Search

SARM1 induces axonal degeneration in response to various insults and is therefore considered an attractive drug target for the treatment of neuro-degenerative diseases as well as for brain and spinal cord injuries. SARM1 activity depends on the integrity of the protein's SAM domains, as well as on the enzymatic conversion of NAD + to ADPR (ADP Ribose) products by the SARM1's TIR domain. Therefore, inhibition of either SAM or TIR functions may constitute an effective therapeutic strategy. However, there is currently no SARM1-directed therapeutic approach available because of an insufficient structural and mechanistic understanding of this protein. In this study, we found that SARM1 assembles into an octameric ring. This arrangement was not described before in other SAM proteins, but is reminiscent of the apoptosome and inflammasome—well-known apoptotic ring-like oligomers. We show that both SARM1 and the isolated tandem SAM1–2 domains form octamers in solution, and electron microscopy analysis reveals an octameric ring of SARM1. We determined the crystal structure of SAM1–2 and found that it also forms a closed octameric ring in the crystal lattice. The SAM1–2 ring interactions are mediated by complementing "lock and key" hydrophobic grooves and inserts and electrostatic charges between the neighboring protomers. We have mutated several interacting SAM1–2 interfaces and measured how these mutations affect SARM1 apoptotic activity in cultured cells, and in this way identified critical oligomerization sites that facilitate cell death. These results highlight the importance of oligomerization for SARM1 function and reveal critical epitopes for future targeted drug development. In this model for SARM1 structure and mechanism of activation, SARM1 forms an octameric ring. We hypothesize that in the inactive form of SARM1, the enzymatic TIR domains are held apart by the auto-inhibiting ARM domains, and that SARM1 activation (possibly by nicotinamide mononucleotide) involves the release of auto-inhibitory interactions that allows functional dimerization of TIR domains. Unlabelled Image • SARM1 is arranged as a homo-octameric ring. • 2.5-Å resolution crystal structure of the human SARM1 SAM1-2 domains. • The structure reveals a closed octamer ring - an arrangement not described before in other SAM proteins, but reminiscent of the well-known apoptosome and inflammasome. • Biochemical and functional experiments demonstrate the importance of SAM1-2-mediated oligomers to SARM1 function. [ABSTRACT FROM AUTHOR]

The Eph receptor family is the largest subfamily of receptor tyrosine kinases and well-known for their pivotal roles in axon guidance, synaptogenesis, artery/venous differentiation and tumorigenesis, etc. Activation of the Eph receptor needs multimerization of the receptors. The intracellular C-terminal SAM domain of Eph receptor was reported to mediate self-association of Eph receptors via the homo SAM–SAM interaction. In this study, we systematically expressed and purified the SAM domain proteins of all fourteen Eph receptors of Mus musculus in Escherichia coli. The FPLC (fast protein liquid chromatography) results showed the recombinant SAM domains were highly homogeneous. Using CD (circular dichroism) spectrometry, we found that the secondary structure of all the SAM domains was typically alpha helical folded and remarkably similar. The thermo-stability tests showed that they were quite stable in solution. SEC-MALS (size exclusion chromatography coupled with multiple angle light scattering) results illustrated 200 μM Eph SAM domains behaved as good monomers in the size-exclusion chromatography. More importantly, DLS (dynamic light scattering) results revealed the overwhelming majority of SAM domains was not multimerized in solution either at 200 μM or 2000 μM protein concentration, which indicating the SAM domain alone was not sufficient to mediate the polymerization of Eph receptor. In summary, our studies provided the systematic biochemical characterizations of the Eph receptor SAM domains and implied their roles in Eph receptor mediated signaling pathways. [ABSTRACT FROM AUTHOR]

GAREM1 (Grb2-associated regulator of Erk/MAPK1) is an adaptor protein that is involved in the epidermal growth factor (EGF) pathway. The nuclear localization of GAREM1 depends on the nuclear localization sequence (NLS), which is located at the N-terminal CABIT (cysteine-containing, all in Themis) domain. Here, we identified 14-3-3ε as a GAREM-binding protein, and its binding site is closely located to the NLS. This 14-3-3 binding site was of the atypical type and independent of GAREM phosphorylation. Moreover, the binding of 14-3-3 had an effect on the nuclear localization of GAREM1. Unexpectedly, we observed that the CABIT domain had intramolecular association with the C-terminal SAM (sterile alpha motif) domain. This association might be inhibited by binding of 14-3-3 at the CABIT domain. Our results demonstrate that the mechanism underlying the nuclear localization of GAREM1 depends on its NLS in the CABIT domain, which is controlled by the binding of 14-3-3 and the C-terminal SAM domain. We suggest that the interplay between 14-3-3, SAM domain and CABIT domain might be responsible for the distribution of GAREM1 in mammalian cells. [ABSTRACT FROM AUTHOR]

Abstract: Mutations in either the Bicaudal-C or the Anks6 gene which encode the Bicc1 and SamCystin proteins respectively cause formation of renal cysts in rodent models of polycystic kidney disease, however their role in the mammalian kidney is unknown. Immunolocalization studies demonstrated that, unlike many other PKD-related proteins, SamCystin and Bicc1 do not localize to the primary cilia of cultured kidney cells. Epitope-tagged recombinant SamCystin and Bicc1 proteins were transiently transfected into inner medullary collecting duct (IMCD) cells and co-immunoprecipitated. The results showed that SamCystin self-associates, Bicc1 and SamCystin interact, the mutation responsible for PKD in the Han:SPRD-Cy rat disrupts the self-association of SamCystin but not the Bicc1–SamCystin interaction, and RNA may be an important component of the Bicc1–SamCystin complex. These studies provide the first evidence that Bicc1 and SamCystin interact at the protein level suggesting that they function in a common molecular pathway that when perturbed, is involved in cystogenesis. [Copyright &y& Elsevier]

The yeast Vts1 SAM (sterile alpha motif) domain is a member of a new class of SAM domains that specifically bind RNA. To elucidate the structural basis for RNA binding, the solution structure of the Vts1 SAM domain, in the presence of a specific target RNA, has been solved by multidimensional heteronuclear NMR spectroscopy. The Vts1 SAM domain retains the “core” five-helix-bundle architecture of traditional SAM domains, but has additional short helices at N and C termini, comprising a small substructure that caps the core helices. The RNA-binding surface of Vts1, determined by chemical shift perturbation, maps near the ends of three of the core helices, in agreement with mutational data and the electrostatic properties of the molecule. These results provide a structural basis for the versatility of the SAM domain in protein and RNA-recognition. [Copyright &y& Elsevier]

Ste11, a homologue of mammalian MAPKKKs, together with its binding partner Ste50 works in a number of MAPK signaling pathways of Saccharomyces cerevisiae. Ste11/Ste50 binding is mediated by their sterile α motifs or SAM domains, of which homologues are also found in many other intracellular signaling and regulatory proteins. Here, we present the solution structure of the SAM domain or residues D37–R104 of Ste11 and its interactions with the cognate SAM domain-containing region of Ste50, residues M27–Q131. NMR pulse-field-gradient (PFG) and rotational correlation time measurements (τc) establish that the Ste11 SAM domain exists predominantly as a symmetric dimer in solution. The solution structure of the dimeric Ste11 SAM domain consists of five well-defined helices per monomer packed into a compact globular structure. The dimeric structure of the SAM domain is maintained by a novel dimer interface involving interactions between a number of hydrophobic residues situated on helix 4 and at the beginning of the C-terminal long helix (helix 5). The dimer structure may also be stabilized by potential salt bridge interactions across the interface. NMR H/2H exchange experiments showed that binding of the Ste50 SAM to the Ste11 SAM very likely involves the positively charged extreme C-terminal region as well as exposed hydrophobic patches of the dimeric Ste11 SAM domain. The dimeric structure of the Ste11 SAM and its interactions with the Ste50 SAM may have important roles in the regulation and activation of the Ste11 kinase and signal transmission and amplifications through the Ste50–Ste11 complex. [Copyright &y& Elsevier]

The PNT (or Pointed) domain, present within a subset of the Ets family of transcription factors, is structurally related to the larger group of SAM domains through a common tertiary arrangement of four α-helices. Previous studies have shown that, in contrast to the PNT domain from Tel, this domain from Ets-1 contains an additional N-terminal helix integral to its folded structure. To further investigate the structural plasticity of the PNT domain, we have used NMR spectroscopy to characterize this domain from two additional Ets proteins, Erg and GABPα. These studies both define the conserved and variable features of the PNT domain, and demonstrate that the additional N-terminal helix is also present in GABPα, but not Erg. In contrast to Tel and Yan, which self-associate to form insoluble polymers, we also show that the isolated PNT domains from Ets-1, Ets-2, Erg, Fli-1, GABPα, and Pnt-P2 are monomeric in solution. Furthermore, these soluble PNT domains do not associate in any pair-wise combination. Thus these latter Ets family PNT domains likely mediate interactions with additional components of the cellular signaling or transcriptional machinery. [Copyright &y& Elsevier]