Laminins are major cell-adhesive proteins of basement membranes that interact with integrins in a divalent cation-dependent manner. Laminin-511 consists of alpha 5, beta 1, and gamma 1 chains, of which three laminin globular domains of the alpha 5 chain (alpha 5/LG1-3) and a Glu residue in the C-terminal tail of chain gamma 1(gamma 1-Glu1607) are required for binding to integrins. However, it remains unsettled whether the Glu residue in the gamma 1 tail is involved in integrin binding by coordinating the metal ion in the metal ion-dependent adhesion site of beta 1 integrin (beta 1-MIDAS), or by stabilizing the conformation of alpha 5/LG1-3. To address this issue, we examined whether alpha 5/LG1-3 contain an acidic residue required for integrin binding that is as critical as the Glu residue in the gamma 1 tail; to achieve this, we undertook exhaustive alanine substitutions of the 54 acidic residues present in alpha 5/LG1-3 of the E8 fragment of laminin-511 (LM511E8). Most of the alanine mutants possessed alpha 6 beta 1 integrin binding activities comparable with wild-type LM511E8. Alanine substitution for alpha 5-Asp3198 and Asp3219 caused mild reduction in integrin binding activity, and that for alpha 5-Asp3218 caused severe reduction, possibly resulting from conformational perturbation of a5/LG1-3. When alpha 5-Asp3218 was substituted with asparagine, the resulting mutant possessed significant binding activity to alpha 6 beta 1 integrin, indicating that alpha 5-Asp3218 is not directly involved in integrin binding through coordination with the metal ion in 01-MIDAS. Given that substitution of gamma 1-Glu1607 with glutamine nullified the binding activity to alpha 6 beta 1 integrin, these results, taken together, support the possibility that the critical acidic residue coordinating the metal ion in beta 1-MIDAS is Glu1607 in the gamma 1 tail, but no such residue is present in alpha 5/LG1-3. (C) 2017 Elsevier Inc. All rights reserved.