Structural investigations connect the disordered N-terminal extension of HypB to the activities of HypB and SlyD in E. coli.

Abstract

The activities of [NiFe]-hydrogenase enzymes, which are critical to many microbes, require insertion of a Ni(II) ion into the bimetallic catalytic center. Delivery of Ni(II) to [NiFe]-hydrogenases depends, in part, on the metallochaperone HypB, which lies at the center of a Ni(II) transfer pathway that includes the metal storage protein SlyD and the metallochaperone HypA. SlyD is a source of Ni(II) ions for HypB, whereas Ni(II) from HypB is transferred to HypA. In this work, we examine how the intrinsically disordered N-terminal extension (NTE) of HypB modulates the action of the HypB GTPase domain (G-domain). The NTE contains a high-affinity Ni(II) binding site, while the G-domain contains a lower affinity Ni(II) binding site that is affected by binding of guanine nucleotides. The HypB G-domain is also affected by SlyD and provides Ni(II) to HypA. Our NMR data show that, although disordered, the HypB NTE possesses residual structure and makes transient interactions with the HypB G-domain and with SlyD. A set of common residues in the center of the NTE are affected by SlyD and G-domain binding, and also by binding of Ni(II) to the high-affinity site located at the N terminus of the protein. The NTE interacts with residues in or near the Ni(II)- and GDP-binding sites in the G-domain, which are also affected when SlyD binds the NTE. Thus, the data showcase a complex interaction network between HypB and SlyD, and provide molecular details regarding how the HypB NTE links the activities of the HypB G-domain and SlyD.