An autonomous folding unit mediates the assembly of two-stranded coiled coils.

Subunit oligomerization of many proteins is mediated by coiled-coil domains. Although the basic features contributing to the thermodynamic stability of coiled coils are well understood, the mechanistic details of their assembly have not yet been dissected. Here we report a 13-residue sequence pattern that occurs with limited sequence variations in many two-stranded coiled coils and that is absolutely required for the assembly of the Dictyostelium discoideum actin-bundling protein cortexillin I and the yeast transcriptional activator GCN4. The functional relationship between coiled-coil "trigger" sequences was manifested by replacing the intrinsic trigger motif of GCN4 with the related sequence from cortexillin I. We demonstrate that these trigger sequences represent autonomous helical folding units that, in contrast to arbitrarily chosen heptad repeats, can mediate coiled-coil formation. Aside from being of general interest for protein folding, trigger motifs should be of particular importance in the protein de novo design.

[1]  P S Kim,et al.  Subdomain folding of the coiled coil leucine zipper from the bZIP transcriptional activator GCN4. , 1994, Biochemistry.

[2]  James C. Hu,et al.  Sequence requirements for coiled-coils: analysis with lambda repressor-GCN4 leucine zipper fusions. , 1990, Science.

[3]  A. Mackay,et al.  Crystals of glutamine synthetase from Escherichia coli. , 1975, Journal of molecular biology.

[4]  R. Hodges,et al.  Demonstration of Coiled-Coil Interactions within the Kinesin Neck Region Using Synthetic Peptides , 1997, The Journal of Biological Chemistry.

[5]  C. Matthews,et al.  Probing the folding mechanism of a leucine zipper peptide by stopped-flow circular dichroism spectroscopy. , 1995, Biochemistry.

[6]  V. Muñoz,et al.  Submillisecond kinetics of protein folding. , 1997, Current opinion in structural biology.

[7]  D. Parry,et al.  α‐Helical coiled coils and bundles: How to design an α‐helical protein , 1990 .

[8]  R. Hodges.  Boehringer Mannheim award lecture 1995. La conference Boehringer Mannheim 1995. De novo design of alpha-helical proteins: basic research to medical applications. , 1996, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[9]  M. Steinmetz,et al.  Cortexillins, Major Determinants of Cell Shape and Size, Are Actin-Bundling Proteins with a Parallel Coiled-Coil Tail , 1996, Cell.

[10]  R. Hodges,et al.  Amino-acid sequence of rabbit skeletal tropomyosin and its coiled-coil structure. , 1972, Proceedings of the National Academy of Sciences of the United States of America.

[11]  H. Gutfreund,et al.  Physical Biochemistry , 1970, Nature.

[12]  L. Mueller,et al.  Characterization of a new four‐chain coiled‐coil: Influence of chain length on stability , 1995, Protein science : a publication of the Protein Society.

[13]  P. S. Kim,et al.  The C-peptide helix from ribonuclease A considered as an autonomous folding unit. , 1987, Cold Spring Harbor symposia on quantitative biology.

[14]  W. DeGrado,et al.  Protein design, a minimalist approach. , 1989, Science.

[15]  R. Kammerer,et al.  Native chick laminin-4 containing the beta 2 chain (s-laminin) promotes motor axon growth , 1996, The Journal of cell biology.

[16]  Structural stability of short subsequences of the tropomyosin chain , 1995, Biopolymers.

[17]  M. Steinmetz,et al.  A distinct 14 residue site triggers coiled‐coil formation in cortexillin I , 1998, The EMBO journal.

[18]  Tobin R. Sosnick,et al.  The role of helix formation in the folding of a fully α‐helical coiled coil , 1996 .

[19]  A Wlodawer,et al.  Structure of ribonuclease A: results of joint neutron and X-ray refinement at 2.0-A resolution. , 1982, Biochemistry.

[20]  Y H Chen,et al.  Determination of the helix and beta form of proteins in aqueous solution by circular dichroism. , 1974, Biochemistry.

[21]  H. Sambrook.  Molecular cloning : a laboratory manual. Cold Spring Harbor, NY , 1989 .

[22]  P. S. Kim,et al.  X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil. , 1991, Science.

[23]  A. Holtzer,et al.  Thermal unfolding in a GCN4-like leucine zipper: 13C alpha NMR chemical shifts and local unfolding curves. , 1997, Biophysical journal.

[24]  A. Lupas.  Coiled coils: new structures and new functions. , 1996, Trends in biochemical sciences.

[25]  H. Sweeney,et al.  Spare the rod, spoil the regulation: necessity for a myosin rod. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[26]  D. Woolfson,et al.  Buried polar residues and structural specificity in the GCN4 leucine zipper , 1996, Nature Structural Biology.

[27]  C. Vinson,et al.  Leucine is the most stabilizing aliphatic amino acid in the d position of a dimeric leucine zipper coiled coil. , 1997, Biochemistry.

[28]  A. Lupas,et al.  Predicting coiled coils from protein sequences , 1991, Science.

[29]  F. Crick,et al.  The packing of α‐helices: simple coiled‐coils , 1953 .

[30]  Terrence G. Oas,et al.  The energy landscape of a fast-folding protein mapped by Ala→Gly Substitutions , 1997, Nature Structural Biology.

[31]  L Serrano,et al.  Favourable native-like helical local interactions can accelerate protein folding. , 1997, Folding & design.

[32]  C. Mant,et al.  Protein design using model synthetic peptides. , 1988, Peptide research.

[33]  R. L. Baldwin,et al.  Helix stabilization by Glu-...Lys+ salt bridges in short peptides of de novo design. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[34]  A Kolinski,et al.  Prediction of the folding pathways and structure of the GCN4 leucine zipper. , 1994, Journal of molecular biology.

[35]  C. Mant,et al.  α-Helical Protein Assembly Motifs* , 1997, The Journal of Biological Chemistry.

[36]  P. S. Kim,et al.  A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants. , 1993, Science.

[37]  R. Hodges,et al.  Effect of chain length on the formation and stability of synthetic alpha-helical coiled coils. , 1994, Biochemistry.

[38]  Luis Serrano,et al.  Elucidating the folding problem of helical peptides using empirical parameters , 1994, Nature Structural Biology.

[39]  R. Kammerer,et al.  Tenascin-C Hexabrachion Assembly Is a Sequential Two-step Process Initiated by Coiled-coil α-Helices* , 1998, The Journal of Biological Chemistry.

[40]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[41]  C. Kurland,et al.  Co-variation of tRNA abundance and codon usage in Escherichia coli at different growth rates. , 1996, Journal of molecular biology.

[42]  A. Mclachlan,et al.  Tropomyosin coiled-coil interactions: evidence for an unstaggered structure. , 1975, Journal of molecular biology.