Abstract
Synthesis of active Klebsiella aerogenes urease requires four accessory proteins to generate, in a GTP-dependent process, a dinuclear nickel active site with the metal ions bridged by a carbamylated lysine residue. The UreD and UreF accessory proteins form stable complexes with urease apoprotein, comprised of UreA, UreB, and UreC. The sites of protein-protein interactions were explored by using homobifunctional amino group-specific chemical cross-linkers with reactive residues being identified by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) of tryptic peptides. On the basis of studies of the UreABCD complex, UreD is capable of cross-linking with UreB Lys(9), UreB Lys(76), and UreC Lys(401). Furthermore UreD appears to be positioned over UreC Lys(515) according to decreased reactivity of this residue compared with its reactivity in UreD-free apoprotein. Several UreB-UreC and UreC-UreC cross-links also were observed within this complex; e.g. UreB Lys(76) with the UreC amino terminus, UreB Lys(9) with UreC Lys(20), and UreC Lys(515) with UreC Lys(89). These interactions are consistent with the proximate surface locations of these residues observed in the UreABC crystal structure. MALDI-TOF MS analyses of UreABCDF are consistent with a cross-link between the UreF amino terminus and UreB Lys(76). On the basis of an unexpected cross-link between UreB Lys(76) and UreC Lys(382) (distant from each other in the UreABC structure) along with increased side chain reactivities for UreC Lys(515) and Lys(522), UreF is proposed to induce a conformational change within urease that repositions UreB and potentially could increase the accessibility of nickel ions and CO(2) to residues that form the active site.
Highlights
The hydrolysis of urea to form carbonic acid and two molecules of ammonia is catalyzed by urease, an enzyme found in all plants and many species of algae, fungi, and bacteria (1)
UreABC and the larger urease apoprotein complexes of K. aerogenes are partially activated by incubation with nickel ions and bicarbonate with full activity achieved by additional inclusion of GTP and UreE (25)
We observed two mass spectrometry (MS) features consistent with UreD cross-linking to UreB Lys[76], one compatible with UreD attaching to UreC Lys[401] and another suggesting that UreD can link to UreB Lys[9]
Summary
0.03 Ϫ0.03 Ϫ0.11 Ϫ0.13 Ϫ0.08 Ϫ0.08 Ϫ0.51 Ϫ0.33 Ϫ0.32 Ϫ0.25 Ϫ0.73 Ϫ0.05 Ϫ0.17 Ϫ0.05 Ϫ0.07 Ϫ0.53 Ϫ0.61. Yeast two-hybrid studies of the H. pylori system identified interactions between UreD (alternatively named UreH in that microorganism) and both UreA (equivalent to the fusion of UreA plus UreB in most bacteria) and UreF (31, 32). Additional evidence was consistent with the following interactions in H. pylori: UreA-UreA, UreAUreB (where UreB is equivalent to UreC in other bacteria), and UreE-UreG. In this study we examined accessory protein-urease interactions for two stable complexes, UreABCD and UreABCDF, of K. aerogenes by using a chemical cross-linking approach. C8H12O2 denotes cross-linking between two peptides by the reagent. We tentatively identify specific sites of cross-linking between UreD and both UreB and UreC. We provide evidence for a previously undescribed interaction between UreF and UreB and identify the putative sites of cross-linking between these proteins. We establish several UreF-dependent changes in reactivity of the urease side chains with crosslinking reagents consistent with a significant conformational change occurring in the presence of UreF that may be relevant to metallocenter assembly
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