Switch I-dependent allosteric signaling in a G-protein chaperone-B12 enzyme complex

Abstract

G-proteins regulate various processes ranging from DNA replication and protein synthesis to cytoskeletal dynamics and cofactor assimilation and serve as models for uncovering strategies deployed for allosteric signal transduction. MeaB is a multifunctional G-protein chaperone, which gates loading of the active 5'-deoxyadenosylcobalamin cofactor onto methylmalonyl-CoA mutase (MCM) and precludes loading of inactive cofactor forms. MeaB also safeguards MCM, which uses radical chemistry, against inactivation and rescues MCM inactivated during catalytic turnover by using the GTP-binding energy to offload inactive cofactor. The conserved switch I and II signaling motifs used by G-proteins are predicted to mediate allosteric regulation in response to nucleotide binding and hydrolysis in MeaB. Herein, we targeted conserved residues in the MeaB switch I motif to interrogate the function of this loop. Unexpectedly, the switch I mutations had only modest effects on GTP binding and on GTPase activity and did not perturb stability of the MCM-MeaB complex. However, these mutations disrupted multiple MeaB chaperone functions, including cofactor editing, loading, and offloading. Hence, although residues in the switch I motif are not essential for catalysis, they are important for allosteric regulation. Furthermore, single-particle EM analysis revealed, for the first time, the overall architecture of the MCM-MeaB complex, which exhibits a 2:1 stoichiometry. These EM studies also demonstrate that the complex exhibits considerable conformational flexibility. In conclusion, the switch I element does not significantly stabilize the MCM-MeaB complex or influence the affinity of MeaB for GTP but is required for transducing signals between MeaB and MCM.

Keywords: GTPase; adenosylcobalamin (AdoCbl); allosteric regulation; electron microscopy (EM); low molecular weight G-protein.

Figure 1.

Nucleotide-gated transfer of AdoCbl from ATR to MCM. AdoCbl transfer is driven by the binding of ATP to a vacant site in the ATR homotrimer and gated by GTP hydrolysis catalyzed by MeaB. AdoCbl bound to ATR is in a five-coordinate “base-off” conformation (λ_max = 458 nm) and switches to a six-coordinate “base-off/His-on” (λ_max = 527 nm) conformation in the MCM active site.

Figure 2.

Structure of MeaB and comparison of its active site with orthologous proteins. A, structure of MeaB protomer with GMPPNP bound (Protein Data Bank code 4JYB) in which the switch I (yellow), II (magenta), and III (green) elements are highlighted. B–D, close-ups of the active site in MeaB·GMPPNP (B), CblA·GDP (Protein Data Bank code 2WWW) (C), and IcmF·GDP·AdoCbl (Protein Data Bank code 4XC6) (D). Shown in stick representation in all panels are the bound guanosine nucleotides (blue). Magnesium cations are shown as gray spheres, and switch II residues, Val¹⁵⁶ in MeaB and Val²⁴⁴ in CblA, are shown as magenta spheres. Conserved switch I residues are labeled in boldface font.

Figure 3.

The impact of switch I mutations on the chaperone functions of MeaB. A–C, spectral changes in MCM in the presence of its substrate methylmalonyl-CoA and in the absence of MeaB (A), the presence of MeaB (B), or the presence of MeaB and GMPPNP (C). The reaction mixtures contained 5 mm methylmalonyl-CoA in 0.1 m potassium phosphate buffer, pH 7.5, containing 10 mm MgCl₂ and were incubated at 20 °C for 70 min. The black and red traces in each panel represent the initial (t = 0 min) and final (t = 70 min) spectra, and the gray spectra were recorded at intermediate time points (every 2 min), and only a subset is shown for clarity. A and B show an increase in absorbance at 351 nm, which is indicative of OH₂Cbl formation. D, change in absorption at 351 nm (at 70 min) representing MCM inactivation in the absence or presence of wild-type (WT) or mutant MeaBs. E, ATP-driven release or transfer of AdoCbl from ATR to the MCM–MeaB complex (containing wild-type or mutant MeaBs) was monitored in the presence of GMPPNP. F, displacement of cob(II)alamin from MCM·cob(II)alamin–MeaB (wild type or switch I mutants) following addition of GMPPNP under anaerobic conditions. The data in D–F are the average of at least two independent experiments (±S.D.).

Figure 4.

Stoichiometry and visualization of the MCM–MeaB complex. A, isolated wild-type and MeaB mutants (25 μm) were mixed with MCM (25 μm) in 50 mm HEPES, 0.3 m KCl, 3 mm MgCl₂, pH 8.0, and GMPPNP (250 μm) and injected onto a size exclusion column coupled to a MALS detector. The black traces represent the normalized refractive index (left axes). The red lines indicate the molecular mass as detected by MALS (right axes). The wild-type complex eluted at an elution volume of 8.7 ml and had a molecular mass (Mw) of 359 kDa and a hydrodynamic radius (Rh) of 7.3 nm. The molecular mass of the complex suggests a ratio of 2:1 MCM:MeaB. Complexes of MCM with each of the switch I MeaB mutants showed essentially the same stoichiometry, and analyses of complexes containing D92A or K106A MeaB are shown as representative data. B, selected reference-free 2D class averages of the MCM–MeaB·GMPPNP complex show a mixture of conformations ranging from closed to open. C, MCM and MeaB densities were identified by comparison with their crystal structures (MCM, Protein Data Bank code 1REQ; MeaB·GMPPNP, Protein Data Bank code 4JYB) and manually positioned to make a model in Chimera (not to scale) and shown with 2D back-projections for comparison.

Figure 5.

Schematic representations of the interaction between G-protein chaperones and client mutases. The G-domains are in blue, mutase substrate domains are in green, and the B₁₂-domains are in red. The GTP-binding sites are shown as yellow spheres. The organization of the stand-alone or fusion proteins is shown above the known or predicted architecture of the complexes. A, the M. extorquens MCM only has one active site per αβ heterodimer. The inactive β-subunit is in gray. MeaB is an α₂ homodimer with two GTP-binding sites. B, IcmF is a fusion protein with AdoCbl, MeaI (G-domain), and substrate-binding domains fused in the direction of N to C terminus as shown. The crystal structure reveals that IcmF exists as a homodimer with the G-domains split into monomers, each interacting with an active α-subunit. C, human MCM is an α₂ homodimer with two active subunits. The organization of the human complex is proposed to be two MCMs to one CblA and is presently unknown.