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.

Allosteric Regulation of Oligomerization by a B12 Trafficking G-Protein Is Corrupted in Methylmalonic Aciduria

“…The newly formed AdoCbl product is then directly transferred from ATR to MCM in a process that is gated by the GTPase activity of CblA (Padovani et al, 2008). Detailed structural enzymology insights into the mitochondrial B 12 pathway have emerged primarily from our studies on the homologous proteins in Methylobacterium extorquens (Campanello et al, 2017;Lofgren et al, 2013aLofgren et al, , 2013bPadovani and Banerjee, 2006b, 2009a, 2009bPadovani et al, 2006;Padovani et al, 2008) and on IcmF, a fusion between a sister isobutyryl-CoA mutase and its G-protein GTPase activity GTPase activity chaperone Banerjee, 2012a, 2012b;Cracan et al, 2010;Jost et al, 2015aJost et al, , 2015bLi et al, 2017).…”

“…As a member of the P-loop GTPases (Leipe et al, 2002), CblA communicates with MCM via conformationally mobile switch I and II loops that classically function as loaded springs for signaling via nucleotide binding-and hydrolysisresponsive conformational changes (Wittinghofer and Vetter, 2011). While the conserved residues in the switch I and II loops in MeCblA are not essential for GTP hydrolysis, they are important for allosteric signaling to and from MCM and for suppressing its intrinsic GTPase activity (Campanello et al, 2017;Lofgren et al, 2013a).…”

“…Furthermore, whereas human MCM is an a 2 homodimer (Froese et al, 2010) and each of the two B 12 -binding subunits can potentially interact with one CblA, MeMCM is an a/b heterodimer and binds one AdoCbl. The proteins form a 2MeMCM-1MeCblA complex (hereafter referred to as the M 2 C 1 complex, Figure 1B) as visualized by negative stain electron microscopy (EM) analysis (Campanello et al, 2017). IcmF exhibits yet another architecture in which the G-domains reside at opposite ends of the a 2 IcmF dimer while the mutase domains build the interface (Jost et al, 2015a(Jost et al, , 2015b.…”

Mechanistic Insights into the Metal-Dependent Activation of ZnII-Dependent Metallochaperones

“…This proposal is consistent with Zn II coordination adjacent to the G2 motif, which defines the switch 1 (Walker A) region that is known to stabilize the transition state necessary for GTP hydrolysis in other P-loop GTPases. 15,16 Nucleotide Specificity and Hydrolysis.…”

“…5 signature loops (G1-G5) involved in nucleotide binding and hydrolysis ( Figure 1A) and consist of metallochaperones implicated in metal or metallocofactor maturation, where loops G2 and G3 act as molecular switches required for activity. [15][16][17] Generally, G3E family members are thought not to deliver the metal directly to the target protein ( Figure 1B,i) with a set of accessory proteins often required for metalloenzyme maturation. UreG and HypB indirectly activate urease and hydrogenase, respectively, by inserting Ni II into an accessory complex [18][19][20][21][22]ii), while MeaB gates the transfer of adenosylcobalamin between methylmalonyl-CoA mutase and adenosyltransferase.…”