Photon Factory Activity Report 2018 #36 (2019)

BL-1A/2016G018

Structure Biology of [NiFe] Hydrogenase Maturation Proteins
Sunghark KWON1, Satoshi WATANABE1,2, Yuichi NISHITANI1, Takumi KAWASHIMA1
and Kunio MIKI1,*1
Department of Chemistry, Graduate School of Science, Kyoto University,
Sakyo-ku, Kyoto, 606-8502, Japan
2
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University,
Katahira 2-1-1, Aoba-ku, Sendai, 980-8577, Japan
1 Introduction[NiFe] Hydrogenases catalyse the oxidation of hydrogenmolecules (H2) and the reduction of hydrogen ions (H+).
They consist of two (large and small) subunits, and thelarge subunit contains a Ni-Fe cluster at the active site. Theformation of the metal cluster does not occur
spontaneously and requires a maturation process involving
specific accessory proteins such as Hyp proteins (HypA,HypB, HypC, HypD, HypE, and HypF). After the metal
insertion, a maturation endopeptidase is involved in thecleavage of the C-terminal residues of the large subunit. It
is also known that a Ni ion is necessary for the substrate
recognition by endopeptidase.
A Ni chaperone HypA is responsible, together withHypB, for Ni insertion into the active site. The structuresof HypA and the HypA-HypB complex have been
determined, but details of the interaction mechanism
between an immature large subunit (HyhL) and HypA
remain obscure. Structures of HyhL in complex with Ni
delivery proteins are indispensable for understanding how
HypA interacts with HyhL for the Ni insertion and how it
affects HyhL in the maturation process. We havedetermined crystal structures of HypA in complex withHyhL from a hyperthermophilic archaeon, Thermococcus
kodakarensis [1].

between Cys65 of HyhL and His2 of HypA is 12.2 Å,
which is likely enough to deliver Ni into the active site withadequate movement. Comparison of the structures ofimmature HyhL provides structural insights into theconformational changes after the metal insertion steps.
Remarkably, the C-terminal extension of immature HyhL,
which is cleaved in the mature form, adopts a b-strand
adjacent to its N-terminal b-strands. The position of the Cterminal extension corresponds to that of the N-terminal ofthe mature large subunit, preventing the access ofendopepdidase to the cleavage site of HyhL. Thesefindings suggest that the Ni insertion into the active siteinduces spatial rearrangement of both N- and C-terminalsof HyhL, which is an important step for incorporation ofthe Ni-Fe cluster in the maturation process of [NiFe]
Hydrogenases.

2 Experimental
The HyhL–HypA complex was prepared by mixing
HyhL and HypA at a 1:1.4 molar ratio, and the mixture waspurified using SEC. Crystals of the HyhL–HypA complex
were obtained by using PEG 3350 as a precipitant in two
forms with space groups C222 and P23 at 20 °C and 4 °C,respectively. The structure was solved by the molecularreplacement method and refined at 3.30 Å and 3.24 Å
resolution for orthorhombic and cubic crystals.
3 Results and Discussion
Figure 1 shows the crystal structure of the HyhL-HypA
complex, which revealed two major binding modes ofHyhL and HypA: hydrophobic interactions and b-sheet
formation. It was also observed that the N-terminal tail ofHyhL interacts with the C-terminal b-strand of HypA,
forming an extended b-sheet with the three b-strands of
HypA. These interactions bring the metal binding sites of
HypA and HyhL close to each other and the distanceFig. 1: Structure of the immature large subunit, HyhL andHypA from Thermococcus kodakarensis
Reference[1] S. Kwon, S. Watanabe, Y. Nishitani, T. Kawashima, T.
Kanai, H. Atomi, and K. Miki, Proc. Natl. Acad. Sci.
USA, 115, 7045-7050 (2018).
* miki@kuchem.kyoto-u.ac.jp