Abstract
The sterile alpha motif (SAM) domain is a protein module found in many diverse signaling proteins. SAM domains in some systems have been shown to self-associate. Previous crystal structures of an EphA4-SAM domain dimer (Stapleton, D., Balan, I., Pawson, T., and Sicheri, F. (1999) Nat. Struct. Biol. 6, 44-49) and a possible EphB2-SAM oligomer (Thanos, C. D., Goodwill, K. E., and Bowie, J. U. (1999) Science 283, 833-836) both revealed large interfaces comprising an exchange of N-terminal peptide arms. Within the arm, a conserved hydrophobic residue (Tyr-8 in the EphB2-SAM structure or Phe-910 in the EphA4-SAM structure) is anchored into a hydrophobic cleft on a neighboring molecule. Here we have solved a new crystal form of the human EphB2-SAM domain that has the same overall SAM domain fold yet has no substantial intermolecular contacts. In the new structure, the N-terminal peptide arm of the EphB2-SAM domain protrudes out from the core of the molecule, leaving both the arm (including Tyr-8) and the hydrophobic cleft solvent-exposed. To verify that Tyr-8 is solvent-exposed in solution, we made a Tyr-8 to Ala-8 mutation and found that the EphB2-SAM domain structure and stability were only slightly altered. These results suggest that Tyr-8 is not part of the hydrophobic core of the EphB2-SAM domain and is conserved for functional reasons. Cystallographic evidence suggests a possible role for the N-terminal arm in oligomerization. In the absence of a direct demonstration of biological relevance, however, the functional role of the N-terminal arm remains an open question.
Highlights
The sterile ␣ motif (SAM)1 domain is approximately 70 amino acids long and conserved in over a hundred diverse proteins, including the Eph family of tyrosine kinase receptors [1, 2]
The helical content observed in solution is in reasonable agreement with the 60% helical content seen in the EphB2 crystal structure. These results indicate that each of the Eph receptor SAM domains can fold, independent of the intact receptor
Analytical ultracentrifugation was used to evaluate the oligomeric state of the three isolated Eph receptor SAM domains (Table I)
Summary
The sterile ␣ motif (SAM)1 domain is approximately 70 amino acids long and conserved in over a hundred diverse proteins, including the Eph family of tyrosine kinase receptors [1, 2]. Stein et al [23] reported that the binding of low molecular weight protein-tyrosine phosphatase to the EphB1 receptor tyrosine kinase is abrogated by a Y929F mutation in the SAM domain [23]. Four SAM domain structures have been reported, including the SAM domain from the Ets-1 transcription factor, the SAM domains from two Eph receptors, EphA4 and EphB2, and a C-terminal SAM domain from the p53 homolog, p73 (16, 26 –28).
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