TABLE 2.
Structural statistics
The notation of the NMR structures is as follows: <SA> are the final 100 simulated annealing structures for the IIAChb* (H89E)-HPr complex and <SAphos> are the final 70 simulated annealing structures for the corresponding phosphoryl transition state.
| <SA> | <SAphos> | ||
|---|---|---|---|
| Number of experimental NMR restraints | |||
| Intermolecular interproton distance restraints | 42 | ||
| IIAChb* intramolecular interproton distance restraintsa | 276 × 3 | ||
| IIAChb* torsion angle restraintsa | 245 × 3 | ||
| HPr torsion angle restraintsb | 36 | ||
| 1DNH RDCs for IIAChb*a | 84 × 3 | ||
| 1DNC′ RDCs for IIAChb*a | 85 × 3 | ||
| 1DCαC′ RDCs for IIAChb*a | 83 × 3 | ||
| 13Cα/13Cβ chemical shift restraints for IIAChb*a | 195 × 3 | ||
| Experimental restraints | |||
| R.m.s. deviation from interproton distance restraints (Å)c | 0.010 ± 0.002 | 0.011 ± 0.001 | |
| R.m.s. deviation from torsion angle restraints (°)c | 0.25 ± 0.04 | 0.25 ± 0.04 | |
| R.m.s. deviation from 13Cα/13Cβ shift restraints (ppm) | 1.12 ± 0.02/0.66 ± 0.02 | 1.12 ± 0.02/0.63 ± 0.01 | |
| 1DNH RDC R-factor (%)d | 7.4 ± 0.1 | 7.4 ± 0.08 | |
| 1DNC′ RDC R-factor (%)d | 17.1 ± 1.3 | 16.8 ± 0.97 | |
| 1DCαC′ RDC R-factor (%)d | 16.2 ± 0.9 | 15.9 ± 0.52 | |
| Deviations from idealized covalent geometrye | |||
| Bonds (Å) | 0.006 ± 0 | 0.006 ± 0 | |
| Angles (degree) | 0.63 ± 0 | 0.63 ± 0 | |
| Impropers (degree) | 0.63 ± 0 | 0.62 ± 0 | |
| Measures of structural qualityf | |||
| Intermolecular repulsion energy (kcal mol−1) | 1.3 ± 0.4 | 1.4 ± 0.4 | |
| Intermolecular Lennard-Jones energy (kcal mol−1) | −16.2 ± 5.2 | −16.7 ± 4.5 | |
| Number of bad contacts per 100 residues | 3.6 ± 1.1 | 3.1 ± 1.1 | |
| % Residues in most favorable region of Ramachandran map | 94.5 ± 1.0 | 94.3 ± 1.3 | |
| Coordinate precision of the complex (Å)g | |||
| Backbone (N, Cα, C′, O) atoms | 0.56 | 0.49 | |
| Interfacial sidechain heavy atoms of IIAChb* and HPrg | 1.25 | 1.07 | |
| Side chain heavy atoms of IIAChb* | 1.32 | 1.24 | |
a The intramolecular experimental restraints (NOE-derived interproton distances, torsion angles, 13Cα/13Cβ chemical shifts and residual dipolar couplings) for IIAChb* in the complex are taken from the structure determination of free IIAChb*. The interproton distance restraints comprise 82 × 3 sequential (|i − j| = 1), 86 × 3 medium range (1 <|i − j| ≤ 5), and 30 × 3 long range (|i − j| > 5) intrasubunit restraints, 78 × 3 intersubunit distances and 154 × 3 distance restraints for 77 × 3 backbone hydrogen bonds within the three helices. The torsion angle restraints comprise 87 × 3 φ, 82 × 3 ψ, 76 × 3 χ angles.
b The torsion angle restraints for HPr comprise 26 interfacial side chain torsion angles, supplemented, in the case of the calculations of the phosphoryl transition state, by 5 φ and 5 ψ backbone torsion angles (derived from chemical shifts in the complex using TALOS+ (58)) for residues 13–17 encompassing the active site His-15. The side chains of HPr given torsional degrees of freedom comprise residues 11–17, 20–21, 23–24, 27, 45–48, 51–57, and 85.
c None of the structures exhibit interproton distance violations >0.3 Å or torsion angle violations >5°.
d The RDC R-factor (70) is defined as the ratio of the r.m.s. deviation between observed and calculated values and the expected r.m.s. deviation for a random distribution of vectors. The latter is given by [2Da2 (4+3η2)/5]1/2, where Da and η are the magnitude of the alignment tensor and the rhombicity, respectively. The values of Da and η for the free IIAChb* trimer are −12.1 Hz and 0, respectively (note the rhombicity for a symmetric trimer is always 0). The R-factor scales between 0 and 100%.
e The His-P-His phosphoryl transition state formed between the Nϵ2 atom of His-89 of subunit A of IIAChb* and the Nδ1 atom of His-15 of HPr is calculated using the same experimental restraints as those used for the unphosphorylated complex with the addition of covalent geometry restraints to describe the pentacoordinate phosphoryl group in a trigonal bypiramidal geometry (37): rNϵ2(His89)-P, rNδ1(His15)-P ≤ 3.5 Å, rP-O = 1.48 Å; Nδ1(His-15)-P-Nϵ2(His-89) = 180°, Nδ1(His-15)-P-O = 90°, Nϵ2(His-89)-P-O = 90°, Cγ(His-15)-Nδ1(His-15)-P = 127.35°, Cϵ1(His-15)-Nδ1(His-15)-P = 127.35°, Cδ2(His-89)-Nϵ2(His-89)-P = 126.35°, Cϵ1(His-89)-Nϵ2(His-89)-P = 126.35°. In addition improper torsion angle restraints are used to ensure that the phosphorus atom lies in the same plane as the imidazole rings of both His-89A and His-15.
f The intermolecular repulsion energy is given by the value of the quartic van der Waals repulsion term calculated with a force constant of 4 kcal mol−1·Å−4 and a van der Waals radius scale factor of 0.78 (61). The intermolecular Lennard-Jones van der Waals interaction energy is calculated using the CHARMM19/20 parameters and is not included in the target function used to calculate the structures. The number of bad contacts per 100 residues and the percentage of residues in the most favorable region of the Ramachandran plot are calculated using PROCHECK (71). The φ/ψ, χ1/χ2, χ1 and χ3/χ4 PROCHECK g-factors are 0.76 ± 0.04, 0.60 ± 0.06, 0.21 ± 0.10, and 0.42 ± 0.07, respectively.
g Defined as the average r.m.s. difference between the final ensemble of simulated annealing structures and the mean coordinates positions.