| Abstract | The sterile alpha motifs or SAM domains are small ( approximately 70 amino acids) protein-protein interaction modules that are involved in diverse functions ranging from cell signaling, transcription regulation, and scaffolding. The Ste11 protein kinase in the mitogen-activated protein kinase (MAPK) signaling cascades of the budding yeast is regulated by a SAM domain located at the N-terminus of the full-length protein. The Ste11 SAM domain forms a symmetrical dimeric structure with an interface stabilized presumably by hydrophobic and ionic interactions. Here, we investigated urea-induced unfolding, using NMR and other optical spectroscopic methods, of the dimeric Ste11 SAM domain and two of the variants, namely, L57R and L60R, each containing a point mutation at the interfacial region. Our results demonstrate that the residue-specific or global unfolding of the Ste11 SAM is highly cooperative without any evidence for folded monomeric or partially folded species. However, replacement of hydrophobic residues with basic residues in the interface caused considerable changes in the stability and folding of the Ste11 SAM domain. The native dimeric structure of the L60R mutant protein is severely affected as indicated by a high propensity toward aggregation. On the other hand, the L57R mutant, although retaining the native structure, shows a dramatic decrease in the conformational stability as revealed by urea-induced denaturation and amide proton exchange studies. Furthermore, isothermal titration calorimetry and intrinsic tryptophan fluorescence experiments demonstrate that the L57R interacts with the cognate SAM domain from Ste50 with reduced affinity, while the L60R protein is devoid of any detectable binding activity. These results demonstrate that the interfacial residues of the dimeric SAM domain of Ste11 are critically involved in its structural stability and binding to the Ste50 SAM domain |
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