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S100A1 utilizes different mechanisms for interacting with calcium-dependent and calcium-independent target proteins
A Landar1, R R Rustandi, D J Weber
1Department of Pharmacology, College of Medicine, University of South Alabama, Mobile 36688, USA.
Abstract:
While previous studies have identified target proteins that interact with S100A1 in a calcium-dependent manner as well as target proteins that interact in a calcium-independent manner, the molecular mechanisms of S100A1-target protein interaction have not been elucidated. In this study, point and deletion mutants of S100A1 were used to investigate the contribution of carboxyl terminal amino acids to S100A1 interaction with calcium-dependent and calcium-independent target proteins. First, a recombinant rat S100A1 protein (recS100A1) expressed in bacteria exhibited physical and chemical properties indistinguishable from native S100A1. Next, proteins lacking the carboxyl-terminal nine residues of recS100A1 (Delta85-93), or containing alanine substitutions at Phe 88 (F88A), Phe 89 (F89A), or Trp 90 (W90A), both Phe 88 and Phe 89 (F88/89A), or all three aromatic residues (F88/89A-W90A) were recombinantly expressed. Like recS100A1, F88A, F89A, and W90A proteins interacted with phenyl-Sepharose in a calcium-dependent manner. However, the Delta85-93 protein did not interact with phenyl-Sepharose, indicating that a phenyl-Sepharose-binding region (PSBR) of recS100A1 had been disrupted. The F88/89A and F88/89A-W90A proteins exhibited reduced calcium-dependent interaction with phenyl-Sepharose when compared with recS100A1, demonstrating that the carboxyl-terminal aromatic residues Phe 88, Phe 89, and Trp 90 comprise the PSBR of S100A1. Fluorescence studies showed that the Delta85-93 protein exhibited reduced calcium-dependent interaction with the dodecyl CapZ peptide, TRTK, while W90A bound TRTK with a Kd of 5.55 microM. These results 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 the point and deletion mutant S100A1s was indistinguishable from native S100A1. These results demonstrate that carboxyl-terminal residues are not required for S100A1 modulation of calcium-independent target protein aldolase A. Alltogether, these results indicate that S100A1 utilizes distinct mechanisms for interaction with calcium-independent and calcium-dependent target proteins.
Insights
S100A1 protein interacts differently with calcium-dependent and independent targets. Carboxyl-terminal residues are crucial for calcium-dependent interactions but not for calcium-independent ones, revealing distinct molecular mechanisms.
Area of Science:
- Molecular Biology
- Protein Biochemistry
- Calcium Signaling
Background:
- S100A1 protein interactions with target proteins can be calcium-dependent or independent.
- The precise molecular mechanisms underlying these interactions remain unclear.
Purpose of the Study:
- To investigate the role of S100A1's carboxyl-terminal amino acids in its interactions with target proteins.
- To elucidate the distinct molecular mechanisms of S100A1's calcium-dependent and independent interactions.
Main Methods:
- Recombinant expression of wild-type and mutant S100A1 proteins (point and deletion mutants).
- Biochemical assays including phenyl-Sepharose binding and fluorescence studies with peptides.
- Assessment of S100A1's effect on aldolase A activity.
Main Results:
- Carboxyl-terminal residues (Phe 88, Phe 89, Trp 90) form the phenyl-Sepharose-binding region (PSBR), essential for calcium-dependent interactions.
- The PSBR is indistinguishable from the calcium-dependent target protein-binding site.
- S100A1 mutants lacking these carboxyl-terminal residues did not affect calcium-independent interactions with aldolase A.
Conclusions:
- S100A1 utilizes distinct molecular mechanisms for calcium-dependent and calcium-independent target protein interactions.
- Carboxyl-terminal residues are critical for calcium-dependent binding but dispensable for calcium-independent modulation of aldolase A.