Molecular dynamics of the FixJ receiver domain: movement of the beta4-alpha4 loop correlates with the in and out flip of Phe101

Abstract

FixJ is a two-domain response regulator involved in nitrogen fixation in Sinorhizobium meliloti. Recent X-ray characterization of both the native (unphosphorylated) and the active (phosphorylated) states of the protein identify conformational changes of the beta4-alpha4 loop and the conserved residue Phe101 as the key switches in activation. These structures also allowed investigation of the transition between conformations of this two-component regulatory receiver domain by molecular dynamics simulations. The path for the conformational change was studied with a distance constraint directing the system from one state to the other. The simulations provide evidence for a correlation between the conformation of the beta4-alpha4 loop and the orientation of the residue Phe101. A model presenting the sequence of events during the activation/deactivation process is discussed.

Fig. 1.

General view of the FixJ receiver domain. This structure corresponds to the JN structure to which a phosphoryl group has been added in silico (JN[+P]). Helices α1 to α5 are colored from green to blue, the central β sheet is orange, and the β4–α4 loop is red. Important residues are shown: Pas54, phosphorylated aspartate (phosphate group added in silico), Thr82 at the N-terminal end of the β4–α4 loop and Phe101 in the outside orientation. This figure was prepared using Bobscript (Esnouf 1997) and Raster 3D (Merritt and Bacon 1997).

Fig. 2.

Molecular dynamics of the activation pathway before the PEDC constraint was applied: heating (30 psec), equilibration (200 psec), and free MD (200 psec) steps. (A) Potential energy in kcal•mole⁻¹; (B) mrms distance in Å with regard to the phosphorylated target structure (JNT∼P).

Fig. 3.

Distance and angle analyses showing the sequence of events during the phosphorylation pathway. The PEDC constraint was applied either on residues 3–122 (A–D) or on residues 82–86 (E–H). (A) and (E): distance between the Cα atoms of Thr82 and Val87 (rearrangement of the β4–α4 loop); (B) and (F): pseudodihedral angle (Psdie) formed by Cα atoms of residues 83 to 86 (rearrangement of the β4–α4 loop); (C) and (G): χ₁ dihedral angle of residue Phe101 (rotation of Phe101); (D) and (H): distance in Å between the hydrogen atom of the hydroxyl group of Thr82 and the O2 of the phosphoryl group (formation of a hydrogen bond). Transitions discussed in the text are indicated by black triangles.

Fig. 4.

Views of four intermediate structures during the activation process. The PEDC constraint was applied to residues 3 to 122. The three residues of interest (P: phosphorylated Asp54, T: Thr82, and F: Phe101) as well as neighboring residues are highlighted. (A) Starting structure of the MD under constraints corresponding to the last structure of the free MD run (430 psec); (B) structure showing the rearrangement of the β4–α4 loop (815 psec); (C) structure after the rotation of residue Phe101 into the hydrophobic cavity (870 psec); (D) last structure of the simulation (1200 psec in which the hydrogen bond between Thr82 and the phosphate group is highlighted). Key changes are indicated with blue arrowheads.

Fig. 5.

Distance and angle analyses showing the sequence of events during the dephosphorylation pathway. The PEDC constraint was applied either on residues 3–122 (A–C) or residues 82–86 (D–F). (A) and (D): χ₁ dihedral angle of residue Phe101 (rotation of Phe101); (B) and (E): distance between the Cα atoms of Thr82 and Val87 (rearrangement of the β4–α4 loop); (C) and (F): distance between the hydroxyl group of Thr82 and the CG atom of residue Asp54 (disruption of hydrogen bond). The conformational changes discussed in the text are indicated with black triangles.

Fig. 6.

Proposed model for the activation/deactivation of the FixJ receiver domain. Upon phosphorylation, the β4–α4 loop undergoes a conformational change from a β-turn to an extended conformation (A). This event is followed by the rotation of Phe101 (B). Finally, the loop is stabilized by the formation of a hydrogen bond between Thr82 and the phosphate group (C). After dephosphorylation, residue Phe101 can rotate to the outside (D) and the β4–α4 loop adopts a β-turn conformation that disrupts the hydrogen bond between Thr82 and Asp54 (E).