Identification of a key residue in the conformational stability of acyl carrier protein

Conformational flexibility of acyl carrier protein (ACP) is important for its ability to interact with multiple enzymes in bacterial fatty acid metabolism. We have recently shown that, unlike the prototypical ACP from Escherichia coli, the more acidic Vibrio harveyi ACP is largely unfolded at physiological pH. Mutations D18K, A75H and A75H/D18K were made in recombinant V. harveyi ACP (rACP) to determine the importance of basic residues Lys-18 and His-75 in maintaining the native conformation of E. coli ACP. Both D18K and A75H ACPs were fatty acylated by acyl-ACP synthetase, showing that neither mutation grossly alters tertiary structure. Circular dichroism (CD) indicated that rACP refolded upon addition of MgCl(2) at 100-fold lower concentrations (<1 mM) than KCl, suggesting that divalent cations stabilize rACP by interaction at specific sites. Surprisingly, mutants A75H and A75H/D18K exhibited native-like conformation in the absence of MgCl(2), while the D18K mutant was comparable to rACP. Moreover, the alpha-helical content of A75H, A75H/D18K and E. coli ACPs was more sensitive than that of rACP or D18K ACP to modification by the histidine-selective reagent diethylpyrocarbonate. Together, these results suggest that the partial positive charge of His-75 may be important in maintaining the conformational stability of E. coli ACP at a neutral pH.     

Structure of a specialized acyl carrier protein essential for lipid a biosynthesis with very long-chain Fatty acids in open and closed conformations

The solution nuclear magnetic resonance (NMR) structures and backbone (15)N dynamics of the specialized acyl carrier protein (ACP), RpAcpXL, from Rhodopseudomonas palustris, in both the apo form and holo form modified by covalent attachment of 4'-phosphopantetheine at S37, are virtually identical, monomeric, and correspond to the closed conformation. The structures have an extra α-helix compared to the archetypical ACP from Escherichia coli, which has four helices, resulting in a larger opening to the hydrophobic cavity. Chemical shift differences between apo- and holo-RpAcpXL indicated some differences in the hinge region between α2 and α3 and in the hydrophobic cavity environment, but corresponding changes in nuclear Overhauser effect cross-peak patterns were not detected. In contrast to the NMR structures, apo-RpAcpXL was observed in an open conformation in crystals that diffracted to 2.0 ? resolution, which resulted from movement of α3. On the basis of the crystal structure, the predicted biological assembly is a homodimer. Although the possible biological significance of dimerization is unknown, there is potential that the resulting large shared hydrophobic cavity could accommodate the very long-chain fatty acid (28-30 carbons) that this specialized ACP is known to synthesize and transfer to lipid A. These structures are the first representatives of the AcpXL family and the first to indicate that dimerization may be important for the function of these specialized ACPs.     

Dynamics in an unusual acyl carrier protein from a ladderane lipid-synthesizing organism

Anaerobic ammonium-oxidizing (anammox) bacteria express a distinct acyl carrier protein implicated in the biosynthesis of the highly unusual “ladderane” lipids these organisms produce. This “anammox-specific” ACP, or amxACP, shows several unique features such as a conserved FF motif and an unusual sequence in the functionally important helix III. Investigation of the protein's structure and dynamics, both in the crystal by ensemble refinement and by MD simulations, reveals that helix III adopts a rare six-residue-long 3₁₀-helical conformation that confers a large degree of conformational and positional variability on this part of the protein. This way of introducing structural flexibility by using the inherent properties of 3₁₀-helices appears unique among ACPs. Moreover, the structure suggests a role for the FF motif in shielding the thioester linkage between the protein's prosthetic group and its acyl cargo from hydrolysis.     

A mechanism based protein crosslinker for acyl carrier protein dehydratases

Recent advances in the structural study of fatty acid synthase (FAS) and polyketide synthase (PKS) biosynthetic enzymes have illuminated our understanding of modular enzymes of the acetate pathway. However, one significant and persistent challenge in such analyses is resolution of the acyl carrier protein (ACP), a small (～9 kDa) protein to which biosynthetic intermediates are tethered throughout the biosynthetic cycle. Here we report a chemoenzymatic crosslinking strategy in which the installation of a historical suicide substrate scaffold upon the 4′-phosphopantetheine (PPant) arm of the ACP is used to capture the active site of acyl carrier protein dehydratase (DH) domains in FAS. Through the synthesis of a small panel of related probes we identify structural features essential for ACP–DH crosslinking, and apply gel-based assays to demonstrate the stability as well as purification strategies for isolation of the chemoenzymatically modified ACP. Applying these carrier protein crosslinking techniques to the structural analysis of FAS and PKS complexes has the potential to provide snapshots of these biosynthetic assembly lines at work.     

A GX2GX3G motif facilitates acyl chain sequestration by Saccharomyces cerevisiae acyl carrier protein

Saccharomyces cerevisiae acyl carrier protein (ScACP) is a component of the large fungal fatty acid synthase I (FAS I) complex. ScACP comprises two subdomains: a conserved ACP domain that shares extensive structural homology with other ACPs and a unique structural domain. Unlike the metazoan type I ACP that does not sequester the acyl chain, ScACP can partially sequester the growing acyl chain within its hydrophobic core by a mechanism that remains elusive. Our studies on the acyl-ScACP intermediates disclose a unique ¹⁸⁸GX₂GX₃G¹⁹⁵ sequence in helix II important for ACP function. Complete loss of sequestration was observed upon mutation of the three glycines in this sequence to valine (G188V/G191V/G195V), while G191V and G188V/G191V double mutants displayed a faster rate of acyl chain hydrolysis. Likewise, mutation of Thr216 to Ala altered the size of the hydrophobic cavity, resulting in loss of C₁₂- chain sequestration. Combining NMR studies with insights from the crystal structure, we show that three glycines in helix II and a threonine in helix IV favor conformational change, which in turn generate space for acyl chain sequestration. Furthermore, we identified the primary hydrophobic cavity of ScACP, present between the carboxyl end of helix II and IV. The opening of the cavity lies between the second and third turns of helix II and loop II. Overall, the study highlights a novel role of the GX₂GX₃G motif in regulating acyl chain sequestration, vital for ScACP function.     

ATP binding is critical for the conformational change from an open to closed state in archaeal group II chaperonin

Group II chaperonins, found in archaea and in eukaryotic cytosol, do not have a co-chaperonin corresponding to GroES. Instead, it is suggested that the helical protrusion extending from the apical domain acts as a built-in lid for the central cavity and that the opening and closing of the lid is regulated by ATP binding and hydrolysis. However, details of this conformational change remain unclear. To investigate the conformational change associated with the ATP-driven cycle, we conducted protease sensitivity analyses and tryptophan fluorescence spectroscopy of alpha-chaperonin from a hyperthermophilic archaeum, Thermococcus strain KS-1. In the nucleotide-free or ADP-bound state, the chaperonin, especially in the helical protrusion region, was highly sensitive to proteases. Addition of ATP and ammonium sulfate induced the transition to the relatively protease-resistant form. The fluorescence intensity of the tryptophan residue introduced at the tip of the helical protrusion was enhanced by the presence of ATP or ammonium sulfate. We conclude that ATP binding induces the conformational change from the lid-open to lid-closed form in archaeal group II chaperonin.     

Flexible docking allowing induced fit in proteins: Insights from an open to closed conformational isomers

Here we dock a ligand onto a receptor surface allowing hinge-bending domain/substructural movements. Our approach mimics and manifests induced fit in molecular recognition. All angular rotations are allowed on the one hand, while a conformational space search is avoided on the other. Rather than dock each of the molecular parts separately with subsequent reconstruction of the consistently docked molecules, all parts are docked simultaneously while still utilizing the position of the hinge from the start. Like pliers closing on a screw, the receptor automatically closes on its ligand in the best surface-matching way. Movements are allowed either in the ligand or in the larger receptor, hence reproducing induced molecular fit. Hinge bending movements are frequently observed when molecules associate. There are numerous examples of open versus closed conformations taking place upon binding. Such movements are observed when the substrate binds to its respective enzyme. In particular, such movements are of interest in allosteric enzymes. The movements can involve entire domains, subdomains, loops, (other) secondary structure elements, or between any groups of atoms connected by flexible joints. We have implemented the hinges at points and at bonds. By allowing 3-dimensional (3-D) rotation at the hinge, several rotations about (consecutive or nearby) bonds are implicitly taken into account. Alternatively, if required, the point rotation can be restricted to bond rotation. Here we illustrate this hinge-bending docking approach and the insight into flexibility it provides on a complex of the calmodulin with its M13 ligand, positioning the hinges either in the ligand or in the larger receptor. This automated and efficient method is adapted from computer vision and robotics. It enables utilizing entire molecular surfaces rather than focusing a priori on active sites. Hence, allows attaining the overall optimally matching surfaces, the extent and type of motions which are involved. Here we do not treat the conformational flexibility of side-chains or of very small pieces of the molecules. Therefore, currently available methods addressing these issues and the method presented here, are complementary to each other, expanding the repertoire of computational docking tools foreseen to aid in studies of recognition, conformational flexibility and drug design. Proteins 32:159–174, 1998. © 1998 Wiley-Liss, Inc.     

An approach to the semisynthesis of acyl carrier protein

A scheme has been devised for the preparation of semisynthetic derivatives of acyl carrier protein (ACP). Acetylated synthetic ACP_1–6 is coupled via its activated pentachlorophenol ester to native ACP (7–77), which had previously been acetylated and converted to the S-5′-dithiobis(2-nitrobenzoate)(DTNB) derivative. Removal of the DTNB moiety after the coupling yielded active ACP in good yield.     

Probing the interactions of an acyl carrier protein domain from the 6-deoxyerythronolide B synthase

The assembly‐line architecture of polyketide synthases (PKSs) provides an opportunity to rationally reprogram polyketide biosynthetic pathways to produce novel antibiotics. A fundamental challenge toward this goal is to identify the factors that control the unidirectional channeling of reactive biosynthetic intermediates through these enzymatic assembly lines. Within the catalytic cycle of every PKS module, the acyl carrier protein (ACP) first collaborates with the ketosynthase (KS) domain of the paired subunit in its own homodimeric module so as to elongate the growing polyketide chain and then with the KS domain of the next module to translocate the newly elongated polyketide chain. Using NMR spectroscopy, we investigated the features of a structurally characterized ACP domain of the 6‐deoxyerythronolide B synthase that contribute to its association with its KS translocation partner. Not only were we able to visualize selective protein–protein interactions between the two partners, but also we detected a significant influence of the acyl chain substrate on this interaction. A novel reagent, CF₃‐S‐ACP, was developed as a ¹⁹F NMR spectroscopic probe of protein–protein interactions. The implications of our findings for understanding intermodular chain translocation are discussed.     

A dynamic model for the structure of acyl carrier protein in solution

The determination of solution structures of proteins using two-dimensional NMR data is commonly based on the assumption that the structure can be represented by a single rigid conformer. We present here a procedure whereby this assumption can be relaxed and illustrate its application to acyl carrier protein from Escherichia coli, a small negatively charged protein with no internal disulfide bonds. The methodology rests on a model having two distinct conformers in dynamic equilibrium. Use of this two-state model results in a dramatic improvement in fit to cross-relaxation-derived distance constraints and a substantial lowering of molecular mechanics energies for individual conformers of acyl carrier protein. The two-state model retains the three-helix motif previously identified on the basis of a one-state structure, but substantial motion of loop regions and the C-terminal peptide, as well as partial disruption of the second helix, is suggested to occur. Support for the existence of these motions can be found in amide exchange rate and spin relaxation time data.     

NMR-pseudoenergy approach to the solution structure of acyl carrier protein

A method for protein structure determination from two-dimensional NMR cross-relaxation data is presented and explored by using short amino acid segments from acyl carrier protein as a test case. The method is based on a molecular mechanics program and incorporates NMR distance constraints in the form of a pseudoenergy term that accurately reflects the distance-dependent precision of NMR cross-relaxation data. When it is used in an indiscriminant fashion, the method has a tendency to produce structures representing local energy minima near starting structures, rather than structures representing a global energy minimum. However, stepwise inclusion of energy terms, beginning with a function heavily weighted by backbone distance constraints, appears to simplify the potential energy surface to a point where convergence to a common backbone structure from a variety of starting structures is possible. In the case of the segment from residues 3 to 15 in acyl carrier protein, a nearly perfect alpha-helix is produced starting with a linear chain, an alpha-helical chain, or a chain having residues with alternating linear and alpha-helical backbone torsional angles. In the case of the segment from residues 26 to 36 a structure having a right-handed loop is produced.     

Deciphering the key residues in Plasmodium falciparum beta-ketoacyl acyl carrier protein reductase responsible for interactions with Plasmodium falciparum acyl carrier protein

The type II fatty acid synthase (FAS) pathway of Plasmodium falciparum is a validated unique target for developing novel antimalarials, due to its intrinsic differences from the typeI pathway operating in humans. beta-Ketoacyl acyl carrier protein (ACP) reductase (FabG) performs the NADPH-dependent reduction of beta-ketoacyl-ACP to beta-hydroxyacyl-ACP, the first reductive step in the elongation cycle of fatty acid biosynthesis. In this article, we report intensive studies on the direct interactions of Plasmodium FabG and Plasmodium ACP in solution, in the presence and absence of its cofactor, NADPH, by monitoring the change in intrinsic fluorescence of P.falciparum FabG (PfFabG) and by surface plasmon resonance. To address the issue of the importance of the residues involved in strong, specific and stoichiometric binding of PfFabG to P.falciparum ACP (PfACP), we mutated Arg187, Arg190 and Arg230 of PfFabG. The activities of the mutants were assessed using both an ACP-dependent and an ACP-independent assay. The affinities of all the PfFabG mutants for acetoacetyl-ACP (the physiological substrate) were reduced to different extents as compared to wild-type PfFabG, but were equally active in biochemical assays with the substrate analog acetoacetyl-CoA. Kinetic analysis and studies of direct binding between PfFabG and PfACP confirmed the identification of Arg187 and Arg230 as critical residues for the PfFabG-PfACP interactions. Our studies thus reveal the significance of the positively charged/hydrophobic patch located adjacent to the active site cavities of PfFabG for interactions with PfACP.     

Nonorganellar acyl carrier protein from oleaginous yeast is a homologue of ribosomal protein P2

Acyl carrier protein (ACP) is responsible for carrying the growing fatty acid chain from one enzyme active site to the next during fatty acid biosynthesis. Here we report the identification, purification, immunocytochemical localization, and cloning of ACP from the oleaginous yeast, Rhodotorula glutinis. The soluble fraction of this organism can synthesize triacylglycerol and is able to accept the acyl group from acyl-ACP for the synthesis. The ACP, cloned from the system, showed a significant similarity with ribosomal protein P2. Expression and characterization of the recombinant protein showed that the ACP was acylated in vitro. The recombinant protein was post-translationally modified, since it was observed in [14C]beta-alanine labeling and matrix-assisted laser desorption mass spectroscopic analysis. Site-directed mutants were generated to identify a serine residue responsible for phosphopantetheinylation and found that mutation of serine 59 to alanine abrogated the fatty acylation ability of the protein. These results demonstrate that a novel modification of ribosomal protein P2 allows it to act as an acyl carrier protein and participate in acylation reactions.     

A conformational transition between an open and closed form of human pancreatic lipase revealed by a monoclonal antibody

The interfacial activation of human pancreatic lipase (HPL) probably involves the motion of a lid covering the active site of the enzyme. Here we observed that the presence of either bile salts or a small proportion of water-miscible organic solvents (called activator compounds) considerably enhances the enzymatic activity of HPL on a monomeric solution of tripropionin. This finding suggests that the activator compounds may favor the opening of the lid. This hypothesis was checked by comparing the immunoreactivity of HPL and HPL with a mini-lid (HPL(-lid)) towards anti-HPL monoclonal antibodies (mAbs), in the presence and absence of the activator compounds. A single conformational mAb (248-31) fails to immunoprecipitate HPL in the presence of activator compounds and HPL covalently inhibited with diethyl p-nitrophenyl phosphate (DP.HPL). This loss of recognition of HPL by mAb 248-31 was probably due to the motion of the lid, since HPL(-lid) was always recognized in the presence or absence of activator compounds. Furthermore, two other mAbs (81-23 and 146-40) immunoprecipitated HPL similarly whether or not the activator compounds were present. MAb 248-31 therefore specifically recognizes HPL in the closed but not the open conformation.     

Essential role of the donor acyl carrier protein in stereoselective chain translocation to a fully reducing module of the nanchangmycin polyketide synthase

Incubation of recombinant module 2 of the polyether nanchangmycin synthase (NANS), carrying an appended thioesterase domain, with the ACP-bound substrate (2RS)-2-methyl-3-ketobutyryl-NANS_ACP1 (2-ACP1) and methylmalonyl-CoA in the presence of NADPH gave diastereomerically pure (2S,4R)-2,4-dimethyl-5-ketohexanoic acid (4a). These results contrast with the previously reported weak discrimination by NANS module 2+TE between the enantiomers of the corresponding N-acetylcysteamine-conjugated substrate analogue (±)-2-methyl-3-ketobutyryl-SNAC (2-SNAC), which resulted in formation of a 5:3 mixture of 4a and its (2S,4S)-diastereomer 4b. Incubation of NANS module 2+TE with 2-ACP1 in the absence of NADPH gave unreduced 3,5,6-trimethyl-4-hydroxypyrone (3) with a k(cat) of 4.4 ± 0.9 min?1 and a k(cat)/K(m) of 67 min?1 mM?1, corresponding to a ～2300-fold increase compared to the k(cat)/K(m) for the diffusive substrate 2-SNAC. Covalent tethering of the 2-methyl-3-ketobutyryl thioester substrate to the NANS ACP1 domain derived from the natural upstream PKS module of the nanchangmycin synthase significantly enhanced both the stereospecificity and the kinetic efficiency of the sequential polyketide chain translocation and condensation reactions catalyzed by the ketosynthase domain of NANS module 2.     

pH-induced conformational transition of H. pylori acyl carrier protein: insight into the unfolding of local structure

Acyl carrier protein (ACP) is a small acidic protein and its primary structure is highly conserved in various bacterial sources. Despite its small size, it interacts with diverse proteins associated with many biosynthetic pathways. The three-dimensional structure of H. pylori ACP and its structural characteristics were clarified using NMR and CD spectroscopy. H. pylori ACP consists of four helices connected by different sized loops. The helices correspond to residues L3-Q14 (alphaI), S36-G50 (alphaII), D56-E60 (alphaIII), and V65-K76 (alphaVI). The size of each helix differs slightly from that of homologous ACPs. However, H. pylori ACP showed a distinct pH-dependent conformational characteristic: at neutral pH, it adopts a partially unfolded structure, while it has a tight fold at pH 6. The chemical shift perturbation and (1)H-(15)N steady state NOE analysis at both pH 6 and 7 showed that the local change of structural components occurred mainly around loop II, and this change was reflected by the changes of the residues Ile 54 and Asp 56. Examination of the structure showed that the network of Glu 47, Ile 54, Asn 75, and Lys 76 is very important for the structural stability. The pH-dependent folding process shows a kind of cooperativity, since all the residues involved in the conformational transitions are contiguous and in spatial proximity.