Molecular functions of intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs), such as molecular recognition and cellular signaling, are ascribed to dynamic changes in the conformational space in response to binding of target molecules. Sortase, a transpeptitase in Gram-positive bacteria, has an IDR in a loop which undergoes a disordered-to-ordered transition (called "disordered loop"), accompanying a tilt of another loop ("dynamic loop"), upon binding of a signal peptide and a calcium ion. In this study, all-atom conformational ensembles of sortase were calculated for the four different binding states (with/without the peptide and with/without a calcium ion) by the multiscale enhanced sampling (MSES) simulation to examine how the binding of the peptide and/or calcium influences the conformational ensemble. The MSES is a multiscale and multicopy simulation method that allows an enhanced sampling of the all-atom model of large proteins including explicit solvent. A 100 ns MSES simulation of the ligand-free sortase using 20 replicas (in total 2 μs) demonstrated large flexibility in both the disordered and dynamic loops; however, their distributions were not random but had a clear preference which populates the N-terminal part of the disordered loop near the bound form. The MSES simulations of the three binding states clarified the allosteric mechanism of sortase: the N- and C-terminal parts of the disordered loop undergo a disorder-to-order transition independently of each other upon binding of the peptide and a calcium ion, respectively; however, upon binding of both ligands, the two parts work cooperatively to stabilize the bound peptide.