The structure of vanadium nitrogenase reveals an unusual bridging ligand

Transcription

1 SUPPLEMENTARY INFORMATION The structure of vanadium nitrogenase reveals an unusual bridging ligand Daniel Sippel and Oliver Einsle Lehrstuhl Biochemie, Institut für Biochemie, Albert-Ludwigs-Universität Freiburg, Albertstrasse 21, Freiburg, Germany, Freiburg Research Institute for Advanced Studies (FRIAS), Albertstrasse 19, Freiburg, Germany, and BIOSS Centre for Biological Signalling Studies, Schänzlestrasse 1, Freiburg, Germany 1

2 SUPPLEMENTARY RESULTS Supplementary Figure 1: Subunit structure of A. vinelandii VFe protein. Stereo representations of the subunits VnfD (a), VnfK (b) and VnfG (c), coloured from blue at the N- terminus to red at the C-terminus. The location within the VFe heterohexamer is indicated in the scheme on the right. Note the close structural relationship between VnfD and VnfK. 2

3 Supplementary Figure 2: Structural homologies within the nitrogenase enzyme family. Structural alignments of VFe protein (black) with a, nitrogenase MoFe protein NifD 2 K 2 (PDB- ID 3U7Q, red), b, MoFe protein maturation factor NifE 2 N 2 (PDB-ID 3PDI, blue), and c, the light-independent protochlorophyllide reductase (LPOR) BchN 2 B 2 from Rhodobacter capsulatus (PDB-ID 3AEK, green). MoFe protein aligns with a root-mean-squared deviation of all atom positions of 1.97 Å to VFe protein, while NifEN, a scaffold protein for a crucial assembly step of FeMo cofactor, aligns with 2.60 Å. For LPOR, which presumably represents a phylogenetic ancestor to nitrogenase, the deviation is 4.64 Å. 3

4 Supplementary Figure 3: Mg 2+ site in vanadium nitrogenase. a, The place of an interstitial Ca 2+ ion in MoFe protein is taken by Mg 2+ in VFe protein. The ion (green) is coordinated by residues E70 (at 2.0 Å distance) and D314 (at 2.1 Å distance) from the two different VnfK subunits of the heterohexamer, and the octahedral ligand field is completed by four water molecules (red). One VnfD subunit is near the binding site, extending residues K413 and K414 towards the site. The 2F o F c electron density map is contoured at the 1s level and coloured according to the different protein chains. 4

5 Supplementary Figure 4: Interference of the VnfG subunit with complex formation. The docking site for Fe protein is defined through several crystal structures of A. vinelandii Mo nitrogenase. 1-4 Due to the high degree of conservation of this interface, analogous interactions can be modelled for V nitrogenase for a, the complex of VFe protein with nucleotide-free Fe protein (green), b, the complex with AMPPCP-bound Fe protein and c, the complex with ADP-bound Fe protein. In all cases, no clash of physical interaction with the additional VnfG subunit of VFe protein is observed. Subunits of VFe protein are colored as in Fig. 1. 5

6 Supplementary Figure 5: Electron density at FeV cofactor. a, FeV cofactor of vanadium nitrogenase, with a 2F o F c electron density map contoured at the 2s level (stereo image). b, Detail of the coordination of a carbonate anion to bridge iron ions Fe4 and Fe5. At the center of FeV cofactor the interstitial carbide (black) is well defined. c, View along the threefold pseudosymmetry axis of FeV cofactor. The ligand is slightly twisted, likely due to the tight hydrogen bond interaction with the protein chain (Fig. 4). 6

7 Supplementary Figure 6: Candidate ligands and electron density properties. d, Candidate molecules considered to explain the observed ligand density. Carbonate and nitrate are isoelectronic, but nitrate is ruled out, as its presence would inhibit nitrogenase expression. Bicarbonate, acetate and carbamate would contain a longer single bond, which is not in line with the refined bond distances in the ligand. e, Evaluation of electron density for the atoms of the ligand. Electron density was integrated within r = 0.7 Å around each atom in the structure, yielding profiles for the elements carbon, nitrogen and oxygen that follow a Gaussian distribution. The electron density for the central atom of the ligand would fit either of the light atoms (green line), while the outer atoms are heavier (red line), implying oxygen. 7

8 Supplementary Figure 7: Maturation factors in the vnf gene cluster. The vnf cluster of A. vinelandii consists of five operons with at least 17 open reading frames. 5 Three previously uncharacterized genes, vnfp1, vnfp2 and vnfp3, may act in concert to remove belt sulfide S3A for subsequent replacement with carbonate. VnfH, and vnfdkg are the structural genes for Fe protein and VFe protein, respectively, while vnfenx encode a maturation scaffold for V insertion and addition of homocitrate. Unannotated reading frames are unique to the vnf cluster. 8

9 Supplementary Table 1 Data collection, phasing and refinement statistics Native MAD data Data collection Space group P1 P1 Cell dimensions a, b, c (Å) 75.3, 79.8, , 79.4, α, β, γ ( ) 84.1, 72.6, , 72.9, 75.0 Peak Inflection Wavelength (Å) Resolution (Å) ( ) ( ) ( ) R sym or R merge (1.048) (0.310) (0.450) 6 R p.i.m (0.780) (0.080) (0.114) 7 CC (1/2) (0.494) (0.988) (0.976) I / σi 10.4 (1.0) 26.0 (8.1) 22.1 (5.7) Completeness (%) 96.1 (92.3) 91.0 (83.6) 90.6 (80.5) Redundancy 3.5 (3.6) 19.0 (17.7) 18.9 (17.6) Refinement Resolution (Å) No. reflections 460,593 (32,710) R work / R free / No. atoms 19,130 Protein 16,777 Ligand/ion 104 Water 2,249 B-factors (Å 2 ) Protein Ligand/ion Water R.m.s deviations Bond lengths (Å) Bond angles ( ) *Values in parentheses are for highest-resolution shell. 9

10 Supplementary References 1. Schindelin, H., Kisker, C., Sehlessman, J.L., Howard, J.B. & Rees, D.C. Structure of ADP AIF - 4 -stabilized nitrogenase complex and its implications for signal transduction. Nature 387, (1997). 2. Schmid, B. et al. Biochemical and structural characterization of the cross-linked complex of nitrogenase: Comparison to the ADP-AIF - 4 -stabilized structure. Biochemistry 41, (2002). 3. Owens, C.P., Katz, F.E., Carter, C.H., Luca, M.A. & Tezcan, F.A. Evidence for Functionally Relevant Encounter Complexes in Nitrogenase Catalysis. J. Am. Chem. Soc. 137, (2015). 4. Tezcan, F.A. et al. Nitrogenase complexes: Multiple docking sites for a nucleotide switch protein. Science 309, (2005). 5. Setubal, J.C. et al. Genome Sequence of Azotobacter vinelandii, an Obligate Aerobe Specialized To Support Diverse Anaerobic Metabolic Processes. J. Bacteriol. 191, (2009). 6. Weiss, M. & Hilgenfeld, R. On the use of the merging R factor as a quality indicator for X-ray data. J. Appl. Crystallogr. 30, (1997). 7. Karplus, P.A. & Diederichs, K. Linking Crystallographic Model and Data Quality. Science 336, (2012). 10