Autoinhibition and signaling by the switch II motif in the G-protein chaperone of a radical B12 enzyme.
ABSTRACT: MeaB is an accessory GTPase protein involved in the assembly, protection, and reactivation of 5'-deoxyadenosyl cobalamin-dependent methylmalonyl-CoA mutase (MCM). Mutations in the human ortholog of MeaB result in methylmalonic aciduria, an inborn error of metabolism. G-proteins typically utilize conserved switch I and II motifs for signaling to effector proteins via conformational changes elicited by nucleotide binding and hydrolysis. Our recent discovery that MeaB utilizes an unusual switch III region for bidirectional signaling with MCM raised questions about the roles of the switch I and II motifs in MeaB. In this study, we addressed the functions of conserved switch II residues by performing alanine-scanning mutagenesis. Our results demonstrate that the GTPase activity of MeaB is autoinhibited by switch II and that this loop is important for coupling nucleotide-sensitive conformational changes in switch III to elicit the multiple chaperone functions of MeaB. Furthermore, we report the structure of MeaB·GDP crystallized in the presence of AlFx(-) to form the putative transition state analog, GDP·AlF4(-). The resulting crystal structure and its comparison with related G-proteins support the conclusion that the catalytic site of MeaB is incomplete in the absence of the GTPase-activating protein MCM and therefore unable to stabilize the transition state analog. Favoring an inactive conformation in the absence of the client MCM protein might represent a strategy for suppressing the intrinsic GTPase activity of MeaB in which the switch II loop plays an important role.
Project description: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.
Project description:Fidelity during cofactor assembly is essential for the proper functioning of metalloenzymes and is ensured by specific chaperones. MeaB, a G-protein chaperone for the coenzyme B12-dependent radical enzyme methylmalonyl-CoA mutase (MCM), uses the energy of GTP binding, hydrolysis or both to regulate cofactor loading into MCM, protect MCM from inactivation and rescue MCM that is inactivated during turnover. Typically, G proteins signal to client proteins using the conformationally mobile switch I and II loops. Crystallographic snapshots of MeaB reported herein reveal a new switch III element that has substantial conformational plasticity. Using alanine-scanning mutagenesis, we demonstrate that the switch III motif is critical for bidirectional signal transmission of the GTPase-activating protein activity of MCM and the chaperone functions of MeaB in the MeaB-MCM complex. Mutations in the switch III loop identified in patients corrupt this interprotein communication and lead to methylmalonic aciduria, an inborn error of metabolism.
Project description:Heterotrimeric G protein alpha (G alpha) subunits possess intrinsic GTPase activity that leads to functional deactivation with a rate constant of approximately 2 min(-1) at 30 degrees C. GTP hydrolysis causes conformational changes in three regions of G alpha, including Switch I and Switch II. Mutation of G202-->A in Switch II of G alpha(i1) accelerates the rates of both GTP hydrolysis and conformational change, which is measured by the loss of fluorescence from Trp-211 in Switch II. Mutation of K180-->P in Switch I increases the rate of conformational change but decreases the GTPase rate, which causes transient but substantial accumulation of a low-fluorescence G alpha(i1).GTP species. Isothermal titration calorimetric analysis of the binding of (G202A)G alpha(i1) and (K180P)G alpha(i1) to the GTPase-activating protein RGS4 indicates that the G202A mutation stabilizes the pretransition state-like conformation of G alpha(i1) that is mimicked by the complex of G alpha(i1) with GDP and magnesium fluoroaluminate, whereas the K180P mutation destabilizes this state. The crystal structures of (K180P)G alpha(i1) bound to a slowly hydrolyzable GTP analog, and the GDP.magnesium fluoroaluminate complex provide evidence that the Mg(2+) binding site is destabilized and that Switch I is torsionally restrained by the K180P mutation. The data are consistent with a catalytic mechanism for G alpha in which major conformational transitions in Switch I and Switch II are obligate events that precede the bond-breaking step in GTP hydrolysis. In (K180P)G alpha(i1), the two events are decoupled kinetically, whereas in the native protein they are concerted.
Project description:The methylmalonyl Co-A mutase-associated GTPase MeaB from Methylobacterium extorquens is involved in glyoxylate regulation and required for growth. In humans, mutations in the homolog methylmalonic aciduria associated protein (MMAA) cause methylmalonic aciduria, which is often fatal. The central role of MeaB from bacteria to humans suggests that MeaB is also important in other, pathogenic bacteria such as Mycobacterium tuberculosis. However, the identity of the mycobacterial MeaB homolog is presently unclear. Here, we identify the M. tuberculosis protein Rv1496 and its homologs in M. smegmatis and M. thermoresistibile as MeaB. The crystal structures of all three homologs are highly similar to MeaB and MMAA structures and reveal a characteristic three-domain homodimer with GDP bound in the G domain active site. A structure of Rv1496 obtained from a crystal grown in the presence of GTP exhibited electron density for GDP, suggesting GTPase activity. These structures identify the mycobacterial MeaB and provide a structural framework for therapeutic targeting of M. tuberculosis MeaB.
Project description:In animals, cobalamin (Cbl) is a cofactor for methionine synthase and methylmalonyl-CoA mutase (MCM), which utilizes methylcobalamin and 5'-deoxyadenosylcobalamin (AdoCbl), respectively. The cblA complementation class of inborn errors of Cbl metabolism in humans is one of three known disorders that affect AdoCbl synthesis. The gene responsible for cblA has been identified in humans (MMAA) as well as its homolog (meaB) in Methylobacterium extorquens. Recently, it has been reported that human MMAA plays an important role in the protection and reactivation of MCM in vitro. However, the physiological function of MMAA is largely unknown. In the present study, we isolated the cDNA encoding MMAA from Euglena gracilis Z, a photosynthetic flagellate. The deduced amino acid sequence of the cDNA shows 79%, 79%, 79% and 80% similarity to human, mouse, Danio rerio MMAAs and M. extorquens MeaB, respectively. The level of the MCM transcript was higher in Cbl-deficient cultures of E. gracilis than in those supplemented with Cbl. In contrast, no significant differences were observed in the levels of the MMAA transcript under the same two conditions. No significant difference in MCM activity was observed between Escherichia coli that expressed either MCM together with MMAA or expressed MCM alone.
Project description:RheB GTPase is a Ras-related molecular switch, which regulates the mTOR signaling pathway by cycling between the active [guanosine triphosphate (GTP)] state and inactive [guanine diphosphate (GDP)] state. Impairment of GTPase activity because of mutations in several small GTPases is known to be associated with several cancers. The conventional GTPase mechanism such as in H-Ras requires a conserved glutamine (Q64) in the switch-II region of RheB to align the catalytic water molecule for efficient GTP hydrolysis. The conformation of this conserved glutamine is different in RheB, resulting in an altered conformation of the entire switch-II region. Studies on the atypical switch-II conformation in RheB revealed a distinct, noncanonical mode of GTP hydrolysis. An RheB mutant Y35A was previously shown to exclusively enhance the intrinsic GTPase activity of RheB, whereas the Y35A-D65A double mutant was shown to reduce the elevated GTPase activity. Here, we have used all-atom molecular dynamics (MD) simulations for comprehensive understanding of the conformational dynamics associated with the fast (Y35A) and slow (Y35A-D65A) hydrolyzing mutants of RheB. Using a combination of starting models from PDB structures and in-silico generated mutant structures, we discuss the observed conformational deviations in wild type (WT) versus mutants. Our results show that a number of interactions of RheB with phosphates of GTP as well as Mg2+ are destabilized in Y35A mutant in the switch-I region. We report distinct water dynamics at the active site of WT and mutants. Furthermore, principal component analysis showed significant differences in the conformational space sampled by the WT and mutants. Our observations provide improved understanding of the noncanonical GTP hydrolysis mechanism adopted by RheB and its modulation by Y35A and Y35A-D65A mutants.
Project description:Engineering of the methylmalonyl-CoA (mmCoA) metabolite node of the Saccharopolyspora erythraea wild-type strain through duplication of the mmCoA mutase (MCM) operon led to a 50% increase in erythromycin production in a high-performance oil-based fermentation medium. The MCM operon was carried on a 6.8kb DNA fragment in a plasmid which was inserted by homologous recombination into the S. erythraea chromosome. The fragment contained one uncharacterized gene, ORF1; three MCM related genes, mutA, mutB, meaB; and one gntR-family regulatory gene, mutR. Additional strains were constructed containing partial duplications of the MCM operon, as well as a knockout of ORF1. None of these strains showed any significant alteration in their erythromycin production profile. The combined results showed that increased erythromycin production only occurred in a strain containing a duplication of the entire MCM operon including mutR and a predicted stem-loop structure overlapping the 3' terminus of the mutR coding sequence.
Project description:Crystal structures of Galpha(i) (and closely related family member Galpha(t)) reveal much of what we currently know about G protein structure, including changes which occur in Switch regions. Galpha(t) exhibits a low rate of basal (uncatalyzed) nucleotide exchange and an ordered Switch II region in the GDP-bound state, unlike Galpha(i), which exhibits higher basal exchange and a disordered Switch II region in Galpha(i)GDP structures. Using purified Galpha(i) and Galpha(t), we examined the intrinsic tryptophan fluorescence of these proteins, which reports conformational changes associated with activation and deactivation of Galpha proteins. In addition to the expected enhancement in tryptophan fluorescence intensity, activation of GalphaGDP proteins was accompanied by a modest but notable red shift in tryptophan emission maxima. We identified a cation-pi interaction between tryptophan and arginine residues in the Switch II of Galpha(i) family proteins that mediates the observed red shift in emission maxima. Furthermore, amino-terminal myristoylation of Galpha(i) resulted in a less polar environment for tryptophan residues in the GTPase domain, consistent with an interaction between the myristoylated amino terminus and the GTPase domain of Galpha proteins. These results reveal unique insights into conformational changes which occur upon activation and deactivation of G proteins in solution.
Project description:Itaconate is an immunometabolite with both anti-inflammatory and bactericidal effects. Its coenzyme A (CoA) derivative, itaconyl-CoA, inhibits B12-dependent methylmalonyl-CoA mutase (MCM) by an unknown mechanism. We demonstrate that itaconyl-CoA is a suicide inactivator of human and Mycobacterium tuberculosis MCM, which forms a markedly air-stable biradical adduct with the 5'-deoxyadenosyl moiety of the B12 coenzyme. Termination of the catalytic cycle in this way impairs communication between MCM and its auxiliary repair proteins. Crystallography and spectroscopy of the inhibited enzyme are consistent with a metal-centered cobalt radical ~6 angstroms away from the tertiary carbon-centered radical and suggest a means of controlling radical trajectories during MCM catalysis. Mycobacterial MCM thus joins enzymes in the glyoxylate shunt and the methylcitrate cycle as targets of itaconate in pathogen propionate metabolism.
Project description:Protein synthesis by the ribosome requires the translocation of transfer RNAs and messenger RNA by one codon after each peptide bond is formed, a reaction that requires ribosomal subunit rotation and is catalyzed by the guanosine triphosphatase (GTPase) elongation factor G (EF-G). We determined 3 angstrom resolution x-ray crystal structures of EF-G complexed with a nonhydrolyzable guanosine 5'-triphosphate (GTP) analog and bound to the Escherichia coli ribosome in different states of ribosomal subunit rotation. The structures reveal that EF-G binding to the ribosome stabilizes switch regions in the GTPase active site, resulting in a compact EF-G conformation that favors an intermediate state of ribosomal subunit rotation. These structures suggest that EF-G controls the translocation reaction by cycles of conformational rigidity and relaxation before and after GTP hydrolysis.