The studies described in this proposal focus on the mechanism of activation of bacterial urease, a bacterial virulence factor. The urease apoprotein (UreABC) serves as the scaffold for creation of a novel lysine carbamate-bridged, dinuclear nickel active site metallocenter. Our working model of the assembly process requires the action of a GTP-dependent molecular chaperone (made up of UreD, UreF, and UreG accessory proteins) along with the participation of a metallochaperone (UreE) that delivers Ni. We seek to understand the structure and function of each urease accessory protein and to elucidate the mechanism by which these proteins participate in this unique GTP-dependent process of metallocenter assembly. Our objectives are to test specific hypotheses regarding the roles of the urease accessory proteins. Thus, we will (A) Carry out structural analyses of the individual UreD, UreF, and UreG components along with the UreDFG heterotrimer. (B) Unravel the sequential binding kinetics associated with formation of the UreD-UreABC, UreDF-UreABC, and UreDFG-UreABC species. (C) Establish the overall conformation of these complexes by small angle X-ray scattering methods. (D) Identify the specific sites of protein:protein interaction by developing an innovative """"""""protein footprinting"""""""" technology that should be widely applicable to other systems. (E) Explore the timing and stability of lysine carbamylation within these complexes by comparing the incorporation of radiolabeled CO2 with and without trapping by diazomethane. (F) Seek to stabilize the putative UreE:UreG interaction by use of mutants and exchange-inert metal ions. (G) Test for the creation of a high affinity Ni-binding site at the UreE:UreG interface by using isothermal titration calorimetry and other metal-binding approaches. (H) Examine the effect of MgGTP and its analogues on conformational changes associated with metal transfer into UreABC. The general processes involved in urease metallocenter biosynthesis are likely to apply to numerous other systems, including the activation of many metalloenzymes of medical interest. Significantly, our focus on understanding urease activation will uncover common principles that can be applied to these less tractable systems. In addition, we will expand our knowledge regarding the biochemistry of nickel. ? ? ?
| Martin-Diaconescu, Vlad; Joseph, Crisjoe A; Boer, Jodi L et al. (2017) Non-thiolate ligation of nickel by nucleotide-free UreG of Klebsiella aerogenes. J Biol Inorg Chem 22:497-503 |
| Macomber, Lee; Minkara, Mona S; Hausinger, Robert P et al. (2015) Reduction of urease activity by interaction with the flap covering the active site. J Chem Inf Model 55:354-61 |
| Farrugia, Mark A; Wang, Beibei; Feig, Michael et al. (2015) Mutational and Computational Evidence That a Nickel-Transfer Tunnel in UreD Is Used for Activation of Klebsiella aerogenes Urease. Biochemistry 54:6392-401 |
| Boer, Jodi L; Mulrooney, Scott B; Hausinger, Robert P (2014) Nickel-dependent metalloenzymes. Arch Biochem Biophys 544:142-52 |
| Farrugia, Mark A; Han, Linjie; Zhong, Yueyang et al. (2013) Analysis of a soluble (UreD:UreF:UreG)2 accessory protein complex and its interactions with Klebsiella aerogenes urease by mass spectrometry. J Am Soc Mass Spectrom 24:1328-37 |
| Farrugia, Mark A; Macomber, Lee; Hausinger, Robert P (2013) Biosynthesis of the urease metallocenter. J Biol Chem 288:13178-85 |
| Boer, Jodi L; Hausinger, Robert P (2012) Klebsiella aerogenes UreF: identification of the UreG binding site and role in enhancing the fidelity of urease activation. Biochemistry 51:2298-308 |
| Carter, Eric L; Proshlyakov, Denis A; Hausinger, Robert P (2012) Apoprotein isolation and activation, and vibrational structure of the Helicobacter mustelae iron urease. J Inorg Biochem 111:195-202 |
| Carter, Eric L; Tronrud, Dale E; Taber, Scott R et al. (2011) Iron-containing urease in a pathogenic bacterium. Proc Natl Acad Sci U S A 108:13095-9 |
| Macomber, Lee; Hausinger, Robert P (2011) Mechanisms of nickel toxicity in microorganisms. Metallomics 3:1153-62 |
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