Packing of apolar side chains enables accurate design of highly stable membrane proteins

Abstract

The features that stabilize the structures of membrane proteins remain poorly understood. Polar interactions contribute modestly, and the hydrophobic effect contributes little to the energetics of apolar side-chain packing in membranes. Disruption of steric packing can destabilize the native folds of membrane proteins, but is packing alone sufficient to drive folding in lipids? If so, then membrane proteins stabilized by this feature should be readily designed and structurally characterized-yet this has not been achieved. Through simulation of the natural protein phospholamban and redesign of variants, we define a steric packing code underlying its assembly. Synthetic membrane proteins designed using this code and stabilized entirely by apolar side chains conform to the intended fold. Although highly stable, the steric complementarity required for their folding is surprisingly stringent. Structural informatics shows that the designed packing motif recurs across the proteome, emphasizing a prominent role for precise apolar packing in membrane protein folding, stabilization, and evolution.

Fig. 1.. Noncovalent forces in MP folding.

(A) Polar interactions are known to stabilize MP structures. (B) vdW packing is abundant in the folded state, but similar interactions with membrane lipids occur in the unfolded state; it is unknown whether packing alone can drive MP folding. (C) Overview of the multipronged approach used.

Fig. 2.. All-atom MD simulation of PLN, a pentameric TM α-helical bundle, illuminates critical apolar packing interactions.

(A) Final simulation frame for full-length PLN in a POPC bilayer. Water molecules are hidden. (B) The TM domains of published NMR structures of PLN (PDB IDs: 1ZLL, red; 2KYV, blue) compared with our MD simulation. RMSF of backbone atoms versus the simulation medoid is displayed as ribbon color. (C) Snapshot showing the water-filled central cavity and rapidly fluctuating polar side-chain interactions at the splayed N-terminal third of the TM-spanning α helices. (D) Central cavity size within each helical bundle versus membrane depth. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

Fig. 3.. Design and structural characterization of PLN-like pentameric protein PL5.

(A) Sequence of PLN and PL5. PLN’s polar region (orange) and apolar region (yellow) highlighted; membrane-spanning α helix underlined (residues 24 to 52). The seven-residue α-helical repeat is labeled “abcdefg”; LxxIxxx motif, green. Red arrows indicate polar-to-apolar mutations. Bold Xs denote EtGly residues. (B) EtGly is approximately isosteric to Ser and Cys. (C) Snapshot from a 1.0-μs MD simulation of PL5 in a POPC bilayer. (D) PL5 shows conformational rigidity by C_α RMSD versus medoid frame (residues 5 to 29). (E) Equilibrium analytical ultracentrifugation, PL5 at 58 μM and 79 μM in 33 mM myristyl sulfobetaine micelles, globally fit to a single species model. Apparent molecular weight = 4.74 monomers. Pent., pentamer; Obs., observed. (F) SDS-PAGE of PL5 in reducing conditions shows a single oligomeric state, resistant to heating (95°C, 30 min) in 2% LDS, 8 M urea. Similar to PLN, PL5 exhibits aberrant gel migration. Figure S2 confirms that the slower band is pentameric. (G) PL5, PL5_EtG, and PL5_EtG3 have similar oligomeric distributions. (H) The pentameric x-ray structure of PL5 (PDB ID 6MQU) closely matches the MD-refined design model (cyan). (I) Well-packed side chains are well-resolved in the 2F_o-F_c electron density map (σ = 1.0). Elongated density is present only at divergent ends of the bundle.

Fig. 4.. Side-chain steric packing at PL5’s symmetric helix-helix interface.

(A) The pairwise interaction of helices, symmetrically repeated, provides the primary stabilization for PL5. (B) High geometric complementarity of interacting residues across the helix-helix interface, roughly in layers: the a/g and e/g layers. (C and D) Axial view of side-chain packing of individual layers. (E) A potential stereochemical code required for pentameric assembly. At the e/d layer, the C_α-C_β bond vector of each amino acid points outward from the helix-helix interface (β_out), whereas the C_β-C_γ bond vector faces inward (γ_in). This suggests that a heavy atom (e.g., N, C, O, S) at the gauche⁺ position is required for tight interhelical packing. (F) In the a/g layer, the opposite is true; the C_α-C_β bond vector points inward (β_in) and the C_β-C_γ bond vector faces outward (γ_out).

Fig. 5.. Design of pentameric MPs from first principles of steric packing.

(A) Helical wheel diagram of a pentameric bundle, the identity of the e position at the intersubunit interface. (B) Given the proposed steric code in Fig. 4E, Ile, Val, and Cys should facilitate favorable packing at position e, whereas Leu and Ala are expected to be unfavorable. (C) Aligned sequences of e-series peptides, PLN, and PL5. Leu is fixed at each g, whereas e is systematically varied. (D to F) SDS-PAGE of peptides under different detergent, temperature, and incubation conditions; the PL5_EtG3 peptide is abbreviated as Et₃. After incubating in the indicated micelle and temperature in the presence of 4 mM of reducing agent tris(2-carboxyethyl)phosphine (TCEP), 2 μg was loaded. After heating, fresh TCEP was added before electrophoresis to 4 mM final. EQ, equilibrium. (G and H) The pentameric x-ray crystal structure of the mini e-Val peptide (1.9-Å resolution, gold). The 2F_o-F_c electron density map (σ = 1.5) highlights layers of e/g (G) and a/g (H) packing layers in the central LxxIVxL repeat; Val at e, blue.

Fig. 6.. Helix-helix packing motifs stabilizing the designed TM bundles are common across the membrane proteome.

(A) Adjacent helices that make up the repeated helix-helix interface in mini e-Val were decomposed into successive 18-residue fragments. Backbone atoms were used to search for close structural matches (<0.85 Å RMSD) within a nonredundant database of MP experimental structures. (B) The helix-helix geometries in mini e-Val are found frequently in nature, within MPs of diverse architectures and functions. GPCR, G protein–coupled receptor. (C) Amino acids enriched in the fragments (P < 0.05) at equivalent position in e-Val are plotted in WebLogo format (55), with asterisks denoting amino acids enriched at >3 standard deviations (P < 0.003). Steric bulk of enriched amino acids at d, e, and g are similar to those in e-Val, and all are consistent with our proposed steric code. A total of nine positions have at least one amino acid enriched with P < 0.003. The binomial probability associated with finding even one position with this level of enrichment is 0.03, whereas the probability for finding nine positions thusly enriched is 1.1 × 10⁻²¹.