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 phi 29 replication protein pi self-interacts in vitro, ge nerating highly ordered structures. Specifically, the 53-amino acid protein p1 Delta N33, which retains the sequence of p1 spanning amino acids Met(34 ) to Lys(85), assembles into two-dimensional protofilament sheets. The regi on of protein p1 located between residues Glu(38) and Asn(65) presumably fo rms an alpha -helical coiled-coil structure. Here we have examined the role of this coiled-coil sequence in the formation of protofilament sheets. Usi ng 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 p1 Delta N33 assembly into sheets. Remarkably, replacemen t of Leu(46) by Val shifts the pathway of molecular assembly, leading to th e formation of filamentous polymers similar to 10 nn in diameter. These res ults show, for the first time, that a short coiled-coil moth can mediate pr otein assembly into protofilament sheet structures.