Abstract
The sterile alpha motif (SAM) is a 65-70-amino acid domain found in over 300 proteins that are involved in either signal transduction or transcriptional activation and repression. SAM domains have been shown to mediate both homodimerization and heterodimerization and in some cases oligomerization. Here, we present the solution structure of the SAM domain of the Saccharomyces cerevisiae protein, Ste50p. Ste50p functions as a modulator of the mitogen-activated protein kinase (MAPK) cascades in S. cerevisiae, which control mating, pseudohyphal growth, and osmo-tolerance. This is the first example of the structure of a SAM domain from a MAPK module protein. We have studied the associative behavior of Ste50p SAM in solution and shown that it is monomeric. We have examined the SAM domain from Ste11p, the MAPK kinase kinase that associates with Ste50p in vivo, and shown that it forms dimers with a self-association K(d) of approximately 0.5 mm. We have also analyzed the interaction of Ste50p SAM with Ste11p SAM and the effects of mutations at Val-37, Asp-38, Pro-71, Leu-73, Leu-75, and Met-99 of STE50 on the heterodimerization properties of Ste50p SAM. We have found that L73A and L75A abrogate the Ste50p interaction with Ste11p, and we compare these data with the known interaction sites defined for other SAM domain interactions.
Highlights
The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
We have examined the SAM domain from Ste11p, the mitogen-activated protein kinase (MAPK) kinase kinase that associates with Ste50p in vivo, and shown that it forms dimers with a self-association Kd of ϳ0.5 mM
Been demonstrated that Ste50p is absolutely required for the Sho1p/Ste11p control of the HOG pathway and that Ste50p may be an integral part of the Ste11p MAPKK kinase (MAPKKK) [6]
Summary
Protein Expression and Purification—The SAM domains from Ste50p (residues 27–108) and Ste11p (residues 14 – 86) were amplified by PCR from a preparation of genomic yeast DNA. A mixed labeled sample of Ste50p SAM was generated by mixing a 1:1 ratio of unlabeled: 13C,15N-labeled protein and adding 8 M guanidine hydrochloride to a final concentration of 6 M. Ste11p SAM-Ste50p SAM Titration—A titration was performed by adding aliquots of unlabeled Ste50p SAM domain to 500 l of 0.65 mM 15N-labeled Ste11p SAM and concentrating the sample back to 500 l. A similar titration was performed by adding aliquots of unlabeled Ste11p SAM to 550 l of 0.3 mM 15N-labeled Ste50p SAM and concentrating the sample back to 550 l. Analytical Ultracentrifugation—Sedimentation equilibrium analysis was performed using a Beckman Optima XL-I analytical ultracentrifuge For both Ste11p SAM and Ste50p SAM, three samples were run simultaneously, with protein concentrations of 1, 2, and 3 mg/ml, respectively.
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