Mutational and Computational Evidence That a Nickel-Transfer Tunnel in UreD Is Used for Activation of Klebsiella aerogenes Urease

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• 作者：Mark A. Farrugia ; Beibei Wang ; Michael Feig ; Robert P. Hausinger
• 刊名：Biochemistry
• 出版年：2015
• 出版时间：October 20, 2015
• 年：2015
• 卷：54
• 期：41
• 页码：6392-6401
• 全文大小：517K
• ISSN：1520-4995

文摘

Nickel-containing urease from Klebsiella aerogenes requires four accessory proteins for proper active site metalation. The metallochaperone UreE delivers nickel to UreG, a GTPase that forms a UreD/UreF/UreG complex, which binds to urease apoprotein via UreD. Prior in silico analysis of the homologous, structurally characterized UreH/UreF/UreG complex from Helicobacter pylori identified a water tunnel originating at a likely nickel-binding motif in UreG, passing through UreF, and exiting UreH, suggestive of a role for the channel in providing the metal to urease apoprotein for its activation; however, no experimental support was reported for the significance of this tunnel. Here, specific variants were designed to disrupt a comparable 34.6 脜 predicted internal tunnel, alternative channels, and surface sites for UreD. Cells producing a set of tunnel-disrupting variants of UreD exhibited greatly reduced urease specific activities, whereas other mutants had no appreciable effect on activity. Affinity pull-down studies of cell-free extracts from tunnel-disrupting mutant cultures showed no loss of UreD interactions with urease or UreF/UreG. The nickel contents of urease samples enriched from activity-deficient cultures were decreased, while zinc and iron incorporation increased. Molecular dynamics simulations revealed size restrictions in the internal channels of the UreD variants. These findings support the role of a molecular tunnel in UreD as a direct facilitator of nickel transfer into urease, illustrating a new paradigm in active site metallocenter assembly.