BD simulation results

Simulation details

Channeling rate

Electrostatic potentials at GLN and GLU binding sites

Brownian dynamics simulation trajectories

Simulation details

  • Brownian motions of charged and uncharged probes of different radii (0.1-1.4 A) were simulated in the presence of rigid protein.
  • Starting point for simulations was a 3A vicinity of the geometric center of the following atoms of HisH active site residues:

    • SG_CYS_B_84, NE2_HIS_B_178, OE1_GLU_B_180 and OE2_GLU_B_180 (or chains D or F for other copies of HisHF).
  • End (target) point of simulations was variable (defined by reaction distance) vicinity of the geometric center of the following atoms of HisF active site residues:

    • ND2_ASN/ASP_A_11, OD1_ASP_A_130 and OD2_ASP_A_130 (or chans C or E).
  • To simulate only the passage of a substrate through the channel (exclude trajectories reaching the end point through the solvent), the trajectories were stopped when the substrate moves further than 20 A from the center of HisHF . This ensures that no trajectory approaches the end point without passing the channel.
  • Additional simulations were done with trajectory stopping distance of 400 A. The rates from these simulations were 3-4 times larger than the rates when trajectories truncated at 20 A, i.e. there is ca 1 channeling trajectory per 3-4 successfull trajectories.
  • A few simulations were done with the different choice of the starting point (NE2 of bound Gln, CD of bound Glu, active site center defined by PASS) - no significant changes in channeling rates.
  • Ammonia has a dipole moment 1.47 D (comparable to the dipole moment of water molecule,1.855 D). Modeling it as dipole (as having 2 or 4 charges) did not result in large differences from neutral probe.







    • Channeling rate - approaching the target point to within 14 A (K99 is passed) and 5 A (S101 is passed)
    Structure
    chains
    Comments
    largest channeling probe (A)
    channeling rate for a neutral probe (%)
    channeling rate for a charged probe (%)
    aicar
    AB
    1.1 / 0.8
    11.3 / 2.7
    54.4 / 1.2
    CD
    ligand removed
    0.9 / 0.8
    11.3 / 2.1
    16.9 / 0.3
    EF
    1.2 / 0.8
    13.0 / 2.9
    5.8 / 0.2
    gln
    AB
    ligand removed
    0.8 / 0.7
    11.8 / 2.3
    10.7 / 1.1
    CD
    0.7 / 0.7
    11.1 / 2.3
    6.0 / 0.3
    EF
    ligand removed
    0.7 / 0.7
    10.4 / 2.2
    7.8 / 1.7
    glut
    AB
    ligand removed
    1.1 / 0.7
    10.9 / 2.5
    42.7 / 1.6
    CD
    ligand removed
    1.2 / 0.8
    10.8 / 2.2
    43.4 / 1.2
    EF
    1.1 / 1.1
    11.8 / 3.9
    43.4 / 7.3
    imgp-gln
    AB
    ligand removed
    0.8 / 0.8
    11.8 / 2.5
    10.3 / 2.3
    CD
    0.7 / 0.7
    10.3 / 2.1
    17.1 / 0.2
    EF
    ligands removed
    0.7 / 0.7
    11.2 / 2.6
    12.5 / 7.5
    1jvn
    AA
    acivicin left
    1.0 / 0.8
    29.0 / 10.6
    -
    BB
    acivicin left
    0.8 / 0.7
    27.2 / 10.9
    -
    aicar
    AB
    T78L
    1.1 / 0.8
    10.9 / 2.2
    -
    AB
    T78L+S101I
    1.1 / 0.5
    10.6 / 1.6
    -
    AB
    R5A
    1.0 / 0.8
    11.0 / 1.9
    -

    The difference between the structures seen from simulations :

  • channel in the aicar and glut structures is more open than in gln and imgp+gln
  • aicar and glut structures have open salt link tetrad for ammonium ion, which passes salt link 4 times more often than neutral ammonia does. Aicar EF structure is different from AB and CD (has different residues 23-24);
  • in the gln structure salt link tetrad seems to be closed for ammonium. This correlates with the observation of hydrogen bondng between OH_Tyr_B138 and NZ_Lys_A99 in the gln structure..
  • Yeast structure 1jvn has larger channeling rate, because the starting point is more buried (but still accessible for the probes ~ 0.5 A) .
  • Mutation influence is different for different probes. Maximal influence of mutations are the rate decrease by a factor of 1.4 forT78L (probe radius 0.4 A), 4.6 for T78L+S101I (probe radius 0.5 A), 1.7 for R5A (probe radius 0.7 A)








    • Electrostatic potentials at GLN and GLU binding sites

    Below: electrostatic potential computed for gln-AB structure and mapped onto the surface. GLN substrate (right) is not used in calculations, but put back for visualisation purposes. GLU substrate (left) is taken from glu-AB structure (superposed to gln-AB).

    Below: electrostatic potential computed for glu-AB structure and mapped onto the surface. GLU substrate is not used in calculations, but put back for visualisation purposes. GLN substrate is taken from gln-AB structure (superposed to glu-AB):

    From these images it is not clear why GLU is moved to its pocket, which is even more negative than GLN's pocket.

    From hydrogen donor/acceptor pairs analysis one can see that there is 1 hydrogen bond donor for GLU substrate in GLU pocket: NE2_Gln_A123 (at 3.4 A from OXT of GLU) and one potential donor ND1_His_B178 (at 3.43 A from OE2 of GLU, it is the only reason for blue spot in the GLU binding pocket).
    In the GLN pocket there are more donors for GLU : N_Gly_B52 (for OE2), NE2_GlnB88 (for OXT), N_ThrB142 (for O), N_Tyr_B143 (for OXT). I.e. from this viewpoint, GLN pocket is more favourable for glutamate than GLU pocket.

    I have done electrostatic binding free energy calculations for GLU bound to HisHF. GLU bound to GLN pocket of gln structure was modeled simply replacing NE2 to OE2 .
    Structure Chain Pocket Energy (van der Waals dielectric surface) (kcal/mole) Energy (connoly dielectric surface) (kcal/mole)
    gln AB GLN -3.6 +1.5
    EF GLN -1.9 +7.3
    glut AB GLU +2.8 +22.0
    CD GLU +3.7 +27.1

    Again, with any dielectric surface treatment, for glutamate, GLU pocket is less favourable than GLN pocket.


    Brownian dynamics simulation trajectories

    There are 2 trajectories shown as yellow and red taken from ca 1-2 ns long simulations. Trajectories start at the lower part





    R.Gabdoulline