Structure of [NiFe] Hydrogenase Maturation Protein HypE from Escherichia coli and Its Interaction with HypF

Abstract

Hydrogenases are enzymes involved in hydrogen metabolism, utilizing H 2 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-Å 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 K d (dissociation constant) of ϳ400 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.

Structural Basis for the Reaction Mechanism of S-Carbamoylation of HypE by HypF in the Maturation of [NiFe]-Hydrogenases

“…5A). The two HypE molecules form a homodimer via the N-terminal domain as in the previously reported crystal structures of HypE from other organisms (12,19,20). In contrast to the HypE dimers of D. vulgaris and E. coli, however, the swapping of the N-terminal segment between two HypE subunits was not found in the C. subterraneus ortholog, whereas the overall structures of the dimers are conserved among organisms regardless of the swapping (Fig.…”

“…Molecular Interaction in the HypE-HypF Complex-The complex formation of HypE and HypF were confirmed by size exclusion chromatography, where the complex was eluted as a dimer of heterodimers as reported for the E. coli orthologs of HypE and HypF (19). The structure of the HypE-HypF complex revealed that the two HypF molecules associated with the opposite sides of the HypE homodimer, resulting in an elongated right-handed helical shape of the complex with a length of ϳ180 Å and width of ϳ75 Å (Fig.…”

“…While crystal structures of HypF from Escherichia coli have been reported for the N-terminal ACP domain (residues 1-91) (18) and for the rest of the C-terminal region (residues 92-750) (17), the intact structure of HypF is not yet available. On the other hand, the crystal structures of the full-length HypE from three different organisms have already been reported (12,19,20).…”

Crystal structures of the carbamoylated and cyanated forms of HypE for [NiFe] hydrogenase maturation

“…The aspartate residue in the DX 4 GAXP motif is involved in holding Mg 2+ ions and nucleotides. The crystal structures of HypE have been reported from Thermococcus kodakarensis (9), Desulfovibrio vulgaris (21), Escherichia coli (22), and Caldanaerobacter subterraneus (23). The structures of all of the other Hyp proteins have also been reported (9,(24)(25)(26)(27)(28)(29)(30)(31)(32).…”

Preparation and Characterization of Bacterial Protein Complexes for Structural Analysis

“…This thiocarbamate then undergoes dehydration to a cyano moiety in an ATP-dependent reaction catalyzed by HypE (Blokesch et al, 2004b). Previously, we have determined the crystal structure of E. coli HypE and characterized its interaction with HypF using several approaches (Rangarajan et al, 2008). The affinity of the two proteins for one another was measured using both SPR and ITC, giving a K d of approximately 400 nM.…”