The structure of urease activation complexes examined by flexibility analysis, mutagenesis, and small-angle X-ray scattering.
Conformational changes of Klebsiella aerogenes urease apoprotein (UreABC)(3) induced upon binding of the UreD and UreF accessory proteins were examined by a combination of flexibility analysis, mutagenesis, and small-angle X-ray scattering (SAXS). ProFlex analysis of urease provided evidence that the major domain of UreB can move in a hinge-like motion to account for prior chemical cross-linking results. Rigidification of the UreB hinge region, accomplished through a G11P mutation, reduced the extent of urease activation, in part by decreasing the nickel content of the mutant enzyme, and by sequestering a portion of the urease apoprotein in a novel activation complex that includes all of the accessory proteins. SAXS analyses of urease, (UreABC-UreD)(3), and (UreABC-UreDF)(3) confirm that UreD and UreF bind near UreB at the periphery of the (UreAC)(3) structure. This study supports an activation model in which a domain-shifted UreB conformation in (UreABC-UreDF)(3) allows CO(2) and nickel ions to gain access to the nascent active site.
Quiroz-Valenzuela
Soledad
S
Department of Biochemistry & Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
Sukuru
Sai Chetan K
SC
Hausinger
Robert P
RP
Kuhn
Leslie A
LA
Heller
William T
WT
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D021902:Enterobacter aerogenes / Q000201:enzymology; D004789:Enzyme Activation; D008954:Models, Biological; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009532:Nickel / Q000032:analysis; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053838:Scattering, Small Angle; D014510:Urease / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014961:X-Ray Diffraction
D017931:DNA Primers; D004269:DNA, Bacterial; D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D009532:Nickel; D014510:Urease