Structural basis of a Ni acquisition cycle for [NiFe] hydrogenase by Ni-metallochaperone HypA and its enhancer

Significance The metal ions in proteins are correctly incorporated by specific metallochaperones. However, it remains unclear how metallochaperones regulate their metal binding affinity during acquisition of correct metal ions and deliver them to target proteins. In this study, we have determined the crystal structures of a transient complex between a Ni metallochaperone HypA and its partner ATPase protein HypB AT, which incorporate a Ni ion into [NiFe] hydrogenase. The structures reveal that HypB AT induces conformational change of HypA through complex formation, leading to formation of a Ni binding site. Consequently, the Ni-binding affinity of HypA is increased from micromolar to nanomolar range (by ∼600-fold). These results indicate that HypB AT functions as a metallochaperone enhancer, which regulates metal binding affinity of metallochaperones. The Ni atom at the catalytic center of [NiFe] hydrogenases is incorporated by a Ni-metallochaperone, HypA, and a GTPase/ATPase, HypB. We report the crystal structures of the transient complex formed between HypA and ATPase-type HypB (HypB AT) with Ni ions. Transient association between HypA and HypB AT is controlled by the ATP hydrolysis cycle of HypB AT, which is accelerated by HypA. Only the ATP-bound form of HypB AT can interact with HypA and induces drastic conformational changes of HypA. Consequently, upon complex formation, a conserved His residue of HypA comes close to the N-terminal conserved motif of HypA and forms a Ni-binding site, to which a Ni ion is bound with a nearly square-planar geometry. The Ni binding site in the HypAB AT complex has a nanomolar affinity ( K d = 7 nM), which is in contrast to the micromolar affinity ( K d = 4 µM) observed with the isolated HypA. The ATP hydrolysis and Ni binding cause conformational changes of HypB AT, affecting its association with HypA. These findings indicate that HypA and HypB AT constitute an ATP-dependent Ni acquisition cycle for [NiFe]-hydrogenase maturation, wherein HypB AT functions as a metallochaperone enhancer and considerably increases the Ni-binding affinity of HypA.