X-Ray Crystallographic Analysis of NifB with a Full Complement of Clusters: Structural Insights into the Radical SAM-Dependent Carbide Insertion During Nitrogenase Cofactor Assembly

Abstract

NifB is an essential radical SAM enzyme required for the assembly of an 8Fe core of the nitrogenase cofactor. Herein, we report the X-ray crystal structures of Methanobacterium thermoautotrophicum NifB without (apo MtNifB) and with (holo MtNifB) a full complement of three [Fe₄ S₄ ] clusters. Both apo and holo MtNifB contain a partial TIM barrel core, but unlike apo MtNifB, holo MtNifB is fully assembled and competent in cofactor biosynthesis. The radical SAM (RS)-cluster is coordinated by three Cys, and the adjacent K1- and K2-clusters, representing the precursor to an 8Fe cofactor core, are each coordinated by one His and two Cys. Prediction of substrate channels, combined with in silico docking of SAM in holo MtNifB, suggests the binding of SAM between the RS- and K2-clusters and putative paths for entry of SAM and exit of products of SAM cleavage, thereby providing important mechanistic insights into the radical SAM-dependent carbide insertion concomitant with cofactor core formation.

Keywords: carbide insertion; cofactors; nitrogenases; radical SAM enzyme; structural biology.

Figure 1.

Crystal structures of holo and apo MtNifB. Ribbon presentations of the structures of (a) holo and (c) apo MtNifB and schematic presentations of the secondary structural elements of (b) holo and (d) apo MtNifB. PYMOL was used to prepare the ribbon presentations.^[22] The subunits of holo and apo MtNifB are colored light blue and orange, respectively (a, c). The RS-, K1- and K2-clusters are illustrated as ball-and-stick models, with the atoms colored as follows: Fe, orange; S, yellow; O, red; C, grey (a, c). The 2Fo-Fc electron density maps of the representative regions of holo MtMifB and apo MtNifB are shown in Figure S3. The α-helices (labeled H) and β-strands (labeled S) are depicted as yellow tubes and light blue arrows, respectively, and consecutively numbered starting from the N-terminus of the protein; whereas the connecting loops are shown as black lines (b, d). The ligands of the RS-, K1- and K2-clusters are depicted as red, blue and yellow ovals, respectively (b, d). The disordered regions of apo MtNifB are rendered transparent (d). The B-factor putty representation and the B-factor plot of holo MtNifB are shown in Figure S4.

Figure 2.

The three [Fe₄S₄] clusters of holo MtNifB. The structure and ligand coordination of the (a) RS- and (b) K1- and K2-clusters are depicted and colored as described in Figure 1a. The 2Fo-Fc electron density maps (upper, grey mesh, contoured at 1.0 σ), overlaid with the Fo-Fc electron density omit maps (upper, green mesh, contoured at 3.0 σ) or the anomalous difference Fourier maps (lower, red mesh, contoured at 3.0 σ), clearly indicate the presence of all three [Fe₄S₄] clusters in holo MtNifB (a, b). PYMOL was used to generate this figure.^[22] See Table S2 for cluster occupancy.

Figure 3.

Predicted substrate channels in holo MtNifB. (a) Surface and (b, c) ribbon presentations of holo MtNifB with the top three channels predicted by CAVER 3.0.3^[23] shown as strings of blue (Channel 1), green (Channel 2) and red (Channel 3) spheres, respectively, and the entrances to these channels indicated by arrows of corresponding colors. All three channels extend from the protein surface to K2, with Channel 1 being mostly perpendicular to β-strands S2, S5, S8 and S9 (b), and Channels 2 and 3 being mostly parallel to β-strands S8 and S9 (c). Note the close location of the entrance of Channel 1 to the RS-cluster (b). PYMOL was used to generate this figure.^[22] The peptides and clusters of holo MtNifB are depicted and colored as described in Figure 1a.

Figure 4.

Model of holo MtNifB bound with SAM. (a) Close-up of the modeled SAM-binding site between the RS-cluster and the K1/K2-modules and (b, c) overlay of Channel 1 (b) and Channels 2 and 3 (c) with the SAM-binding region. PYMOL was used to generate this figure.^[22] Binding of SAM was modeled using GLIDE^[24] in Maestro 12.4.^[25] Based on our model, SAM interacts with the following conserved residues in holo MtNifB that are part of the structural motifs that bind SAM in other known radical SAM (rSAM) enzymes^[8]: (i) Gly⁹⁰, part of a ‘GDA sequence’ that corresponds to the ‘GGE motif’ of the ‘β-strand 2 region’ that anchors the amino group of SAM in other rSAM enzymes; (ii) Asn¹⁹⁴, the location of which corresponds to the ‘β-strand 5 region’ that interacts with one ribose hydroxyl group of SAM in other rSAM enzymes; and (iii) Pro²²⁵, the location of which corresponds to ‘the β-strand 6 region’ that ligates adenine moiety in other rSAM enzymes. Additionally, SAM is further stabilized through unique interactions with other residues/elements in holo MtNifB: (i) Gly⁵⁷, which interacts with the carboxylate group of SAM in a location corresponding to the ‘β-strand 1 region’ in other rSAM enzymes; and (ii) two Fe atoms, one each from the K1- and K2-clusters, which interact with one ribose hydroxyl group in a location previously unseen in other rSAM enzymes. Ligation of SAM by these cluster Fe atoms could result from an ‘inward’ folding of the N- and C-termini into the barrel region of holo MtNifB, which allows these termini to participate in the binding of the K1- and K2-clusters, positioning them in place for the subsequent interaction with SAM. Such an ‘inward’ folding of the two termini of the protein has not been observed in other rSAM enzymes, which may be uniquely required for the reaction between SAM and the substrate K1/K2 clusters during the process of carbide insertion in holo MtNifB. The clusters and SAM are shown in ball-and-stick presentations, and the coordinating ligands are shown as sticks. The atoms are colored as described in Figure 1a, and the channels are depicted as described in Figure 3. The peptides are shown in ribbon presentation and rendered transparent in the background.