A conserved trimerization motif controls the topology of short coiled coils

Abstract

In recent years, short coiled coils have been used for applications ranging from biomaterial to medical sciences. For many of these applications knowledge of the factors that control the topology of the engineered protein systems is essential. Here, we demonstrate that trimerization of short coiled coils is determined by a distinct structural motif that encompasses specific networks of surface salt bridges and optimal hydrophobic packing interactions. The motif is conserved among intracellular, extracellular, viral, and synthetic proteins and defines a universal molecular determinant for trimer formation of short coiled coils. In addition to being of particular interest for the biotechnological production of candidate therapeutic proteins, these findings may be of interest for viral drug development strategies.

Fig. 1.

X-ray crystal structure of ccCor1. Side view with the N terminus on top. Side chains and backbones are shown as sticks and cartoon representation, respectively. Oxygen and nitrogen atoms are colored in red and blue, and carbon atoms are shown in cyan, green, and yellow for monomers A, B, and C, respectively. Residues Arg-450–Glu-455 that form the structural motif are shown with van der Waals spheres. For the sequence of ccCor1 see Fig. 4.

Fig. 2.

Prominent structural motif seen in the ccCor1 trimer. (A) Side view of the salt-bridge network (indicated by yellow dots) formed between Arg-450, Asp-452′, and Glu-455′ and the water-mediated hydrogen bond between Glu-455′:Oε2 and Arg-450:O. (B) End-on view of the a3 layer showing the shielding of the Leu-451 residues from solvent by the aliphatic side-chain moieties of Arg-450. (C) End-on view of the d3 layer showing the hydrophobic packing between the Leu-454 and the aliphatic side-chain moieties of the Glu-455 residues. Side chains of residues are shown as stick representation and van der Waals spheres (B and C), the water molecule as a small red sphere (A), and the three monomers are shown as Cα traces. Colors of atoms and monomers are the same as in Fig. 1.

Fig. 3.

CD analysis of WT ccCor1 and variants. Far-UV CD spectra recorded at 5°C (A) and thermal unfolding profiles recorded by CD at 222 nm (B) of ccCor1 (•), ccCor1-R450A (○), ccCor1-R450Nle (□), and ccCor1-R450K (▵). The experiments were carried out at 35 μM peptide concentration (monomer) in PBS.

Fig. 4.

The ccCor1 sequence Arg-450–Glu-455 is conserved in intracellular (I), extracellular (II), transmembrane (III), viral (IV), and synthetic (V) proteins containing short three-stranded parallel coiled-coil domains. Sequence alignments of mouse coronin 1A (mCor-1A) with the coiled-coil domains of human heat shock factor-binding protein 1 (hHSBP1), chicken tenascin C (chTN-C), human matrilin-1 (hMat-1), human type XVII collagen a1 (hXVII), influenza hemagglutinin (HA2), HIV-1 envelope glycoprotein (HIVgp41), and the synthetic ccβ-p coiled coil are shown. Heptad repeats are shown in blocks of seven amino acids, and residues at positions a and d are underlined. The conserved residues forming the trimerization motif in ccCor1 (Fig. 2) are shown in bold and colored according to their physicochemical properties: blue, positively charged; red, negatively charged; green, hydrophobic. The deduced consensus sequence is shown at the bottom. For a full alignment, see Fig. 7.

Fig. 5.

The trimerization motif is structurally conserved. (A) Frequency of occurrence of the sequence pattern R-[ILVM]-X-X-[ILV]-E in a structurally verified set of short (≤50 amino acid residues) parallel (black bars) and antiparallel (gray bars) coiled coils. (B) Superimposition of trimerization motifs found in ccCor1 (red), simian immunodeficiency virus gp41 (blue; PDB ID code 1qbz), visna TM (yellow, PDB ID code 1jek), ccβ-p (magenta; PDB ID code 1s9z), p4 (cyan; PDB ID code 1kyc), and influenza hemagglutinin (salmon; PDB ID code 1eo8) is shown in stereo and in line representation.