BACKGROUND: The gastric pathogen Helicobacter pylori relies on nickel-containing urease and hydrogenase enzymes in order to colonize the host. Incorporation of Ni(2+) into urease is essential for the function of the enzyme and requires the action of several accessory proteins, including the hydrogenase accessory proteins HypA and HypB and the urease accessory proteins UreE, UreF, UreG and UreH. METHODS: Optical biosensing methods (biolayer interferometry and plasmon surface resonance) were used to screen for interactions between HypA, HypB, UreE and UreG. RESULTS: Using both methods, affinity constants were found to be 5nM and 13nM for HypA-UreE and 8μM and 14μM for UreG-UreE. Neither Zn(2+) nor Ni(2+) had an effect on the kinetics or stability of the HypA-UreE complex. By contrast, addition of Zn(2+), but not Ni(2+), altered the kinetics and greatly increased the stability of the UreE-UreG complex, likely due in part to Zn(2+)-mediated oligomerization of UreE. Finally our results unambiguously show that HypA, UreE and UreG cannot form a heterotrimeric protein complex in vitro; instead, HypA and UreG compete with each other for UreE recognition. GENERAL SIGNIFICANCE: Factors influencing the pathogen's nickel budget are important to understand pathogenesis and for future drug design.
BACKGROUND: The gastric pathogen Helicobacter pylori relies on nickel-containing urease and hydrogenase enzymes in order to colonize the host. Incorporation of Ni(2+) into urease is essential for the function of the enzyme and requires the action of several accessory proteins, including the hydrogenase accessory proteins HypA and HypB and the urease accessory proteins UreE, UreF, UreG and UreH. METHODS: Optical biosensing methods (biolayer interferometry and plasmon surface resonance) were used to screen for interactions between HypA, HypB, UreE and UreG. RESULTS: Using both methods, affinity constants were found to be 5nM and 13nM for HypA-UreE and 8μM and 14μM for UreG-UreE. Neither Zn(2+) nor Ni(2+) had an effect on the kinetics or stability of the HypA-UreE complex. By contrast, addition of Zn(2+), but not Ni(2+), altered the kinetics and greatly increased the stability of the UreE-UreG complex, likely due in part to Zn(2+)-mediated oligomerization of UreE. Finally our results unambiguously show that HypA, UreE and UreG cannot form a heterotrimeric protein complex in vitro; instead, HypA and UreG compete with each other for UreE recognition. GENERAL SIGNIFICANCE: Factors influencing the pathogen's nickel budget are important to understand pathogenesis and for future drug design.
Authors: Joy Concepcion; Krista Witte; Charles Wartchow; Sae Choo; Danfeng Yao; Henrik Persson; Jing Wei; Pu Li; Bettina Heidecker; Weilei Ma; Ram Varma; Lian-She Zhao; Donald Perillat; Greg Carricato; Michael Recknor; Kevin Du; Huddee Ho; Tim Ellis; Juan Gamez; Michael Howes; Janette Phi-Wilson; Scott Lockard; Robert Zuk; Hong Tan Journal: Comb Chem High Throughput Screen Date: 2009-09 Impact factor: 1.339
Authors: Robert W Herbst; Iva Perovic; Vlad Martin-Diaconescu; Kerrie O'Brien; Peter T Chivers; Susan Sondej Pochapsky; Thomas C Pochapsky; Michael J Maroney Journal: J Am Chem Soc Date: 2010-08-04 Impact factor: 15.419
Authors: Jonathan L McMurry; Tohru Minamino; Yukio Furukawa; Joshua W Francis; Stephanie A Hill; Katy A Helms; Keiichi Namba Journal: PLoS One Date: 2015-08-05 Impact factor: 3.240