Assembly of the type 1 procollagen molecule: Selectivity of the interactions between the alpha 1(I)- and alpha 2(I)-carboxyl propeptides

Assembly of the heterotrimeric procollagen I molecule is initiated by inter actions between the carboxyl propeptide domains of the completed nascent pr o alpha chains. The [pro alpha 1(I)](2)[pro alpha 2(I)] heterotrimer is the predominant molecule, with much smaller amounts of stable [pro alpha 1(I)] (3) homotrimer also being formed, However, the [pro alpha 2(1)](3) homotrim er has not been detected, raising questions as to the mechanism of chain as sembly and why [pro alpha 2(1)](3) homotrimers are not formed. These questi ons have been examined here by expressing the intact and amino- or carboxyl -terminal truncated C-propeptides of the pro alpha chains recombinantly in bacteria and in a coupled transcription/translation reticulocyte lysate sys tem. Their interactions were studied in vitro by binding analyses and in vi vo by using the yeast two-hybrid system. The C-pro alpha 1(1) interacted wi th itself, and with C-pro alpha 2(I), as expected. Surprisingly, the C-pro alpha 2(I) also interacted with itself, both in vitro and in vive. While th e interaction of C-pro alpha 2(I) with itself and C-pro alpha 1(I) in vitro was strong, these interactions were weaker in vivo. Deletion of 36 amino a cids from the C-terminal domain of C-pro alpha 1 had no effect on its bindi ng to intact self or intact C-pro alpha 2, but the same deletion in C-pro a lpha 2 completely abolished its binding to intact C-pro alpha 2 and to C-pr o alpha 1. Comparable N-terminal deletions in C-pro alpha 1 or C-pro alpha 2 diminished, but did not abolish, their binding to intact C-pro alpha 1 an d C-pro alpha 2, In the yeast two-hybrid system, C-pro alpha 2 interacted w ith itself more weakly than with C-pro alpha 1. Molecular modeling and circ ular dichroism analyses showed that C-pro alpha 1 and C-pro alpha 2 have di fferent folded structures and stability. Studies with antibodies specific t o the C-pro alpha 1 and alpha 2 peptides showed them to precipitate differe nt, specific. and distinct cell proteins from fibroblast lysates. The C-pro alpha 2(I) antibody complexed with more cell proteins. We hypothesize that the lack of pro alpha 2(I) homotrimers is not due to the inability of the C-pro alpha 2(I) to interact with itself, but rather to the competing prese nce of other cell proteins. The specificity of these interactions may resid e in conformational differences in N- and C-terminal sequences of the two p ropeptides or in their different folding patterns.