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42724-AC3
Analysis of SlyD, A New Component of Hydrogenase Metallocenter Assembly in E. Coli
Several years ago we identified a new component of nickel homeostasis in Escherichia coli, a protein called SlyD, and demonstrated that it has a role in the biosynthetic pathway of [NiFe]-hydrogenase enzymes. The objectives of this grant are to characterize the properties of SlyD and to examine its role in the hydrogenase metallocenter assembly process. The initial proposal was broken down into three sets of goals, and progress has been made in all three areas.
Characterization of the metal-binding activity of SlyD.
SlyD is a 196-residue protein with an unusual metal-binding sequence in the C-terminal domain, with 15 histidines, 6 cysteines, and 7 aspartate/glutamates in the final 49 residues of the protein. Although it is known that this sequence can bind metal ions, the molecular details of this interaction are unknown. Experiments using several methods revealed that SlyD binds up to five-six nickel ions with a range of affinities. At least one of these ions is bound very tightly, with a picomolar affinity, and three are bound very weakly with high micromolar affinities. Analysis of SlyD1-146 revealed that two of the weakly bound ions are bound to this protein, demonstrating that not all of the metal sites are in the C-terminal domain. To define the coordination of the metal-binding sites we are using x-ray absorption spectroscopy (XAS) in collaboration with Prof. Ingrid Pickering and Prof. Graham George at the University of Saskatchewan. Biochemical analysis, in combination with the XAS studies, is being used to examine the nickel complexes of SlyD and site-directed mutants. The first set of mutations involves changing pairs of cysteines to alanines, and our initial experiments suggest that one pair of cysteines is involved in the tight metal-binding site.
Determination of which properties of SlyD are required for hydrogenase biosynthesis and whether metal binding regulates the other activities.
In addition to metal binding, SlyD is also a peptidyl-prolyl isomerase (PPIase) and folding chaperone, and our experiments demonstrated that these activities are independent of each other. To define what SlyD is doing during hydrogenase biosynthesis, mutations or truncations were designed to disrupt these activities and then the purified proteins were tested in vitro for the appropriate activity. In parallel, the same mutants were introduced into a ?slyD strain of E. coli on an inducible vector to determine the effect on hydrogenase biosynthesis. The SlyD1-146 is not active in vivo, demonstrating that the metal-binding domain is essential. Similarly, the mutant ?107-111, which disrupts the chaperone activity, is also inactive in vivo. In contrast, mutants deficient in the PPIase activity are at least partially functional in vivo.
Evaluation of the metallochaperone activity of SlyD.
SlyD forms a complex with HypB, an essential accessory protein for hydrogenase biosynthesis that binds several nickel ions. We have mapped the HypB binding site to residues 107-111 of SlyD, which may explain why this mutant is not functional in vivo. In an unexpected result, we discovered that SlyD also activates metal release from HypB. Metal activation by SlyD requires direct complex formation with HypB as well as the metal-binding domain of SlyD but not the PPIase activity. Preliminary experiments suggest that nickel is transferred from HypB to SlyD, but more experiments are needed to examine this process and to determine it occurs even in the presence of competitors. Experiments are also underway to examine the function of the SlyD-HypB complex in vivo, and to determine if SlyD has a role on nickel storage as originally proposed.
Impact.
An examination of SlyD enhances our understanding of prokaryotic nickel homeostasis and hydrogenase biosynthesis. In addition to potential medical or biotechnology applications, SlyD is a very interesting protein with respect to its biological and bioinorganic chemistry as well as its physiological function. It has provided challenges for the people working on this project, including graduate students Alistair Dias and Harini Kaluarachchi, research technician Kathleen Zhang, and postdoctoral fellow Michael Leach. These trainees are learning a variety of methods and applying an interdisciplinary approach as they examine SlyD and its properties. For example, this grant has provided the opportunity for Alistair and Harini to learn how to perform XAS experiments with our collaborators and to bring that expertise back to this lab. Furthermore, research on this protein has lead to several new projects. For example, the discovery that SlyD activates metal release from HypB reveals that SlyD is not just a metal storage and delivery protein, as we had originally thought. The fact that SlyD has an active role in this pathway indicates that more information is needed on the complex between the two proteins and how SlyD influences HypB structure and metal binding, studies that will be part of a recently funded grant proposal from a Canadian federal agency.
