C-terminal Sterile Alpha Motif Research Articles

Structural analysis of the SAM domain of the Arabidopsis mitochondrial tRNA import receptor

Mitochondria are membrane bound organelles of endosymbiotic origin with limited protein coding capacity. The import of nuclear-encoded protein and nucleic acids is required and essential for maintaining organelle mass, number and activity. As plant mitochondria do not encode all the necessary tRNA types required, the import of cytosolic tRNA is vital for organelle maintenance. Recently, two mitochondrial outer membrane proteins, named Tric1 and Tric2, for tRNA import component, were shown to be involved in the import of cytosolic tRNA. Tric1/2 binds tRNAala via conserved residues in the C-terminal Sterile Alpha Motif (SAM) domain. Here we report the X-ray crystal structure of the Tric1 SAM domain. We identified the ability of the SAM domain to form a helical superstructure with 6 monomers per helical turn and key amino acid residues responsible for its formation. We determined that the oligomerization of Tric1 SAM domain may play a role in protein function whereby mutation of Gly241 introducing a larger side chain at this position disrupted the oligomer and resulted in the loss of RNA binding capability. Furthermore, complementation of Arabidopsis thaliana Tric1/2 knockout lines with a mutated Tric1 failed to restore the defective plant phenotype. AlphaFold2 structure prediction of both the SAM domain and Tric1 support a cyclic pentameric or hexameric structure. In the case of a hexameric structure a pore of sufficient dimensions to transfer tRNA across the mitochondrial membrane is observed. Our results highlight the importance of oligomerization of Tric1 for protein function.

The C-terminal SAM domain of p73 binds to the N terminus of MDM2

BackgroundThe p53, p63 and p73 proteins belong to the p53 family of transcription factors, playing key roles in tumour suppression. The α-splice variant of p73 (p73α) has at its C terminus a sterile alpha motif (SAM); this domain, SAMp73, formed by five helices (α1 to α5), is thought to mediate in protein-protein interactions. The E3-ligase MDM2 binds to p73 at its N terminus transactivation domain (TA), but it does not promote its degradation via ubiquitination; however, the details of such MDM2/p73 interaction are not fully known. MethodsWe studied the binding of SAMp73 with N-terminal MDM2, by several biophysical techniques, namely, fluorescence, far-UV circular dichroism (CD), NMR and bio-layer interferometry (BLI). ResultsOur results obtained by fluorescence, T2-relaxation measurements and BLI show that there was binding between both proteins with a dissociation constant of ~10 μM. Furthermore, the binding region of SAMp73 involved mainly residues in the major α-helix, α5, and the nearby α4, as shown by HSQC-NMR. The binding was so specific that an isolated peptide comprising α4 and α5 helices of SAMp73, α4α5, did also bind to the N terminus of MDM2, although with weaker affinity than the entire domain. ConclusionsA new interaction between MDM2 and SAMp73 has been found, which could have potential therapeutic applications in cancers involving inactivated p53. General significanceA novel interaction between the C-terminal SAM of p73 and N-terminal MDM2 is described. The interaction could be used to modulate the functions where the wild-type, intact p73 is involved.

The Sam-Sam interaction between Ship2 and the EphA2 receptor: design and analysis of peptide inhibitors

The lipid phosphatase Ship2 represents a drug discovery target for the treatment of different diseases, including cancer. Its C-terminal sterile alpha motif domain (Ship2-Sam) associates with the Sam domain from the EphA2 receptor (EphA2-Sam). This interaction is expected to mainly induce pro-oncogenic effects in cells therefore, inhibition of the Ship2-Sam/EphA2-Sam complex may represent an innovative route to discover anti-cancer therapeutics. In the present work, we designed and analyzed several peptide sequences encompassing the interaction interface of EphA2-Sam for Ship2-Sam. Peptide conformational analyses and interaction assays with Ship2-Sam conducted through diverse techniques (CD, NMR, SPR and MST), identified a positively charged penta-amino acid native motif in EphA2-Sam, that once repeated three times in tandem, binds Ship2-Sam. NMR experiments show that the peptide targets the negatively charged binding site of Ship2-Sam for EphA2-Sam. Preliminary in vitro cell-based assays indicate that -at 50 µM concentration- it induces necrosis of PC-3 prostate cancer cells with more cytotoxic effect on cancer cells than on normal dermal fibroblasts. This work represents a pioneering study that opens further opportunities for the development of inhibitors of the Ship2-Sam/EphA2-Sam complex for therapeutic applications.

Trifluoroethanol-induced conformational transition of the C-terminal sterile alpha motif (SAM) of human p73

The alpha splice variant of p73 (p73α), a homologue of the tumour suppressor p53, has at its C terminus a sterile alpha motif (SAM); this domain, SAMp73, is involved in lipid binding and it is thought to mediate in protein-protein interactions. As SAMp73 is a 68-residue-long helical bundle, it could be a good model to study the (2,2,2-trifluoroethanol) TFE-induced conformational transitions of α-helical proteins. Furthermore, as SAMp73 binds to lipids through a well-known polypeptide patch, we can test whether TFE is a good mimic of lipids and membranes. To address those questions, we used several biophysical probes, namely, fluorescence, circular dichroism, 1D, 2D and 3D-NMR spectroscopies, and dynamic light scattering. The TFE-induced conformational transition of SAMp73 was complex, involving several species as detected by the biophysical probes. The last TFE-induced transition occurred at a concentration of TFE of ∼20% (v/v), where the protein lost its compactness. None of those TFE-induced species accumulated during the two-state folding of SAMp73 in aqueous solution. The final state at 40% TFE was highly helical, but its structure was not rigid. For SAMp73, TFE did not properly mimic a membrane-like environment, since at very low TFE concentrations, other residues, together with those known to interact with lipids, were also affected by the co-solvent. Comparison with studies on isolated peptides, comprising the helical regions of SAMp73, suggests that peptides were good models of the intact protein in TFE.

Bicc1 Polymerization Regulates the Localization and Silencing of Bound mRNA.

Loss of the RNA-binding protein Bicaudal-C (Bicc1) provokes renal and pancreatic cysts as well as ectopic Wnt/β-catenin signaling during visceral left-right patterning. Renal cysts are linked to defective silencing of Bicc1 target mRNAs, including adenylate cyclase 6 (AC6). RNA binding of Bicc1 is mediated by N-terminal KH domains, whereas a C-terminal sterile alpha motif (SAM) self-polymerizes in vitro and localizes Bicc1 in cytoplasmic foci in vivo. To assess a role for multimerization in silencing, we conducted structure modeling and then mutated the SAM domain residues which in this model were predicted to polymerize Bicc1 in a left-handed helix. We show that a SAM-SAM interface concentrates Bicc1 in cytoplasmic clusters to specifically localize and silence bound mRNA. In addition, defective polymerization decreases Bicc1 stability and thus indirectly attenuates inhibition of Dishevelled 2 in the Wnt/β-catenin pathway. Importantly, aberrant C-terminal extension of the SAM domain in bpk mutant Bicc1 phenocopied these defects. We conclude that polymerization is a novel disease-relevant mechanism both to stabilize Bicc1 and to present associated mRNAs in specific silencing platforms.

Functional relationship between CABIT, SAM and 14-3-3 binding domains of GAREM1 that play a role in its subcellular localization

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.

O-GlcNAcylation prevents aggregation of the Polycomb group repressor polyhomeotic.

The glycosyltransferase Ogt adds O-linked N-Acetylglucosamine (O-GlcNAc) moieties to nuclear and cytosolic proteins. Drosophila embryos lacking Ogt protein arrest development with a remarkably specific Polycomb phenotype, arising from the failure to repress Polycomb target genes. The Polycomb protein Polyhomeotic (Ph), an Ogt substrate, forms large aggregates in the absence of O-GlcNAcylation both in vivo and in vitro. O-GlcNAcylation of a serine/threonine (S/T) stretch in Ph is critical to prevent nonproductive aggregation of both Drosophila and human Ph via their C-terminal sterile alpha motif (SAM) domains in vitro. Full Ph repressor activity in vivo requires both the SAM domain and O-GlcNAcylation of the S/T stretch. We demonstrate that Ph mutants lacking the S/T stretch reproduce the phenotype of ogt mutants, suggesting that the S/T stretch in Ph is the key Ogt substrate in Drosophila. We propose that O-GlcNAcylation is needed for Ph to form functional, ordered assemblies via its SAM domain.

T-cell death following immune activation is mediated by mitochondria-localized SARM

Following acute-phase infection, activated T cells are terminated to achieve immune homeostasis, failure of which results in lymphoproliferative and autoimmune diseases. We report that sterile α- and heat armadillo-motif-containing protein (SARM), the most conserved Toll-like receptors adaptor, is proapoptotic during T-cell immune response. SARM expression is significantly reduced in natural killer (NK)/T lymphoma patients compared with healthy individuals, suggesting that decreased SARM supports NK/T-cell proliferation. T cells knocked down of SARM survived and proliferated more significantly compared with wild-type T cells following influenza infection in vivo. During activation of cytotoxic T cells, the SARM level fell before rising, correlating inversely with cell proliferation and subsequent T-cell clearance. SARM knockdown rescued T cells from both activation- and neglect-induced cell deaths. The mitochondria-localized SARM triggers intrinsic apoptosis by generating reactive oxygen species and depolarizing the mitochondrial potential. The proapoptotic function is attributable to the C-terminal sterile alpha motif and Toll/interleukin-1 receptor domains. Mechanistically, SARM mediates intrinsic apoptosis via B cell lymphoma-2 (Bcl-2) family members. SARM suppresses B cell lymphoma-extra large (Bcl-xL) and downregulates extracellular signal-regulated kinase phosphorylation, which are cell survival effectors. Overexpression of Bcl-xL and double knockout of Bcl-2 associated X protein and Bcl-2 homologous antagonist killer substantially reduced SARM-induced apoptosis. Collectively, we have shown how T-cell death following infection is mediated by SARM-induced intrinsic apoptosis, which is crucial for T-cell homeostasis.

The C-terminal sterile alpha motif (SAM) domain of human p73 is a highly dynamic protein, which acquires high thermal stability through a decrease in backbone flexibility

The α-splice variant of p73 (p73α), a homologue of the tumour suppressor p53, has close to its C terminus a sterile alpha motif (SAM), SAMp73, that is involved in protein-biomolecule interactions. The conformational stability of SAMp73 is low (∼5 kcal mol(-1)), although its thermal stability is high. To explain this high thermostability, we studied the dynamics of SAMp73 over a wide range of GdmCl (guanidine hydrochloride) concentrations and temperatures by NMR relaxation, NMR hydrogen-exchange (HX) and fluorescence lifetime approaches. The slowest exchanging residues of SAMp73 belong to the helical regions, and they did exchange by a global unfolding process. Moreover, SAMp73 was very flexible, with most of its amide protons affected by slow μs-ms conformational exchange. Within this time scale, the residues of SAMp73 with the largest exchange rates (R(ex)) were involved in binding with other molecules; therefore, the flexibility in the μs-ms range was associated with biological functions. As the [GdmCl] increased, the pico-to-nanosecond flexibility of the backbone amide protons raised, but it did so differently depending on the residue. We were able to obtain, for the first time, the linear [GdmCl]-variation of the local conformational entropies, m(S(i)), which ranged from 5.3 to 0.3 cal mol(-1) K(-1) M(-1), similar to those measured by using macroscopic techniques in other proteins. Conversely, the temperature dependence of the pico-to-nanosecond dynamics of the backbone amide protons of SAMp73 indicates that the flexibility of some residues decreased with the temperature; these results explain the high thermostability of the protein.

SUMO modification of the Polycomb group protein Sex comb on midleg (Scm) counteracts its function as a repressor of homeotic genes

The Drosophila protein Sex Comb on Midleg (Scm) is a member of the Polycomb group (PcG), a set of transcriptional repressors that maintain silencing of homeotic genes during development. We have found that the SUMO conjugating enzyme Ubc9 interacts with the Scm C‐terminal Sterile alpha motif (SAM) domain and this interaction is crucial for the efficient sumoylation of Scm. Scm is associated with the major Polycomb response element (PRE) of the homeotic gene Ultrabithorax (Ubx) and efficient PRE recruitment requires an intact Scm SAM domain. Scm is found in discrete intranuclear foci known as PcG bodies and global changes in sumoylation disrupt formation of these structures. Sumoylation‐defective Scm mutants are recruited to the PRE at higher than normal levels and globally perturbing sumoylation augments the Scm‐PRE association. In the metathorax, Ubx expression promotes haltere formation and suppresses wing development. When SUMO levels are reduced we observe decreased expression of Ubx and partial haltere‐to‐wing transformation phenotypes. These observations suggest that SUMO negatively regulates Scm function by impeding its recruitment to the Ubx major PRE.

A role for zinc in postsynaptic density asSAMbly and plasticity?

Chemical synapses are asymmetric cell junctions that mediate communication between neurons. Multidomain scaffolding proteins of the Shank family act as major organizing elements of the "postsynaptic density"--that is, the cytoskeletal protein matrix associated with the postsynaptic membrane. A recent study has shown that the C-terminal sterile alpha-motif or "SAM domain" of Shank3 (also known as ProSAP2) can form two-dimensional sheets of helical fibers. Assembly and packaging of these fibers are markedly enhanced by the presence of Zn2+ ions. Zn2+ can be released together with glutamate from synaptic vesicles and can enter the postsynaptic cell through specific ionotropic receptors. Based on these observations, we propose a new model of synaptic plasticity in which Zn2+ influx directly and instantly modulates the structure and function of the postsynaptic density.

Mechanisms of transcriptional repression of cell-cycle G2/M promoters by p63.

p63 is a developmentally regulated transcription factor related to p53, which activates and represses specific genes. The human AEC (Ankyloblepharon–Ectodermal dysplasia-Clefting) and EEC (Ectrodactyly–Ectodermal dysplasia–Cleft lip/palate) syndromes are caused by missense mutations of p63, within the DNA-binding domain (EEC) or in the C-terminal sterile alpha motif domain (AEC). We show here that p63 represses transcription of cell-cycle G2/M genes by binding to multiple CCAAT core promoters in immortalized and primary keratinocytes. The CCAAT-activator NF-Y and ΔNp63α are associated in vivo and a conserved α-helix of the NF-YC histone fold is required. p63 AEC mutants, but not an EEC mutant, are incapable to bind NF-Y. ΔNp63α, but not the AEC mutants repress CCAAT-dependent transcription of G2/M genes. Chromatin immunoprecipitation recruitment assays establish that the AEC mutants are not recruited to G2/M promoters, while normally present on 14-3-3σ, which contains a sequence-specific binding site. Surprisingly, the EEC C306R mutant activates transcription. Upon keratinocytes differentiation, NF-Y and p63 remain bound to G2/M promoters, while HDACs are recruited, histones deacetylated, Pol II displaced and transcription repressed. Our data indicate that NF-Y is a molecular target of p63 and that inhibition of growth activating genes upon differentiation is compromised by AEC missense mutations.

C‐terminal synaptic targeting elements for postsynaptic density proteins ProSAP1/Shank2 and ProSAP2/Shank3

Synapses are specialized contact sites mediating communication between neurons. Synaptogenesis requires the specific assembly of protein clusters at both sides of the synaptic contact by mechanisms that are barely understood. We studied the synaptic targeting of multi-domain proteins of the ProSAP/Shank family thought to serve as master scaffolding molecules of the postsynaptic density. In contrast to Shank1, expression of green-fluorescent protein (GFP)-tagged ProSAP1/Shank2 and ProSAP2/Shank3 deletion constructs in hippocampal neurons revealed that their postsynaptic localization relies on the integrity of the C-termini. The shortest construct that was perfectly targeted to synaptic sites included the last 417 amino acids of ProSAP1/Shank2 and included the C-terminal sterile alpha motif (SAM) domain. Removal of 54 residues from the N-terminus of this construct resulted in a diffuse distribution in the cytoplasm. Altogether, our data delineate a hitherto unknown targeting signal in both ProSAP1/Shank2 and ProSAP2/Shank3 and provide evidence for an implication of these proteins and their close homologue, Shank1, in distinct molecular pathways.

Equilibrium unfolding of the C-terminal SAM domain of p73.

The sterile alpha motif (SAM) domain is a protein module of approximately 65 to 70 amino acids found in many diverse proteins whose functions range from signal transduction to transcriptional repression. The alpha splice variant of p73 (p73 alpha), a homologue of the tumor suppressor p53, has close to its C-terminus a SAM motif. Here, we report the folding equilibrium properties of the p73 alpha SAM domain (SAMp73) by using different biophysical techniques (circular dichroism, fluorescence, and Fourier transform infrared spectroscopies, and differential scanning calorimetry). Those probes indicate that SAMp73 folds via a two-state mechanism. Fluorescence experiments performed at different pHs showed two titrations: the first one due to an acid residue (with a pK(a) = 4.5 +/- 0.3) and the second due to deprotonation of tyrosine residues. The conformational stability of the protein upon chemical denaturation was determined over the pH range 3 to 10. The maximum conformational stability is DeltaG = 5.7 +/- 0.4 kcal x mol(-1) (at 25 degrees C) and occurs in a broad maximum, with little variation, between pH 6 and 10. The high melting temperature of SAMp73 (T(m) = 93.5 degrees C), despite its moderate conformational stability at 25 degrees C, can be ascribed to its low heat capacity change upon unfolding, DeltaC(p), which is estimated to be around 915 cal x K(-1) x mol(-1) at 25 degrees C and only around 543 cal x K(-1) x mol(-1) at the T(m). The implications of the temperature-dependent nature of DeltaC(p) are discussed in relation to the thermal stability of proteins as opposed to their conformational stability at room temperature.

Somatostatin Receptor Interacting Protein Defines a Novel Family of Multidomain Proteins Present in Human and Rodent Brain

By using the yeast two-hybrid system we identified a novel protein from the human brain interacting with the C terminus of somatostatin receptor subtype 2. This protein termed somatostatin receptor interacting protein is characterized by a novel domain structure, consisting of six N-terminal ankyrin repeats followed by SH3 and PDZ domains, several proline-rich regions, and a C-terminal sterile alpha motif. It consists of 2185 amino acid residues encoded by a 9-kilobase pair mRNA; several splice variants have been detected in human and rat cDNA libraries. Sequence comparison suggests that the novel multidomain protein, together with cortactin-binding protein, forms a family of cytoskeletal anchoring proteins. Fractionation of rat brain membranes indicated that somatostatin receptor interacting protein is enriched in the postsynaptic density fraction. The interaction of somatostatin receptor subtype 2 with its interacting protein was verified by overlay assays and coimmunoprecipitation experiments from transfected human embryonic kidney cells. Somatostatin receptor subtype 2 and the interacting protein display a striking overlap of their expression patterns in the rat brain. Interestingly, in the hippocampus the mRNA for somatostatin receptor interacting protein was not confined to the cell bodies but was also observed in the molecular layer, suggesting a dendritic localization of this mRNA.

Proline-Rich Synapse-Associated Proteins ProSAP1 and ProSAP2 Interact with Synaptic Proteins of the SAPAP/GKAP Family

We have recently isolated a novel proline-rich synapse-associated protein-1 (ProSAP1) that is highly enriched in postsynaptic density (PSD). A closely related multidomain protein, ProSAP2, shares a highly conserved PDZ (PSD-95/discs-large/ZO-1) domain (80% identity), a ppI domain that mediates the interaction with cortactin, and a C-terminal SAM (sterile alpha-motif) domain. In addition, ProSAP2 codes for five ankyrin repeats and a SH3 (Src homology 3) domain. Transcripts for both proteins are coexpressed in many regions of rat brain, but show a distinct expression pattern in the cerebellum. Using the PDZ domains of ProSAP1 and 2 as bait in the yeast two-hybrid system, we isolated several clones of the SAPAP/GKAP (SAP90/PSD-95-associated protein/guanylate kinase-associated protein) family. The association of the proteins was verified by coimmunoprecipitation and cotransfection in HEK cells. Therefore, proteins of the ProSAP family represent a novel link between SAP90/PSD-95 bound membrane receptors and the cytoskeleton at glutamatergic synapses of the central nervous system.

P53 Family members p63 and p73 are SAM domain-containing proteins.

Homologs of the tumor suppressor p53, called p63 and p73, have been identified. The p63 and p73 family members possess a domain structure similar to p53, but contain variable C-terminal extensions. We find that some of the C-terminal extensions contain Sterile Alpha Motif (SAM) domains. SAM domains are protein modules that are involved in protein-protein interactions. Consistent with this role, the C-terminal SAM domains of the p63 and p73 may regulate function by recruiting other protein effectors.