Crystal structure of hydrogenase maturating endopeptidase HycI from Escherichia coli

Biochemical and Biophysical Research Communications 389 (2009) 310–314

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Crystal structure of hydrogenase maturating endopeptidase HycI from Escherichia coli Thirumananseri Kumarevel a,*, Tomoyuki Tanaka a, Yoshitaka Bessho b, Akeo Shinkai a, Shigeyuki Yokoyama b,c,* a b c

RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

a r t i c l e

i n f o

Article history: Received 19 August 2009 Available online 29 August 2009 Keywords: Hydrogenase HycI Maturation Protease Metal ion Calcium binding Cleavage X-ray Escherichia coli X-ray crystallography

a b s t r a c t The maturation of [NiFe]-hydrogenases is a catalyzed process involving the activities of at least seven proteins. The last step consists of the endoproteolytic cleavage of the precursor of the large subunit, after the [NiFe]-metal center has been assembled. The HycI endopeptidase is involved in the C-terminal processing of HycE, the large subunit of hydrogenase 3 from Escherichia coli. Although HycI has been well characterized biochemically, the crystallization of the protein has been quite challenging. Here, we present the crystal structure of HycI at 1.70 Å resolution. The crystal structure resembles the recently reported solution structure (NMR) of the same protein and the holo-HyPD structure of the same family, but a significant conformational change is observed at the L5 loop, as compared with the solution structures of HycI and HyPD. In our crystal structure, three specific metal binding sites (Ca1–3) were identified and these metal ions are possibly involved in the C-terminal cleavage of HycE. Ó 2009 Elsevier Inc. All rights reserved.

Introduction Hydrogenases catalyze the reversible formation of molecular hydrogen (2H++ 2e M H2), and thus contribute to energy metabolism in a variety of microorganisms. They are involved in many biological processes in which hydrogen molecules are produced and consumed. The hydrogenase enzymes are widely distributed in bacteria and archaea [1]. Generally, [NiFe]-hydrogenases are heterodimers consisting of small and large subunits. The small subunit contains up to three Fe–S clusters, which mediate the electron transfer between the active center and the redox partner. The active site is located in the large subunit, and is composed of an iron [Fe] atom and a nickel [Ni] atom coordinated by four cysteine residues [2,3]. In addition, the iron atom is further coordinated by two cyanides and one carbon monoxide [4,5]. The biosynthesis of this metallocenter in the large subunit of the hydrogenase requires the functions of conserved accessory proteins [6,7]. In the case of hydrogenase 3 of Esch-

* Corresponding authors. Fax: +81 791 58 2835 (T. Kumarevel). Address: Systems and Structural Biology Center, Yokohama Institute, RIKEN, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan. Fax: +81 45 503 9195 (S. Yokoyama). E-mail addresses: [email protected] (T. Kumarevel), [email protected] (S. Yokoyama). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.08.135

erichia coli, seven genes, hypABCDEF and hycI, are involved in the regulation and maturation of the large subunit (HycE) [8–10]. Among them, HypA and HypB are involved in the insertion of the nickel atom into the precursor of the large subunit [11,12]. The HypB protein also exhibits GTPase activity and is responsible for nickel delivery [13]. HypC and HypD catalyze iron insertion. HypC acts as a chaperone-like protein that interacts with pre-HypE and maintains a conformation capable of metal incorporation [14–19]. HypE and HypF are required for the synthesis of the cyanide ligand coordinated to the active site iron atom [20,21]. Finally, the endopeptidase, HycI, is responsible for the proteolytic maturation of the precursor of the large subunit. HycI removes a 32 amino acid fragment from the Cterminus of pre-HycE [9,22,23]. In vivo and in vitro analyses clearly showed that the nickel ion is essential for the recognition and cleavage of the large subunit by the endopeptidase [24,25]. Recently, the crystal structures of HypC, HypD, and HypE from Thermococcus kodakaraensis KOD1 were reported, and a cyanation reaction mechanism via unique thiol redox signaling in the hypCDE complex was proposed [26]. Previously, the crystal structure of HypD from E. coli was solved in the holo-form, bound with Cd2+ [8]. The cadmium ion was bound to the proposed nickel ion binding site, and it was penta-coordinated with Glu16, Asp62, His93 and a water molecule in a pseudo-tetragonal arrangement. All of

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these metal ion binding site residues were highly consistent among the homologous proteins, and mutational analyses suggested that these residues were important for catalysis [8]. Recently, the NMR structure of E. coli HycI was reported in the apo-form. The putative metal binding site in HycI was proposed, based on a structural comparison with HypD, although the sequence identity between HycI and HypD is only about 20% [27]. Here, we describe the crystal structure of the E. coli HycI in the presence of Ca2+ ions. Although the overall crystal structure is similar to the solution structure, a significant conformational change was observed at the L5 loop. Interestingly, three specific metal ion binding sites were identified, which may be required for cleavage and maturation of the large subunit of [NiFe]-hydrogenases.

Materials and methods Overexpression and purification of HycI. The selenomethionine (SEMET)-substituted E. coli K-12 HycI was synthesized in a cell-free system as a His-tagged protein with a TEV protease cleavage site. The HycI protein was first absorbed to a HisTrap HP5 column (GE Healthcare Biosciences) in 20 mM Tris–HCl, pH 8.0, containing 500 mM NaCl and 20 mM imidazole, and after the bound protein was eluted by an imidazole gradient (0.02–500 mM), the fractions were analyzed by SDS–PAGE. The protein-containing solutions were pooled and applied to a gel-filtration column (Superdex 200, GE Healthcare Biosciences), which was pre-equilibrated with 20 mM Tris–HCl buffer (pH 8.0), containing 500 mM NaCl and 20 mM imidazole, and the protein was subjected to TEV protease digestion. To remove the histidine tag and the TEV protease from

the mixture, the solution was subsequently purified by chromatography on HisTrap HP5 and HiPrep columns (GE Healthcare Biosciences). The protein was concentrated to 15.84 mg/ml. Crystallization and data collection. Initial crystals of HycI were produced at 20 °C by the sitting drop vapor diffusion method [28], by adding 1 ll of protein solution to 1 ll of well solution, containing 28% polyethylene glycol (PEG) 400, 0.2 M calcium chloride and 0.1 M sodium Hepes (pH 7.5). Diffraction quality crystals of HycI grew within a week. Complete MAD data sets were obtained at 100 K on the RIKEN structural genomics beamline I (BL26B1) at SPring-8, Hyogo, Japan. These crystals belonged to the orthorhombic space group P21212, with cell dimensions a = 87.425, b = 54.984, c = 67.964 Å. The data sets were processed up to 1.70 Å, using the HKL 2000 suite [29] (Table 1). The HycI structure was determined by the multiwavelength anomalous dispersion (MAD) method with the three different wavelength data sets collected at the Se edge, using the program SOLVE [30]. Solvent flattening and initial model building were performed by RESOLVE [30]. Improvement of the partial model derived from RESOLVE was performed with the program ARP/ wARP [31]. The final model was refined and manually fitted using CNS [32] and Coot [33]. The final model with 159 residues, except for the C-terminal three residues, was refined to a crystallographic R-factor of 0.248 (Rfree = 0.258) at 1.7 Å resolution, using synchrotron radiation X-ray data collected at cryo temperature (Table 1). We have modeled three extra residues at the N-terminus, which were derived from the cleavage site, probably due to inefficient protease cleavage. The coordinates and structure factors are available in the Protein Data Bank, under the accession code 2E85.

Table 1 Data collection and refinement statistics of E. coli HycI.

a

Data collection

Remote (low)

Peak

Edge

Wavelength Space group Cell dimensions (Å) No. of molecules (asu) Solvent content Resolution range (Å) Unique reflections Redundancy Completeness (%) Average I/r(I) Rmergeb

1.0000 P21212 a = 87.425, b = 54.984, c = 67.964 2 0.48 50.0–1.70 (1.66–1.70)a 36,372 6.5 (6.5) 98.6 (97.7) 19.54 (5.84) 0.084 (0.231)

0.97921

0.97955

50.0–1.60 (1.66–1.60) 43,195 6.4 (5.7) 98.4 (95.4) 13.33 (6.43) 0.115 (0.229)

50.0–1.60 (1.66–1.60) 43,176 6.4 (5.7) 98.3 (95.4) 18.52 (5.84) 0.089 (0.248)

Refinement statistics Resolution range (Å) Reflections used in the refinement Total number of reflections used for working set R (%)c Total number of reflections used for Rfree Rfree (%)d No. of protein atoms No. of water molecules No. of Ca2+ ions RMSD bond lengths (Å) RMSD bond angles (°) Average B factor (Å2)

20.0–1.70 35,885 34,095 24.8 1790 25.8 2362 284 6 0.006 1.0 14.8

Ramachandran statistics Most favored regions Allowed regions

97.7 2.3

PDB code

2E85

Values in parentheses are for the highest resolution shell. P P P P Rmerge = h i |I(h, i)  hI(h)i|/ h i I(h, i), where I(h, i) is the intensity value of the ith measurement of h and hI(h)i is the corresponding mean value of I(h) for all i measurements. P c R-factor = ||Fobs|  |Fcalc||/|Fobs|, where |Fobs| and |Fcalc| are the observed and calculated structure factor amplitudes, respectively. d Rfree is the same as R-factor, but for a 5% subset of all reflections. b

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Results and discussion Purification and crystallization of HycI We cloned, expressed, and purified the HycI enzyme, as described in the Materials and methods section. N-terminal sequencing of the purified enzyme confirmed that it was the expected protein (data not shown). HycI consists of 156 amino acids (17 kDa). The potential pI of this protein is 3.68. The protein was crystallized successfully in the presence of Ca2+, by the sitting drop vapor diffusion method, as described in the Materials and methods section. In contrast, it was previously reported that attempts to crystallize E. coli HycI were not successful [8]. Actually, our attempts to reproduce the crystal in the absence of Ca2+ were unsuccessful, either with or without any other metal ions, including Ni2+ and Fe2+. Structural description of HycI The crystal structure of HycI was solved by the MAD method at 1.70 Å, and was refined to a final R value of 24.8% and an Rfree value of 25.8% (Table 1). The overall structure of HycI belongs to the a/b family. It consists of five a-helices and five b-strands, arranged as an a/b sandwich type in the order of b1-a1-b2-a2-b3-b4-a3-a4b5-a5 in the primary structure (Fig. 1A and B). Although the gel-filtration experiments showed that HycI exists as a monomer in solution [9], the asymmetric subunit contains two molecules, with overall dimensions of 66  24  28 Å. In the dimer, the two molecules are related by a non-crystallographic two-fold axis, perpendicular to the plane in Fig. 1B. The total solvent area buried upon dimerization was 826 Å2 (10%) per molecule. At the dimer interface, the residue contacts were identified between the two molecules, A and B: A47Ile–B92Met, A91Asn–B44Glu, A92Met–B97Leu, A95Asn–B51Arg, A96Tyr–B51Arg, and A104Asp–B96Tyr. We unambiguously identified a few huge peaks (>6r level) in the electron density map. We suspected that these densities corresponded to either calcium or chloride ion. Analyses of the 191 calcium- and chloride ion-containing, high resolution crystal structures (1.89–1.91 Å) revealed that the average calcium–ligand distances were 2.5–2.52 Å (minimum and maximum, respectively), and the average chloride–ligand distances were 3.16–3.26 Å [35]. Based on the modes of coordination as well as the ligand–ion distances, we concluded that the densities corresponded to a bound Ca2+ ion, which is derived from the crystallization buffer (CaCl2). In total, we added seven Ca2+ ions to the refined structure. Among these seven identified metal ions, three of them were located in each monomer and one was at the dimer interface. Detailed analyses of the metal ions are described below. Structural comparison A structural comparison between the present HycI crystal and the recently reported solution (NMR) structure in the apo-form [27] (PDB ID, 2I8L) is shown in Fig. 1C. Although the overall topology seems to be very similar throughout the structures, significant changes are observed in the L5 loop, which connects the a3 and a4-helices. The following residues, including Asp80–Asn95, in the solution structure formed a long loop (L5). Specifically, in the solution structure, residues 84–92 showed extra length in the loop, whereas the same residues formed the loop, but it was bent towards the L4 loop in the crystal structure (Fig. 1C). This is the only significant difference observed between the absence and presence of metal ions (the apo- and holo-forms, respectively, of HycI). A structural similarity search of HycI, using the DALI program [36], readily identified a few other protein structures with reasonably good Z-scores, such as 14.4 for hydrogenase 2 maturation protease,

HypD (PDB ID, 1cfz), 9.0 for peptidyl tRNA hydrolase (2pth), 7.5 for mtaSAH nucleosidase (1jys), 7.4 for spore protease (1c8b), 7.2 for pyrrolidone carboxyl peptidase (1a2z), 7.1 for amp nucleosidase (1t8r), and 6.9 for 50 -methylthioadenosine nucleosidase (2h8g), and the sequence identity falls between 7% and 20% among these proteins. The crystal structure of E. coli HypD (PDB ID, 1cfz) was superimposed on the solution and crystal structures of HycI, for comparison (Fig. 1D). Although HypD shows similar overall packing in the core structural regions, significant diversity was observed in two places. In the first, although the L4 loop seems to be the same length in both HypD and the holo-form of HycI, in particular, the Cd2+ binding site and the geometry of the three key residues constituting the putative active site significantly differ from those of the two structures. Mutagenesis experiments revealed that Asp16, Asp62, and His90 in HycI were important for the nickel binding and the catalysis role [8]. Based on the sequence alignment, it was proposed that the nickel binding residues in HycI correspond to Glu16, Asp62, and His93 in HypD [8]. As shown in Fig. 1E, Asp16 and Asp62 assume similar conformations to those observed for Glu16 and Asp62 in HypD, but His90 in the holo-form of HycI is 6 Å away from the position corresponding to that of His93 in HypD. The same residue, His90, in the solution structure is located in the L5 loop, which is far away from the other two residues (Asp16 and Asp62), as well as about 9 Å away from His93 in HypD, and is moved up by 10 Å, as compared to His90 in the crystal structure of HycI. The second notable difference is observed around the C-terminal regions. In HycI, the C-terminus points to the putative nickel-binding site and does not form regular secondary structures, whereas it forms a few short secondary structures in HypD and points away from the protein core structure. The C-terminal region of HycI in solution as well as in the crystal adopts a similar conformation, and we suggest that this may be the probable conformation in solution. The HypD C-terminal conformation may not be the preferred conformation in solution, and this might be due to the contacts with the neighboring molecule, as previously suggested [8]. Metal ion binding The in vivo and in vitro analyses revealed that the nickel ion is essential for substrate recognition and cleavage of the large subunit (HycE) by the endopeptidase [24,25]. Based on the metal ion–ligand interactions, it is likely that two of the metal ions in each monomer (Ca1 and Ca2) and the dimer-interface metal ion (Ca3) are specific. The Ca1 ion is bound with the OD1 (2.6 Å) and OD2 (2.5 Å) of Asp46, the OD1 (2.2 Å) of Asn45, the backbone oxygens of Ala42 and Gly39 (2.3 and 2.3 Å), and a couple of water molecules (2.5 Å) (Fig. 1F). As shown in Fig. 1G, the Ca2 ion is bound with the OD1s (2.3 Å) of Asp16 and Asp62, the backbone oxygen of Ala63 (2.6 Å) and three water molecules (2.4 Å). The Ca3 ion bonded to the OD1 of Asp99 (2.2 Å, molecule A), the OE1s of Gln100 and Glu103 (2.3 Å, molecule B) and three water molecules (Fig. 1H). The other two metal ions (non-specific) are bound to Asp116 and Glu140. Among the three specific sites, the Ca2 ion is bound at the same site where the Cd2+ ion is also observed in HypD. The HycI Asp16 and Asp62 residues correspond to the HypD Glu16, and Asp62 residues. His93 is involved in the metal ion coordination in HypD, whereas in HycI, the corresponding residue, His90, is away from the binding site. In the case of the apo-form of HycI in solution, His90 is far away from the binding site. In addition, Ala63 is bound to the Ca2+ ion. In either case, His90 is not required for the metal ion coordination of HycI. This is also consistent with the mutagenesis analyses of HycI, in which the Asp16Asn, Asp62Asn, and Asp62Met mutants were completely inactive and the His90Gln mutant was about fivefold less active [8] (E. Theodoratou, Univer-

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Fig. 1. Crystal structure and analysis of HycI. (A) A view of the HycI monomer. HycI is shown in a ribbon model with labels for a-helices, b-strands, and loop regions. The Nand C-termini of HycI are indicated by N and C, respectively. (B) Dimer representation of HycI, formed by the non-crystallographic two-fold axis in the asymmetric unit (asu). The bound Mg2+ ion is shown in a cpk model. (C) The 3D-structural comparison of HycI. Superposition of the HycI structure with its solution structure (NMR). The crystal and solution structures of HycI are colored red and blue, respectively. (D) Structural comparison of HycI (crystal and NMR) with HypD of the same protein family. The crystal and solution structures of HycI are colored as described above, and the HypD is colored yellow. (E) Stereo view of the Cd2+ binding site in HypD. The coloring scheme is the same as in (D). (F–H) A close-up view of the Ca2+ binding sites in HycI. The three bound Ca2+ sites, Ca1 (F), Ca2 (G), and Ca3 (H), are shown. Hydrogen bonds are indicated by dotted lines. The protein residues are represented by ball-and-stick models colored by atom type (nitrogen, blue; carbon, green; oxygen, red). The Ca2+ and water molecules are represented by cpk models in green and red, respectively. The electron density around the metal ion Ca1 was contoured at the 3r level, whereas in the cases of Ca2 and Ca3, they were contoured at the 1.5r level. The figures were generated using Pymol [34]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

sität München, Personal communication). Taken together, Asp16 and Asp62 are very important for the metal ion coordinations. We have identified a unique metal ion binding site, Ca1. This Ca1 site is composed of four protein residues and two water molecules. The Ca3 ion is located at the dimer-interface. The Gln100 and Glu103 backbones are also shifted by 3 Å, as compared with those in the solution structure, in order to coordinate with the metal ions. A backbone movement by 3–5 Å is also observed around this binding site in HypD, as compared with HycI. As a consequence of this metal ion involvement in the dimer-interface, the L5 loop might have changed its conformation in order to provide more stability as well as suitability for the peptidase catalysis for the substrate. Although we do not have direct evidence for the other two metal ion binding site residues (Ca1 and Ca3), the Ni2+ and Cd2+ titration experiments using the two-dimensional NMR analyses revealed that the residues at or around the metal ion binding site, especially Asp16–Gly19, Gly40–Glu44, Val61–Asp65, Asp77–Leu97, and the C-terminus, undergo significant conforma-

tional changes [27]. The Ca1 and Ca3 ions have been identified for the first time in the crystal structure. Therefore, based on the structural as well as 2D-NMR and mutagenic analyses, it seems that all three of the Ca2+ ion binding sites are important for the enzymatic activity and the maturation of the large subunit of the hydrogenase, HycE, from the precursor protein; and however, these Ca2+ ion binding sites are needs to be clarified by further experiments.

Acknowledgments The authors thank C. Kuroishi for cloning of hycI gene. This work was supported by the RIKEN Structural Genomic/Proteomics Initiative (RSGI) of the National Project on Protein Structural and Functional Analyses, Ministry of Education, Culture, Sports, Science and Technology of Japan. The authors thank Dr. Tetsuya Ishikawa for his moral support and encouragement.

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