Structure of the cyanobacterial Magnesium Chelatase H subunit determined by single particle reconstruction and small-angle X-ray scattering

Abstract

The biosynthesis of chlorophyll, an essential cofactor for photosynthesis, requires the ATP-dependent insertion of Mg(2+) into protoporphyrin IX catalyzed by the multisubunit enzyme magnesium chelatase. This enzyme complex consists of the I subunit, an ATPase that forms a complex with the D subunit, and an H subunit that binds both the protoporphyrin substrate and the magnesium protoporphyrin product. In this study we used electron microscopy and small-angle x-ray scattering to investigate the structure of the magnesium chelatase H subunit, ChlH, from the thermophilic cyanobacterium Thermosynechococcus elongatus. Single particle reconstruction of negatively stained apo-ChlH and Chl-porphyrin proteins was used to reconstitute three-dimensional structures to a resolution of ∼30 Å. ChlH is a large, 148-kDa protein of 1326 residues, forming a cage-like assembly comprising the majority of the structure, attached to a globular N-terminal domain of ∼16 kDa by a narrow linker region. This N-terminal domain is adjacent to a 5 nm-diameter opening in the structure that allows access to a cavity. Small-angle x-ray scattering analysis of ChlH, performed on soluble, catalytically active ChlH, verifies the presence of two domains and their relative sizes. Our results provide a basis for the multiple regulatory and catalytic functions of ChlH of oxygenic photosynthetic organisms and for a chaperoning function that sequesters the enzyme-bound magnesium protoporphyrin product prior to its delivery to the next enzyme in the chlorophyll biosynthetic pathway, magnesium protoporphyrin methyltransferase.

FIGURE 1.

Proto binding and enzyme activity of the magnesium chelatase ChlH subunit from T. elongatus. A, the purity of the apo-ChlH subunit is indicated by the elution trace from the HPLC gel filtration column (black line, detection of absorption at 280 nm; red line, detection at 398 nm), and the SDS-PAGE analysis (inset). The two lower traces show the analysis of the sample where ChlH and D_IX were premixed prior to gel filtration. B, plot of ChlH fluorescence at 350 nm in arbitrary units (A.U.) as a function of D_IX concentration. The curve fits the experimental data to a single substrate binding model. C, Mg chelatase assays run in duplicate with H, I, and D subunits from Synechocystis as a positive control (blue), and I and D subunits alone as a negative control (green). The data in red show the progress of a Mg chelatase reaction with the T. elongatus ChlH subunit mixed with the Synechocystis ChlI and ChlD subunits.

FIGURE 2.

Electron microscopy and 3D reconstruction of negatively stained apo-ChlH and ChlH-D_IX particles. A and D, electron micrographs of apo-ChlH and ChlH-D_IX samples, respectively. Scale bar = 100 nm (A and D). B and E, 36 boxed single molecules of apo-ChlH and ChlH-D_IX samples, respectively. The box size is 25 nm × 25 nm. C, reconstruction of apo-ChlH. Top row, six selected averaged two-dimensional classes; center row, 3D reconstructions viewed at Euler angles corresponding to those assigned to the class averages above; bottom row, corresponding reprojections from the 3D models. F, reconstruction of apo-ChlH with details as for C.

FIGURE 3.

A and B, 3D models of apo-ChlH (cyan) and the ChlH-D_IX complex (red), calculated at a cutoff resolution of 30 Å. The threshold value of each model was adjusted to correspond to the molecular mass of ChlH. In the center and bottom rows, the molecules are rotated successively by 90° about the z axis. The handedness for each of the apo-ChlH and ChlH-D_IX models that gave the best fit to the SAXS model in Figs. 5 and 6 was chosen. C and D, 3D models of BchH taken from Ref. . The apo-BchH structure is in cyan (C), and the porphyrin complex is in red (D). In the center and bottom rows, the molecules are rotated successively by 90° about the z axis. Scale bar = 10 nm. The 3D models were generated using Chimera (64).

FIGURE 4.

Identification of the N terminus of ChlH by labeling with nitrilotriacetic acid-nanogold. A gallery of electron micrographs of negatively stained apo-ChlH proteins labeled with 5 nm-diameter gold particles (black) that attach to the N-terminal His₆ tag on ChlH. Each box is 30 nm × 30 nm.

FIGURE 5.

SAXS data collected for Synechocystis apo-ChlH reveal a structure with two domains. A, collected solution scattering data for Synechocystis apo-ChlH. The plot contains merged scattering curves collected from 2 mg/ml ChlH at a camera length of 4.5 m and 6.9 mg/ml at 1 m. A typical fitted line from a GASBOR run is shown in red. The axes are the logarithm of scattering intensity and the scattering vector q. B (inset), pair distribution function p(r) for Synechocystis ChlH with a D_max value of 170 Å. C and D, side and top views of the consensus 3D model of Synechocystis ChlH representing the average of ab initio models restored with GASBOR. Molecular graphics images and approximate distances were generated with PyMOL.

FIGURE 6.

Superposition of the 3D reconstruction of apo-ChlH from T. elongatus (cyan) from EM analysis and the apo-ChlH from Synechocystis (yellow) from the SAXS analysis. The superposition is viewed from three angles to emphasize the similarity between the two structural models. The handedness of the 3D reconstruction was selected arbitrarily for the best fit.

FIGURE 7.

Deletion of the N-terminal 565 residues from ChlH abolishes Mg chelatase activity but leaves porphyrin binding unaffected. A, linear map of ChlH from Synechocystis showing the locations of the deletion (dotted line), the head region identified in EM and SAXS studies (red), and the region not found in BchH (cyan) (see also supplemental Fig. S1). B, SDS-PAGE of purified recombinant His-tagged wild-type ChlH and the N-terminal deletion of ChlH. C, plot of ChlH fluorescence at 350 nm as a function of the concentration of D_IX (▴) and MgD_IX (△). The curve fits the experimental data to a single substrate binding model. The K_D values are 5.66 ± 0.74 and 3.51 ± 0.68 μm, respectively. D, the effect of Gun4 on Mg chelatase activity for the wild-type ChlH-I-D enzyme (●), a mutant ChlH/gun5-I-D enzyme (○), and the N-terminal deletion of ChlH plus ChlI and D (▴).