Volume 27, Issue 1, Pages 29-40 (July 2007) Crystal Structures of [NiFe] Hydrogenase Maturation Proteins HypC, HypD, and HypE: Insights into Cyanation Reaction by Thiol Redox Signaling  Satoshi Watanabe, Rie Matsumi, Takayuki Arai, Haruyuki Atomi, Tadayuki Imanaka, Kunio Miki  Molecular Cell  Volume 27, Issue 1, Pages 29-40 (July 2007) DOI: 10.1016/j.molcel.2007.05.039 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Crystal Structure of HypC from T. kodakaraensis (A) Overall structure of HypC in a ribbon representation. The OB fold domain is shown in green; the C-terminal α helix is shown in pink. (B) Surface representations of HypC. (Left panel) Electrostatic surface potential with positively (blue) and negatively (red) charged residues (Nicholls et al., 1991). (Middle panel) Hydrophobic residues (green). (Right panel) Conserved regions: completely conserved residues are colored in blue; conserved residues are colored in light blue. (C) A superposition of the Cα backbone of the three HypC molecules in the asymmetric unit. Molecular Cell 2007 27, 29-40DOI: (10.1016/j.molcel.2007.05.039) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Overall Structure of HypE from T. kodakaraensis (A) Ribbon representation of the overall structure of HypE. The two α/β domains A and B and the C-terminal tail are shown in green, magenta, and cyan, respectively. The connecting loop between domains A and B is colored light brown. The conserved motifs I, II, III, and IV are colored red, yellow, blue, and orange, respectively. (B) Topology diagram of HypE. The conserved motifs are shown in the same colors as those used in (A). (C) Top (right panel) and side (left) views of the HypE dimer. A crystallographic 2-fold axis is indicated as an ellipse. (D and E) Electron density of a simulated annealing omit map (omitting residues 328–338) around the C-terminal tail in the absence of ATP (D) or in the presence of ATP (E) is shown at 2.5σ (blue). (F) Stereo view of the interaction between the PR(V/I)C motif and conserved residues in the inward form. The prime indicates the residues of the other subunit in the HypE dimer. Broken orange and yellow lines show hydrogen bonds and van der Waals contacts, respectively. Molecular Cell 2007 27, 29-40DOI: (10.1016/j.molcel.2007.05.039) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Overall Structure of HypD from T. kodakaraensis (A) Ribbon representation of the overall structure of HypD. The two α/β domains I and II, and the Fe-S cluster binding domain are shown in green, magenta, and blue, respectively. The conserved motifs are highlighted by different colors: CGXH motif, red; GPGCPVC motif, cyan; GFETT motif, orange; and PXHVS motif, yellow, respectively. Cysteine residues and the [4Fe-4S] cluster are represented in a stick-and-sphere model. (B) The topology diagram of HypD. The colors used in the presentation of the conserved motifs are the same as those used in (A). Molecular Cell 2007 27, 29-40DOI: (10.1016/j.molcel.2007.05.039) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Conserved Region of HypD (A) Stereo view of the assembly of the conserved motifs in a stick representation. The conserved motifs are shown in different colors: CGXH motif, pink; GPGCPVC motif, cyan; GFETT motif, orange; and PXHVS motif, yellow. Essential cysteine residues are colored in green. (B) Stereo view of a surface representation of HypD. Identical and conserved residues among HypD proteins are colored in orange and light orange, respectively. The orientation is identical to that in Figure 3A. (C) Stereo view of a surface representation of the opposite side of (B). Molecular Cell 2007 27, 29-40DOI: (10.1016/j.molcel.2007.05.039) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Unstable Disulfide Bonds of HypD Electron densities are shown for the 2Fo − Fc map at 1.5σ in light gray and for the Fo − Fc map in red (+3.0σ) and blue (−3.0σ), respectively. (A) 100% oxidized model of the disulfide bond SS2 (Cys325- Cys354). (B) 100% oxidized model of the disulfide bond SS1 (Cys66-Cys69). (C) A mixture of 60% oxidized and 40% reduced structure of SS2. (D) Mixture of 60% oxidized and 40% reduced structure of SS1. Molecular Cell 2007 27, 29-40DOI: (10.1016/j.molcel.2007.05.039) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 [4Fe-4S] Cluster of HypD (A) Stereo view of the FTR-like [4Fe-4S] cluster of HypD. The electron density of a simulated annealing omit map around the [4Fe-4S] cluster is shown at 4.5σ (blue). (B) Redox cascade between the [4Fe-4S] cluster and two disulfide bonds. Sulfur atoms in the dithiol form of cysteine residues are shown in magenta. Molecular Cell 2007 27, 29-40DOI: (10.1016/j.molcel.2007.05.039) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 7 Proposed Reaction Pathway of the Cyanation by Thiol Redox Signaling See text. Molecular Cell 2007 27, 29-40DOI: (10.1016/j.molcel.2007.05.039) Copyright © 2007 Elsevier Inc. Terms and Conditions