Neutralization of Acidic Residues in Helix II Stabilizes the Folded Conformation of Acyl Carrier Protein and Variably Alters Its Function with Different Enzymes

Abstract

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 Mg 2؉ or upon fatty acylation. Mg 2؉ binding to sites A or B independently stabilized native ACP conformation, whereas mutant SA/SB was folded in the absence of Mg 2؉ , 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. Acyl carrier protein (ACP)2 is an acidic and highly conserved protein typically consisting of 70 -100 residues and is essential for bacterial growth, communication, and pathogenesis. ACP is responsible for supplying acyl groups for the biosynthesis of a plethora of bacterial molecules, including fatty acids (1), phospholipids (2), lipid A (3), lipoic acid (4), hemolysin (5), acyl homoserine lactones involved in quorum-sensing (6), and the aldehyde substrate of luciferase in bioluminescent bacteria such as Vibrio harveyi (7). Other functions of ACP and its homologues include the production of membrane-derived oligosaccharides (8), rhizobial nodulation signaling factors (9), polyketide (10) and non-ribosomal peptide antibiotics (11), and lipoteichoic acid (12). The list of ACP-binding partners continues to expand based on proteomic efforts (13). The requirement for ACP in these diverse processes suggests that interactions between ACP and its partner enzymes must be specific. Information about how individual amino acid residues contribute to ACP conformation and interactions with functionally diverse enzymes will provide insight into the design of novel antimicrobial agents against ACP-dependent targets that are essential for bacterial growth and pathogenesis.Structural analyses of type II ACPs from Escherichia coli and several other bacterial species reveal a common three-helix bun...

Electrospray ionization mass spectra of acyl carrier protein are insensitive to its solution phase conformation

“…Strategic placement of basic residues in ACP can influence its solution conformation; e.g., mutation of Ala-75 to His results in a more compact helical ACP at neutral°pH°due°to°charge°neutralization° [24].°Recently, we have shown that site-directed mutagenic neutralization of seven acidic divalent cation-binding residues at both ends of Helix II also increases helical content and decreases°the°hydrodynamic°radius°of°ACP° [26].°This ACP mutant (SA/SB) is expressed predominantly in the apo-form and cannot be further stabilized by divalent cation°binding.°As°shown°in°Figure°1c,°apo-SA/SB exhibited significantly greater helical content than aporACP at neutral pH and was only slightly unfolded even at pH 9.5. However, the CSD of this mutant exhibited only a modest change over this pH range, with average net charge decreasing from 8.0 at pH 9.5 to 7.6°at°pH°4.4°(Figure°2b).°Moreover,°this°profile°was very similar to that of rACP, consistent with earlier observations that the CSD of a protein does not necessarily°reflect°its°net°charge°in°solution° [15],°which°in°this case would be considerably lower for SA/SB (total of 15 acidic residues) relative to rACP (22 acidic residues) at neutral and higher pH values.…”

“…Myristoyl-ACP was prepared enzymatically from holo-ACP by incubation with myristic acid (14:0), ATP, and V. harveyi acyl-ACP synthetase as detailed elsewhere [25]. Site-directed mutagenesis of rACP and preparation of mutant SA/SB, in which seven central conserved acidic amino acid residues are replaced with their corresponding neutral amide residues, is described in reference [26].°Similar methods were used to replace Leu at position 46 with Trp to introduce an intrinsic fluorescence probe sensitive to ACP folding (Gong and Byers, manuscript in preparation). Residue numbering corresponds to that of native V. harveyi ACP (SwissProt accession no.…”

Structural Insights into the Acyl Intermediates of the Plasmodium falciparum Fatty Acid Synthesis Pathway

“…This region is already known to be recognized by a number of enzymes, e.g. ␤-ketoacyl-(ACP) reductase (37,38), acyl carrier protein synthase (39), protein acyltransferase HlyC (40), acyl-ACP synthetase, UDP-N-acetyl glucosamine acyltransferase (41), and ␤-ketoacyl-ACP synthase III (42). Outward movement of helix III in combination with acyl chain insertion generates strain on the ACP molecule, as revealed from slight changes in C ␣ and C ␤ of residues present at the bottom opening of the hydrophobic core in tetradecanoyl-ACP.…”

Intein-mediated Cyclization of Bacterial Acyl Carrier Protein Stabilizes Its Folded Conformation but Does Not Abolish Function

“…To further examine the effect of cyclization on ACP structure in solution, we used a variety of biophysical methods to analyze the conformation of both cycL46W and linL46W in response to Mg 2ϩ ions, which are known to induce a folded helical conformation upon binding to V. harveyi ACP at physiological pH (10). The apo forms of linear ACPs were used in these studies, as cyclic ACP is predominantly expressed in this form (see below).…”

“…For example, Vibrio harveyi ACP is largely unfolded at neutral pH, but its helical conformation can be stabilized by charge neutralization (i.e. at low pH or by residue replacements) (13), by binding of divalent cations to helix II (10), or by interaction with partner enzymes (15). Molecular dynamic simulations (16) and NMR experiments (17,18) have indicated greatest mobility in the long loop I connecting helices I and II, in the helix II-helix III region, and at the N and C termini, which are in close proximity.…”