The Ca2+-dependent interaction between complement serine proteases C1r and C1s is mediated by their alpha regions, encompassing the major part of their N-terminal CUB-EGF-CUB (where EGF is epidermal growth factor) module array. In order to define the boundaries of the C1r domain(s) responsible for Ca2+ binding and Ca2+-dependent interaction with C1s and to assess the contribution of individual modules to these functions, the CUB, EGF, and CUB-EGF fragments were expressed in eucaryotic systems or synthesized chemically. Gel filtration studies, as well as measurements of intrinsic Tyr fluorescence, provided evidence that the CUB-EGF pair adopts a more compact conformation in the presence of Ca2+. Ca2+-dependent interaction of intact C1r with C1s was studied using surface plasmon resonance spectroscopy, yielding KD values of 10.9-29.7 nM. The C1r CUB-EGF pair bound immobilized C1s with a higher KD (1.5-1.8 microM), which decreased to 31.4 nM when CUB-EGF was used as the immobilized ligand and C1s was free. Half-maximal binding was obtained at comparable Ca2+ concentrations ranging from 5 microM with intact C1r to 10-16 microM for C1ralpha and CUB-EGF. The isolated CUB and EGF fragments or a CUB + EGF mixture did not bind C1s. These data demonstrate that the C1r CUB-EGF module pair (residues 1-175) is the minimal segment required for high affinity Ca2+ binding and Ca2+-dependent interaction with C1s and indicate that Ca2+ binding induces a more compact folding of the CUB-EGF pair.