Structural basis of a Ni acquisition cycle for [NiFe] hydrogenase by Ni-metallochaperone HypA and its enhancer

Significance

The metal ions in proteins are correctly incorporated by specific metallochaperones. However, it remains unclear how metallochaperones regulate their metal binding affinity during acquisition of correct metal ions and deliver them to target proteins. In this study, we have determined the crystal structures of a transient complex between a Ni metallochaperone HypA and its partner ATPase protein HypB_AT, which incorporate a Ni ion into [NiFe] hydrogenase. The structures reveal that HypB_AT induces conformational change of HypA through complex formation, leading to formation of a Ni binding site. Consequently, the Ni-binding affinity of HypA is increased from micromolar to nanomolar range (by ∼600-fold). These results indicate that HypB_AT functions as a metallochaperone enhancer, which regulates metal binding affinity of metallochaperones.

Abstract

The Ni atom at the catalytic center of [NiFe] hydrogenases is incorporated by a Ni-metallochaperone, HypA, and a GTPase/ATPase, HypB. We report the crystal structures of the transient complex formed between HypA and ATPase-type HypB (HypB_AT) with Ni ions. Transient association between HypA and HypB_AT is controlled by the ATP hydrolysis cycle of HypB_AT, which is accelerated by HypA. Only the ATP-bound form of HypB_AT can interact with HypA and induces drastic conformational changes of HypA. Consequently, upon complex formation, a conserved His residue of HypA comes close to the N-terminal conserved motif of HypA and forms a Ni-binding site, to which a Ni ion is bound with a nearly square-planar geometry. The Ni binding site in the HypAB_AT complex has a nanomolar affinity (K_d = 7 nM), which is in contrast to the micromolar affinity (K_d = 4 µM) observed with the isolated HypA. The ATP hydrolysis and Ni binding cause conformational changes of HypB_AT, affecting its association with HypA. These findings indicate that HypA and HypB_AT constitute an ATP-dependent Ni acquisition cycle for [NiFe]-hydrogenase maturation, wherein HypB_AT functions as a metallochaperone enhancer and considerably increases the Ni-binding affinity of HypA.

Approximately one-half of all cellular proteins require specific metal ions for proper function, which are delivered by specific metallochaperones (1, 2). However, the mechanisms of correct acquisition and delivery to target proteins of many metallochaperones remain poorly understood. [NiFe] hydrogenases harbor a complex metal cofactor, NiFe(CN)₂CO, in their active sites (3). This cofactor catalyzes reversible H₂ production. The Ni atom in the NiFe(CN)₂CO cofactor is bound to four thiolate groups, two of which also bridge the Fe(CN)₂CO group (4, 5). NiFe(CN)₂CO biosynthesis requires specific maturation machinery, in which six Hyp proteins (HypA–HypF) play key roles (6, 7). Four Hyp proteins (HypC–HypF) are involved in the biosynthesis and incorporation of the Fe(CN)₂CO group (8–15). After Fe insertion, HypA and HypB insert the Ni ion into the hydrogenase large subunit (16).

HypA is a Ni-metallochaperone that binds to a Ni ion with micromolar affinity (17–19), and its structure consists of a Ni-binding domain (NiBD) and a Zn-binding domain (ZnBD) (20, 21). The NiBD contains a highly conserved MHE motif that is essential for Ni binding at the N terminus (20, 22). HypB consists of a common GTPase domain and a less conserved metal-binding region (23–25). Recently, ATPase-type HypB (HypB_AT, previously abbreviated as mmHypB) proteins were identified from Thermococcales (26). GTPase and ATPase types of HypB belong to the SIMIBI class NTPase family and share a similar architecture, despite their low sequence similarity (27). HypA and HypB form a transient complex in the Ni insertion process (17, 22, 28, 29). In the Escherichia coli system, Ni transfer occurs from HypB to HypA (30). However, the functional relationship between HypA and HypB or HypB_AT for the maturation process is not fully understood. Here, we determined the crystal structures of the HypA–HypB_AT complex from Thermococcus kodakarensis, providing the structural basis of concerted actions of these proteins for Ni insertion in the [NiFe]-hydrogenase maturation process.

Results

ATP-Dependent Interaction Between HypA and HypB_AT.

HypB_AT is an ATPase, showing a significant ATPase activity (Fig. 1A). The ATPase activity of HypB_AT was further increased by threefold in the presence of HypA, suggesting that HypA enhances the ATPase activity of HypB_AT through interaction with HypB_AT.

Fig. 1.

Interaction between HypA and HypB_AT. (A) The ATP hydrolysis activities of HypB_AT, HypA, and the mixture of HypB_AT and HypA were measured by a colorimetric method that measures the amount of inorganic phosphate from ATP hydrolysis. Error bars represent SDs of three measurements. (B) SEC elution profiles of equal molar mixtures of HypA and HypB_AT in the absence of nucleotides (blue) or in the presence of ATPγS (red) or ADP (green).

We examined interactions between the two proteins in the presence or absence of nucleotide by size exclusion chromatography (SEC) (Fig. 1B and Fig. S1). ADP or ATPγS-bound HypB_AT forms a stable homodimer in solution. However, elution volume of nucleotide-free HypB_AT corresponded to a molecular mass between monomeric and dimeric state (∼40 kDa), suggesting that nucleotide-free HypB_AT exists in rapid equilibrium between monomer and dimer. SEC experiments showed that ATPγS-bound HypB_AT weakly interacts with HypA, forming a binary complex at a molar ratio of 2:2 (Fig. S1A). However, nucleotide-free or ADP-bound HypB_AT did not show complex formation with HypA (Fig. S1 B and C). These results indicate that association and dissociation of HypA and HypB_AT is regulated by the ATP hydrolysis cycle of HypB_AT.

Overall Structure of the HypA–HypB_AT Complex.

To elucidate the molecular mechanisms underlying HypA–HypB_AT interactions, we determined the crystal structure of the HypAB_AT complex at 1.63–3.10 Å resolution in the presence of (i) ATPγS and Ni ions; (ii) AMPPCP (a nonhydrolyzed ATP analog) and Ni ions; and (iii) only AMPPCP (Fig. 2A and Table S1). The overall architectures of these three states are similar, despite small differences in local conformations (Fig. S2). In the case of the presence of ATPγS and Ni ions, although ATPγS was slowly hydrolyzed in the crystal, its overall architecture was retained, owing to tight crystal packing (Fig. S3).

Fig. 2.

Crystal structure of the HypA–HypB_AT complex. (A) Overall structure of the HypA–HypB_AT complex. Green and yellow, monomers in the HypB_AT dimer; purple and pink, HypA molecules with Zn (cyan) and Ni (orange) atoms. The ADP molecules are shown in a sphere model. (B) Interactions between HypA monomer and HypB_AT dimer. Three interfaces are shown in black boxes. (C) Close-up view of interface 1. The α1 helix of HypB_AT is shown in a ribbon model and the NiBD of HypA is shown in a molecular surface model. Key residues involved at interface 1 are shown in a stick model. A hydrogen bond is shown as a dashed line. (D) Close-up view of interface 2. The ZnBD of HypA is shown in a ribbon model and the hydrophobic cleft of HypB_AT is shown in a molecular surface model. Salt bridges and hydrogen bonds are shown as dashed lines. (E) Close-up view of interface 3.

The HypB_AT monomer structure consists of a central eight-stranded β-sheet (β1–β8) flanked by nine α-helices (α1–α9) (Fig. S4A), and forms a homodimer. The AMPPCP/ADP molecules are sandwiched at the dimer interface. The HypA structure consists of the NiBD and ZnBD (Fig. S4B). The ZnBD is composed of two α-helices, a short three-stranded β-sheet (β3, β4, and β5) and a zinc finger motif (CXXCXnCPXC), in which a Zn ion is coordinated by four cysteine residues (Fig. S4C). The NiBD is composed of two α-helices (α1 and α2) and a three-stranded β-sheet (β1, β2, and β6). In the presence of Ni ions, the conserved MHE motif binds a Ni ion (details described below).

The structure of the HypAB_AT complex shows a heterotetrameric structure containing two HypA and two HypB_AT molecules (Fig. 2A). The two HypA molecules are bound to the opposite surface of the ATP-binding site of the HypB_AT dimer. The HypAB_AT complex is formed through three interfaces (Fig. 2B). At interface 1, the HypB_AT α1 helix is unwound from Pro6, and residues 2–6 adopt β-strand conformations, interacting with a hydrophobic patch between the α1 helix and β6 strand in the HypA-NiBD (Fig. 2B, Upper and C). At interface 2, the α3, η4, and α4 helices in the HypA-ZnBD interact with the β6-α4 and α3-β4 loops of HypB_AT through hydrophobic interactions and several hydrogen bonds (Fig. 2B, Lower and D). Interface 3 involves hydrogen bonds between residues surrounding the HypA zinc finger motif and the α5′ and α6´ helices of HypB_AT (where the prime indicates the second HypB_AT in the dimer) (Fig. 2B, Lower and E), suggesting that the dimeric form of HypB_AT is required for complex formation with HypA. The residues involved in the three interfaces are well conserved in both proteins (Figs. S5 and S6).

Complex Formation Creates a Ni-Binding Site with Nanomolar Affinity.

Upon complex formation, HypA undergoes striking conformational changes (Fig. 3A, Fig. S5, and Movie S1). In a superposition of the isolated form of HypA with that in the HypAB_AT complex, there is steric and electrostatic repulsion between HypA-Glu119 and HypB_AT-Glu109, and between HypA-Asp117 and HypB_AT-Glu177´ (Movie S1). To avoid the repulsion, the ZnBD in the complex is substantially rotated clockwise, and the η2, η3, and η4 3₁₀ helices are moved toward HypB_AT (Fig. 3A, Right)_, leading to formation of the interfaces 2 and 3 (Fig. 2 D and E). This movement disrupts a hydrophobic core formed by Phe89, Ile97, Phe99, and Phe107 in the isolated state. Consequently, Phe99 is flipped out, and Ile93 moves into the hydrophobic core, resulting in formation of the α3 and α4 helices. These conformational changes bring a conserved His residue (HypA-His98) close to the conserved MHE motif, forming a Ni-binding site (Fig. 3B). The Ni ion bound to this site has a nearly square-planar geometry, coordinated by the amine nitrogen of Met1, the amide nitrogen and Nδ of His2, and Nε of His98. Oε of Glu3 also exhibits van der Waals interactions (3.8 Å) with the Ni ion, further supporting the coordination of the Ni ion.

Fig. 3.

HypAB_AT complex formation creates a Ni-binding site with nanomolar affinity. (A) Comparison of the ZnBD of HypA in the free state (Left) and in the complex (Right). (B) Close-up view of the Ni-binding site in the HypAB_AT complex. A simulated annealing Fo-Fc omit map at 3 σ and an anomalous difference Fourier map at 25 σ is shown in gray and blue, respectively. Ni-ligand distances are shown in angstroms. (C) ITC raw data (Upper) and binding isotherm data (Lower) for titration of Ni ions into HypA (Left), HypB_AT with ATPγS (Middle), and mixtures of HypA and HypB_AT with ATPγS (Right).

To determine how HypB_AT affects the Ni-binding property of HypA, we performed isothermal titration calorimetry (ITC) experiments (Fig. 3C and Table S2). HypA bound to a Ni ion with a K_d value of 4.1 µM (Fig. 3C, Left), but no Ni binding to ATPγS-bound HypB_AT was observed (Fig. 3C, Middle). Furthermore, the ITC data for equal molar mixtures of HypA and HypB_AT with ATPγS revealed a strong and a weak binding site (Fig. 3C, Right). The best-fit parameters obtained by a two-site model were K_d1 = 7.3 nM and K_d2 = 6.6 µM, for the strong and weak sites, respectively. Under the experimental conditions, HypAB_AT complex and isolated HypA and HypB_AT were observed in the sample cell (Fig. 1B). The binding parameters for the weak site (K_d2) were comparable to those of isolated HypA. The strong binding site (K_d1) with nanomolar affinity corresponds to the observed Ni-binding site in the HypAB_AT complex. These results indicate that complex formation increases the Ni-binding affinity of HypA from micromolar to nanomolar range by formation of the Ni-binding site with the square-planar coordination.

Conformational Changes by ATP Hydrolysis and Ni Binding.

Conformational changes induced by ATP hydrolysis of HypB_AT account for the ATP-dependent interaction of HypB_AT with HypA. Despite lack of the structures of ATP-bound HypB_AT alone, HypB_AT in the HypAB_AT complex can be regarded as an “ATP-bound state,” because only the HypB_AT bound to ATP or ATP analogs interacts with HypA. Upon ATP hydrolysis, the Walker A and B motifs undergo outward and inward rotations, respectively (Fig. 4A and Fig. S3). These conformational changes displace the β6-α4 loop and α4 helix of HypB_AT and, consequently, affect interface 2 of HypB_AT (Fig. 4B). In the HypAB_AT complex, the hydrophobic cleft at interface 2 accommodates Phe99, Ile100, and Val103 of HypA (Figs. 2D and 4B, Left). However, interface 2 of ADP-bound HypB_AT alone shows a flat molecular surface (Fig. 4B, Middle). The nucleotide-free state of HypB_AT also shows a different molecular surface at interface 2 (Fig. 4B, Right). In addition, the ADP-bound HypB_AT dimer adopts a more open configuration (Fig. 4C), affecting interface 3. Therefore, conformational changes induced by ATP hydrolysis impair the interactions of HypB_AT with HypA by disrupting interfaces 2 and 3.

Fig. 4.

Conformational changes of HypB_AT by ATP hydrolysis and Ni binding. (A) Superposition of the Cα backbone of the ATP-bound (light blue) and ADP-bound (pink) states of HypB_AT. The structure of the ATP-bound state is derived from that in complex with HypA. The Walker A (WA) and Walker B (WB) motifs, β6-α4 loop and α4 helix are indicated with blue (ATP bound) and red (ADP bound) lines. Curved arrows show conformational changes induced by ATP hydrolysis. (B) Comparison of the molecular surface of the ATP-bound (complex with HypA), ADP-bound, and nucleotide-free states of HypB_AT. Residues at interface 2 are shown in red. (C) Superposition of Cα backbone of the ATP-bound (blue) and ADP-bound (pink) states of the HypB_AT dimer. Arrows show conformational changes induced by ATP hydrolysis. (D) Superposition of Cα backbone of the Ni-free (blue) and Ni-bound (red) states of HypA in complex with HypB_AT. The ZnBDs of the two structures are superimposed on each other. Curved arrows show conformational changes induced by Ni binding. (E) Superposition of Cα backbone of the nickel-free (green and blue) and nickel-bound (red and magenta) states of the HypAB_AT complex.

Ni binding to the HypAB_AT complex also affects the HypA–HypB_AT complex interfaces. Comparison of the Ni-free and Ni-bound structures shows that upon Ni binding, the HypA-NiBD is slightly rotated and the N terminus moves toward His98 to form the Ni-binding site (Fig. 4D). This conformational change slightly displaces the NiBD of HypA from HypB_AT (Fig. 4E), suggesting that Ni binding to the HypAB_AT complex weakens the interactions. However, the ZnBD in the Ni-bound state is nearly identical to that in the Ni-free state. The observation that the coordination of Zn site in Helicobacter pylori HypA (HpHypA) is dynamic, depending on pH and Ni binding (19) appears to be a specific property of HpHypA, because the His residues involved in Zn binding in HpHypA are not conserved among other HypA homologs including T. kodakarensis HypA.

Discussion

The present study shows that the transient complex formation between HypA and HypB_AT induces conformational changes of HypA, resulting in >560-fold enhancement of the Ni binding affinity of HypA. The association and dissociation of the HypAB_AT complex are regulated by the ATP-hydrolysis cycle of HypB_AT. Based on the present findings, we propose the following mechanism for an ATP-dependent Ni acquisition cycle in [NiFe]-hydrogenase maturation (Fig. 5). ATP-bound HypB_AT interacts with HypA and induces conformational changes in the ZnBD of HypA. Consequently, the conserved His residue comes close to the MHE motif, forming the Ni-binding site with nanomolar affinity. HypA rapidly traps a Ni ion with the nanomolar affinity site. Upon Ni binding, the conformational changes in HypA affect its association with HypB_AT, and complex formation stimulates ATP hydrolysis of HypB_AT, thereby disrupting the complex interfaces, leading to release of HypA from HypB_AT. Subsequently, HypA reassumes the micromolar affinity form, either spontaneously or by association with the hydrogenase large subunit, and inserts the Ni ion into the large subunit (31, 32). Sequence analysis suggests that the functional roles of HypB_AT in the ATP-dependent Ni acquisition cycle are conserved among all HypB_AT proteins, although HypB_AT homologs including that from T. kodakarensis do not have a Ni binding site like those found in the conserved G domain of HypB (Fig. S6).

Fig. 5.

Schematic for an ATP-dependent Ni acquisition cycle for [NiFe]-hydrogenase maturation. HypA, MHE motif, and His98 of HypA are indicated in purple, gray, and green, respectively. The second HypA is omitted for clarity. The HypB_AT dimer is shown in green and yellow, and the [NiFe] hydrogenase (H2ase) large subunit (LS) is shown in brown.

The structures of the HypAB_AT complex determined herein provide insight into the interactions of HypB with HypA. A HypB-binding site of HpHypA is located at the cleft between the α1 helix and β6 strand (29), which is consistent with interface 1 of HypA. Hydrophobic residues at the N-terminal or linker region of HypB were shown to be involved in its interaction with HypA (28, 29). Thus, interface 1 is likely to be conserved among all HypA and HypB protein types.

The metal-binding affinities of metallochaperones are tightly controlled during the trafficking of specific metal ions. Micromolar affinity is preferable for delivering metal ions into target proteins, but is insufficient for acquiring metal ions at low concentration in the cytoplasm (33). In this study, HypB_AT increased the Ni-binding affinity of HypA from the micromolar to nanomolar range. Because nanomolar affinity is sufficient for specific binding for the Ni ion, HypB_AT appears to function as a metallochaperone enhancer that increases the metal-binding affinity of the target metallochaperone (HypA). The concept of metallochaperone enhancer can account for the previous generic observations that disruption of the hypB_AT gene resulted in a significant defect in [NiFe]-hydrogenase maturation (26). Without HypB_AT, HypA cannot adopt the high-affinity form for Ni ions and, therefore, fails to acquire and transfer Ni ions. This defect can be restored by Ni supplementation, because increasing the cellular Ni concentration allows HypA to deliver Ni ions to the hydrogenase large subunit. Thus, metallochaperone enhancer is essential for effective acquisition and delivery of specific metal ions by metallochaperone. We believe that these findings will provide a general model for the functional regulation of metallochaperones.

Materials and Methods

For crystallization, the HypAB_AT complex sample was prepared by mixing the two proteins at an equal molar concentration and 1 mM ATP analog (ATPγS or AMPPCP) with or without 1 mM NiCl₂ for 1–2 h at room temperature. Crystals of the HypAB_AT complex were obtained by using MgCl₂ and PEG 3350 as precipitates. The X-ray diffraction data were collected at the BL1A and AR-NE3A beamlines at the Photon Factory and the BL41XU beamline at SPring-8 and were processed with the HKL2000 package (34). The structure of the HypAB_AT complex was determined by the molecular replacement method with Molrep (35) and Phaser (36), using the previously determined HypA (PDB ID code 3A43) and HypB_AT (PDB ID code 3VX3) as search models. The structures were refined with PHENIX (37) and validated with MolProbity (38). The Ni atoms in the crystal structures were confirmed with dataset collected at the peak and edge wavelengths of Ni (Table S1). More detailed experimental procedures are described in SI Materials and Methods.