Computational prediction of atomic structures of helical membrane proteins aided by EM maps

Abstract

Integral membrane proteins pose a major challenge for protein-structure prediction because only approximately 100 high-resolution structures are available currently, thereby impeding the development of rules or empirical potentials to predict the packing of transmembrane alpha-helices. However, when an intermediate-resolution electron microscopy (EM) map is available, it can be used to provide restraints which, in combination with a suitable computational protocol, make structure prediction feasible. In this work we present such a protocol, which proceeds in three stages: 1), generation of an ensemble of alpha-helices by flexible fitting into each of the density rods in the low-resolution EM map, spanning a range of rotational angles around the main helical axes and translational shifts along the density rods; 2), fast optimization of side chains and scoring of the resulting conformations; and 3), refinement of the lowest-scoring conformations with internal coordinate mechanics, by optimizing the van der Waals, electrostatics, hydrogen bonding, torsional, and solvation energy contributions. In addition, our method implements a penalty term through a so-called tethering map, derived from the EM map, which restrains the positions of the alpha-helices. The protocol was validated on three test cases: GpA, KcsA, and MscL.

FIGURE 1

Schematic flow chart of our prediction methodology.

FIGURE 2

Plots displaying the 20% (12,700) lowest-scoring conformations that were energy-minimized during the predictions of (a) GpA, (b) KcsA, and (c) MscL. RMSD values, in Å, refer to the backbone atoms. Energy values are in kcal/mol. A logarithmic scale was used on the energy axis to see more detail of the low-energy conformations. In each case, the origin of the energy scale has been set at 25 kcal/mol lower than the minimum energy. The numbers indicate the rankings of the indicated solutions (see text). On the lower right of each plot, the (minimized) energy of each experimental structure is shown. Due to unresolvable clashes, there are many low-RMSD conformations that span a wide range of energy values, especially in cases b and c.

FIGURE 3

GpA. (a) Isocontour surface of the simulated density map obtained from the NMR structure. (b) Tethering map, used as a restraint during the minimization stage. (c) Solvent-accessibility map, contoured at level 0.8. Displayed for reference are the two TM helices. (d) Side view and (e) top view of the dimer. The NMR structure is in blue, and our prediction (lowest-energy conformation) is in red. The backbone RMSD of this prediction is 0.89 Å with respect to the NMR structure. (f) Closeup of a helix-packing region near the center, where the fit of side chains is close to the NMR structure. (g) Closeup of a region facing the lipid, where, due to lack of packing constraints, the predicted side chains deviated from the NMR structure. Panel heights are: first row, 56 Å; second row, 50 Å; and third row, 12 Å.

FIGURE 4

KcsA. (a) Isocontour surface of the simulated density map obtained from the x-ray structure, including the loops and pore helices. (b) Tethering map, used as a restraint during the minimization stage. (c) Solvent-accessibility map, contoured at level 0.8. Displayed for reference are two TM helices of a subunit. (d) Side view of a subunit and (e) top view of the tetramer. The crystal structure is in blue, and our prediction (lowest-energy conformation) is in red. The backbone RMSD of this prediction is 1.59 Å with respect to the crystal structure. (f) Closeup of a helix-helix interface where the predicted side-chain packing is close to the crystal structure. (g) Closeup of a region facing the lipid, where, due to lack of packing constraints, the predicted side chains deviated from the crystal structure. Note that the fourfold symmetry of the side chains is almost perfect, even though it was not imposed. Panel heights are: first row, 80 Å; second row, 70 Å; and third row, 12 Å.

FIGURE 5

MscL. (a) Isocontour surface of the simulated density map obtained from the x-ray structure. (b) Tethering map, used as a restraint during the minimization stage. (c) Solvent-accessibility map, contoured at level 0.8. Displayed for reference are two TM helices of a subunit. (d) Side view of a subunit and (e) top view of the pentamer. The crystal structure is in blue, and our prediction (lowest-energy conformation) is in red. The backbone RMSD of this prediction is 1.88 Å with respect to the crystal structure. (f) Closeup of a helix-helix interface where the predicted side-chain packing is close to the crystal structure. (g) Closeup of a portion of the shorter helix, for which the prediction exhibits a screw motion along the backbone relative to the crystal structure, but in such a way that there is an approximate substitution of side chains. The fivefold symmetry of the side chains is nearly perfect. Panel heights are: first row, 60 Å; second row, 60 Å; and third row, 12 Å.