Helicobacter pylori hydrogenase accessory protein HypA and urease accessory protein UreG compete with each other for UreE recognition

Abstract

Background The gastric pathogen Helicobacter pylori relies on nickel-containing urease and hydrogenase enzymes in order to colonize the host. Incorporation of Ni2+ 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 5 nM and 13 nM for HypA–UreE and 8 µM and 14 µM for UreG-UreE. Neither Zn2+ nor Ni2+ had an effect on the kinetics or stability of the HypA–UreE complex. By contrast, addition of Zn2+, but not Ni2+, altered the kinetics and greatly increased the stability of the UreE–UreG complex, likely due in part to Zn2+-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.

Structure of UreG/UreF/UreH Complex Reveals How Urease Accessory Proteins Facilitate Maturation of Helicobacter pylori Urease

“…Affinity pull-downs showed that K. aerogenes UreG and UreE form a complex in the presence of nickel [22]. The strength of the interaction between UreG and UreE was measured using surface plasmon resonance and biolayer interferometry techniques [40]. These observations all support the idea of a nickel transfer between UreE and UreG via protein–protein interaction.…”

UreE-UreG Complex Facilitates Nickel Transfer and Preactivates GTPase of UreG in Helicobacter pylori

“…4). A previous study demonstrated that both HypA and UreG compete with each other for UreE (48). We show here that preference of UreE toward HypA and UreG is elaborately tuned in biological systems and that UreE has a tendency to bind HypA in the absence of GTP and Mg 2ϩ , whereas it binds UreG in the presence of GTP and Mg 2ϩ .…”

Nickel Ligation of the N-Terminal Amine of HypA Is Required for Urease Maturation in Helicobacter pylori

“…56 Under acidic conditions, HypAB could provide a higher level of Ni incorporation into UreE by 1) increasing the local concentration of Ni by formation of a complex with HypAB, or 2) by simply enhancing the affinity of UreE for Ni in a complex with HypAB. The former mechanism is suggested by the known HypA-UreE complex formation, 16, 57 and by protein structural changes that accompany changes in the Zn site structure at pH 6.3. 18, 24 The latter mechanism is supported by the increase in Ni affinity that occurs in the HypAB complex in T. kodakaraensis.…”