Protein interactions and localization of the Escherichia coli accessory protein HypA during nickel insertion to [NiFe] hydrogenase

Abstract

Nickel delivery during maturation of Escherichia coli [NiFe] hydrogenase 3 includes the accessory proteins HypA, HypB, and SlyD. Although the isolated proteins have been characterized, little is known about how they interact with each other and the hydrogenase 3 large subunit, HycE. In this study the complexes of HypA and HycE were investigated after modification with the Strep-tag II. Multiprotein complexes containing HypA, HypB, SlyD, and HycE were observed, consistent with the assembly of a single nickel insertion cluster. An interaction between HypA and HycE did not require the other nickel insertion proteins, but HypB was not found with the large subunit in the absence of HypA. The HypA-HycE complex was not detected in the absence of the HypC or HypD proteins, involved in the preceding iron insertion step, and this interaction is enhanced by nickel brought into the cell by the NikABCDE membrane transporter. Furthermore, without the hydrogenase 1, 2, and 3 large subunits, complexes between HypA, HypB, and SlyD were observed. These results support the hypothesis that HypA acts as a scaffold for assembly of the nickel insertion proteins with the hydrogenase precursor protein after delivery of the iron center. At different stages of the hydrogenase maturation process, HypA was observed at or near the cell membrane by using fluorescence confocal microscopy, as was HycE, suggesting membrane localization of the nickel insertion event.

FIGURE 1.

HypAStr activity and protein complex formation. A, crude cell lysates from wild-type MC4100 cells or DPABF (hypA ATG → TAA, ΔhybF) cells grown with or without the pBAD plasmid containing the hypA or hypAStr gene were prepared and tested for hydrogenase activity using benzyl viologen as the electron acceptor in an anaerobic solution assay containing 4% hydrogen gas. The results represent the average of three independent experiments, and error bars indicate ± 1 S.D. B, shown is a Western blot of the pulldown of HypAStr from DPABF using a Strep-tactin-Sepharose column. The expected molecular masses of the hydrogenase proteins are: HypAStr (14.3 kDa), HypB (31.6 kDa), SlyD (20.8 kDa), and hydrogenase isoform 3 large subunit HycE (65 kDa). The HypB protein is sensitive to proteolytic degradation, resulting in an additional 21-kDa fragment. SlyD typically migrates slower than expected on SDS-PAGE. Samples were reduced with β-mercaptoethanol and boiled before resolution on a 12.5% SDS-polyacrylamide gel followed by Western blotting with the indicated polyclonal antibodies.

FIGURE 2.

HycEStr or HypAStr protein complexes. Cells were exposed to a cell permeable cross-linker before lysis, and pulldown assays were performed by using a Strep-tactin-Sepharose column. Samples were reduced with β-mercaptoethanol and boiled before resolution on 10% (HycEStr pulldown assays) or 12.5% (HypAStr pulldown assays) SDS-polyacrylamide gels followed by Western blotting with the indicated antibodies. A, HD705 producing HycEStr is shown. B, DPABF producing HypAStr is shown.

FIGURE 3.

HypA protein complexes formed in the absence of HypB, SlyD, or hydrogenase 1–3 large subunits. Cells were exposed to a cell permeable cross-linker before lysis, and pulldown assays were performed by using a Strep-tactin-Sepharose column. Samples were reduced with β-mercaptoethanol and boiled before resolution on 10% (HycEStr pulldown assays) or 12.5% (HypAStr pulldown assays) SDS-polyacrylamide gels followed by Western blotting with the indicated antibodies. A, MC4100, DPABF, DHP-B, and ΔslyD strains producing HycEStr are shown. B, ΔhyaB ΔhybC ΔhycE, DHP-B, and ΔslyD strains producing HypAStr are shown. C, ΔhyaB ΔhybC ΔhycE expressing HypAStr is shown.

FIGURE 4.

The interaction between HypA and HycE occurs after iron insertion. Wild-type MC4100 cells or DHP-C (ΔhypC) cells producing HycEStr were exposed to a cell permeable cross-linker before lysis, and pulldown assays were performed by using a Strep-tactin-Sepharose column. Samples were reduced with β-mercaptoethanol and boiled before resolution on 10% SDS-polyacrylamide gels followed by Western blotting with the indicated antibodies.

FIGURE 5.

An intact NikABCDE transporter enhances the formation of a HypA-HycE complex. HycEStr was produced in MC4100 cells or HYD723 with or without supplementation of the growth media with 0.5 mm nickel. A, cell extracts were prepared and tested for hydrogenase activity using benzyl viologen as the electron acceptor in an anaerobic solution assay containing 4% hydrogen gas. The results represent the average of three independent experiments, and error bars indicate ± 1 S.D. Shown is an anti-StrepMAB Western blot with processed and unprocessed HycEStr (B) and anti-HypA Western (C) blot. For B and C, cells were exposed to a cell permeable cross-linker before lysis, and pulldown assays were performed by using a Strep-tactin-Sepharose column. Samples were reduced with β-mercaptoethanol and boiled 10% SDS-polyacrylamide gels before resolution on 10%SDS-polyacrylamide gel.

FIGURE 6.

Co-eluting proteins with HypAStr, HypBStr, or HycEStr. The elution fractions from Strep-tactin-Sepharose pulldown assays from lysates of DPABF-producing HypAStr (A), DHP-B-producing HypBStr (B), and HD705-producing HycEStr (C) from non-cross-linked cells were resolved on 12.5% (A and B) and 10% SDS-polyacrylamide gels followed by staining with Coomassie Blue dye (C). Selected protein bands were cut from the gels and extracted by digestion with trypsin followed by LC-MS/MS for identification. Proteins identified by LC-MS/MS (supplemental Table S1) are labeled, and the asterisk indicates the bait protein used for each experiment.

FIGURE 7.

HycEStr and HypAStr are localized near the cell membrane. Shown are confocal fluorescence microscopy images of HD705 (A) and HYD723 (B) producing HycEStr, DPABF (C), HYD723 (D), and ΔhyaB ΔhybC ΔhycE (E) producing HypAStr, SlyD from MC4100 cells (F), and BL21(DE3) (G) producing YjiXStr. Cells were stained with BODIPY® lipid probe 558/568 (left panels, red) to visualize cell membranes and either Alexa Fluor® 633 streptavidin conjugate to visualize Strep-tag II HycE, HypA, and YjiX proteins or Alexa Fluor® goat anti-mouse secondary antibody to probe SlyD monoclonal antibodies (middle panels, green). The overlay of both fluorescent images is shown on the right. See also supplemental Fig. S8 for fluorescence profiles. Bars indicate 1 μm.