Abstract
HlyB functions as an adenosine triphosphate (ATP)-binding cassette (ABC) transporter that enables bacteria to secrete toxins at the expense of ATP hydrolysis. Our previous work, based on potential energy profiles from combined quantum mechanical and molecular mechanical (QM/MM) calculations, has suggested that the highly conserved H-loop His residue H662 in the nucleotide binding domain (NBD) of E. coli HlyB may catalyze the hydrolysis of ATP through proton relay. To further test this hypothesis when entropic contributions are taken into account, we obtained QM/MM minimum free energy paths (MFEPs) for the HlyB reaction, making use of the string method in collective variables. The free energy profiles along the MFEPs confirm the direct participation of H662 in catalysis. The MFEP simulations of HlyB also reveal an intimate coupling between the chemical steps and a local protein conformational change involving the signature-loop residue S607, which may serve a catalytic role similar to an Arg-finger motif in many ATPases and GTPases in stabilizing the phosphoryl-transfer transition state.
Highlights
As a member of adenosine triphosphate (ATP)-binding cassette (ABC) transporters [1], Haemolysin B (HlyB) mediates secretion of the 107 kD pore-forming toxin Haemolysin A (HlyA) from gram-negative bacteria in an ATP-dependent manner [2]
As the C-loop Ser under this hypothesis changes its local conformation in a way similar to that of an “arginine finger” seen in many GTP/ATP hydrolyzing enzymes [15,16], we suggest that it acts as a “serine finger” for linking specific protein motion to efficient catalysis in HlyB
The general acid catalysis (GAC) reaction mechanism is in accord with the pH dependence of the ATPase activity reported for HlyB, which shows maximal ATPase activities at pH = 7 [3], a condition necessary for His to exist predominantly as a singly protonated neutral species to activate the proton relay pathway
Summary
As a member of ATP-binding cassette (ABC) transporters [1], Haemolysin B (HlyB) mediates secretion of the 107 kD pore-forming toxin Haemolysin A (HlyA) from gram-negative bacteria in an ATP-dependent manner [2]. The NBD pair in HlyB is a highly conserved molecular motor that binds and hydrolyzes ATP, energizing the substrate translocation across the TMDs through conformational coupling [3]. In E. coli cells, to ensure efficient couplings among these domains, HlyB-NBDs work as an ATPase that catalyzes ATP hydrolysis at a rate constant of kcat = 0.2 s−1 [3], reducing the half-life of ATP from ~100 days (k = 8 × 10−8 s−1 ) [4] in aqueous solution to about 3.5 s to meet the kinetic demand of translocating HlyA through bacterial membranes. A molecular-level understanding of how HlyB-NBDs achieve this remarkable catalytic capability has been developed, thanks to a wealth of information accumulated from structural. HlyB-NBDs achievethat thisfunctioning remarkable HylB-NBDs catalytic capability [3,5,6], genetic [7], andunderstanding biochemical [3,5,6,7]
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