Energetics of Interaction between the G-protein Chaperone, MeaB, and B12-dependent Methylmalonyl-CoA Mutase

Tetyana Labunska,Dominique Padovani,Ruma Banerjee

doi:10.1074/jbc.m600047200

Tetyana Labunska, Dominique Padovani + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m600047200

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Abstract

MeaB is an auxiliary protein that supports the function of the radical B(12)-dependent enzyme, methylmalonyl-CoA mutase, although its precise role is not understood. Mutations in the human homolog of MeaB, MMAA, lead to methylmalonic aciduria, an inborn error of metabolism that can be fatal. To obtain insights into the function of this recently discovered protein, we have characterized the entropic and enthalpic contributions to DeltaGdegree (assoc) for complexation of MeaB (in the presence and absence of nucleotides) with methylmalonyl-CoA mutase (in the presence and absence of cofactor). The dissociation constant for binding of methylmalonyl-CoA mutase and MeaB ranges from 34 +/- 4 to 524 +/- 66 nm, depending on the combination of nucleotide and mutase form. Holomutase binds MeaB 15-fold more tightly when the nonhydrolyzable GTP analog, GMPPNP, is bound versus GDP. In contrast, the apomutase binds MeaB with similar affinity in the presence of either nucleotide. Our studies reveal that a large structural rearrangement accompanies interaction between these proteins and buries between approximately 4000 and 8600A(2) of surface area, depending on the combination of ligands in the active sites of the two proteins. Furthermore, we demonstrate that MeaB binds GTP and GDP with similar affinity (K(d) of 7.3 +/- 1.9 and 6.2 +/- 0.7 microm, respectively at 20 degrees C) and has low intrinsic GTPase activity (approximately 0.04 min(-1) at 37 degrees C), which is stimulated approximately 100-fold by methylmalonyl-CoA mutase. These studies provide insights into the energetics of interaction between the radical enzyme methylmalonyl-CoA mutase and MeaB, which are discussed.

Highlights

The most recently described member of this subfamily of GTPases is MeaB, which is strongly associated in bacterial operons with methylmalonyl-CoA mutase [8], a B12-dependent isomerase that catalyzes the rear
An ortholog of MeaB, MMAA, is found in humans and is the locus of mutations associated with the cblA class of inborn errors of cobalamin disorders that leads to methylmalonic aciduria [11, 12]
We demonstrate that the low intrinsic GTPase activity of MeaB is stimulated ϳ100-fold by methylmalonyl-CoA mutase, and the identity of the nucleotide as well as the presence or absence of the B12 cofactor modulate the affinity between the two proteins

Summary

EXPERIMENTAL PROCEDURES

AdoCbl, GTP, GMPPNP, GDP, cGMP, GMP, AMPPNP, and ATP were purchased from Sigma. Methylmalonic acid was purchased from. The column was calibrated using gel filtration standards from Bio-Rad. To analyze complex formation between MeaB and methylmalonylCoA mutase, 16 ␮g of apoenzyme was mixed with 10 ␮g of MeaB either alone or in the presence of (i) 50 ␮M AdoCbl or (ii) 50 ␮M AdoCbl and 1 mM methylmalonyl-CoA at 20 °C in 50 mM HEPES, pH 8, 300 mM KCl, 10 mM MgCl2, 5% glycerol. The effect of methylmalonyl-CoA mutase (apo- or holo-form) on the GTPase activity of MeaB was analyzed by incubating the complex (containing 10 ␮M of each protein) in 50 mM HEPES, pH 8, 300 mM KCl, 10 mM MgCl2, 5% glycerol with 5 mM GTP at 37 °C. (mol1⁄7K) for binary protein-protein interactions) and ⌬S oother, which is related to Rth because Rth ϭ ⌬S oother/Ϫ5.6 e.u. [17]

RESULTS

34 Ϯ 4 524 Ϯ 66

DISCUSSION