The Extended C-Terminal α-Helix of the HypC Chaperone Restricts Recognition of Large Subunit Precursors by the Hyp-Scaffold Machinery during [NiFe]-Hydrogenase Maturation in Escherichia coli

Abstract

Members of the HypC protein family are chaperone-like proteins that play a central role in the maturation of [NiFe]-hydrogenases (Hyd). Escherichia coli has a second copy of HypC, called HybG, and, as a component of the HypDEF maturation scaffold, these proteins help synthesize the NiFe-cofactor and guide the scaffold to its designated hydrogenase large subunit precursor. HypC is required to synthesize active Hyd-1 and Hyd-3, while HybG facilitates Hyd-2 and Hyd-1 synthesis. To identify determinants on HypC that allow it to discriminate against Hyd-2, we made amino acid exchanges in 3 variable regions, termed VR1, VR2, and VR3, of HypC, that make it more similar to HybG. Region VR3 includes a HypC-specific C-terminal α-helical extension, and this proved particularly important in preventing the maturation of Hyd-2 by HypC. Truncation of this extension on HypC increased Hyd-2 activity in the absence of HybG, while retaining maturation of Hyd-3 and Hyd-1. Combining this truncation with amino acid exchanges in VR1 and VR2 of HypC negatively affected the synthesis of active Hyd-1. The C-terminus of E. coli HypC is thus a key determinant in hindering Hyd-2 maturation, while VR1 and VR2 appear more important for Hyd-1 matu­ration.

Exchange of a Single Amino Acid Residue in the HybG Chaperone Allows Maturation of All H2-Activating [NiFe]-Hydrogenases in Escherichia coli

“…It is important to note that in all experiments described in the current study, HypC and HybG both carried a C -terminal Strep-tag II. The presence of this StrepII-tag does not interfere with the functionality of either protein with respect to Hyd precursor maturation ( Blokesch et al, 2004 ; Soboh et al, 2013 ; Thomas et al, 2018 ). Consequently, in the interest of convenience, we will henceforth generally refer to these proteins without mentioning the tag.…”

“…Due to the lack of structural information on the HybG-pre-HybC or HypC-pre-HycE complexes, we must rely on the knowledge gained from in vivo studies performed with E. coli ( Hartwig et al, 2015 ; Thomas et al, 2018 ), and from structural analyses of HypC and HypD from Thermococcus kodakarensis ( Watanabe et al, 2007 , 2012 ), to gain insight into potentially key residues or motifs involved in distinguishing client proteins.…”

[NiFe] Hydrogenase Accessory Proteins HypB–HypC Accelerate Proton Conversion to Enhance the Acid Resistance and d-Lactic Acid Production of Escherichia coli

“…Mature [NiFe] hydrogenase can catalyze the reversible reaction of hydrogen from hydrogen to protons and electrons, 2H + + 2e – ⇌ H 2 . − Even some soluble hydrogenases can reversibly catalyze the electrons in NADH to produce hydrogen molecules from two protons by modulating the redox potential. , This mechanism offers the possibility to reverse the accumulated protons in a timely manner, thus sharing the stress of intracellular depletion or the exocytosis of protons and allowing the cell to maintain metabolite activity under acidic stress. The maturation process of this kind of hydrogenase Hyd-3 is mainly involved with six Hyp proteins (HypA, HypB, HypC, HypD, HypE, and HypF) and SlyD (Figure A).…”