Klebsiella aerogenes UreF: Identification of the UreG Binding Site and Role in Enhancing the Fidelity of Urease Activation

Abstract

The Ni-containing active site of Klebsiella aerogenes urease is assembled through the concerted action of the UreD, UreE, UreF, and UreG accessory proteins. UreE functions as a metallochaperone that delivers Ni to a UreD–UreF–UreG complex bound to urease apoprotein, with UreG serving as a GTPase during enzyme activation. This study focuses on the role of UreF, previously proposed to act as a GTPase activating protein (GAP) of UreG. Sixteen conserved UreF surface residues that may play roles in protein–protein interactions were independently changed to Ala. When produced in the context of the entire urease gene cluster, cell-free extracts of nine site-directed mutants had less than 10% of the wild-type urease activity. Enrichment of the variant forms of UreF, as the UreE-F fusion proteins, uniformly resulted in copurification of UreD and urease apoprotein, whereas UreG bound to only a subset of the species. Notably, weakened interaction with UreG correlated with the low-activity mutants. The affected residues in UreF map to a distinct surface on the crystal structure, defining the UreG binding site. In contrast to the hypothesis that UreF is a GAP, the UreD–UreF–UreG–urease apoprotein complex containing K165A UreF exhibited significantly greater levels of GTPase activity than that containing the wild-type protein. Additional studies demonstrated the UreG GTPase activity was largely uncoupled from urease activation for the complex containing this UreF variant. Further experiments with these complexes provided evidence that UreF gates the GTPase activity of UreG to enhance the fidelity of urease metallocenter assembly, especially in the presence of the noncognate metal Zn.

Analysis of a Soluble (UreD:UreF:UreG)2 Accessory Protein Complex and Its Interactions with Klebsiella aerogenes Urease by Mass Spectrometry

“…The Strep -tag II version of (UreABC) 3 :UreDFG was formed in cells transformed with pKKG containing ureDABCEFG where ureG is fused to the codons for Strep -tag II [25]. This complex was purified by use of a Strep -tactin column followed by Superdex 200 gel filtration chromatography, as previously described [25].…”

“…This complex was purified by use of a Strep -tactin column followed by Superdex 200 gel filtration chromatography, as previously described [25]. …”

Biosynthesis of the Urease Metallocenter

“…A role as a GTPase-activating protein was suggested for UreF (41), but the UreH:UreF:UreG structure (which shows UreF binding UreG opposite the GTP site) (Fig. 2) and experimental evidence derived from mutagenesis and GTPase activity studies (42) argue against this proposal. For example, a urease activation complex containing a UreF variant exhibited enhanced GTPase activity compared with the complex with wild-type accessory protein.…”

“…Furthermore, UreABC:UreD:UreE-UreF:UreG is made in cells producing the UreE-UreF fusion protein (38). Mutagenesis studies identified Asp-80 as a key UreG residue involved in this interaction (48) and defined several residues along one face of UreF as the UreG-docking site (42). Using standard activation conditions, ϳ60% of the nascent active sites in UreABC: UreD:UreF:UreG become active (54).…”

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

“…8 Ka UreF alone is insoluble. 9 Ka UreG, which exists as a soluble monomer that binds one equivalent of nickel or zinc with similar affinities, 10, 11 acts as a GTPase during urease maturation, although the precise role of GTP hydrolysis in this process remains unclear. Ka UreG residues associated with a highly conserved Cys-X-His motif are hypothesized to function as the metal binding site, but substitution of one or both of these residues does not abolish metal binding.…”