Table 1.
Data collection and refinement statistics.
Figure 1.
AMP transfer catalyzed by Fic proteins and their inhibition-relieved variants.
Autoradiography of VbhA/VbhT(FIC), SoFic and NmFic (wt, wild type; E/G, E->G mutant) after incubation with radioactively labeled α-32P-ATP.
Figure 2.
Crystal structures of wild-type Fic proteins representing classes I to II in complex with ATP substrate.
(A) VbhA/VbhT(FIC), (B) SoFic. Structures are shown in cartoon representation (red, FIC core as defined by PFAM; yellow, active site loop and N-terminal end of helix α5; dark-green, inhibitory helix αinh). In (A), the fold of the antitoxin is shown in dark-green and steel-blue. Selected residues are shown in full with the inhibitory glutamate (E24 or E73, respectively) colored in dark. The 2Fo-Fc simulated annealing omit maps covering the ligand are contoured at 1.1 σ. In both structures, the orientation of the α-phosphate prevents nucleophilic attack of a putative target side-chain hydroxyl onto the ATP substrate, since the position inline with the scissile Pα-O3α bond (magenta star) is unattainable. C) Stereo view of the superposition of the ATP nucleotides shown in panel A and B with AMPPNP from the complex structure of the class III NmFic protein (PDB 3S6A [8]) within the active site of the VbhA/VbhT(FIC) complex (same as in panel A). The nucleotides of the various complexes are distinguished by their colors (white for the ATP bound to VbhA/VbhT(FIC), green for the ATP bound to SoFic, and blue for the AMPPNP of the NmFic complex. Note that the AMPPNP γ-phosphate in NmFic is found disordered [8] and therefore not shown. The residues of the HxFx(D/E)GNGRxxR Fic signature motif are labeled, the two glycine and the two arginine residues are distinguished by a "1" or "2" in brackets. The phenylalanine (not shown) is part of the hydrophobic core. The inhibitory glutamate from αinh is labeled as Einh.
Figure 3.
Crystal structures of E->G mutated Fic proteins representing classes I to III in complex with substrate or substrate analog.
A, VbhAE24G/VbhT(FIC) in complex with ATP/Mg2+; B, SoFicE73G, C, NmFicE186G, both in complex with AMPPNP/Mg2+. Representation as in Fig. 2 with magnesium ions shown as magenta spheres. The 2Fo-Fc simulated annealing omit maps covering the nucleotide/Mg2+ ligands are contoured at 1.1 σ. D, Stereo view of the superposition of the ligand structures shown in panels B and C onto the VbhAE24G/VbhT(FIC) complex (same as in panel A). Note that the nucleotides of the various complexes are distinguished by their carbon color (VbhAE24G/VbhT(FIC) ATP in green, SoFicE73G AMPPNP in orange and NmFicE186G AMPPNP in pink). The residues of the HxFx(D/E)GNGRxxR signature motif are labeled as in Fig. 2C with the phenylalanine not shown. Also shown is the modifiable hydroxyl side-chain Y32 of Cdc42 (blue) after superposition of the IbpA(FIC2)/Cdc42 complex [4] onto VbhAE24G/VbhT(FIC). For the superposition, only the Fic active site loops were used. The α-phosphate moieties appear well-suited for in-line attack of the target hydroxyl group (broken line in magenta).
Figure 4.
Comparison of triphosphate nucleotide structures as bound to wild-type and E->G mutated Fic proteins from class I to III.
Stereo views of the ligand structures after superposition of the FIC domains (not shown). Also shown is the inhibitory glutamate of the wild-type structures. A, ATP as bound to VbhA/VbhT wild-type (white) and the E24G mutant (dark green). B, ATP and AMPPNP as bound to SoFic wild-type (green) and the E73G mutant (orange), respectively. C, AMPPNP as bound to NmFic (blue) and the E186G mutant (pink). Note that the AMPPNP γ-phosphate in NmFic is found disordered [8] and therefore not shown.
Figure 5.
Sequence independent registration of peptide or target protein to the FIC flap.
The bound peptide/protein segment (blue) and the target dock (brown) are shown in full. Main chain-main chain H-bonds are depicted as stippled lines. A, Product complex of IbpA(Fic2) with Cdc42 target [4]. Tyrosine 32 from the switch1 region of Cdc42 is adenylylated. B, VbhA/VbhT(FIC) complexed with residues 203 to 206 of a symmetry related molecule. The 2Fo-Fc simulated annealing omit map covering the residues 203 to 206 is contoured at 1.1 σ. Note that the preceding 7 residues are disordered and not shown. C, SoFic complexed with residues 0 to 4 of a symmetry related molecule (PDB 3EQX) [16]. The side-chains of residues 0, 1 and 4 are disordered and not displayed for clarity reason. Note that Y32 in panel A, V203 in panel B and W3 in panel C are in equivalent positions.