Salt dependence conformational stability of the dimeric SAM domain of MAPKKK Ste11 from budding yeast : a native-state H/D exchange NMR Study

Abstract

The sterile α motif, also called the SAM domain, is known to form homo or heterocomplexes that modulate diverse biological functions through regulation of specific protein-protein interactions. The MAPK pathway of budding yeast Saccharomyces cerevisiae comprises a three-tier kinase system akin to mammals. The MAPKKK Ste11 protein of yeast contains a homodimer SAM domain, which is critical for transmitting cues to the downstream kinases. The structural stability of the dimeric Ste11 SAM is maintained by hydrophobic and ionic interactions at the interfacial amino acids. Urea induced equilibrium unfolding process of the Ste11 SAM domain is cooperative without evidence of any intermediate states. The native state H/D exchange under sub-denaturing conditions is a useful method for the detection of intermediate states of proteins. In the present study, we investigated the effect of ionic strength on the conformational stability of the dimer using the H/D exchange study. The hydrogen exchange behavior of the Ste11 dimer under physiological salt concentration reveals two metastable partially folded intermediate states, which may be generated by a sequential and cooperative unfolding of the five helices of the fold. These intermediates appear to be the dominant species for the dynamic and reversible unfolding of the Ste11 SAM domain, underlining a significant pathway for its folding kinetics via hydrophobic collapse. In contrast, higher ionic concentration eliminates this cooperativity between the stabilizing pairs of helices.