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. 2015 May 15;290(20):12908-18.
doi: 10.1074/jbc.M115.646521. Epub 2015 Mar 27.

PqqD is a novel peptide chaperone that forms a ternary complex with the radical S-adenosylmethionine protein PqqE in the pyrroloquinoline quinone biosynthetic pathway

John A Latham  1 , Anthony T Iavarone  2 , Ian Barr  1 , Prerak V Juthani  1 , Judith P Klinman  3
Affiliations

PqqD is a novel peptide chaperone that forms a ternary complex with the radical S-adenosylmethionine protein PqqE in the pyrroloquinoline quinone biosynthetic pathway

John A Latham et al. J Biol Chem. .

Abstract

Pyrroloquinoline quinone (PQQ) is a product of a ribosomally synthesized and post-translationally modified pathway consisting of five conserved genes, pqqA-E. PqqE is a radical S-adenosylmethionine (RS) protein with a C-terminal SPASM domain, and is proposed to catalyze the formation of a carbon-carbon bond between the glutamate and tyrosine side chains of the peptide substrate PqqA. PqqD is a 10-kDa protein with an unknown function, but is essential for PQQ production. Recently, in Klebsiella pneumoniae (Kp), PqqD and PqqE were shown to interact; however, the stoichiometry and KD were not obtained. Here, we show that the PqqE and PqqD interaction transcends species, also occurring in Methylobacterium extorquens AM1 (Me). The stoichiometry of the MePqqD and MePqqE interaction is 1:1 and the KD, determined by surface plasmon resonance spectroscopy (SPR), was found to be ∼12 μm. Moreover, using SPR and isothermal calorimetry techniques, we establish for the first time that MePqqD binds MePqqA tightly (KD ∼200 nm). The formation of a ternary MePqqA-D-E complex was captured by native mass spectrometry and the KD for the MePqqAD-MePqqE interaction was found to be ∼5 μm. Finally, using a bioinformatic analysis, we found that PqqD orthologues are associated with the RS-SPASM family of proteins (subtilosin, pyrroloquinoline quinone, anaerobic sulfatase maturating enzyme, and mycofactocin), all of which modify either peptides or proteins. In conclusion, we propose that PqqD is a novel peptide chaperone and that PqqD orthologues may play a similar role in peptide modification pathways that use an RS-SPASM protein.

Keywords: PqqD; PqqE; peptide biosynthesis; peptide interaction; protein complex; quinone; small-angle x-ray scattering (SAXS).

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Figures

FIGURE 1.
FIGURE 1.
Abbreviated PQQ biosynthesis. Formation of PQQ from conserved PqqA glutamate and tyrosine residues.
FIGURE 2.
FIGURE 2.
Structure of XcPqqD. The XcPqqD (PDB code 3G2B) dimer is depicted in ribbon form with each monomer differentiated by color. Each elongated monomer consists of a three α-helix bundle and a β-turn-β domain connected by a β-strand. Phosphate ligands from crystallization buffer are shown in orange. The box indicates the portion of the crystal structure used to model a compact XcPqqD monomer.
FIGURE 3.
FIGURE 3.
SEC and SAXS analysis of KpPqqD. A, the analytical size exclusion chromatogram of purified KpPqqD monitoring tryptophan absorbance at 280 nm shows a single peak with an elution volume that corresponds to a 14.9-kDa protein, calculated from the standard curve in B. B, the standard curve of the molecular weight standards shows a linear relationship with elution volume (black points and line). The calculated molecular weight for KpPqqD is shown by the red triangle. C, the similarity of the SAXS profiles for KpPqqD at all concentrations studied (1, 2, 3, 4, and 5 mg/ml: black, violet, dark blue, blue, and cyan, respectively) is independent of protein concentration. D, the plot of the radius of gyration as a function of KpPqqD shows its independence of protein concentration. E, experimental SAXS profile of KpPqqD (○) does not agree with the XcPqqD SAXS profiles calculated from the dimer (cyan line) or monomer (blue line) crystal structures. However, the SAXS profile for KpPqqD does agree with the compact model of XcPqqD (red line), constructed as described in the text and illustrated in Fig. 4B. F, the Guinier plot shows the linearity of the low q region of the experimental KpPqqD SAXS data extrapolated to zero scattering angle (red line), indicating that the protein has not aggregated.
FIGURE 4.
FIGURE 4.
Solution structure of KpPqqD. Ab initio SAXS reconstruction of KpPqqD (mesh) shown as side-on views. A, the ribbon structure of the elongated XcPqqD monomer (PDB code 3G2B) was docked into the SAXS envelope demonstrating that the SAXS-reconstructed envelope and the crystal structure are not in agreement. B, the ribbon structure of the compact XcPqqD model, taken from the β1 and β2 strands from one polypeptide chain and the β3, α1, α2, and α3 motifs from the second polypeptide chain, could be docked into the reconstructed envelope.
FIGURE 5.
FIGURE 5.
Nano-Q-TOF-ESI-MS characterization shows PqqD and PqqE form a 1:1 complex. Native mass spectra of K. pneumoniae (left panels) or M. extorquens AM1 (right panels) PqqE with and without PqqD. A, the spectrum of KpPqqD shows a single population with a molecular mass of 10.4 kDa calculated from the charge state distribution. B, likewise, the spectrum of KpPqqE shows a monomer population with a molecular mass of 45.9 kDa calculated from the charge state distribution. C, in the presence of KpPqqD, a new charge state distribution, corresponding to a mass of 56.6 kDa, is formed consistent with a 1:1 KpPqqD-KpPqqE complex. D, the MePqqD native mass spectrum shows a monomer population with a molecular mass of 10.4 kDa calculated from the charge state distribution. E, the native mass spectrum of MePqqE shows two sets of charge state distributions with masses of 43.6 and 86.9 kDa, indicating that both the monomeric and dimeric species are present. F, in the presence of MePqqD, a 54.0-kDa charge state distribution is formed corresponding to a 1:1 MePqqE-MePqqD complex.
FIGURE 6.
FIGURE 6.
Binding of PqqCD and PqqA observed by SPR and ITC. A representative dataset (His6-MePqqCD as ligand and MePqqA as analyte) showing that: A, the SPR response increases as MePqqA concentration increases (blue to violet) and B, the steady state fits were used to calculate the dissociation constant (error bars represent standard deviation of three independent experiments. C, ITC was used to independently verify the dissociation constants and determine the molar ratio. The representative raw isotherm traces (top panel) and the integral data points (bottom panel) are shown for an experiment with MePqqA and His6-MePqqCD (black) and a control experiment lacking His6-MePqqCD (blue). As discussed under “Experimental Procedures,” the first injection for each experiment was omitted in data analysis. The single-site model fit curve generated for the experimental data is shown in red.
FIGURE 7.
FIGURE 7.
Nano-Q-TOF-ESI-MS characterization of the MePqqA-MePqqD-MePqqE complex. The native mass spectrum of a solution containing 50 μm MePqqA, 50 μm MePqqD, and 10 μm MePqqE shows two sets of charge state distributions. The 12–13+ charged 43.6 kDa ions correspond to the MePqqE monomer and the new 13–15+ charged 57.0 kDa ions correspond to a ternary 1:1:1 MePqqA-MePqqD-MePqqE complex.
FIGURE 8.
FIGURE 8.
Representative sequence similarity network for the SPASM domain family. Each node (oval) represents a group of sequences where each sequence in the node is at least 60% identical to the representative sequence that defines the node (computed by the CD-HIT program (33)). The 1,882 nodes shown in this network represent over 20,000 sequences. Each edge (lines) that connects two nodes represents a BLAST similarity score of an -log(E-value) of 45 or greater, alignments of lengths between 340 and 450 amino acids (typical length of RS-SPASM proteins) and an average sequence identity of 35%. Cytoscape was used to visualize the network yFiles in an organic layout. The gene contexts of representative sequences were examined for the presence of a PqqD homologue and a peptide within a ±5-gene region on the same DNA strand. A node is colored blue if the representative sequence was fused to or found nearby a PqqD orthologue and a peptide was present in the gene context. A node is colored red if the representative sequence was fused to or found nearby a PqqD orthologue but a peptide was not found in the gene context. A node is colored yellow if the representative sequence was not fused to or found nearby a PqqD orthologue and a peptide was found in the gene context. A node is colored in gray if the representative sequence is not fused to or found nearby a PqqD orthologue and a peptide was not found in the gene context or the gene information was unavailable. Nodes colored in black were not analyzed.
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