Abstract
The 14-3-3 family of intracellular proteins are dimeric, multifunctional adaptor proteins that bind to and regulate the activities of many important signaling proteins. The subunits within 14-3-3 dimers are predicted to be stabilized by salt bridges that are largely conserved across the 14-3-3 protein family and allow the different isoforms to form heterodimers. Here, we have examined the contributions of conserved salt-bridging residues in stabilizing the dimeric state of 14-3-3ζ. Using analytical ultracentrifugation, our results revealed that Asp21 and Glu89 both play key roles in dimer dynamics and contribute to dimer stability. Furthermore, hydrogen-deuterium exchange coupled with mass spectrometry showed that mutation of Asp21 promoted disorder in the N-terminal helices of 14-3-3ζ, suggesting that this residue plays an important role in maintaining structure across the dimer interface. Intriguingly, a D21N 14-3-3ζ mutant exhibited enhanced molecular chaperone ability that prevented amorphous protein aggregation, suggesting a potential role for N-terminal disorder in 14-3-3ζ's poorly understood chaperone action. Taken together, these results imply that disorder in the N-terminal helices of 14-3-3ζ is a consequence of the dimer-monomer dynamics and may play a role in conferring chaperone function to 14-3-3ζ protein.
Highlights
The 14-3-3 family of intracellular proteins are dimeric, multifunctional adaptor proteins that bind to and regulate the activities of many important signaling proteins
Our results revealed that Asp[21] and Glu[89] both play key roles in dimer dynamics and contribute to dimer stability
The 14-3-3 crystal structure revealed that 620 Å2 is buried at the dimer interface and suggested that salt bridges across this interface hold the monomer subunits together (15)
Summary
The 14-3-3 family of intracellular proteins are dimeric, multifunctional adaptor proteins that bind to and regulate the activities of many important signaling proteins. A D21N 14-3-3 mutant exhibited enhanced molecular chaperone ability that prevented amorphous protein aggregation, suggesting a potential role for N-terminal disorder in 14-3-3’s poorly understood chaperone action. Taken together, these results imply that disorder in the N-terminal helices of 14-3-3 is a consequence of the dimer– monomer dynamics and may play a role in conferring chaperone function to 14-3-3 protein. Salt bridges control 14-3-3 conformation and dynamics tional role of these individual salt bridges in dimer stability has not been formally tested Distinct from their phosphoserine-binding capabilities, 14-3-3 proteins possess molecular chaperone activity, preventing temperature-induced aggregation of target proteins (8) in a manner similar to that of small heat-shock proteins (sHsps)[5] (9). The data reveal hitherto unknown features of 14-3-3 protein structure and hint at important aspects of 14-3-3 biology in relation to its chaperone function
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