γ-Glutamyltransferase (GGT) is an enzyme that uses γ-glutamyl compounds as substrates and catalyzes their transfer to a water molecule or an acceptor substrate with varied physiological function in bacteria, plants, and animals. Crystal structures of GGT are known for different species and in different states of the chemical reaction; however, the structural dynamics of the substrate binding to the catalytic site of GGT are unknown. Here, we modeled Escherichia coli GGT's glutamine binding by using a swarm of accelerated molecular dynamics (aMD) simulations. Characterization of multiple binding events identified three structural binding motifs composed of polar residues in the binding pocket that govern glutamine binding into the active site. Simulated open and closed conformations of a lid-loop protecting the binding cavity suggest its role as a gating element by allowing or blocking substrates entry into the binding pocket. Partially open states of the lid-loop are accessible within thermal fluctuations, while the estimated free energy cost of a complete open state is 2.4 kcal/mol. Our results suggest that both specific electrostatic interactions and GGT conformational dynamics dictate the molecular recognition of substrate-GGT complexes.