Abstract:The two-component system (TCS) is an important signal transduction component for most
bacteria. This signaling pathway is mediated by histidine kinases via autophosphorylation between P1 and
P4 domains. Taking chemotaxis protein CheA as a model of TCS, the autophosphorylation mechanism of
the TCS histidine kinases has been investigated in this study by using a computational approach integrated
homology modeling, ligand-protein docking, protein-protein docking, and molecular dynamics (MD)
simulations. Four nanosecond-scale MD simulations were performed on the free P4 domain, P4-ATP,
P4-TNPATP, and P1-P4-ATP complexes, respectively. Upon its binding to the binding pocket of P4
with a folded conformation, ATP gradually extends to an open state with help from a water molecule.
Meanwhile, ATP forms two hydrogen bonds with His413 and Lys494 at this state. Because of the lower
energy of the folded conformations, ATP shrinks back to its folded conformations, leading to the rupture of
the hydrogen bond between ATP and Lys494. Consequently, Lys494 moves away from the pocket entrance,
resulting in an open of the ATP lid of P4. It is the open state of P4 that can bind tightly to P1, where the
His45 of P1 occupies a favorable position for its autophosphorylation from ATP. This indicates that ATP is
not only a phosphoryl group donor but also an activator for CheA phosphorylation. Accordingly, a mechanism
of the autophosphorylation of CheA is proposed as that the ATP conformational switch triggers the opening
of the ATP lid of P4, leading to P1 binding tightly, and subsequently autophosphorylation from ATP to P1.