Investigating the tolerance of coiled-coil peptides to nonheptad sequence inserts.

Coiled-coil motifs foster a wide variety of protein-protein interactions. Canonical coiled coils are based on 7-residue repeats, which guide the folding and assembly of amphipathic alpha-helices. In many cases such repeats remain unbroken for tens to hundreds of residues. However, the sequences of an increasing number of putative and characterised coiled coils digress from this pattern. We probed the consequences of nonheptad inserts using a designed leucine-zipper system. The parent peptide, SKIP0, which had four contiguous heptads, was confirmed as a parallel homodimer by circular dichroism spectroscopy and analytical ultracentrifugation. Seven daughter peptides were constructed in which 1 to 7 alanine residues were inserted between the central heptads of SKIP0. Like SKIP0, SKIP7 formed a stable helical dimer, but the other peptides were highly destabilised, with the order of dimer stability SKIP4 >> SKIP5 > SKIP6 > SKIP3 > SKIP2 > SKIP1. These results are consistent with an extended theory of coiled-coil assembly in which coiled-coil-compatible motifs are based on 3- and 4-residue spacings and most notably heptad (7-residue) and hendecad (11-residue) repeats. Thus, they help explain why in natural sequences, inserts after canonical heptad repeats most commonly comprise 4 residues. Possible biological roles for nonheptad inserts are discussed.

[1]  Derek N. Woolfson,et al.  Sticky-end assembly of a designed peptide fiber provides insight into protein fibrillogenesis. , 2000 .

[2]  R. L. Baldwin,et al.  Aromatic side-chain contribution to far-ultraviolet circular dichroism of helical peptides and its effect on measurement of helix propensities. , 1993, Biochemistry.

[3]  D. Woolfson,et al.  Predicting oligomerization states of coiled coils , 1995, Protein science : a publication of the Protein Society.

[4]  P. V. von Hippel,et al.  Calculation of protein extinction coefficients from amino acid sequence data. , 1989, Analytical biochemistry.

[5]  K. Lechtreck.  Analysis of striated fiber formation by recombinant SF-assemblin in vitro. , 1998, Journal of molecular biology.

[6]  D N Woolfson,et al.  Coiled-coil assembly by peptides with non-heptad sequence motifs. , 1997, Folding & design.

[7]  Thomas Kreis,et al.  Guidebook to cytoskeletal and motor proteins , 1999 .

[8]  J. Skehel,et al.  Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. , 2000, Annual review of biochemistry.

[9]  J Kuriyan,et al.  Crystal structure of the nucleotide exchange factor GrpE bound to the ATPase domain of the molecular chaperone DnaK. , 1997, Science.

[10]  D. Baker,et al.  Segmented alpha-helical coiled-coil structure of the protein giardin from the Giardia cytoskeleton. , 1988, Journal of molecular biology.

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

[12]  D V Holberton,et al.  Sequence and structure of a new coiled coil protein from a microtubule bundle in Giardia. , 1993, Journal of molecular biology.

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

[14]  W. Weissenhorn,et al.  Crystal structure of the Ebola virus membrane fusion subunit, GP2, from the envelope glycoprotein ectodomain. , 1998, Molecular cell.

[15]  C. Pace,et al.  How to measure and predict the molar absorption coefficient of a protein , 1995, Protein science : a publication of the Protein Society.

[16]  J. Janin,et al.  Surface and inside volumes in globular proteins , 1979, Nature.

[17]  J. Marshall,et al.  Giardia gene predicts a 183 kDa nucleotide-binding head-stalk protein. , 1995, Journal of cell science.

[18]  Arthur J. Rowe,et al.  Analytical ultracentrifugation in biochemistry and polymer science , 1992 .

[19]  J. Skehel,et al.  Structure of influenza haemagglutinin at the pH of membrane fusion , 1994, Nature.

[20]  D. Baker,et al.  Segmented α-helical coiled-coil structure of the protein giardin from the Giardia cytoskeleton , 1988 .

[21]  W Baumeister,et al.  Hyperthermostable surface layer protein tetrabrachion from the archaebacterium Staphylothermus marinus: evidence for the presence of a right-handed coiled coil derived from the primary structure. , 1996, Journal of molecular biology.

[22]  R. Ruigrok,et al.  Tetrameric coiled coil domain of Sendai virus phosphoprotein , 2000, Nature Structural Biology.

[23]  M L Johnson,et al.  Analysis of data from the analytical ultracentrifuge by nonlinear least-squares techniques. , 1981, Biophysical journal.

[24]  P. Lyu,et al.  CD Spectroscopy and the Helix-Coil Transition in Peptides and Polypeptides , 1996 .

[25]  A. Fersht,et al.  Quantitative determination of helical propensities from trifluoroethanol titration curves. , 1994, Biochemistry.

[26]  Lumb,et al.  A buried polar interaction imparts structural uniqueness in a designed heterodimeric coiled coil , 1998, Biochemistry.

[27]  D. Parry,et al.  Heptad breaks in α‐helical coiled coils: Stutters and stammers , 1996 .

[28]  D. Parry,et al.  Heptad breaks in alpha-helical coiled coils: stutters and stammers. , 1996, Proteins.

[29]  J Walshaw,et al.  Socket: a program for identifying and analysing coiled-coil motifs within protein structures. , 2001, Journal of molecular biology.

[30]  M. Melkonian,et al.  SF-assemblin, the structural protein of the 2-nm filaments from striated microtubule associated fibers of algal flagellar roots, forms a segmented coiled coil , 1993, The Journal of cell biology.

[31]  A. Dunker,et al.  Aromatic and Cystine Side-Chain Circular Dichroism in Proteins , 1996 .

[32]  P. S. Kim,et al.  High-resolution protein design with backbone freedom. , 1998, Science.

[33]  D N Woolfson,et al.  Sticky-end assembly of a designed peptide fiber provides insight into protein fibrillogenesis. , 2000, Biochemistry.

[34]  A. Lupas.  Prediction and analysis of coiled-coil structures. , 1996, Methods in enzymology.

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

[36]  P S Kim,et al.  Structural characterization of the human respiratory syncytial virus fusion protein core. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[37]  M. Melkonian,et al.  The cruciated microtubule-associated fibers of the green alga Dunaliella bioculata consist of a 31 kDa SF-assemblin. , 1996, Journal of cell science.

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

[39]  M. Mann,et al.  Analysis of the Saccharomyces Spindle Pole by Matrix-assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry , 1998, The Journal of cell biology.

[40]  G. Fasman.  Circular Dichroism and the Conformational Analysis of Biomolecules , 1996, Springer US.

[41]  R. Kammerer,et al.  Crystal structure of a naturally occurring parallel right-handed coiled coil tetramer , 2001, Nature Structural Biology.

[42]  T. N. Bhat,et al.  The Protein Data Bank , 2000, Nucleic Acids Res..

[43]  A. Lustig,et al.  Fibritin encoded by bacteriophage T4 gene wac has a parallel triple-stranded alpha-helical coiled-coil structure. , 1994, Journal of molecular biology.