Structure of [NiFe] hydrogenase maturation protein HypE from Escherichia coli and its interaction with HypF

Abstract

Hydrogenases are enzymes involved in hydrogen metabolism, utilizing H2 as an electron source. [NiFe] hydrogenases are heterodimeric Fe-S proteins, with a large subunit containing the reaction center involving Fe and Ni metal ions and a small subunit containing one or more Fe-S clusters. Maturation of the [NiFe] hydrogenase involves assembly of nonproteinaceous ligands on the large subunit by accessory proteins encoded by the hyp operon. HypE is an essential accessory protein and participates in the synthesis of two cyano groups found in the large subunit. We report the crystal structure of Escherichia coli HypE at 2.0-A resolution. HypE exhibits a fold similar to that of PurM and ThiL and forms dimers. The C-terminal catalytically essential Cys336 is internalized at the dimer interface between the N- and C-terminal domains. A mechanism for dehydration of the thiocarbamate to the thiocyanate is proposed, involving Asp83 and Glu272. The interactions of HypE and HypF were characterized in detail by surface plasmon resonance and isothermal titration calorimetry, revealing a Kd (dissociation constant) of approximately 400 nM. The stoichiometry and molecular weights of the complex were verified by size exclusion chromatography and gel scanning densitometry. These experiments reveal that HypE and HypF associate to form a stoichiometric, hetero-oligomeric complex predominantly consisting of a [EF]2 heterotetramer which exists in a dynamic equilibrium with the EF heterodimer. The surface plasmon resonance results indicate that a conformational change occurs upon heterodimerization which facilitates formation of a productive complex as part of the carbamate transfer reaction.

FIG. 1.

Cartoon representation of HypE. (a) Stereo view of the monomer. The domains are individually colored (N terminal: helices, cyan; strands, magenta; loops, salmon; C terminal: helices, red; strands, yellow; loops, green). The “bulge” corresponding to residues 64 to 71 is colored blue. This figure and subsequent figures were prepared using PyMol (www.pymol.org). (b) HypE dimer viewed along the twofold axis colored as described above. The active sites are indicated with arrows. (c) Superposition of the C-terminal domain of HypE (gray) with the N-terminal domain of HypF (cyan) shown in a cartoon representation. The parts of HypE that share the fold with HypF are colored in magenta. (d) Surface representation of the active site of HypE formed at the interface of monomers A (cyan) and B (salmon). The N-terminal helix (pink) of monomer A and the C-terminal loop (light green) of monomer B are situated proximal to each other and contribute toward the active site (shown by arrow). (e) Stereo view of the active site of HypE with modeled ADP positioned based on the superposition of HypE with PurL. One monomer is depicted in cartoon format while the second monomer is shown in surface representation. (f) Blowup of the putative carboxamide binding site situated near the C-terminal cysteine (Cys336).

FIG. 1.

Cartoon representation of HypE. (a) Stereo view of the monomer. The domains are individually colored (N terminal: helices, cyan; strands, magenta; loops, salmon; C terminal: helices, red; strands, yellow; loops, green). The “bulge” corresponding to residues 64 to 71 is colored blue. This figure and subsequent figures were prepared using PyMol (www.pymol.org). (b) HypE dimer viewed along the twofold axis colored as described above. The active sites are indicated with arrows. (c) Superposition of the C-terminal domain of HypE (gray) with the N-terminal domain of HypF (cyan) shown in a cartoon representation. The parts of HypE that share the fold with HypF are colored in magenta. (d) Surface representation of the active site of HypE formed at the interface of monomers A (cyan) and B (salmon). The N-terminal helix (pink) of monomer A and the C-terminal loop (light green) of monomer B are situated proximal to each other and contribute toward the active site (shown by arrow). (e) Stereo view of the active site of HypE with modeled ADP positioned based on the superposition of HypE with PurL. One monomer is depicted in cartoon format while the second monomer is shown in surface representation. (f) Blowup of the putative carboxamide binding site situated near the C-terminal cysteine (Cys336).

FIG. 1.

Cartoon representation of HypE. (a) Stereo view of the monomer. The domains are individually colored (N terminal: helices, cyan; strands, magenta; loops, salmon; C terminal: helices, red; strands, yellow; loops, green). The “bulge” corresponding to residues 64 to 71 is colored blue. This figure and subsequent figures were prepared using PyMol (www.pymol.org). (b) HypE dimer viewed along the twofold axis colored as described above. The active sites are indicated with arrows. (c) Superposition of the C-terminal domain of HypE (gray) with the N-terminal domain of HypF (cyan) shown in a cartoon representation. The parts of HypE that share the fold with HypF are colored in magenta. (d) Surface representation of the active site of HypE formed at the interface of monomers A (cyan) and B (salmon). The N-terminal helix (pink) of monomer A and the C-terminal loop (light green) of monomer B are situated proximal to each other and contribute toward the active site (shown by arrow). (e) Stereo view of the active site of HypE with modeled ADP positioned based on the superposition of HypE with PurL. One monomer is depicted in cartoon format while the second monomer is shown in surface representation. (f) Blowup of the putative carboxamide binding site situated near the C-terminal cysteine (Cys336).

FIG. 2.

Proposed reaction mechanism for HypE. The reaction intermediates that are formed starting from free cysteine (Cys336) are the thiocarbamate after reaction with carbamylated AMP in the presence of HypE before (I) and after (Ia) activation, thiocarbamic phosphoryl anhydride (or iminophosphate intermediate) (II), and thiocyanate (III)—the final product. Glu272 is proposed to act in the deprotonation of the amino nitrogen of the substrate (I), and Asp58 is proposed to act in the deprotonation of the imino nitrogen (II).

FIG. 3.

Characterization of HypE-HypF interactions. (a) SEC: elution profiles of HypE (dots) and HypF (dashes) and an equal mixture of HypE and HypF by mass (solid line). Calculated molecular weights averaged from at least three runs (Table 2) confirm a monomeric HypF, a dimeric HypE, and a 2:2 complex of HypE and HypF. The elution volumes of four molecular mass standards are indicated on the top x axis. (b) Quantitation of HypE and HypF from the major elution peak of the HypE-HypF complex (solid line, marked by an arrow) following SEC. Lanes 1 to 5 of the SDS gel are increasing amounts of the HypF (left panel) and HypE (right panel) standards, lane 6 is empty, and lanes 7 to 10 are different amounts of the HypE and HypF complex from the peak fraction. Estimates from two independent measurements gave an average HypE/HypF ratio of 1.17 ± 0.11. (c) Cross-linking by BS³ (top gel) and EDC (bottom gel) analyzed by SDS-PAGE: lanes 1 and 2 are cross-linked HypE and HypF, lane 3 is HypE and HypF, lane 4 is a broad-range molecular mass marker (from top, 250, 130, 100, 70, 55, 35, and 27 kDa), lanes 5 and 6 are cross-linked HypE, lane 7 is HypE alone, lanes 8 and 9 are cross-linked HypF, and lane 10 is HypF alone. Bands c1 to c4 correspond to cross-linked HypE and HypF complexes.

FIG. 4.

SPR analysis of HypF binding to GST-HypE. (a) Varied contact times (60, 150, 375, or 1,500 s) of 500 nM His-HypF over approximately 300 RU HypE-GST noncovalently captured over a 7,400-RU rabbit anti-GST antibody surface. The binding sensograms are normalized to the start of the dissociation phase to highlight the change of kinetic values with time. (b) Global fit of 2-min (light gray) and 10-min (dark gray) injections of HypF-His (2 μM to 62.5 nM) over 300 RU HypE-GST noncovalently captured using a 7,400-RU rabbit anti-GST antibody surface. The sensograms were blank subtracted, double referenced, and normalized to account for slight variations in HypE-GST capture. Evaluation of the curves was performed using a global fit of both the 2- and 10-min injections using a conformational change model in the BiaEvaluation software v3.2. The modeled curves are shown as the black lines and fit to the experimental data with χ² < 2 in this example. Three independent runs were performed and resulted in a K_d(eq) of 464 ± 94 nM.

FIG. 5.

Model for HypE-HypF interaction. The HypE monomer-dimer E₂ equilibrium is represented by reaction I. E₂ and F interact to form an unstable tetrameric intermediate [FEEF]* (reaction II). [FEEF]* rearranges to form a stable tetramer [EF]₂ (reaction III), which allows proximity of the putative active sites of E and F. Alternatively, the E dimer in [FEEF]* would dissociate to form EF (reaction IV). The protein concentration-dependent heterodimer [EF]-heterotetramer [EF]₂ equilibrium is represented by reaction V. “ap” refers to the acylphosphatase domain of HypF.