Mobile loop dynamics in adenosyltransferase control binding and reactivity of coenzyme B12

Abstract

Cobalamin is a complex organometallic cofactor that is processed and targeted via a network of chaperones to its dependent enzymes. AdoCbl (5'-deoxyadenosylcobalamin) is synthesized from cob(II)alamin in a reductive adenosylation reaction catalyzed by adenosyltransferase (ATR), which also serves as an escort, delivering AdoCbl to methylmalonyl-CoA mutase (MCM). The mechanism by which ATR signals that its cofactor cargo is ready (AdoCbl) or not [cob(II)alamin] for transfer to MCM, is not known. In this study, we have obtained crystallographic snapshots that reveal ligand-induced ordering of the N terminus of Mycobacterium tuberculosis ATR, which organizes a dynamic cobalamin binding site and exerts exquisite control over coordination geometry, reactivity, and solvent accessibility. Cob(II)alamin binds with its dimethylbenzimidazole tail splayed into a side pocket and its corrin ring buried. The cosubstrate, ATP, enforces a four-coordinate cob(II)alamin geometry, facilitating the unfavorable reduction to cob(I)alamin. The binding mode for AdoCbl is notably different from that of cob(II)alamin, with the dimethylbenzimidazole tail tucked under the corrin ring, displacing the N terminus of ATR, which is disordered. In this solvent-exposed conformation, AdoCbl undergoes facile transfer to MCM. The importance of the tail in cofactor handover from ATR to MCM is revealed by the failure of 5'-deoxyadenosylcobinamide, lacking the tail, to transfer. In the absence of MCM, ATR induces a sacrificial cobalt-carbon bond homolysis reaction in an unusual reversal of the heterolytic chemistry that was deployed to make the same bond. The data support an important role for the dimethylbenzimidazole tail in moving the cobalamin cofactor between active sites.

Keywords: cobalamin; cofactor; crystal structure; kinetics; trafficking.

Fig. 1.

Cobalamin binding to and functions of ATR. (A) In the presence of ATP, ATR binds cob(II)alamin in an unfavorable 4-c geometry. Following a one electron reduction, ATR catalyzes the adenosylation of cob(I)alamin to form 5-c base-off AdoCbl (455 nm), which is transferred to the MCM-CblA complex with concomitant hydrolysis of GTP. In the absence of AdoCbl transfer, the newly formed Co-C bond is weakened in the ternary ATR•AdoCbl•PPPi complex leading to a species with an absorption maximum at 440 nm. In the presence of oxygen, Co-C bond cleavage is observed with concomitant formation of hydroperoxyadenosine (Ado-OOH) and cob(II)alamin, with an absorption maximum at 464 nm, which upon reduction, can serve in another cycle of AdoCbl synthesis. Alternatively, further oxidation of cob(II)alamin leads to aquocobalamin, which is released into solution due to its weak affinity for ATR. (B) Increasing concentrations of ATR were added to cob(II)alamin (50 µM, black) in anaerobic Buffer A at 25 °C in the presence of 5 mM ATP. Spectra were recorded 5 min after each addition (gray lines). Binding of cob(II)alamin to ATR•ATP results in a strong absorption peak at 464 nm (red, final spectrum). (C) The change in absorbance at 464 nm (in B) versus ATR monomer concentration yielded K_D = 0.44 ± 0.08 µM (mean ± SD, n = 3). (D) Increasing concentration of ATR were added to AdoCbl (30 µM, black) in Buffer A at 25 °C and spectra were recorded 5 min after each addition (gray lines). ATR binding resulted in a spectral shift from 525 nm to 455 nm (red, final spectrum). (E) The change in absorbance at 525 nm (in D) versus ATR monomer concentration yielded K_D = 0.92 ± 0.1 µM (mean ± SD, n = 4).

Fig. 2.

ATP and PPPi influence the EPR spectrum of Mtb ATR-bound cob(II)alamin. (A and B) EPR spectra of cob(II)alamin (100 µM) in Buffer A in the absence (A) or presence (B) of ATR (100-µM trimer). The singlets in the high-field region (lines) and the broad peak at ∼2,600 G (arrow) indicate a minor population of 5-c cob(II)alamin in which axial DMB nitrogen ligand is replaced by oxygen from H₂O. The major population with triplet superhyperfine structures corresponds to free cob(II)alamin. (C) Addition of 5 mM PPPi to the sample in B resulted in an increase in the fraction of 5-c cob(II)alamin with a water ligand indicated by the increase in intensity at ∼2,700 G (arrow) and well-resolved hyperfine singlets in the high field region (vertical lines). (D) Addition of 5 mM ATP to B resulted in a spectrum that is typical of 4-c cob(II)alamin. Two of the three hyperfine coupling constants are indicated by vertical lines.

Fig. 3.

Structural basis of AdoCbl binding and Co-C bond cleavage. (A, B, and E) Surface representation of the structures of apo-ATR (A), ATR•AdoCbl (B), and ATR•cob(II)alamin•PPPi (E). The subunits are in blue, gray, and yellow and B₁₂ is in a ball-and-stick display. The progressive ordering of the N-terminal loop is best seen in the blue subunit, which cups B₁₂ in the ATR•cob(II)alamin•PPPi structure. (C) Close-up of the ATR•AdoCbl active site shows that the N-terminal β-hairpin (blue) sits above the 5′-dAdo moiety of AdoCbl (yellow stick display). The mobile loop (108–123) and the C-terminal tail form the floor of the B₁₂ binding pocket. (D and F) Fo–Fc (2.5 σ) simulated annealing omit maps of ATR•AdoCbl without (D) or with (F) PPPi (orange sticks), which binds in the predicted ATP site. The shortest distance between a PPPi oxygen and the C5′ atom in dAdo is 3.2 Å.

Fig. 4.

Structural basis for cob(II)alamin binding and activation. (A) Close-up of the ATR•cob(II)alamin•PPPi active site showing interactions between the N-terminal residues (3 to 29), B₁₂ and PPPi, which are bound between adjacent subunits (blue and gray). The purple and green spheres represent Mg²⁺ and K⁺, respectively. (B) Surface view of the ATR•cob(II)alamin •PPPi structure shows that the ordered N terminus forces the DMB tail into an extended position. (C) A Fo–Fc omit map of B₁₂, PPPi and water are shown at 2.5 σ. (D) An overlay of ATR•cob(II)alamin•PPPi (blue) and ATR•AdoCbl•PPPi (yellow) reveals differences in the N terminus and the 78–93 loop and the position of the corrin ring in cob(II)alamin (yellow sticks) due to interactions between acetamide group “A” with PPPi and propionamide group “B” with Thr-6. The extended position of the DMB tail (yellow), contrasts with its tucked position in the AdoCbl structure (red).

Fig. 5.

Regulation of cofactor translocation from ATR to MCM. (A) AdoCbl transfer from ATR to MCM•CblA•GTP. Transfer of AdoCbl to MCM in the presence of CblA leads to a shift from 455 nm (5-c) to 525 nm (6-c) but no further change upon addition of PPPi. (Lower left) ATR (15-µM trimer) was loaded with AdoCbl (15 µM, Inset) (black) before addition of MCM (30 µM), CblA (60-µM dimer) and GTP (1 mM) and incubated for 20 min at 25 °C. Spectra were recorded every minute (gray traces) and the final spectrum is shown in red. (Lower right) PPPi (5 mM) was added after 20 min and incubated at 25 °C; the spectrum was recorded after 20 min (red). No spectroscopic changes were observed, indicating that AdoCbl had transferred to MCM. (B) AdoCbi does not transfer from ATR to MCM•CblA•GTP. Subsequent addition of PPPi under aerobic conditions leads to Co-C bond homolysis in ATR-bound AdoCbi and to the formation of a mixture of cob(II)alamin and H₂OCbl. (Lower left) ATR (15 µM trimer) was loaded with AdoCbi (15 µM, Inset) (black) before addition of MCM (30 µM), CblA (60 µM) and GTP (1 mM) in Buffer A and incubated for 20 min at 25 °C. Spectra were recorded every minute (gray traces) and the final spectrum is shown in red. (Lower right) PPPi (5 mM) was added after 20 min and incubated at 25 °C; the spectrum was recorded after 20 min (red). The increase in absorption at 466 nm corresponds to cob(II)alamin resulting from Co-C bond homolysis of AdoCbi bound to ATR and the subsequent oxidation product, H₂OCbl (355 nm).

Fig. 6.

Model for AdoCbl translocation from ATR to MCM. The N terminus of ATR is ordered by ATP into a cup for cob(II)alamin binding and forces the DMB tail into a hydrophobic side pocket. This conformation favors the reductive adenosylation reaction. In the product complex, the first approximately eight residues are disordered, which accommodates the tucked-tail conformation and increases solvent accessibility to the cofactor. The histidine residue on MCM, which serves as the lower ligand to AdoCbl, serves an important role in its translocation.