Fdc1, the homodimeric ferulic acid decarboxylase from Aspergillus niger, depends on the prenylated flavin cofactor prFMN for its non-oxidative decarboxylation chemistry. A combined native mass spectrometry, ion-mobility MS and hydrogen-deuterium exchange MS study (Beveridge et al., 2016) showed that prFMN binding stabilizes and compacts the Fdc1 dimer and produces both local and distal (allosteric) conformational tightening, but left the underlying mechanism unresolved: the equilibrium prFMN occupancy in solution was not measured (gas-phase cofactor loss during electrospray could not be excluded), no atomistic model of the apo state has been published or released (apo-Fdc1 resists crystallization and HDX gives only peptide-level information), the supporting simulations were 10 ns in vacuo rather than solution-phase, and the residue-level route by which local cofactor coordination causes distal tightening was not identified. The problem is to provide a residue-resolved, solution-phase mechanistic account of prFMN-induced structural reorganization of Fdc1, comprising four jointly linked aspects.

Background

Fdc1 is a fungal homodimeric enzyme (dimer mass ~112.3 kDa) from Aspergillus niger that reversibly decarboxylates phenylacrylic acids such as ferulic acid. It belongs to the UbiD enzyme family, whose members all depend on prFMN (prenylated flavin mononucleotide), a heavily modified flavin carrying an extra isoprenoid-derived ring that the flavin prenyltransferase UbiX/Pad1 installs on FMN. In its oxidized iminium form, prFMN is a prosthetic group that catalyses decarboxylation through a reversible 1,3-dipolar cycloaddition between the substrate and an azomethine-ylide form of the cofactor, the first enzymatic 1,3-dipolar cycloaddition to be described (Payne et al., 2015). Atomic-resolution crystal structures of holo A. niger Fdc1, with the cofactor bound next to a Mn2+ ion that coordinates the prFMN phosphate, established the active-site chemistry; however, no crystals of the cofactor-free (apo) A. niger enzyme could be obtained, so all crystallographic information reports the cofactor-bound state. A parallel structure of apo Saccharomyces cerevisiae FDC1 (Bhuiya et al., 2015; PDB 4S13, determined before prFMN was known) shows a broadly similar preorganized fold in a different organism and crystal environment, but no solution dynamics and no cofactor-bound comparison; its cofactor-independent mechanistic proposal was superseded by the prFMN cycloaddition mechanism.

Beveridge et al. (2016) compared apo-Fdc1 (produced by expressing fdc1 alone in E. coli) with holo-Fdc1 (co-expressed with UbiX) using three mass-spectrometry methods: native electrospray MS, which weighs intact non-denatured complexes and reveals stoichiometry; ion-mobility MS (IM-MS), in which gas-phase ions drift through helium so that the rotationally averaged collision cross section (CCS, in square angstroms) reports on overall compactness; and hydrogen-deuterium exchange MS (HDX-MS), which measures the solvent accessibility and dynamics of individual peptide regions through their time-dependent deuterium uptake. They found that prFMN binding stabilizes the dimer (monomer peaks disappear), shifts CCS distributions toward more compact conformers, and causes both local decreases in deuterium uptake near the cofactor- and Mn2+-binding sites and distal decreases far from the cofactor site, i.e. an overall conformational tightening, while the dimer interface is essentially unchanged. The mechanistic interpretation remained open in four ways: (i) the native spectra show a mixture of dimers with zero, one, and two bound cofactors, and the singly-to-doubly-bound intensity ratio rises from about 0.66 to about 0.75 when the desolvation (sample-cone) voltage is raised from 60 to 200 V, implicating gas-phase cofactor loss, so the true equilibrium occupancy in solution was never directly measured; (ii) no atomistic model of the apo state has been published or released (the short in-vacuo cofactor-removed simulations accompanying the reported holo runs are noted only as 'data not shown'), only peptide-level HDX differences relative to the holo crystal coordinates, leaving the registries and lengths of destabilized helices and loops undetermined; (iii) the supporting molecular dynamics (MD) simulations were only 10 ns and run in vacuo to mimic gas-phase behaviour, far from the millisecond ion-mobility timescale and from solution-phase dynamics; and (iv) the residue-level route by which local prFMN coordination chemistry causes the distal conformational tightening was not identified. Later UbiD-family work established that prFMN-binding-domain motion between open and closed states accompanies cofactor binding in some family members (HudA/PA0254, and VdcCD where the small VdcD subunit allosterically activates the UbiD-like VdcC), but none of it resolves the solution-phase apo-to-holo mechanism for Fdc1 itself.

Problem Statement

Determine, at residue resolution and in solution, how binding of the prFMN cofactor reorganizes the homodimeric ferulic acid decarboxylase Fdc1 from Aspergillus niger. This single objective comprises four jointly linked aspects, each an ingredient of the same mechanism:

  1. The equilibrium occupancy and stoichiometry of prFMN binding to Fdc1 in aqueous solution under near-native conditions, meaning an aqueous medium whose pH, ionic strength, temperature, Mn2+ status, and protein and prFMN concentrations are explicitly stated, anchored to the conditions of the source (purified protein in 25 mM Tris pH 7.5, 100 mM NaCl, 1 mM MnCl2; native MS sprayed from 100 mM ammonium acetate, pH 6.8), and established by an approach that separates true solution speciation from gas-phase cofactor loss during electrospray and desolvation (the source data show a zero-/one-/two-cofactor mixture by native MS and a cone-voltage-dependent singly/doubly-bound ratio, but no direct solution measurement).

  2. An atomistic description of the apo-Fdc1 ensemble in solution, including the registry and lengths of the helices and loops that HDX-MS shows to be destabilized relative to the holo crystal coordinates (PDB entries such as 4ZA4), going beyond peptide-level exchange data.

  3. The kinetic pathway of the apo-to-holo conformational transition and the role of solvent in it, replacing the 10 ns in-vacuo simulations with an account relevant to solution-phase dynamics and to the timescales accessible to experiment.

  4. The causal, residue-by-residue allosteric communication pathway by which local prFMN and Mn2+ coordination chemistry at the cofactor-binding site produces the distal conformational tightening detected by HDX-MS, together with its mechanistic link to the observed stabilization and compaction of the dimer.

The mechanism sought is a single coherent structural-dynamical account of how prFMN binding reorganizes Fdc1 that jointly reproduces the observed apo-versus-holo differences (dimer stabilization, CCS compaction, local and distal uptake changes, unchanged dimer interface); the four aspects above are jointly constitutive of that mechanism, so no one of them alone is sufficient. A single partial determination such as an apo structure or an occupancy measurement addresses only one aspect of the mechanism and does not by itself establish how prFMN binding reorganizes Fdc1.

References

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  2. Payne KAP, White MD, Fisher K, Khara B, Bailey SS, Parker D, Rattray NJW, Trivedi DK, Goodacre R, Beveridge R, Barran P, Rigby SEJ, Scrutton NS, Hay S, Leys D. New cofactor supports α,β-unsaturated acid decarboxylation via 1,3-dipolar cycloaddition. Nature 522:497-501 (2015). doi:10.1038/nature14560, https://doi.org/10.1038/nature14560
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