S100A1 utilizes different mechanisms for interacting with calcium-dependent and calcium-independent target proteins

While previous studies have identified target proteins that interact with S 100A1 in a calcium-dependent manner as well as target proteins that interac t in a calcium-independent manner, the molecular mechanisms of S100A1-targe t protein interaction have not been elucidated. In this study, point and de letion mutants of S100A1 were used to investigate the contribution of carbo xyl terminal amino acids to S100A1 interaction with calcium-dependent and c alcium-independent target proteins. First, a recombinant rat S100A1 protein (recS100A1) expressed in bacteria exhibited physical and chemical properti es indistinguishable from native S100A1. Next, proteins lacking the carboxy l-terminal nine residues of recS100A1 (Delta 85-93), or containing alanine substitutions at Phe 88 (F88A), Phe 89 (F89A), or Trp 90 (W90A), both Phe 8 8 and Phe 89 (F88/89A), or all three aromatic residues (F88/89A-W90A) were recombinantly expressed. Like recS100A1, F88A, F89A, and W90A proteins inte racted with phenyl-Sepharose in a calcium-dependent manner. However, the De lta 85-93 protein did not interact with phenyl-Sepharose, indicating that a phenyl-Sepharose-binding region (PSBR) of recS100A1 had been disrupted. Th e F88/89A and F88/89A-W90A proteins exhibited reduced calcium-dependent int eraction with phenyl-Sepharose when compared with recS100A1, demonstrating that the carboxyl-terminal aromatic residues Phe 88, Phe 89, and Trp 90 com prise the PSBR of S100A1. Fluorescence studies showed that the Delta 85-93 protein exhibited reduced calcium-dependent interaction with the dodecyl Ca pZ peptide, TRTK, while W90A bound TRTK with a K-d Of 5.55 mu M. These resu lts demonstrate that the calcium-dependent target protein-binding site and the PSBR are indistinguishable. In contrast to the calcium-dependent target TRTK, activation of the calcium-independent target protein aldolase A by t he point and deletion mutant S100A1s was indistinguishable from native S100 A1. These results demonstrate that carboxyl-terminal residues are not requi red for S100A1 modulation of calcium-independent target protein aldolase A. Alltogether, these results indicate that S100A1 utilizes distinct mechanis ms for interaction with calcium-independent and calcium-dependent target pr oteins.