Chemical cross-linking and mass spectrometric identification of sites of interaction for UreD, UreF, and urease.
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.
Chang
Zhenzhan
Z
Departments of Microbiology & Molecular Genetics and Biochemistry & Molecular Biology, Michigan State University, East Lansing, Michigan 48824-4320, USA.
Kuchar
Jason
J
Hausinger
Robert P
RP
D001059:Apoproteins / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002245:Carbon Dioxide / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D021902:Enterobacter aerogenes / Q000201:enzymology*; D007477:Ions; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D009532:Nickel / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014510:Urease / Q000737:chemistry*
D001059:Apoproteins; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D007477:Ions; D010455:Peptides; C086767:UreD protein, Bacteria; C101639:UreF protein, bacteria; D002245:Carbon Dioxide; D009532:Nickel; D014510:Urease; D008239:Lysine