Background: Calnuc is a multidomain Ca2+-binding protein with many interacting partners but whose function is still elusive. Results: Calnuc is a serine protease with its active site catalytic triad present in the C-terminal domain. Conclusion: The serine protease activity of calnuc is allosterically regulated by Zn2-binding and its interaction with G proteinβ subunit. Significance: Novel proteolytic function of calnuc will have vital implications in its physiological role. The functions of calnuc, a novel Ca 2+-binding protein with multiple structural domains and diverse interacting partners, are yet unknown. We demonstrate unknown facets of calnuc, which is a serine protease in which Ser-378 of GXSXG motif, Asp-328 of DTG motif, and His-339 form the "catalytic triad," locating the enzyme active site in the C-terminal region. Analogous to the active site of Zn2 carboxypeptidases, calnuc has two high affinity (Kd= 20 nM), well conserved Zn2-binding sites near its N terminus, although it is inactive as a peptidase. Zn2 binding allosterically and negatively regulates the serine protease activity of calnuc, inhibition being caused by an "open to close" change in its conformation not seen upon Ca2+ binding. Most strikingly, interaction with G proteinβ subunit completely inhibits the enzymatic activity of calnuc. We thus illustrate that G proteins and Zn2 act as two "keys" that control enzymatic activity of calnuc, arresting it in "locked" state. Calnuc, therefore, exists dynamically in two different forms, (i) as a Ca2+-binding protein in Zn2-bound form and (ii) as a protease in Zn2-free form, commissioning it to perform multiple functions. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.