The binding of the gamma-glutamyl carboxylase to its protein substrates is mediated by a conserved 18 amino acid propeptide sequence found in all vitamin K-dependent proteins. We recently found that the apparent affinities of the naturally occurring propeptides for the carboxylase vary over a 100-fold range and that the propeptide of bone Gla protein has severely impaired affinity for the carboxylase [Stanley, T. B., et al. (1999) J. Biol. Chem. 274, 16940-16944 (1)]. Here we report a consensus propeptide sequence that binds tighter (K(i) = 0.43 nM) to the carboxylase than any known propeptide sequence. Comparing the factor IX propeptide to the propeptides of protein C, bone Gla protein, and prothrombin, the weakest binding propeptides, allowed us to predict which residues might be responsible for these substrates' relatively weak binding to the carboxylase. We then made propeptides with the predicted amino acid changes and determined their binding affinities. The reduced binding affinity of these propeptides relative to that of FIX is due to residues -15 in protein C, -10 and -6 in bone Gla protein, and -9 in prothrombin. A role for the -9 position was not previously recognized but is further shown by our identification of a new, naturally occurring mutation at this position in factor IX which causes a warfarin-sensitive hemophilia B phenotype. In addition, we find that propeptides with mutations found in warfarin-sensitive patients have reduced affinity for the carboxylase, suggesting a physiological relevance of propeptide binding affinity.