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 nucleotideselective 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.

Visualization of a radical B 12 enzyme with its G-protein chaperone

“…We now posit that MCM F573S, R616C, G642R, and G648D, as well as MMAA G188R (Table S3) (7), lead to disease because they disrupt the MCM:MMAA interaction, thereby impairing AdoCbl delivery to MCM and causing methylmalonic aciduria. Biochemical data are available to support two of these predictions: the pathogenic G188R mutation in MMAA was shown to inhibit complex formation with human MCM (33) and the pathogenic R616C mutation in MCM was mimicked in a bacterial ortholog and exhibited reduced affinity for MeaB (12).…”

“…This location provides a molecular explanation for the observation that chaperone GTPase activity is affected by binding to a target protein, as observed with MCM: MMAA/MeaB (33,39). Sizable regions of the GTPase active site are disordered in structures of MeaB and MMAA alone, and likely become ordered upon complex formation to assume the positions observed in IcmF.…”

“…The fortuitous fusion of two "stand-alone" proteins into one in IcmF has allowed us to obtain the requisite structural data to interrogate both the signaling pathways and the conformational dynamics related to cofactor delivery. With IcmF serving as a model system, we predict that the human and bacterial MCM:G-protein complexes will form such that their G proteins interact similarly with their mutase domains despite their differences in oligomerization state [bacterial MCM is a heterodimer of an active and an inactive chain (22,23,37) and human MCM is a homodimer (33,38)]. Notably, our structure-based docking model places five pathogenic human mutations-four in human MCM and one in MMAA-at the MCM:MMAA interface.…”

Engineered and Native Coenzyme B12-dependent Isovaleryl-CoA/Pivalyl-CoA Mutase

“…MeaB diminishes the inactivation rate of the mutase ϳ15-and ϳ3-fold in the presence of GTP and GDP, respectively (20). The human ortholog of MeaB is CblA and it is reported to prevent inactivation and increase the enzymatic activity of human MCM (38,39). In IcmF, which has a built-in G-protein chaperone, GTP and GDP protect against inactivation in wild-type but not in F598A IcmF (Fig.…”

Navigating the B12 Road: Assimilation, Delivery, and Disorders of Cobalamin

“…Strikingly, this mutation also corrupts the ability of MeaB to block cob(II)alamin binding to MCM and to promote release of cob(II)alamin from the inactive mutase. Most G-proteins use structural motifs known as switch I and II loops that exhibit conformational sensitivity to nucleotide binding and hydrolysis for communicating with client proteins (76,77). Missense mutations in the switch I and II loops are pathogenic and are located at the surface of MeaB and CblA, which might be important for interactions with the mutase (71,78).…”