AbstractCombined deficiency of factor V and factor VIII (F5F8D) is a bleeding disorder caused by mutations in either LMAN1 or MCFD2. LMAN1 (ERGIC-53) and MCFD2 form a Ca2+-dependent cargo receptor that cycles between the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment for efficient transport of FV/FVIII from the ER to the Golgi. Here we show that the C-terminal EF-hand domains are both necessary and sufficient for MCFD2 to interact with LMAN1. MCFD2 with a deletion of the entire N-terminal non-EF hand region still retains the LMAN1-binding function. Deletions that disrupt core structure of the EF-hand domains abolish LMAN1 binding. Circular dichroism spectroscopy studies on missense mutations localized to different structural elements of the EF-hand domains suggest that Ca2+-induced folding is important for LMAN1 interaction. The EF-hand domains also mediate the interaction with FV and FVIII. However, mutations in MCFD2 that disrupt the tertiary structure and abolish LMAN1 binding still retain the FV/FVIII binding activities, suggesting that this interaction is independent of Ca2+-induced folding of the protein. Our results suggest that the EF-hand domains of MCFD2 contain separate binding sites for LMAN1 and FV/FVIII that are essential for cargo receptor formation and cargo loading in the ER.