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

Abstract

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.X-ray crystallography | metalloprotein | transient complex | metallochaperone A pproximately one-half of all cellular proteins require specific metal ions for proper function, which are delivered by specific metallochaperones (1, 2). However, the mechanisms of correct acquisition and delivery to target proteins of many metallochaperones remain poorly understood.[NiFe] hydrogenases harbor a complex metal cofactor, NiFe(CN) 2 CO, in their active sites (3). This cofactor catalyzes reversible H 2 production. The Ni atom in the NiFe(CN) 2 CO cofactor is bound to four thiolate groups, two of which also bridge the Fe(CN) 2 CO group (4, 5). NiFe(CN) 2 CO biosynthesis requires specific maturation machinery, in which six Hyp proteins (HypA-HypF) play key roles (6, 7). Four Hyp proteins (HypC-HypF) are involved in the biosynthesis and incorporation of the Fe(CN) 2 CO group (8-15). After Fe insertion, HypA and HypB insert the Ni ion into the hydrogenase large subunit (16).HypA is a Ni-metallochaperone that binds to a Ni ion with micromolar affinity (17-19), and its structure consists of a Ni-binding domain (NiBD) and a Zn-binding domain (ZnBD) (20, 21). The NiBD contains a highly conserved MHE motif that is essential for Ni binding at the N terminus (20,22). HypB consists of a common GTPase domain and a less conserved metal-binding region (23)(24)(25). Recently, ATPase-type HypB (HypB AT , previously abbreviated as mmHypB) proteins were identified from Thermococcales (26). GTPase and ATPase types of HypB belong to the SIMIBI class NTPase family and share a similar architecture, despite their low sequence similarity (27). HypA and HypB form a transient complex in the Ni insertion process (17,22,28,29). In the Escherichia coli system, Ni transfer occurs from HypB to HypA (30). However, the functi...

Heterologous complementation studies in Escherichia coli with the Hyp accessory protein machinery from Chloroflexi provide insight into [NiFe]-hydrogenase large subunit recognition by the HypC protein family

“…HypB is involved in the latter stages of [NiFe]-hydrogenase maturation where it functions together with HypA and the peptidyl-prolyl cis/trans isomerase SlyD to deliver the nickel ion to the active site (Maier et al, 1993;Waugh & Boxer, 1986;Zhang et al, 2005). A recent structural study indicates that HypA proteins likely deliver nickel directly to the hydrogenase large subunit, as exemplified for T. kodakarensis (Watanabe et al, 2015); however, Functional conservation of the Hyp machinery T. kodakarensis lacks a classical HypB protein and instead has a functional homologue (Sasaki et al, 2013) that has an 'enhancer'-like role in facilitating delivery of nickel to the active site by the HypA protein. The data presented here nevertheless suggest that the HypB protein might recognize specific amino acid residues on the apo-large subunits of hydrogenases during maturation, and that D. mccartyi CBDB1 HypB lacks the ability to interact effectively with HyaB, the large subunit of Hyd-1.…”

“…Functional conservation of the Hyp machinery that the limitation in Hyd-1 activity was indeed the HypA metallochaperone-enhancer function provided by HypB (Watanabe et al, 2015). While BEF314 alone failed to synthesize active Hyd-1, even in the presence of 100 mM NiCl 2 , the recovery of Hyd-1 after transformation with pSHH23 confirmed that sufficient levels of functional HypC, D, E and HypF proteins from D. mccartyi were synthesized in the heterologous host to deliver active Hyd-1 (Fig.…”

Nickel Ligation of the N-Terminal Amine of HypA Is Required for Urease Maturation in Helicobacter pylori

“…21 The Ni-sites of the HypA-HypB AT complex show four-coordinate sites with the ligation of the N-terminal amine from Met1, backbone amide and side chain imidazole of His2, and the imidazole side chain from His98. Although this conformation of the Ni-site encompasses both the N-terminal ligation and the four-coordinate planar site, it is unlikely to exist in H. pylori HypA.…”

High-affinity metal binding by the Escherichia coli [NiFe]-hydrogenase accessory protein HypB is selectively modulated by SlyD

“…These results add to the growing body of work that demonstrates that the metal selectivity of metallochaperone activity is not dictated by specific metal binding, but instead is achieved through allosteric coupling, often via partner protein complexes, as observed for example in several studies of the HypA-HypB complex. 20–22 …”

“…19 One possible strategy, as outlined in several recent reports, is that partner protein interactions modulate selective metal binding and transfer. 20–22 …”