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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 φ29 replication protein p1 self-interacts in vitro, generating highly ordered structures. Specifically, the 53-amino acid protein p1ΔN33, which retains the sequence of p1 spanning amino acids Met34 to Lys85, assembles into two-dimensional protofilament sheets. The region of protein p1 located between residues Glu38 and Asn65 presumably forms an α-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 Leu46, Met53, and Leu60, but not Leu39, are essential for p1ΔN33 assembly into sheets. Remarkably, replacement of Leu46 by Val shifts the pathway of molecular assembly, leading to the formation of filamentous polymers ∼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.