Assembly of the type 1 procollagen molecule: Selectivity of the interactions between the α1(I)- and α2(I)-carboxyl propeptides

Assembly of the heterotrimeric procollagen I molecule is initiated by interactions between the carboxyl propeptide domains of the completed nascent pro α chains. The [pro α1(I)]₂[pro α2(I)] heterotrimer is the predominant molecule, with much smaller amounts of stable [pro α1(I)]₃ homotrimer also being formed. However, the [pro α2(I)]₃ homotrimer has not been detected, raising questions as to the mechanism of chain assembly and why [pro α2(I)]₃ homotrimers are not formed. These questions have been examined here by expressing the intact and amino- or carboxyl-terminal truncated C- propeptides of the pro α chains recombinantly in bacteria and in a coupled transcription/translation reticulocyte lysate system. Their interactions were studied in vitro by binding analyses and in vivo by using the yeast two- hybrid system. The C-pro αl(I) interacted with itself, and with C-pro α2(I), as expected. Surprisingly, the C-pro α2(I) also interacted with itself, both in vitro and in vivo. While the interaction of C-pro α2(I) with itself and C-pro α1(I) in vitro was strong, these interactions were weaker in vivo. Deletion of 36 amino acids from the C-terminal domain of C-pro α1 had no effect on its binding to intact self or intact C-pro α2, but the same deletion in C-pro α2 completely abolished its binding to intact C-pro α2 and to C-pro α1. Comparable N-terminal deletions in C-pro α1 or C-pro α2 diminished, but did not abolish, their binding to intact C-pro α1 and C-pro α2. In the yeast two-hybrid system, C-pro α2 interacted with itself more weakly than with C-pro α1. Molecular modeling and circular dichroism analyses showed that C-pro α1 and C-pro α2 have different folded structures and stability. Studies with antibodies specific to the C-pro α1 and α2 peptides showed them to precipitate different, specific, and distinct cell proteins from fibroblast lysates. The C-pro α2(I) antibody complexed with more cell proteins. We hypothesize that the lack of pro α2(I) homotrimers is not due to the inability of the C-pro α2(I) to interact with itself, but rather to the competing presence of other cell proteins. The specificity of these interactions may reside in conformational differences in N- and C- terminal sequences of the two propeptides or in their different folding patterns.