Structures of the human GTPase MMAA and vitamin B12-dependent methylmalonyl-CoA mutase and insight into their complex formation

Abstract

Vitamin B(12) (cobalamin, Cbl) is essential to the function of two human enzymes, methionine synthase (MS) and methylmalonyl-CoA mutase (MUT). The conversion of dietary Cbl to its cofactor forms, methyl-Cbl (MeCbl) for MS and adenosyl-Cbl (AdoCbl) for MUT, located in the cytosol and mitochondria, respectively, requires a complex pathway of intracellular processing and trafficking. One of the processing proteins, MMAA (methylmalonic aciduria type A), is implicated in the mitochondrial assembly of AdoCbl into MUT and is defective in children from the cblA complementation group of cobalamin disorders. To characterize the functional interplay between MMAA and MUT, we have crystallized human MMAA in the GDP-bound form and human MUT in the apo, holo, and substrate-bound ternary forms. Structures of both proteins reveal highly conserved domain architecture and catalytic machinery for ligand binding, yet they show substantially different dimeric assembly and interaction, compared with their bacterial counterparts. We show that MMAA exhibits GTPase activity that is modulated by MUT and that the two proteins interact in vitro and in vivo. Formation of a stable MMAA-MUT complex is nucleotide-selective for MMAA (GMPPNP over GDP) and apoenzyme-dependent for MUT. The physiological importance of this interaction is highlighted by a recently identified homoallelic patient mutation of MMAA, G188R, which, we show, retains basal GTPase activity but has abrogated interaction. Together, our data point to a gatekeeping role for MMAA by favoring complex formation with MUT apoenzyme for AdoCbl assembly and releasing the AdoCbl-loaded holoenzyme from the complex, in a GTP-dependent manner.

FIGURE 1.

Dimeric assembly of human and bacterial MUT. Cartoon and surface representations of the hMUT homodimer (a, c) and psMUT heterodimer (b, d, PDB code 4REQ). Catalytic subunits (α) are colored according to domain architecture (N-domain, blue; C-domain, magenta; interdomain belt, yellow). The bacterial regulatory subunit (β) is colored gray. In panels c and d, the cofactor binding pocket and substrate binding channel are indicated by white arrow and black arrow, respectively. mCoA, malonyl-CoA; Cbl, cobalamin; sCoA, succinyl-CoA.

FIGURE 2.

Ligand-induced conformational changes in hMUT. a, active site view of the hMUT_holo structure (cyan), superimposed with the hMUT_apo structure (gray). The bound AdoCbl molecule in the hMUT_holo structure is shown in green sticks. b, Superposition of the hMUT_holo (cyan) and hMUT_ter (pink) structures, viewed from the entrance of the substrate binding channel (shown with substrate analog mCoA in white sticks). Black arrows indicate the displacement of secondary structures in the N-domain upon binding of mCoA. c and d, surface representation of the structures of hMUT_apo (top), hMUT_holo (middle), and hMUT_ter (bottom), highlighting the substrate binding channel (broken arrow), cofactor binding pocket (solid arrow), and part of the flexible interdomain belt (yellow) that becomes ordered upon cofactor binding. For convenient comparison, molecules of mCoA and AdoCbl have been added to all panels. e, substrate binding channel of hMUT is shown with channel-lining residues in the hMUT_holo (cyan lines) and hMUT_ter (pink lines) structures. The bound substrate analog (mCoA) and cofactor (Cbl) are shown in sticks. Two alternative conformations of the cofactor 5′-deoxyadenosyl group (Ado), as revealed in the electron density map, are shown (yellow).

FIGURE 3.

Crystal structure of human MMAA. a, structure of an hMMAA monomer consists of the N-extension (pink), the G-domain (yellow) harboring four conserved sequence motifs for P-loop GTPases (red), and the dimerization arm (blue). b, cartoon representation of the hMMAA nucleotide binding site, bound with GDP (in sticks). Residues forming polar interactions with GDP are shown in lines. Three conserved sequence motifs are colored red.

FIGURE 4.

Dimer assembly in hMMAA and MeaB. a, Cα-superposition of hMMAA (cyan) and MeaB (gray) monomeric subunits, highlighting several loops in the G-domain (L2–L4) that are ordered in the MeaB structure, and the relative displacement of the dimerization arms of the two structures. b and c, surface representations of the hMMAA and MeaB homodimers, highlighting the positions of the nucleotide binding site of each subunit (red arrows). The bound GDP is shown in sticks. The two subunits of each dimer are colored in light and dark shades of cyan (hMMAA) or gray (MeaB). In panel b, the ordered loops L2–L4 in MeaB structure are added onto the structure of hMMAA to illustrate the more complete polypeptide.

FIGURE 5.

Functional and physical interactions between hMMAA and hMUT. a, effect of hMUT on the GTPase activity of hMMAA. Reactions were performed by incubating hMMAA with hMUT_apo (square), hMUT_holo (triangle), or without hMUT (circle). Kinetic parameters of GTP hydrolysis are shown in Table 2. b, gel filtration profile on a HiLoad 10/30 Superdex 200 column. The samples run are hMMAA_GMPPNP:hMUT_apo mixture (black line), hMMAA_GDP:hMUT_apo mixture (gray line), hMMAA_GMPPNP:hMUT_holo (black dashed line), and hMMAA_G188R,GMPPNP:hMUT_apo (gray dashed line). The two complex peaks as well as individual protein peaks are labeled. c, in vivo hMMAA-hMUT interaction. pCMV-hMMAA-Flag (lane 1), pCMV-Flag control (lane 2), or pCMV-hMMAA_G188R-Flag (lane 3) was transfected into HepG2 cells, cross-linked with 1% PFA, immunoprecipitated (IP) with anti-Flag antibody, and immunoblotted (IB) with anti-hMUT or anti-Flag antibody to identify hMUT and hMMAA, respectively.