Structure-based prediction of the stability of transmembrane helix-helix interactions: the sequence dependence of glycophorin A dimerization

Abstract

The ability to predict the effects of point mutations on the interaction of alpha-helices within membranes would represent a significant step toward understanding the folding and stability of membrane proteins. We use structure-based empirical parameters representing steric clashes, favorable van der Waals interactions, and restrictions of side-chain rotamer freedom to explain the relative dimerization propensities of 105 hydrophobic single-point mutants of the glycophorin A (GpA) transmembrane domain. Although the structure at the dimer interface is critical to our model, changes in side-chain hydrophobicity are uncorrelated with dimer stability, indicating that the hydrophobic effect does not influence transmembrane helix-helix association. Our model provides insights into the compensatory effects of multiple mutations and shows that helix-helix interactions dominate the formation of specific structures.

Figure 1

Possible states for a peptide containing a stretch of approximately 18 hydrophobic residues. Water-solvated, unfolded molecules (A) may be inserted in the membrane as α-helices (B) that in turn may associate to form higher-order structures (C).

Figure 2

Thermodynamic cycle for point mutation of a dimerizing transmembrane α-helix. Red arrows indicate the change in association state (monomer to dimer), and blue arrows indicate that one residue is being mutated (triangle to circle). The change in free energy of dimerization caused by a mutation, ΔΔG_mut, may be obtained from experimental data (ΔG₂ − ΔG₁) or by computation (ΔG₄ − ΔG₃).

Figure 3

Correlation table for model III: calculated and experimental dimerization propensities. Binning of the data by experimental (i) and calculated (j) stability enables direct comparison of the scores as integers, where 0 corresponds to no dimerization and 3 corresponds to wild-type dimerization. Model III scores 75 of 105 mutants correctly; the clustering of the data at or near the diagonal is reflected in the R_grp² of 0.760 (see Eq. 2).

Figure 4

Components contributing to calc for six mutants of GpA, with weightings given by Eq. 4, and comparison with experiment. Values for expt are reported relative to the best-fit constant term, 2.7, so that the difference between calc and expt accurately reflects the model error. The series of mutations at Thr-87 shows that one, two, or all three parameters may contribute to reductions in stability. Terms may also offset one another. Substitution Val-80 → Leu results in superior packing (vdw) for one of the available leucine rotamers, but because the other rotamers clash, this substitution incurs a large cost in side-chain rotamer freedom (dsrot). By contrast, the parameter dsrot has a stabilizing effect on the mutant Leu-75 → Ala because the wild-type residue loses rotamer freedom upon dimerization, whereas the mutant side chain does not. This partially offsets the loss of favorable packing (vdw) resulting from this sequence change. Mutation Ile-76 → Leu achieves superior packing to the wild-type interface by rearrangement of the side chain of Leu-75, but this gain is offset by the cost of excluding a side-chain rotamer. The agreement of calc with expt is representative of the fit of the model; the mean squared error is 0.27 for model III and 0.25 for these six examples.

Figure 5

Correlation of calculated dimerization propensity and free energy of dissociation for wild-type GpA and two point mutants. Values for calc, using Eq. 4, are strongly correlated with the free energies of dissociation, ΔG_diss, reported in ref. . The squared correlation coefficient, R², is 0.997 and the slope is 1.04 (±0.03) kcal⁻¹.

Figure 6

Sequences, inferred structures, and calculated and experimental dimer stabilities for insertion mutants of GpA. Sequences of GpA fusion proteins from ref. are aligned by using Thr-87. Inserted or mutated residues are in red; residues aligned with the motif are in large type. Numbering of residues in the panels corresponds to their positions in the motif alignment, not to numbering in the construct. Values for calc and ΔG_app values are computed by using Eq. 4 and 5, respectively. The six panels show the packing at the interface resulting from building these sequences into the GpA wild-type backbone geometry. Molecular surfaces are displayed for wild type, 3A, 4A, and 4A(G79L), where one monomer approaches within 1.5 Å of the other and serve to depict the close packing across the interface. For sequences 1A and 2A, clashes are created upon building in the residues, and the surfaces serve to highlight these steric collisions. (wildtype) The wild-type residues Leu-75, Ile-76, Gly-79, and Val-80 exhibit excellent intermonomer packing. (1A) Introducing Val at position 79 of the motif generates a steric clash. (2A) Either Met at position 79 or Ile at position 75 will clash with any rotamer of the Phe at position 76. (3A) Four weakly disruptive single mutations are placed into motif positions, but our model indicates that these should have compensatory interactions with one another. The Ala at position 79 would clash slightly in the wild-type sequence as described previously (14), but having Ala replace Val at position 80 eliminates this clash and generates good packing. The Phe side chain of position 75 would need to swing away from the interface to avoid a clash in a wild-type context, but the space afforded by the Gly at position 76 enables close packing between the Phe side chain and the backbone of the opposite monomer. (4A) Interactions between Ala residues at 79 and 80 are favorable, as for 3A. Placing Val in position 76 reintroduces the favorable interactions obtained in the wild type by having a β-branched residue at the dyad. (4A(G79L)) As 4A, except that a (wild-type) Leu is placed at position 75 in a new geometry. The rank order of the calculated and predicted dimerization propensities compare well, showing that the stabilities of these mutants can be predicted by using the same rules as were used for the single-point mutants.

Figure 7

Correlation of calculated dimerization propensity and apparent free energy of dissociation for wild type and 33 multiple mutants of GpA. Values for calc, using Eq. 4, are shown to be strongly correlated with the apparent free energies of dissociation, ΔG_app, computed by using Eq. 5 and data from refs. and 23). Excluding the outlier, the squared correlation coefficient, R², is 0.845 and the slope is 0.85 (±0.07) kcal⁻¹. Triangles indicate data points corresponding to sequences depicted in Fig. 6.