Poly-γ-glutamylation of biomolecules

Abstract

Poly-γ-glutamate tails are a distinctive feature of archaeal, bacterial, and eukaryotic cofactors, including the folates and F₄₂₀. Despite decades of research, key mechanistic questions remain as to how enzymes successively add glutamates to poly-γ-glutamate chains while maintaining cofactor specificity. Here, we show how poly-γ-glutamylation of folate and F₄₂₀ by folylpolyglutamate synthases and γ-glutamyl ligases, non-homologous enzymes, occurs via processive addition of L-glutamate onto growing γ-glutamyl chain termini. We further reveal structural snapshots of the archaeal γ-glutamyl ligase (CofE) in action, crucially including a bulged-chain product that shows how the cofactor is retained while successive glutamates are added to the chain terminus. This bulging substrate model of processive poly-γ-glutamylation by terminal extension is arguably ubiquitous in such biopolymerisation reactions, including addition to folates, and demonstrates convergent evolution in diverse species from archaea to humans.

Fig. 1. Cofactor substrates of folylpolyglutamate synthase (folate) or γ-glutamyl ligase (F₄₂₀) bearing variable length poly-γ-glutamate tails.

The glutamyl residues in green indicate the terminus of the growing tail in both molecules.

Fig. 2. 4-Nitro-L-2-aminobutyric acid as a chain terminator of poly-γ-glutamylation in folates and F₄₂₀.

A poly-γ-glutamate tail is shown with variable L-glutamate numbers and terminated by a non-reactive nitro functional group. Calculated masses of nitro-terminated folate and F₄₂₀ molecules are provided for comparison to experimental results – the central number indicates the total residue count in the tail including the chain terminator residue. The masses take into account functional group protonation within the mass spectrometer operating in positive ion mode and are provided to 4 decimal places to match the precision of the high-resolution LC-MS experiment. The table of data provides the identity and experimentally measured masses of substrate, predominant polyglutamylated product, and nitro-terminated product. Enzymes systems investigated are human FPGS (hFPGS), Mycobacterium tuberculosis FPGS (Mtb-FPGS), Archaeoglobus fulgidus CofE (Af-CofE), and Mycobacterium tuberculosis FbiB (Mtb-FbiB).

Fig. 3. Mtb-FPGS-dependent glutamylation of folate-2 with ¹⁵N-glutamate monitored by NMR spectroscopy.

a Reaction scheme for Mtb-FPGS/Tm-DHFR coupled conversion of folate-2 to tetrahydrofolate-3 and subsequent oxidation to N-(4-aminobenzoyl)-(L-Glu)₃. The orange asterisk indicates the ¹⁵N-enriched site. Panels b to e show the amide region of 1D ¹H and 2D ¹H-¹⁵N HSQC spectra for samples as follows. b Reaction sample containing folate-2 and ¹⁵N-glutamate prior to addition of enzyme. c Reaction sample following a 2 h preincubation with Tm-DHFR, followed by incubation with both Tm-DHFR and Mtb-FPGS for 2 h and d 20 h. e Folate-3 standard. Panels f to i show the results of ¹H-¹H correlation experiments confirming addition of ¹⁵N-glutamate at the terminus of the Mtb-FPGS/Tm-DHFR-catalyzed reaction product. f Arrows indicate through-bond (solid line) and through-space (dashed line) correlations observed between the ¹⁵N-labelled amide and other ¹H nuclei in the polyglutamate chain, based on folate-3 assignments. g Comparison of the chemical shifts of ¹H-¹H correlations observed for the ¹⁵N-enriched amide and the 3′ glutamyl amide of folate-3. h Overlay of ¹⁵N-coupled (blue), and ¹⁵N-decoupled (pink) ¹H-¹H TOCSY experiments for the ¹⁵N-enriched amide highlighting correlations with the 3′α, 3′β, and 3′γ protons. i Overlay of ¹⁵N-coupled (purple), and ¹⁵N-decoupled (green) ¹H-¹H NOESY experiments for the ¹⁵N-enriched amide highlighting a correlation with the 2′γ proton.

Fig. 4. High-resolution structures of reaction ligands in the active site of Archaeoglobus fulgidus γ-glutamyl ligase CofE.

a An overlay of F₄₂₀-1 (yellow) and F₄₂₀-2 (cyan) molecules highlighting the bulge in the tail of F₄₂₀-2. GTP/GDP (pink stick model), manganese and sodium cations (white spheres) are located adjacent to the terminal glutamyl binding site. b The F₄₂₀-1 phospholactyl moiety binds in a secondary glutamate binding pocket and its carbonyl closely approaches the γ-phosphate atom of a modelled GTP. c Free L-glutamate binding in the primary glutamate pocket. Extensive hydrogen bonding between the co-substrate and two oxyanion-like holes via α- and γ-carboxylates locks the ligand in place. d The first, basal glutamyl of F₄₂₀-2 occupies the secondary glutamate pocket and projects the carbonyl oxygen of the following cis-amide bond toward the GTP γ-phosphate atom. Polar interactions are shown as dashed white lines with distances labelled in Å units.

Fig. 5. Schematic of the proposed poly-γ-glutamylation mechanism in CofE and its homologue FbiB.

Isoalloxazine and primary glutamate binding sites are depicted as bold, dotted lines, with glutamate or glutamyl residues colored green or blue by their order of addition. In STEP 1, F₄₂₀-0 effects nucleophilic attack on the GTP γ-phosphate to form an acyl intermediate. The phosphate abstracts a proton from the free glutamate amino group and is eliminated from GTP. STEP 2 shows an additional proton abstraction, nucleophilic attack of the glutamate on the acyl phosphate, and the consequent and concerted loss of phosphate as H₂PO₄⁻. STEP 3 depicts the crystal structure model of F₄₂₀-1 binding. STEP 4 and STEP 5 show a second cycle of phosphorylation/activation, proton abstraction, nucleophilic attack, and elimination of H₂PO₄^–. STEP 6 depicts the crystal structure model of F₄₂₀-2. STEPS 4–6 highlight the bulge in the growing poly-glutamyl chain. The schematic has been simplified for clarity and multiple binding, bond making, and bond breaking events have been combined – this does not imply a specific order of binding nor concerted reactions.