Preferred side-chain constellations at antiparallel coiled-coil interfaces

Abstract

Reliable predictive rules that relate protein sequence to structure would facilitate postgenome predictive biology and the engineering and de novo design of peptides and proteins. Through a combination of experiment and analysis of the protein data bank (PDB), we have deciphered and rationalized new rules for helix-helix interfaces of a common protein-folding and association motif, the antiparallel dimeric coiled coil. These interfaces are defined by a specific pattern of interactions among largely hydrophobic side chains often referred to as knobs-into-holes (KIH) packing: a knob from one helix inserts into a hole formed by four residues on the partner. Previous work has focused on lateral interactions within the KIH motif, for example, between an a position on one helix and a d' position on the other in an antiparallel coiled coil. We show that vertical interactions within the KIH motif, such as a'-a-a', are energetically important as well. The experimental and database analyses concur regarding preferred vertical combinations, which can be rationalized as leading to favorable side-chain interactions that we call constellations. The findings presented here highlight an unanticipated level of complexity in coiled-coil interactions, and our analysis of a few specific constellations illustrates a general, multipronged approach to addressing this complexity.

Fig. 1.

Knobs-into-hole packing at antiparallel coiled-coil interfaces. (a and b) Orthogonal views of a knob (light gray) into hole (dark gray) interaction observed in an experimentally determined protein structure (residues 411 and 58–65 of PDB ID code 2ic6). (c) Diagram illustration of b with the heptad register assignment superimposed. Lateral interactions of the knob residue are indicated with red arrows, and vertical interactions with blue arrows.

Fig. 2.

Thioester model system. (a) Design and sequence of N_T-C; Succ = N-terminal succinyl group. Residues a/d′/a′ correspond to mutations sites. (b) Thioester exchange process for N_T-C. The thioester-thiol pair on the left comprises N- (blue) and C-terminal (red) segments, whereas the pair on the right contains the full-length coiled coil and a small thiol.

Fig. 3.

Coiled-coil interactions in thioester model system. (a) Helical-wheel diagram showing the helical regions of N_T-C. (b) Partial helical net for N_T-C. In each diagram, the N-terminal segment is shown in blue and the C-terminal segment is shown in red. Note that the numbering of the mutation sites is different from that in ref. .

Fig. 4.

Partial helical-net diagrams for N_T-C and three mutants used to calculate the discrimination energy (DE). The reported DE value was derived from the thermodynamic information in Table 1.

Fig. 5.

Oakley model system. (a) Helical-wheel diagram showing the helical regions of Oakley's heterodimeric antiparallel coiled coil (25). (b) Partial helical net for the peptides shown in a. In each diagram, the “acid” peptide is shown in blue and the “base” peptide is shown in red. The boxes highlight interactions discussed in the text.

Fig. 6.

Correlation of ΔG_fold/Urea for the Oakley acid–base mutations and ΔG_fold/TE for the corresponding mutations in N_T-C. The line corresponds to a linear regression fit (y = 3.9321x − 3.0308, r² = 0.855).

Fig. 7.

Superposition of structures culled from the PDB for vertical triads containing Ile and Leu in antiparallel two-helix coiled coils. Superpositions were performed by using the MMTSB toolset (41), and rendered in PYMOL (42). The PDB and associated chain and residue identifiers for these structures are given in SI Table 12. For each overlay, a representative backbone is shown to guide the eye (backbone rmsd = 0.33–0.86 Å for the four Ile-Leu combinations at a′aa′).

Fig. 8.

Significant discrimination energies DE_LI(X/Y) for d′ = Leu and d′ = Ala (see Fig. 4). Values were determined to be significant if DE ≥0.4 kcal/mol (two times the expected error). Positive values (blue) indicate a preference for X paired with leucine vertical contacts, whereas negative values (red) indicate a preference for X paired with isoleucine vertical contacts. Note that only one side of the diagonal (dotted line) is shown because DE_LI(X/Y) = −DE_LI(Y/X). These graphs are intended to show qualitatively that the residue at d′ can strongly influence vertical pairing preferences of the residue at a′. DE data are available in SI Tables 7–11.