biostudies-literatureKwon SMinistry of Education, Culture, Sports, Science and Technology7045-7050https://www.ebi.ac.uk/biostudies/studies/S-EPMC6142260biostudies-literatureUnknown115(27)Ni-Fe clusters are inserted into the large subunit of [NiFe] hydrogenases by maturation proteins such as the Ni chaperone HypA via an unknown mechanism. We determined crystal structures of an immature large subunit HyhL complexed with HypA from <i>Thermococcus kodakarensis</i> Structure analysis revealed that the N-terminal region of HyhL extends outwards and interacts with the Ni-binding domain of HypA. Intriguingly, the C-terminal extension of immature HyhL, which is cleaved in the mature form, adopts a β-strand adjacent to its N-terminal β-strands. The position of the C-terminal extension corresponds to that of the N-terminal extension of a mature large subunit, preventing the access of endopeptidases to the cleavage site of HyhL. These findings suggest that Ni insertion into the active site induces spatial rearrangement of both the N- and C-terminal tails of HyhL, which function as a key checkpoint for the completion of the Ni-Fe cluster assembly.Proceedings of the National Academy of Sciences of the United States of AmericaCrystal structures of a [NiFe] hydrogenase large subunit HyhL in an immature state in complex with a Ni chaperone HypA.PMC614226017H0364226291012Kanai TKwon SWatanabe SAtomi HKawashima TNishitani YMiki KfalseCrystal structures of a [NiFe] hydrogenase large subunit HyhL in an immature state in complex with a Ni chaperone HypA.Ni-Fe clusters are inserted into the large subunit of [NiFe] hydrogenases by maturation proteins such as the Ni chaperone HypA via an unknown mechanism. We determined crystal structures of an immature large subunit HyhL complexed with HypA from <i>Thermococcus kodakarensis</i> Structure analysis revealed that the N-terminal region of HyhL extends outwards and interacts with the Ni-binding domain of HypA. Intriguingly, the C-terminal extension of immature HyhL, which is cleaved in the mature form, adopts a β-strand adjacent to its N-terminal β-strands. The position of the C-terminal extension corresponds to that of the N-terminal extension of a mature large subunit, preventing the access of endopeptidases to the cleavage site of HyhL. These findings suggest that Ni insertion into the active site induces spatial rearrangement of both the N- and C-terminal tails of HyhL, which function as a key checkpoint for the completion of the Ni-Fe cluster assembly.2018-01-01T00:00:00Z2018 Jul2024-11-21T10:09:32.661Z2019-03-26T22:33:25ZS-EPMC61422602991504610.1073/pnas.1801955115