Solution Structure of 4′-Phosphopantetheine - GmACP3 fromGeobacter metallireducens: A Specialized Acyl Carrier Protein with Atypical Structural Features and a Putative Role in Lipopolysaccharide Biosynthesis

Abstract

GmACP3 from Geobacter metallireducens is a specialized acyl carrier protein (ACP) whose gene, gmet_2339, is located near genes encoding many proteins involved in lipopolysaccharide (LPS) biosynthesis, indicating a likely function for GmACP3 in LPS production. By overexpression in Escherichia coli, about 50% holo-GmACP3 and 50% apo-GmACP3 were obtained. ApoGmACP3 exhibited slow precipitation and non-monomeric behavior by 15 N NMR relaxation measurements. Addition of 4′-phosphopantetheine (4′-PP) via enzymatic conversion by E. coli holo-ACP synthase, resulted in stable >95% holo-GmACP3 that was characterized as monomeric by 15 N relaxation measurements and had no indication of conformational exchange. We have determined a high-resolution solution structure of holo-GmACP3 by standard NMR methods, including refinement with two sets of NH residual dipolar couplings, allowing for a detailed structural analysis of the interactions between 4′-PP and GmACP3. Whereas the overall four helix bundle topology is similar to previously solved ACP structures, this structure has unique † This work was supported by a grant from the Protein Structure Initiative of the National Institutes of Health (P50 GM 074958).FTICR mass spectrometry was performed in the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the Department of Energy's Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory in Richland, WA.To whom correspondence should be addressed: Department of Chemistry and Biochemistry, 701 East High Street, Miami University, Oxford OH, 45056. michael.kennedy@muohio.edu. Phone: (513) . SUPPORTING INFORMATION AVAILABLE Figures, tables and text containing mass spectrometry data, NMR 1 H-15 N HSQC spectra, GmACP3 homologs, possible functional importance of gmet_2337 and gmet_2340, 4′-PP NOEs, 1 H-15 N HSQC spectrum centered on histidine resonances, DALI hits, and RMSDs between holo-GMACP3 and ACPs structural homologs. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2012 March 8. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript characteristics, including an ordered 4′-PP conformation that places the thiol at the entrance to a central hydrophobic cavity near a conserved hydrogen-bonded Trp-His pair. These residues are part of a conserved WDSLxH/N motif found in GmACP3 and it's orthologs. The helix locations and the large hydrophobic cavity are more similar to medium-and long-chain acyl-ACPs than to other apo-and holo-ACP structures. Taken together, structural characterization along with bioinformatic analysis of nearby genes suggest that GmACP3 is involved in lipid A acylation, possibly by atypical long-chain hydroxy fatty acids, and potentially involved in synthesis of secondary metabolites.Bacterial acyl carrier proteins (ACPs) are conserved abundant proteins that transport acyl intermediate...

Probing the Phosphopantetheine Arm Conformations of Acyl Carrier Proteins Using Vibrational Spectroscopy

“…The IR data were entirely consistent with this hypothesis: the CN stretching band for cyanylated GmACP3 is red-shifted by 6–7 cm –1 compared to the cyanylated ACT ACP (Figure 2 ), in agreement with previous findings that the Ppant arms of these two proteins are in markedly different environments 11 , 31 and confirming that the Ppant* can serve as a site-specific reporter of the Ppant arm’s local environment.…”

The structural role of the carrier protein – active controller or passive carrier

“…Unusually, the terminal thiol of the 4 0 -PP of GmACP3 interacts with a conserved, hydrogen bonded Trp-His pair at the entrance to a hydrophobic cavity. 53 As well as the assignment of unambiguous NOEs between the body of the ACP and the 4 0 -PP group, NMR relaxation studies showed that the amide groups of the 4 0 -PP side arm were rigid and not mobile as observed in other holo-ACPs (Fig. 3F).…”

Sticky swinging arm dynamics: studies of an acyl carrier protein domain from the mycolactone polyketide synthase

“…The Ppant arms of holo ACP species adopt a spectrum of dynamic states, ranging from tightly bound to the carrier protein surface to freely swinging. For example, the prosthetic group of the holo form of an atypical Geobacter metallireducens ACP ( Gm ACP 3 ) is rigid, displaying S 2 order parameter values similar to those for structured backbone sites (∼0.8), together with 23 Ppant-protein NOE connections [ 51 ]. The Plasmodium falciparum type II FAS ACP ( Pf ACP) represents the centre ground, exhibiting two conformers in slow exchange due to different docked orientations of the Ppant moiety; in both states the prosthetic group is semi-rigid, with S 2 values for N 4 and N 8 in the 0.1–0.5 range, but also 6 NOEs between the arm and the protein surface [ 46 ].…”

“…Interestingly, backbone 1 H N / 15 N chemical shift perturbations appear to be less sensitive probes of protein/prosthetic group interactions than Ppant 15 N relaxation parameter measurements or the detection of direct Ppant-protein NOE connections. For mACP 9 , which possesses a highly dynamic arm, the largest detected Δδ av value between the apo and holo forms is ∼0.3 ppm ( Figure 4 A), whereas the largest corresponding shift change for the firmly bound Gm ACP 3 species is only ∼0.4 ppm [ 51 ]. Furthermore, we found that the magnitude of shift changes between apo and holo mACP 9 did not depend in a straightforward way on distance from the point of attachment of the prosthetic group.…”