Table 1

Structural statistics

StructureDipolar coupling R factor, %*rms from intermolecular distance restraints, ÅIntermolecular ELJ, kcal⋅mol−1Backbone atomic rms to original NMR structure of complex
Free x-ray structures
Starting EIN, HPr coordinate frame66.3§79.4028.8
Rigid body docking with all intermolecular interproton distance restraints
  Round 1 rigid body28.90.814.1  × 1071.18
  Round 1 side chain28.90.424.5  × 1011.18
  Round 2 rigid body28.80.372.1  × 1011.17
  Round 2 side chain28.80.37−1.2  × 1011.17
Rigid body docking with nine intermolecular distance restraints**
  Round 1 rigid body28.81.341.5  × 1071.46
  Round 1 side chain28.81.059.3  × 1021.46
  Round 2 rigid body27.80.935.8  × 1021.31
  Round 2 side chain27.80.926.8  × 1021.31
  • * The dipolar coupling factor is defined as the ratio of the measured rms to the expected rms if the N–H vectors were randomly distributed. The latter is given by {2Da2[4 + 3 (Dr/Da)2/5}1/2 (18). 

  • ELJ is the Lennard–Jones van der Waals energy (computed only for the intermolecular contacts) from the CHARMM19/20 empirical energy function. This term is not included in any of the calculations. 

  • Refers to N, Cα, and C′ backbone atoms of residues 2–230 of EIN and residues 1–85 of HPr. 

  • § The dipolar coupling R factor for the free proteins and for EIN and HPr in the initial coordinate frame are obtained by optimization of the orientation of the alignment tensor, keeping the values of Da (−14.3 Hz) and Dr/Da (0.4) fixed to their experimental values. 

  • These values represent the backbone atomic rms difference between the NMR structure of EIN in the complex and the x-ray structure of free EIN, and between the NMR structure of HPr in the complex and the x-ray structure of free HPr. 

  • The protocol of rigid body minimization followed by Powell minimization of the interfacial sidechains is given in the text. 

  • ** The intermolecular distance restraints comprise eight approximate interproton distance restraints corresponding to all the observed intermolecular NH–methyl NOEs, supplemented by a distance restraint to ensure that the Cα–Cα distance between the active–site histidines is less than 12 Å.