Visualizing molecular juggling within a B12-dependent methyltransferase complex

Abstract

Derivatives of vitamin B(12) are used in methyl group transfer in biological processes as diverse as methionine synthesis in humans and CO(2) fixation in acetogenic bacteria. This seemingly straightforward reaction requires large, multimodular enzyme complexes that adopt multiple conformations to alternately activate, protect and perform catalysis on the reactive B(12) cofactor. Crystal structures determined thus far have provided structural information for only fragments of these complexes, inspiring speculation about the overall protein assembly and conformational movements inherent to activity. Here we present X-ray crystal structures of a complete 220 kDa complex that contains all enzymes responsible for B(12)-dependent methyl transfer, namely the corrinoid iron-sulphur protein and its methyltransferase from the model acetogen Moorella thermoacetica. These structures provide the first three-dimensional depiction of all protein modules required for the activation, protection and catalytic steps of B(12)-dependent methyl transfer. In addition, the structures capture B(12) at multiple locations between its 'resting' and catalytic positions, allowing visualization of the dramatic protein rearrangements that enable methyl transfer and identification of the trajectory for B(12) movement within the large enzyme scaffold. The structures are also presented alongside in crystallo spectroscopic data, which confirm enzymatic activity within crystals and demonstrate the largest known conformational movements of proteins in a crystalline state. Taken together, this work provides a model for the molecular juggling that accompanies turnover and helps explain why such an elaborate protein framework is required for such a simple, yet biologically essential reaction.

Figure 1. The overall CFeSP/MeTr complex

Ribbon representation of MeTr homodimer (MeTr₂) in light and dark pink, CFeSP small subunits in orange, and CFeSP large subunit Fe₄S₄ domains in teal and cyan, TIM barrel domains in green and blue, and B₁₂-domains in dark green and dark blue. B₁₂ cofactors in magenta sticks with cobalt as violet spheres. Fe₄S₄ clusters in spheres: Fe in orange and S in yellow.

Figure 2. Comparison of B₁₂ positions in “resting” ChCFeSP, folate-free, and folate-bound CFeSP/MeTr

a, ChCFeSP (grey ribbons, Co of B₁₂: black sphere). b, Folate-free CFeSP/MeTr (green ribbons, Co of B₁₂: green sphere) superimposed with CH₃-H₄folate-bound MeTr (PDB ID: 2E7F, pink ribbons). c, Folate-bound CFeSP/MeTr (orange ribbons, Co of B₁₂: orange sphere). Panels a–c are identical in orientation, B₁₂ sticks coloured C: ribbon colour, O: red, N: blue, and P: orange. d, Superposition of ChCFeSP (grey), folate-free CFeSP/MeTr (green), and folate-bound CFeSP/MeTr (orange) structures in a–c, highlighting one helix (thick ribbons) to show clamping motion (helix axes as straight blue lines) and B₁₂ (sticks) with 12 residue linker (thick ribbons) to B₁₂ domain (surface) to show swinging motion. e, Superposition of B₁₂ and CH₃-H₄folate in d, with Asn199 shown for CFeSP/MeTr structures in sticks (C: ribbon colour, O: red, N: blue). f, Same as e, with 2F_o-F_c density in blue (1.0 σ) and pink mesh (4.0 σ), and F_o-F_c density in green mesh (3.0 σ) for folate-bound CFeSP/MeTr structure. Putative alternative B₁₂ corrin: cyan. g, Superposition of B₁₂ cofactors and CH₃-H₄folate in f.

Figure 3. Methyltransfer activity of CFeSP/MeTr crystals by UV-vis absorption spectroscopy

As-isolated spectra (blue lines) for CFeSP/MeTr crystals and CFeSP in solution similarly have broad features at ~400 nm and ~470 nm arising from the Fe₄S₄ cluster and the B₁₂ corrin. Following established protocols^,,,,,, B₁₂ reduction was achieved with Ti(III) citrate, yielding a sharp 390 nm peak indicative of active Co(I) in both solution and in crystallo spectra (black lines). Further treatment with CH₃-H₄folate yields decreased absorbance at 390 nm and a new peak at 450 nm (red lines), characteristic of the product complex (protein-bound CH₃-Co(III)^,,,). A control reaction (green line) confirms that turnover does not occur from free CH₃-H₄folate without MeTr, and the 450 nm peak indicates that B₁₂ remains CFeSP-bound (free B₁₂ has a peak at ~520 nm instead^,,).

Figure 4. Cartoon model of B₁₂-dependent methyltransfer in CFeSP/MeTr

For simplicity, only one of the two CFeSP heterodimers is shown. Protein domains are colored as in Fig. 1, loops represent linkers, red hexagon is B₁₂, blue rectangle is folate, and transferred methyl group is shown as a yellow sphere. Curved arrows denote “swinging” and “clamping” motions.