A single amino acid substitution within a coiled-coil motif changes the assembly of a 53-amino acid protein from two-dimensional sheets to filamentous structures.
The bacteriophage phi29 replication protein p1 self-interacts in vitro, generating highly ordered structures. Specifically, the 53-amino acid protein p1DeltaN33, which retains the sequence of p1 spanning amino acids Met(34) to Lys(85), assembles into two-dimensional protofilament sheets. The region of protein p1 located between residues Glu(38) and Asn(65) presumably forms an alpha-helical coiled-coil structure. Here we have examined the role of this coiled-coil sequence in the formation of protofilament sheets. Using sedimentation assays and negative-stain electron microscopy analysis, we demonstrate that residues Leu(46), Met(53), and Leu(60), but not Leu(39), are essential for p1DeltaN33 assembly into sheets. Remarkably, replacement of Leu(46) by Val shifts the pathway of molecular assembly, leading to the formation of filamentous polymers approximately 10 nm in diameter. These results show, for the first time, that a short coiled-coil motif can mediate protein assembly into protofilament sheet structures.
Centro de Biologia Molecular Severo Ochoa (Consejo Superior de Investigaciones Cientificas-Universidad Autónoma de Madrid (SIC-UAM)), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
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Bravo
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Serrano-Heras
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D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001407:Bacillus / Q000821:virology; D017103:Bacillus Phages / Q000235:genetics; D015951:Factor Xa / Q000378:metabolism; D008239:Lysine; D008715:Methionine; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000648:ultrastructure; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure*
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C060516:phage phi29 protein p1; D008715:Methionine; D015951:Factor Xa; D008239:Lysine