Calcium enhances the proteolytic activity of BACE1: An in vitro biophysical and biochemical characterization of the BACE1–calcium interaction

BACE1 is a novel type I transmembrane aspartyl protease that has been implicated in the pathogenesis of Alzheimer's disease. Cleavage of the amyloid precursor protein by the β-secretase, BACE1, is the first step in the production of the Aβ peptide and is a prime target for therapeutic intervention. Using circular dichroism, we reveal that the secondary structure of BACE1 in a membrane environment is significantly different from what was determined from the previously resolved crystal structure, and, we provide the first evidence that show differences in stability between the active (pH 4.8) and inactive (pH 7.4) forms of BACE1. In this study we have also examined Ca2+ binding to BACE1, the effect of this binding on the secondary and tertiary structural characteristics of BACE1, and the influence of this binding on the specific activity of the purified protein. Circular dichroism and endogenous tryptophan fluorescence measurements demonstrated that the secondary and tertiary structures, respectively, are sensitive to increasing concentrations of Ca2+. Isothermal titration calorimetry was then used to characterize the Ca2+–BACE1 interaction in more detail. Our results suggest that there is a high affinity of binding (kd = 2.0 × μM) between Ca2+ and BACE1 and that the binding process was exothermic (ΔH = − 3.5 kcal/mol). We also could demonstrate that low concentrations of Ca2+ (μM range) significantly increased the proteolytic activity of BACE1. Collectively, these results identify a direct interaction between BACE1 and Ca2+ and suggest that under physiological conditions, the function(s) of BACE1 must also be influenced by Ca2+.