Molecular basis of coiled-coil formation

Abstract

Coiled coils have attracted considerable interest as design templates in a wide range of applications. Successful coiled-coil design strategies therefore require a detailed understanding of coiled-coil folding. One common feature shared by coiled coils is the presence of a short autonomous helical folding unit, termed ''trigger sequence,'' that is indispensable for folding. Detailed knowledge of trigger sequences at the molecular level is thus key to a general understanding of coiled-coil formation. Using a multidisciplinary approach, we identify and characterize here the molecular determinants that specify the helical conformation of the monomeric early folding intermediate of the GCN4 coiled coil. We demonstrate that a network of hydrogen-bonding and electrostatic interactions stabilize the trigger-sequence helix. This network is rearranged in the final dimeric coiled-coil structure, and its destabilization significantly slows down GCN4 leucine zipper folding. Our findings provide a general explanation for the molecular mechanism of coiled-coil formation.autonomous folding unit ͉ protein folding ͉ trigger sequence ͉ leucine zipper ͉ ␣-helix A lthough coiled coils have been used traditionally as model systems for protein folding studies, the molecular basis of how they fold is still largely unknown. A need for a detailed understanding of coiled-coil folding is relevant, particularly in light of the important functions these structures play in almost all biological processes, as well as the considerable attention coiled coils have recently garnered in a wide range of applications, including basic research, nanomaterials, protein engineering, biotechnology, and medicine (1-13). Furthermore, understanding the folding mechanisms of coiled coils is of fundamental interest to experimentalists and theoreticians challenged by the question of how the sequence of a protein defines its specific 3D structure. Along these lines, we have been interested particularly in coiled-coil ''trigger sequences,'' which encode stable monomeric ␣-helices that are indispensable for coiled-coil formation (14-18). Although there are a few examples of synthetic peptides that fold either into heterodimers (19) or at conditions of extremes of pH (20) without an apparent trigger sequence, the ''trigger site'' concept is generally accepted because these short autonomous helical folding units are structurally and functionally conserved in a large number of native coiled-coil proteins (reviewed in refs. 21-24). Detailed knowledge of the properties of trigger sequences at the molecular level is therefore key to an understanding of coiled-coil formation and function in general.The parallel two-stranded leucine zipper of the yeast transcriptional activator GCN4 is the best-characterized coiled coil and thus represents an excellent paradigm for a comprehensive analysis of the roles of trigger sequences in coiled-coil folding. Our current understanding of the folding kinetics of the GCN4 leucine zipper is based on extensive stopped-flow studies and te...

The tripartite motif coiled-coil is an elongated antiparallel hairpin dimer

“…3C, gray symbols). These results confirm that the center of the coiled-coil helix is critical for proper folding of the TRIM25 dimer, perhaps making up the "trigger site" that directs coiled-coil formation (23)(24)(25).…”

“…Furthermore, the distinctive 7-7-7-7-11-11-11-11-7-7-7-7 pattern of heptad and hendecad repeats in the TRIM25 coiled-coil appears to be conserved, and we speculate that it may be a "signature" of the TRIM family. Studies of dimeric coiled-coils have shown that short sequence elements or "trigger sites" of 7-14 amino acids are critically important for proper folding because they are the first segments to become helical, and therefore nucleate dimerization (23)(24)(25). Once the initial dimer contact is established, the peripheral residues then "zip up" to form the fully folded coiled-coil.…”

Probing the Interaction between the Coiled Coil Leucine Zipper of cGMP-dependent Protein Kinase Iα and the C Terminus of the Myosin Binding Subunit of the Myosin Light Chain Phosphatase

“…These studies identified that PKG-I␣ 1- in the PKG-I␣ and MBS interaction because the PKG-I␤ isoform does not interact with MBS (50,51). The hydrophobic residues that are conformationally affected upon complex formation, Leu 22 , Ile 33 , Leu 36 , and Leu 40 , are localized within the a/d hydrophobic core of the heptad repeat and are shared by both PKG-I␣ and PKG-I␤ supporting a structural role for these residues, a notion that has recently been supported by the extensive structural studies of GCN4 and LZ variants (52,53). In addition, the interaction interface of PKG-I␣ 1-59 ⅐MBS CT42 contains two charged lysines (Lys 37 and Lys 39 ) that are in the e and g positions of the fourth heptad repeat of PKG-I␣ 1-59 , respectively.…”

Unique Features of the Anti-parallel, Heterodimeric Coiled-coil Interaction between Methyl-cytosine Binding Domain 2 (MBD2) Homologues and GATA Zinc Finger Domain Containing 2A (GATAD2A/p66α)

“…A coiled-coil domain can be identified by a regular seven amino acid (a-g) repeat of hydrophobic and charged residues (5,6). In this heptad repeat, a branched hydrophobic residue is present at a and d positions, whereas charged/polar residues are present at e and g. These seven residues form approximately two turns of a typical ␣-helix, generating a hydrophobic face (a and d) bordered by charged/polar residues (e and g).…”