Andrew M. Sydor

Super-Resolution Microscopy: From Single Molecules to Supramolecular Assemblies

Super-resolution microscopy (SRM) methods have allowed scientists to exceed the diffraction limit of light, enabling the discovery and investigation of cellular structures at the nanometer scale, from individual proteins to entire organelles. In this review we survey the application of SRM in elucidating the structure of macromolecules in the native cellular environment. We emphasize how SRM can generate molecular maps of protein complexes and extract quantitative information on the number, size, distribution, and spatial organization of macromolecules. We discuss both the novel information that can be generated through SRM as well as the experimental considerations to examine while conducting such studies. With the increasing popularity of SRM in the biological sciences, this review will serve as a tool to navigate the range of applications and harness the power of SRM to elucidate biological structures.

Effects of metal on the biochemical properties of Helicobacter pylori HypB, a maturation factor of [NiFe]-hydrogenase and urease.

The biosyntheses of the [NiFe]-hydrogenase and urease enzymes in Helicobacter pylori require several accessory proteins for proper construction of the nickel-containing metallocenters. The hydrogenase accessory proteins HypA and HypB, a GTPase, have been implicated in the nickel delivery steps of both enzymes. In this study, the metal-binding properties of H. pylori HypB were characterized, and the effects of metal binding on the biochemical behavior of the protein were examined. The protein can bind stoichiometric amounts of Zn(II) or Ni(II), each with nanomolar affinity. Mutation of Cys106 and His107, which are located between two major GTPase motifs, results in undetectable Ni(II) binding, and the Zn(II) affinity is weakened by 2 orders of magnitude. These two residues are also required for the metal-dependent dimerization observed in the presence of Ni(II) but not Zn(II). The addition of metals to the protein has distinct impacts on GTPase activity, with zinc significantly reducing GTP hydrolysis to below detectable levels and nickel only slightly altering the k(cat) and K(m) of the reaction. The regulation of HypB activities by metal binding may contribute to the maturation of the nickel-containing enzymes.

Relationship between Ni(II) and Zn(II) Coordination and Nucleotide Binding by the Helicobacter pylori [NiFe]-Hydrogenase and Urease Maturation Factor HypB

Background: Helicobacter pylori HypB (HpHypB) is a metal-regulated GTPase essential for the biosynthesis of [NiFe]-hydrogenase and urease. Results: Nickel binding to HpHypB is altered upon nucleotide binding, an effect not observed with zinc. Conclusion: Metal coordination and the GTPase cycle of HpHypB are intimately linked. Significance: These data suggest that HpHypB contributes to metal fidelity in [NiFe]-hydrogenase and urease biosynthesis. The pathogen Helicobacter pylori requires two nickel-containing enzymes, urease and [NiFe]-hydrogenase, for efficient colonization of the human gastric mucosa. These enzymes possess complex metallocenters that are assembled by teams of proteins in multistep pathways. One essential accessory protein is the GTPase HypB, which is required for Ni(II) delivery to [NiFe]-hydrogenase and participates in urease maturation. Ni(II) or Zn(II) binding to a site embedded in the GTPase domain of HypB modulates the enzymatic activity, suggesting a mechanism of regulation. In this study, biochemical and structural analyses of H. pylori HypB (HpHypB) revealed an intricate link between nucleotide and metal binding. HpHypB nickel coordination, stoichiometry, and affinity were modulated by GTP and GDP, an effect not observed for zinc, and biochemical evidence suggests that His-107 coordination to nickel toggles on and off in a nucleotide-dependent manner. These results are consistent with the crystal structure of HpHypB loaded with Ni(II), GDP, and Pi, which reveals a nickel site distinct from that of zinc-loaded Methanocaldococcus jannaschii HypB as well as subtle changes to the protein structure. Furthermore, Cys-142, a metal ligand from the Switch II GTPase motif, was identified as a key component of the signal transduction between metal binding and the enzymatic activity. Finally, potassium accelerated the enzymatic activity of HpHypB but had no effect on the other biochemical properties of the protein. Altogether, this molecular level information about HpHypB provides insight into its cellular function and illuminates a possible mechanism of metal ion discrimination.

Nickel Metallomics: General Themes Guiding Nickel Homeostasis

The nickel metallome describes the distribution and speciation of nickel within the cells of organisms that utilize this element. This distribution is a consequence of nickel homeostasis, which includes import, storage, and export of nickel, incorporation into metalloenzymes, and the modulation of these and associated cellular systems through nickel-regulated transcription. In this chapter, we review the current knowledge of the most common nickel proteins in prokaryotic organisms with a focus on their coordination environments. Several underlying themes emerge upon review of these nickel systems, which illustrate the common principles applied by nature to shape the nickel metallome of the cell.

The Response of Escherichia coli NikR to Nickel: A Second Nickel-Binding Site

The Escherichia coli transcription factor NikR mediates two levels of regulatory control of Ni(II) uptake in response to changes in the levels of available nickel. Despite the evidence that metal binding to two distinct sites on NikR, referred to as the high- and low-affinity Ni(II) sites, is required for Ni(II)-selective DNA binding by the protein, the role of the latter set of Ni(II) ions in the activation of NikR remains controversial, and the position of the putative low-affinity Ni(II)-binding site(s) on NikR has not been determined. In this study we confirm that NikR has a high-affinity Ni(II)-binding site that is maintained upon DNA binding. The ligands of the low-affinity Ni(II)-binding site were examined by using selective chemical modification and mass spectrometry performed in the presence of excess Ni(II) and DNA. We localized this Ni(II) site to a region at the interface between the metal- and DNA-binding domains and identified His48 and His110 as residues that participate in the low-affinity Ni(II)-binding response. Mutation of His48 and His110 to asparagines reduces significantly both NikRs tendency to precipitate in the presence of excess Ni(II) and the affinity of the DNA-bound complex in the presence of excess Ni(II). A complete scheme involving all of the metal-binding sites that contribute to the regulatory function of E. coli NikR in nickel homeostasis is described.

PPP1R35 is a novel centrosomal protein that regulates centriole length in concert with the microcephaly protein RTTN

Centrosome structure, function, and number are finely regulated at the cellular level to ensure normal mammalian development. Here, we characterize PPP1R35 as a novel bona fide centrosomal protein and demonstrate that it is critical for centriole elongation. Using quantitative super-resolution microscopy mapping and live-cell imaging we show that PPP1R35 is a resident centrosomal protein located in the proximal lumen above the cartwheel, a region of the centriole that has eluded detailed characterization. Loss of PPP1R35 function results in decreased centrosome number and shortened centrioles that lack centriolar distal and microtubule wall associated proteins required for centriole elongation. We further demonstrate that PPP1R35 acts downstream of, and forms a complex with, RTTN, a microcephaly protein required for distal centriole elongation. Altogether, our study identifies a novel step in the centriole elongation pathway centered on PPP1R35 and elucidates downstream partners of the microcephaly protein RTTN.