Mutations in the SAM domain of the ETV6-NTRK3 chimeric tyrosine kinase block polymerization and transformation activity

The 12p13 ETV6 (TEL) gene is frequently targeted by chromosomal translocations in human malignancies, resulting in the formation of oncogenic ETV6 gene fusions. Many of the known partner genes encode protein tyrosine kinases (PTKs), generating fusion proteins that function as chimeric PTKs. ETV6-NTRK3 (EN), comprised of the ETV6 SAM domain fused to the NTRK3 PTK, is unique among ETV6 chimeric oncoproteins, as it is expressed in cancers of multiple lineages. We initially hypothesized that, similar to other ETV6-PTK chimeras, SAM-mediated dimerization of EN leads to constitutive activation of the PTK and downstream signaling cascades. However, when the EN SAM domain was replaced with an inducible FK506 binding protein (FKBP) dimerization system, resulting FKBP-NTRK3 chimeras failed to transform NIH 3T3 cells even though PTK activation was preserved. It was recently shown that the ETV6 SAM domain has two potential interacting surfaces, raising the possibility that this domain can mediate protein polymerization. We therefore mutated each EN SAM binding interface in a manner shown previously to abolish self-association of wild-type ETV6. Each mutation completely blocked the ability of EN to polymerize, to activate its PTK, and to transform NIH 3T3 cells. Furthermore, EN itself formed large polymeric structures within cells while mutant EN proteins were present only as monomers. Finally, we observed a dominant negative effect on the transformation of isolated SAM domains coexpressed in EN-transformed cells. Taken together, our results suggest that higher-order polymerization may be a critical requirement for the transformation activity of EN and possibly other ETV6-PTK fusion proteins.     

A Nuclear Localization Signal at the SAM-SAM Domain Interface of AIDA-1 Suggests a Requirement for Domain Uncoupling Prior to Nuclear Import

The neuronal scaffolding protein AIDA-1 is believed to act as a convener of signals arising at postsynaptic densities. Among the readily identifiable domains in AIDA-1, two closely juxtaposed sterile alpha motif (SAM) domains and a phosphotyrosine binding domain are located within the C-terminus of the longest splice variant and exclusively in four shorter splice variants. As a first step towards understanding the possible emergent properties arising from this assembly of ligand binding domains, we have used NMR methods to solve the first structure of a SAM domain tandem. Separated by a 15-aa linker, the two SAM domains are fused in a head-to-tail orientation that has been observed in other hetero- and homotypic SAM domain structures. The basic nuclear import signal for AIDA-1 is buried at the interface between the two SAM domains. An observed disparity between the thermal stabilities of the two SAM domains suggests a mechanism whereby the second SAM domain decouples from the first SAM domain to facilitate translocation of AIDA-1 to the nucleus.     

Polymer-driven crystallization

Obtaining well-diffracting crystals of macromolecules remains a significant barrier to structure determination. Here we propose and test a new approach to crystallization, in which the crystallization target is fused to a polymerizing protein module, so that polymer formation drives crystallization of the target. We test the approach using a polymerization module called 2TEL, which consists of two tandem sterile alpha motif (SAM) domains from the protein translocation Ets leukemia (TEL). The 2TEL module is engineered to polymerize as the pH is lowered, which allows the subtle modulation of polymerization needed for crystal formation. We show that the 2TEL module can drive the crystallization of 11 soluble proteins, including three that resisted prior crystallization attempts. In addition, the 2TEL module crystallizes in the presence of various detergents, suggesting that it might facilitate membrane protein crystallization. The crystal structures of two fusion proteins show that the TELSAM polymer is responsible for the majority of contacts in the crystal lattice. The results suggest that biological polymers could be designed as crystallization modules.