Polymerization of the SAM domain of MAPKKK Ste11 from the budding yeast: Implications for efficient signaling through the MAPK cascades

Abstract

The sterile ␣-motif (SAM) is a protein module ∼70 residues long and mainly involved in the protein-protein interactions of cell signaling and transcriptional repression. The SAM domain of the yeast MAPKKK Ste11 has a well-folded dimeric structure in solution. Interestingly, the well-folded dimer of the Ste11 SAM undergoes a time-dependent self-assembly upon lowering of the pH, leading to the formation of high molecular weight oligomers. The oligomeric structures rapidly disassemble to the well-folded dimer upon reversal of the pH to close to neutral conditions. Circular dichroism (CD) and atomic force microscopy (AFM) experiments demonstrate that the oligomeric structure formed at pH 5.0 appears to be highly helical and has architecture akin to proto-fibrils. Residue-specific kinetics of pH-triggered oligomerization obtained from real-time 15 N-1 H HSQC experiments indicate that the dimer-oligomer transition appears to involve all residues of the well-folded dimeric structure of the Ste11 SAM. Very interestingly, the interactions of the Ste11 and Ste50 SAM domains also lead to the formation of non-homogeneous hetero-complexes with significant populations of high molecular weight aggregates. AFM imaging shows that the Ste11-Ste50 hetero-polymeric aggregates assume the shapes of circular nano-particles with dimensions of 50-60 nanometers (nm), in contrast to the proto-fibrils formed by the Ste11 SAM domain alone. Such intrinsic propensity for dimer to oligomer transition of the Ste50-binding SAM domain of Ste11 may endow the MAPKKK Ste11 with unique functional properties required for efficient and high fidelity signal transduction in the budding yeast. Keywords: SAM domain; Ste11; Ste50; MAPKThe sterile ␣-motif (SAM) domain is a protein-protein interaction module that was first discovered as part of the multi-domain signaling proteins from yeast and within transcription regulators of Drosophila (Ponting 1995). Up to now, SAM domains have been found in >250 regulatory proteins including tyrosine kinases, Ser/Thr kinases, adaptor proteins, and transcription factors (Ponting 1995;Schultz et al. 1997;Kim and Bowie 2003). Three dimensional structures of several SAM domains have been reported, showing a compact fold made of five helices (Slupsky et al. 1998;Chi et al. 1999;Smalla et al. 1999;Stapleton et al. 1999;Thanos et al. 1999;Kim et al. 2001Kim et al. , 2002Mackereth et al. 2002;Bhattacharjya et al. 2004;Grimshaw et al. 2004;Kwan et al. 2004). These structural studies also revealed Article published online ahead of print. Article and publication date are at

The novel SAM domain protein Aveugle is required for Raf activation in the Drosophila EGF receptor signaling pathway

“…In the context of Raf activation, it is possible that polymerization of Ave, together with Cnk and perhaps other SAM domain-containing proteins, leads to the formation of large scaffolding complexes in which the local concentration of Raf and/or its activators is increased. Interestingly, the yeast adaptor protein Ste50, which is required for the activation of a MAPKKK, Ste11 (Ramezani-Rad 2003), induces polymerization of Ste11 through interactions between the SAM domains of the two molecules (Bhattacharjya et al 2005). This may stabilize a complex in which the Ste20 kinase can phosphorylate Ste11 (Ramezani-Rad 2003).…”

SLP-76 Sterile α Motif (SAM) and Individual H5 α Helix Mediate Oligomer Formation for Microclusters and T-cell Activation

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The NMR structure of the murine DLC2 SAM domain reveals a variant fold that is similar to a four-helix bundle

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