substrate, could link the effects of insulin to insulin-stimulated glucose transport (ISGT) via phosphorylation of its effector domain since MARCKS has a role in cytoskeletal rearrangements.

Acyl carrier protein (ACP) is an essential protein in bacterial metabolism, where it acts as the major donor for fatty acyl groups in the synthesis of lipids and other specialized products. As such, ACP is an attractive anti‐microbial drug target. ACP normally encloses attached fatty acyl groups in a hydrophobic pocket within its three mainα‐helices, but the exact mechanism of interaction and acyl transfer from ACP to partner enzymes is poorly understood. ACP interacts with most proteins through its acidic helix II, and acyl transfer must involve partial or full unfolding of ACP in order to present the acyl chain to the partner enzyme. To examine the requirement of ACP unfolding for acyl transfer, we created a cyclic version of Vibrio harveyi ACP using split‐intein technology. Mass spectrometry, circular dichroism and fluorescence studies suggest that cyclic ACP (cACP) is trapped in a folded conformation. In vitro characterization indicates that cACP can be modified by holo‐ACP synthetase yielding an active ACP. Furthermore, in vivo complementation assays suggest that cACP is able to replace the linear wild‐type ACP and support growth. Our results thus suggest that cACP is able to interact with and transfer its acyl chain to its many partner enzymes within the bacterial cell despite constraining of its N‐ and C‐termini. (Supported by CIHR, NSERC, and an IWK Studentship).

Acyl carrier protein (ACP) plays an essential role in the synthesis of bacterial lipids, and is an attractive target for the development of new antibiotic classes. ACP also forms numerous interactions with proteins involved in other cellular processes that have yet to be fully identified or characterized. Using a C‐terminal Tandem Affinity Purification (TAP)‐tagged ACP we identified two ACP binding partners not directly involved in lipid metabolism, AidB and SpoT, that both exhibit increased interaction with ACP in stationary phase. The increase in binding of SpoT, a synthetase and hydrolase of the alarmone guanidine ‐ 5' (tri)diphosphate ‐3'diphosphate, to ACP appears to be due to increased affinity between the two proteins as cells enter stationary phase. In contrast, the increased interaction with AidB, an adaptive response protein, results from elevation in the level of AidB as cells enter stationary phase. AidB levels are also increased upon exposure of cells to alkylating agents. AidB binds DNA directly and we hypothesize that the positively charged DNA binding region of AidB also binds to the negatively charged ACP. AidB binding to ACP may have important functional consequences in DNA repair. To test this, ACP‐AidB protein‐protein interaction domains are being mapped, and we are determining if AidB‐ACP interactions regulate the ability of the cell to respond to DNA damaging agents. (Supported by NSERC)

We have introduced tryptophan as a local fluorescent probe to monitor the conformation of Vibrio harveyi acyl carrier protein (ACP), a small flexible protein that is unfolded at neutral pH but must undergo reversible conformational change during the synthesis and delivery of bacterial fatty acids. Consistent with known 3D structures of ACP, steady-state fluorescence and quenching experiments indicated that Trp at positions 46, 50, and 72 are buried in the hydrophobic core upon Mg2+-induced ACP folding, whereas residues 25 and 45 remain in a hydrophilic environment on the protein surface. Attachment of fatty acids to the phosphopantetheine prosthetic group progressively stabilized the folded conformation of all Trp-substituted ACPs, but longer chains (14:0) were less effective than medium chains (8:0) in shielding Trp from acrylamide quenching in the L46W protein. Interaction with ACP-dependent enzymes LpxA and holo-ACP synthase also caused folding of L46W; fluorescence quenching indicated proximity of Trp-45 in helix II of ACP in LpxA binding. Our results suggest that divalent cations and fatty acylation produce differing environments in the ACP core and also reveal enzyme partner-induced folding of ACP, a key feature of “natively unfolded” proteins.

Electrospray ionization mass spectrometry (ESI-MS) can be used to monitor conformational changes of proteins in solution based on the charge state distribution (CSD) of the corresponding gas-phase ions, although relatively few studies of acidic proteins have been reported. Here, we have compared the CSD and solution structure of recombinant Vibrio harveyi acyl carrier protein (rACP), a small acidic protein whose secondary and tertiary structure can be manipulated by pH, fatty acylation, and site-directed mutagenesis. Circular dichroism and intrinsic fluorescence demonstrated that apo-rACP adopts a folded helical conformation in aqueous solution below pH 6 or in 50% acetonitrile/0.1% formic acid, but is unfolded at neutral and basic pH values. A rACP mutant, in which seven conserved acidic residues were replaced with their corresponding neutral amides, was folded over the entire pH range of 5 to 9. However, under the same solvent conditions, both wild type and mutant ACPs exhibited similar CSDs (6(+)-9(+) species) at all pH values. Covalent attachment of myristic acid to the phosphopantetheine prosthetic group of rACP, which is known to stabilize a folded conformation in solution, also had little influence on its CSD in either positive or negative ion modes. Overall, our results are consistent with ACP as a "natively unfolded" protein in a dynamic conformational equilibrium, which allows access to (de)protonation events during the electrospray process.

Purpose: Human newborn infants have increased susceptibility to gram-negative bacterial infection. Since lipopolysaccharide (LPS) primes polymorphonuclear neutrophils (PMN) to enhance host defense functions, we investigated its effect on adult and newborn PMN in vitro. Methods: PMN were isolated from blood of healthy adults and umbilical cords of full term newborns using dextran and Ficoll-Paque gradient sedimentation. Gel electrophoresis and Western blotting of membranes were used to probe for Mitogen-Activated Protein (MAP) kinase p38 phosphorylation, Toll-like Receptor-4 (TLR-4) and Myeloid Differentiation Factor 88 (MyD88) on isolated PMN membranes using specific antibodies. LPS induced degranulation was assessed using CD66 expression on PMN measured by flow cytometry. Results: We show that p38 phosphorylation in newborn PMN is attenuated in response to LPS stimulation even though adult and newborn PMN have similar amounts of p38 protein. The degree of attenuation in newborn PMN is dependent on the osmolarity of the medium. In addition, LPS-induced degranulation, a process that is p38 dependent, was also absent in newborn PMN. Although the LPS receptor TLR-4 is present at similar levels on newborn and adult PMN, its downstream adaptor protein MyD88 was significantly diminished in newborn PMN compared to adult cells. Conclusions: Although the mechanism of PMN priming by LPS is not fully understood, our results suggest that MyD88 and p38 phosphorylation are important pathways in the process and contribute to attenuated response of newborn PMN to LPS in vitro.

A modified phenol-based protocol and a phenol-free protocol that involves hot SDS extraction followed by TCA precipitation in acetone were qualitatively and quantitatively compared and evaluated on apple peel and strawberry fruit. The phenol protocol resulted in significantly higher protein yields of 2.35 +/- 0.1 and 0.46 +/- 0.06 mg/g of FW from apple and strawberry fruit, respectively, compared to the SDS protocol, which produced 0.74 +/- 0.1 and 0.27 +/- 0.02 mg/g of FW, respectively. 2-DE analysis of apple protein extracts revealed 1422 protein spots associated with the phenol protocol and 849 spots associated with the SDS protocol. Of these, 761 were present only in phenol gels, whereas 23 were exclusive to SDS samples. For strawberry, SDS extraction produced poor-quality spots with a high degree of streaking, indicating possible contamination. The application of a cleanup procedure resulted in a purified protein extract with high-quality spots. 2-DE analysis of strawberry protein extracts revealed 1368 spots for the phenol protocol and 956 spots for the SDS protocol accompanied by the cleanup procedure. Of these, 599 spots were present only in phenol gels, whereas 109 were present only in SDS samples. Spots from each fruit tissue and extraction procedure were selected, and a total of 26 were identified by LC-MS/MS. Overall, this study demonstrates the complexity of protein extraction of fruit tissues and suggests that a phenol-based protein extraction protocol should be used as a standard procedure for recalcitrant fruit tissues, whereas a SDS protocol with or without a cleanup procedure may be used as an alternative protocol.

Acyl carrier protein (ACP) is a universal and highly conserved carrier of acyl intermediates during fatty acid synthesis. In yeast and mammals, ACP exists as a separate domain within a large multifunctional fatty acid synthase polyprotein (type I FAS), whereas it is a small monomeric protein in bacteria and plastids (type II FAS). Bacterial ACPs are also acyl donors for synthesis of a variety of products, including endotoxin and acylated homoserine lactones involved in quorum sensing; the distinct and essential nature of these processes in growth and pathogenesis make ACP-dependent enzymes attractive antimicrobial drug targets. Additionally, ACP homologues are key components in the production of secondary metabolites such as polyketides and nonribosomal peptides. Many ACPs exhibit characteristic structural features of natively unfolded proteins in vitro, with a dynamic and flexible conformation dominated by 3 parallel α helices that enclose the thioester-linked acyl group attached to a phosphopantetheine prosthetic group. ACP conformation may also be influenced by divalent cations and interaction with partner enzymes through its “recognition” helix II, properties that are key to its ability to alternately sequester acyl groups and deliver them to the active sites of ACP-dependent enzymes. This review highlights recent progress in defining how the structural features of ACP are related to its multiple carrier roles in fatty acid metabolism.

Acyl carrier protein (ACP) is a natively unfolded protein that undergoes reversible conformational change as it shuttles fatty acyl groups among a variety of enzymes in the synthesis of bacterial lipids and other products. Folding of ACP upon divalent cation binding or fatty acylation has previously been monitored by intrinsic Trp fluorescence of ACP mutant L46W. In the present study, we found that interaction with UDP‐GlcNAc acyltransferase (LpxA) produced a large fluorescence blue shift of L46W, indicating that ACP also folds upon LpxA binding. LpxA (but not Mg2+) caused a smaller blue shift of mutant A45W, suggesting that this residue may be involved directly in enzyme interaction. Based on Trp fluorescence, LpxA interacted preferentially with holo‐ vs apo‐ACP, while the opposite was true for holo‐ACP synthase (AcpS). Titration with LpxA also caused a 2‐fold increase in fluorescence intensity of ACP labeled with AEDANS at the phosphopantetheine sulfhydryl group (apparent Kd of 2 μM). Addition of Mg2+ reversed this LpxA‐induced fluorescence increase, suggesting that binding of LpxA and divalent cations to ACP results in different phosphopantetheine environments. Fluorescence thus provides insight into the binding and conformational changes that accompany ACP interaction with partner enzymes

Acyl carrier protein (ACP), a small protein essential for bacterial growth and pathogenesis, interacts with diverse enzymes during the biosynthesis of fatty acids, phospholipids, and other specialized products such as lipid A. NMR and hydrodynamic studies have previously shown that divalent cations stabilize native helical ACP conformation by binding to conserved acidic residues at two sites (A and B) at either end of the “recognition” helix II. To examine the roles of these amino acids in ACP structure and function, site-directed mutagenesis was used to replace individual site A (Asp-30, Asp-35, Asp-38) and site B (Glu-47, Glu-53, Asp-56) residues in recombinant Vibrio harveyi ACP with the corresponding amides, along with combined mutations at each site (SA, SB) or both sites (SA/SB). Like native V. harveyi ACP, all individual mutants were unfolded at neutral pH but adopted a helical conformation in the presence of millimolar Mg2+ or upon fatty acylation. Mg2+ binding to sites A or B independently stabilized native ACP conformation, whereas mutant SA/SB was folded in the absence of Mg2+, suggesting that charge neutralization is largely responsible for ACP stabilization by divalent cations. Asp-35 in site A was critical for holo-ACP synthase activity, while acyl-ACP synthetase and UDP-N-acetylglucosamine acyltransferase (LpxA) activities were more affected by mutations in site B. Both sites were required for fatty acid synthase activity. Overall, our results indicate that divalent cation binding site mutations have predicted effects on ACP conformation but unpredicted and variable consequences on ACP function with different enzymes.