The influence of amino acid protonation states on molecular dynamics simulations of the bacterial porin OmpF

Abstract

Several groups, including our own, have found molecular dynamics (MD) calculations to result in the size of the pore of an outer membrane bacterial porin, OmpF, to be reduced relative to its size in the x-ray crystal structure. At the narrowest portion of its pore, loop L3 was found to move toward the opposite face of the pore, resulting in decreasing the cross-section area by a factor of approximately 2. In an earlier work, we computed the protonation states of titratable residues for this system and obtained values different from those that had been used in previous MD simulations. Here, we show that MD simulations carried out with these recently computed protonation states accurately reproduce the cross-sectional area profile of the channel lumen in agreement with the x-ray structure. Our calculations include the investigation of the effect of assigning different protonation states to the one residue, D(127), whose protonation state could not be modeled in our earlier calculation. We found that both assumptions of charge states for D(127) reproduced the lumen size profile of the x-ray structure. We also found that the charged state of D(127) had a higher degree of hydration and it induced greater mobility of polar side chains in its vicinity, indicating that the apparent polarizability of the D(127) microenvironment is a function of the D(127) protonation state.

FIGURE 1

Schematic of the angular-sweep methodology that was used in conjugation with the HOLE (33) algorithm to determine accurate descriptions of SAXA of channel pores. See the “Calculating solvent accessible cross-sectional areas” section for details. The magnitude of as appears in the schematic is set large only for the purpose of visualization. It was set at a value of 0.02 radians for computation purposes.

FIGURE 2

MD backbone RMS fluctuations of OmpF simulated with D¹²⁷ in its charged and neutral states are compared with RMS fluctuations derived from Debye-Waller B-factors. The RMS fluctuations were calculated for all three monomers of each trimer and then averaged to obtain a single representative profile for each trimer. The B-factors derived from the crystal structure were converted into RMS fluctuations using the formula

FIGURE 3

Superimposed backbone structures of OmpF. The time-averaged MD structures of the trajectories generated with a charged D¹²⁷ (magenta) and with a neutral D¹²⁷ (green) are shown superimposed on the x-ray structure. Most of the loops have been clipped to clearly show the structures of the following loops: loops L3 in all trimers, loops L1 of monomer M1 in the x-ray structure and in the average structure of trajectory generated with a charged D¹²⁷, and loops L8 of monomer M3 in the x-ray structure and in the average structure of trajectory generated with a charged D¹²⁷. The backbone atoms of residue D¹²⁷ are highlighted in yellow. The backbone atoms of the PEFGGD fragments in loops L3 (tip of the loop), which were found to move toward the opposite face of the pore in all previous simulations, are highlighted in orange in the x-ray structure. This figure was created using PyMOL (http://pymol.sourceforge.net).

FIGURE 4

Variation of SAXA along the axis of four OmpF monomers: crystal structure, monomer representing the MD trajectory generated with a neutral D¹²⁷, monomer representing the MD trajectory generated with a charged D¹²⁷, and monomer representing the MD trajectory generated for a previous investigation with other charge assignments for the residues (12). The monomers used for generating SAXA profiles were obtained as follows. MD trajectories of the trimers simulated using different charge states of D¹²⁷ and for a previous investigation were first separately aligned to obtain their respective average trimer structures. The three monomers in each trimer were then separately superimposed to obtain average monomer structures for each trajectory. These two monomers were then superimposed onto the crystal structure to eliminate any rotational artifacts in SAXA profiles. The SAXA profiles were then calculated using HOLE (33) along with an angular-sweep algorithm (see Methods for details).

FIGURE 5

Partial view of the x-ray structure (9). Titratable residues E¹¹⁷, E²⁹⁶, and D³¹², which are involved in the interaction of the PEFGGD fragment of loop L3 with the wall of the β-barrel, are shown as stick models. The solid lines on the backbone of loop L3 indicate the ends of the PEFGGD fragment. A block arrow indicates the direction of the movement of this fragment that was seen in all previous simulations. This figure was created using PyMOL (http://pymol.sourceforge.net).

FIGURE 6

Local environment of residue D¹²⁷ as revealed by the x-ray structure (9). Residue names are indicated in black, and the atom names are indicated in gray. Carboxylate oxygen atoms OD1 and OD2 of D¹²⁷ are labeled as 1 and 2, respectively. Distances from these atoms to neighboring proton donors and acceptors, including the crystallographically resolved water oxygen atoms (labeled as H₂O), are indicated in angstrom units. This figure was created using RasMOL.

FIGURE 7

Time-dependent center of mass deviation from x-ray crystal structure of residue R¹⁶⁷ belonging to each of the three monomers (M1, M2, and M3) of the trajectories generated with (a) a charged D¹²⁷ and (b) a neutral D¹²⁷. These deviations were first computed at intervals of 5 ps, and then cubic spline interpolation was used to eliminate the noise in the data associated with rapid fluctuations.

FIGURE 8

Conformational space sampled by residues D¹²⁷ and R¹⁶⁷ in each monomer (M1, M2, and M3) of the trajectory generated with D¹²⁷ in its (a) charged state and (b) neutral state. The carbon atoms are shown in green, oxygen atoms in red, nitrogen atoms in blue, and the protons on the side-chain carboxylate groups in yellow. Note that the figures are drawn with slightly different perspectives to highlight the relative conformations of these residues. The backbone atoms of these residues essentially do not undergo any structural deviation (RMSD < 0.03 Å) and the apparent relative deviations are entirely due to side-chain reorientations. We find that the charged state of D¹²⁷ does better at preventing the side chain of residue R¹⁶⁷ from moving into the lumen of the channel. This figure were created using PyMOL (http://pymol.sourceforge.net).

FIGURE 9

Radial distribution of water around the two carboxylate oxygen atoms of residue D¹²⁷ calculated for the last nanosecond trajectory of both simulations: (a) around OD1 atom and (b) around OD2 atom. Radial distributions were first calculated separately for each monomer and then averaged to obtain single profiles representative of each MD trajectory.

FIGURE 10

Superimposed trajectories of six separate water molecules (colored spheres) that were found to adopt the nearest neighbor position to the OD1 atom of residue D¹²⁷ (drawn as a stick model) during any time of their respective trajectories. Residue D¹²⁷ shown here is charged and belongs to monomer M3 (traced in gray). All simulated water molecules were considered at 50 ps time intervals to identify these six water molecules. This figure was created using PyMOL (http://pymol.sourceforge.net).

FIGURE 11

Evolution of the local environment of D¹²⁷ in all the six monomers of the two trajectories. The distances of all potential proton donors and acceptors relative to the two carboxylate oxygen atoms of D¹²⁷, as indicated in Fig. 6, are shown as a function of simulation time. Horizontal lines represent the distance in the crystal structure. The monomers are labeled accordingly only if they are distinguishable from each other.

FIGURE 12

Average number of ions along the axis of the two trimers: (a) charged D¹²⁷ and (b) neutral D¹²⁷. Positions of the constriction zone and residue D¹²⁷ along the axis of the channel are shown. Irrespective of the protonation state of residue D¹²⁷, there are moderately greater numbers of potassium than chloride ions in the pore, corresponding to the observed selectivity for potassium over chloride conduction in this channel.