Unveiling the activation dynamics of a fold-switch bacterial glycosyltransferase by 19F NMR

Abstract

Fold-switch pathways remodel the secondary structure topology of proteins in response to the cellular environment. It is a major challenge to understand the dynamics of these folding processes. Here, we conducted an in-depth analysis of the α-helix-to-β-strand and β-strand-to-α-helix transitions and domain motions displayed by the essential mannosyltransferase PimA from mycobacteria. Using ¹⁹F NMR, we identified four functionally relevant states of PimA that coexist in dynamic equilibria on millisecond-to-second timescales in solution. We discovered that fold-switching is a slow process, on the order of seconds, whereas domain motions occur simultaneously but are substantially faster, on the order of milliseconds. Strikingly, the addition of substrate accelerated the fold-switching dynamics of PimA. We propose a model in which the fold-switching dynamics constitute a mechanism for PimA activation.

Keywords: 19F NMR; carbohydrate active enzymes; conformational dynamics; enzyme catalysis; glycosyltransferases; protein fold-switching, protein dynamics; protein function; protein structure; relaxation dispersion.

Figure 1.

Crystal structures of PimA. (A) In its inactive state, PimA adopts two conformations (PDB entry 4NC9, apo state of PimA) with different tertiary structures in the reshuffling region (displayed in rainbow color) but identical topology. In the inactive, extended form (orange), two helices (α4 and α5) point away from the N-terminal domain and fold back along hinge loops onto the N-terminal domain in the inactive, compact conformation (yellow). Note that the loop connecting α4 and α5 is only partially resolved in the crystal structure of the inactive, extended state. Transition to the active state (PDB entry 2GEJ, PimA in the presence of GDP-Man) is accompanied by a fold-switch in the reshuffling region (red). In the crystal structures, R144 is shown/modeled as spheres in green. Selected secondary structure elements of PimA, including the reshuffling region (residues 118–163), as observed in the unliganded (inactive) compact and extended conformations and the liganded (active) PimA-GDP-Man complex, are shown. β5, β6, and β7 are shown in blue, light blue, and orange, respectively. (B) ¹⁹F spectra of PimA^{T126C-V359C-R144C-TFA} in 10% D₂O (top), PimA^R144C-TFA in 100% D₂O (center), and PimA^R144C-TFA in 10% D₂O (bottom). (C) ¹⁹F spectra of PimA^R144C-TFA in the absence (blue) and at increasing concentrations of GDP-Man. Intensities are corrected for dilution effects.

Figure 2.

Slow conformational exchange of PimA^R144C-TFA. (A) Intensity attenuation of the active state resonance in absence (black) and presence (orange) of saturating concentrations of GDP-Man upon saturation of the inactive state resonance. (B) Intensity attenuation of the inactive state resonance in the absence (black) and presence (orange) of saturating concentrations of GDP-Man upon the saturation of the active state resonance. Experiments were conducted with 100 μm PimA^R144C-TFA and 500 μm GDP-Man, where applicable. (C) Secondary structure representation of the reshuffling region. The position of the mutation (R144C) is indicated as a green dot. Parameters (p, populations; k_s, slow exchange rates) are obtained from the fit to data in panels A and B.

Figure 3.

Intermediate conformational exchange of the inactive and the active states of PimA^R144C-TFA. (A) Relaxation dispersion of the inactive state resonance in the absence (black) and presence (orange) of saturating concentrations of GDP-Man. (B) Extended-to-compact dynamics in the reshuffling region (extended conformation in orange; compact conformation in yellow). Parameters (p, populations; Δδ, chemical shift difference) obtained from the fit to data in panel A are indicated (black, no ligand; orange, with GDP-Man). (C) Relaxation dispersion of the active state resonance in the absence (black) and presence (orange) of saturating concentrations of GDP-Man. (D) Extended-to-compact dynamics that occur in the reshuffling region (extended conformation in gray; compact conformation in red). Only the compact, active conformation is structurally characterized. Parameters (p, populations; Δδ, chemical shift difference) obtained from the fit to the data in panel C are indicated (black, no ligand; orange, with GDP-Man). Experiments were conducted with 100 μm PimA^R144C-TFA and 500 μm GDP-Man where applicable. All observed exchange rates are on the order of 4000–8000 s⁻¹ (see Table 1 for details).

Figure 4.

Activation mechanism of PimA. Structural changes of PimA upon activation. Initially, almost all inactive PimA molecules are in the compact conformation but are in millisecond exchange with the inactive, extended state. Following the fold-switch, PimA is in the structurally uncharacterized active, extended state and in millisecond exchange with the active, compact state, which is the conformation in which catalysis occurs. The location of R144 is shown/modeled for clarity.