Tomas Obsil

Crystal Structure of the 14-3-3ζ:Serotonin N-Acetyltransferase Complex: A Role for Scaffolding in Enzyme Regulation

Serotonin N-acetyltransferase (AANAT) controls the daily rhythm in melatonin synthesis. When isolated from tissue, AANAT copurifies with isoforms epsilon and zeta of 14-3-3. We have determined the structure of AANAT bound to 14-3-3zeta, an association that is phosphorylation dependent. AANAT is bound in the central channel of the 14-3-3zeta dimer, and is held in place by extensive interactions both with the amphipathic phosphopeptide binding groove of 14-3-3zeta and with other parts of the central channel. Thermodynamic and activity measurements, together with crystallographic analysis, indicate that binding of AANAT by 14-3-3zeta modulates AANATs activity and affinity for its substrates by stabilizing a region of AANAT involved in substrate binding.

Activation and Regulation of Purinergic P2X Receptor Channels

Mammalian ATP-gated nonselective cation channels (P2XRs) can be composed of seven possible subunits, denoted P2X1 to P2X7. Each subunit contains a large ectodomain, two transmembrane domains, and intracellular N and C termini. Functional P2XRs are organized as homomeric and heteromeric trimers. This review focuses on the binding sites involved in the activation (orthosteric) and regulation (allosteric) of P2XRs. The ectodomains contain three ATP binding sites, presumably located between neighboring subunits and formed by highly conserved residues. The detection and coordination of three ATP phosphate residues by positively charged amino acids are likely to play a dominant role in determining agonist potency, whereas an AsnPheArg motif may contribute to binding by coordinating the adenine ring. Nonconserved ectodomain histidines provide the binding sites for trace metals, divalent cations, and protons. The transmembrane domains account not only for the formation of the channel pore but also for the binding of ivermectin (a specific P2X4R allosteric regulator) and alcohols. The N- and C- domains provide the structures that determine the kinetics of receptor desensitization and/or pore dilation and are critical for the regulation of receptor functions by intracellular messengers, kinases, reactive oxygen species and mercury. The recent publication of the crystal structure of the zebrafish P2X4.1R in a closed state provides a major advance in the understanding of this family of receptor channels. We will discuss data obtained from numerous site-directed mutagenesis experiments accumulated during the last 15 years with reference to the crystal structure, allowing a structural interpretation of the molecular basis of orthosteric and allosteric ligand actions.

Role of a pineal cAMP-operated arylalkylamine N-acetyltransferase/14-3-3-binding switch in melatonin synthesis.

The daily rhythm in melatonin levels is controlled by cAMP through actions on the penultimate enzyme in melatonin synthesis, arylalkylamine N-acetyltransferase (AANAT; serotonin N-acetyltransferase, EC 2.3.1.87). Results presented here describe a regulatory/binding sequence in AANAT that encodes a cAMP-operated binding switch through which cAMP-regulated protein kinase-catalyzed phosphorylation [RRHTLPAN → RRHpTLPAN] promotes formation of a complex with 14-3-3 proteins. Formation of this AANAT/14-3-3 complex enhances melatonin production by shielding AANAT from dephosphorylation and/or proteolysis and by decreasing the Km for 5-hydroxytryptamine (serotonin). Similar switches could play a role in cAMP signal transduction in other biological systems.

Structure/function relationships underlying regulation of FOXO transcription factors

The FOXO subgroup of forkhead transcription factors plays a central role in cell-cycle control, differentiation, metabolism control, stress response and apoptosis. Therefore, the function of these important molecules is tightly controlled by a wide range of protein–protein interactions and posttranslational modifications including phosphorylation, acetylation and ubiquitination. The mechanisms by which these processes regulate FOXO activity are mostly elusive. This review focuses on recent advances in structural studies of forkhead transcription factors and the insights they provide into the mechanism of DNA recognition. On the basis of these data, we discuss structural aspects of protein–protein interactions and posttranslational modifications that target the forkhead domain and the nuclear localization signal of FOXO proteins.

Structural basis of 14-3-3 protein functions.

The 14-3-3 proteins, a family of conserved regulatory molecules, participate in a wide range of cellular processes through binding interactions with hundreds of structurally and functionally diverse proteins. Several distinct mechanisms of the 14-3-3 protein function were described, including conformational modulation of the bound protein, masking of its sequence-specific or structural features, and scaffolding that facilitates interaction between two simultaneously bound proteins. Details of these functional modes, especially from the structural point of view, still remain mostly elusive. This review gives an overview of the current knowledge concerning the structure of 14-3-3 proteins and their complexes as well as the insights it provides into the mechanisms of their functions. We discuss structural basis of target recognition by 14-3-3 proteins, common structural features of their complexes and known mechanisms of 14-3-3 protein-dependent regulations.

Hippo/Mst1 Stimulates Transcription of the Proapoptotic Mediator NOXA in a FoxO1-Dependent Manner

The proapoptotic protein Noxa, a member of the BH3-only Bcl-2 protein family, can effectively induce apoptosis in cancer cells, although the relevant regulatory pathways have been obscure. Previous studies of the cytotoxic effects of α-tocopheryl succinate (α-TOS) on cancer cells identified a mechanism whereby α-TOS caused apoptosis requiring the Noxa-Bak axis. In the present study, ab initio analysis revealed a conserved FoxO-binding site (DBE; DAF-16 binding element) in the NOXA promoter, and specific affinity of FoxO proteins to this DBE was confirmed by fluorescence anisotropy. FoxO1 and FoxO3a proteins accumulated in the nucleus of α-TOS-treated cells, and the drug-induced specific FoxO1 association with the NOXA promoter and its activation were validated by chromatin immunoprecipitation. Using siRNA knockdown, a specific role for the FoxO1 protein in activating NOXA transcription in cancer cells was identified. Furthermore, the proapoptotic kinase Hippo/Mst1 was found to be strongly activated by α-TOS, and inhibiting Hippo/Mst1 by specific siRNA prevented phosphorylation of FoxO1 and its nuclear translocation, thereby reducing levels of NOXA transcription and apoptosis in cancer cells exposed to α-TOS. Thus, we have demonstrated that anticancer drugs, exemplified by α-TOS, induce apoptosis by a mechanism involving the Hippo/Mst1-FoxO1-Noxa pathway. We propose that activation of this pathway provides a new paradigm for developing targeted cancer treatments.

Both the N-terminal Loop and Wing W2 of the Forkhead Domain of Transcription Factor Foxo4 Are Important for DNA Binding

FoxO4 belongs to the “O” subset of forkhead transcription factors, which participate in various cellular processes. The forkhead DNA binding domain (DBD) consists of three-helix bundle resting on a small antiparallel β-sheet from which two extended loops protrude and create two wing-like structures. The wing W2 of FoxO factors contains a 14-3-3 protein-binding motif that is phosphorylated by protein kinase B in response to insulin or growth factors. In this report, we investigated the role of the N-terminal loop (portion located upstream of first helix H1) and the C-terminal region (loop known as wing W2) of the forkhead domain of transcription factor FoxO4 in DNA binding. Although the deletion of either portion partly reduces the FoxO4-DBD binding to the DNA, the simultaneous deletion of both regions inhibits DNA binding significantly. Förster resonance energy transfer measurements and molecular dynamics simulations suggest that both studied N- and C-terminal regions of FoxO4-DBD directly interact with DNA. In the presence of the N-terminal loop the protein kinase B-induced phosphorylation of wing W2 by itself has negligible effect on DNA binding. On the other hand, in the absence of this loop the phosphorylation of wing W2 significantly inhibits the FoxO4-DBD binding to the DNA. The binding of the 14-3-3 protein efficiently reduces DNA-binding potential of phosphorylated FoxO4-DBD regardless of the presence of the N-terminal loop. Our results show that both N- and C-terminal regions of forkhead domain are important for stability of the FoxO4-DBD·DNA complex.

Participation of the Lys313-Ile333 Sequence of the Purinergic P2X4 Receptor in Agonist Binding and Transduction of Signals to the Channel Gate

To study the roles of the Lys313-Ile333 ectodomain sequence of the rat P2X4 receptor in ATP binding and transduction of signals to the channel gate, the conserved Lys313, Tyr315, Gly316, Ike317, Arg318, Asp320, Val323, Lys329, Phe330, and Ile333 residues were mutated. Current recordings were done on lifted cells and ATP was applied using an ultrafast solution-switching system. The rates of wild type channel opening and closing in the presence of ATP, but not the rate of washout-induced closing, were dependent on agonist concentration. All mutants other than I317A were expressed in the plasma membrane at comparable levels. The majority of mutants showed significant changes in the peak amplitude of responses and the EC50 values for ATP. When stimulated with the supramaximal (1.4 mm) ATP concentration, mutants also differed in the kinetics of their activation, deactivation, and/or desensitization. The results suggest a critical role of the Lys313 residue in receptor function other than coordination of the phosphate group of ATP and possible contribution of the Tyr315 residue to the agonist binding module. The pattern of changes of receptor function by mutation of other residues was consistent with the operation of the Gly316-Ile333 sequence as a signal transduction module between the ligand binding domain and the channel gate in the second transmembrane domain.

Identification of P2X 4 receptor transmembrane residues contributing to channel gating and interaction with ivermectin

Ivermectin (IVM), a large macrocyclic lactone, specifically enhances P2X4 receptor-channel function by interacting with residues of transmembrane (TM) helices in the open conformation state. In this paper, we used cysteine-scanning mutagenesis of rat P2X4-TMs to identify and map residues of potential importance for channel gating and interaction with IVM. The receptor function was unchanged by mutations in 29 different residues, and among them, the IVM effects were altered in Gln36, Leu40, Val43, Val47, Trp50, Asn338, Gly342, Leu346, Ala349, and Ile356 mutants. The substitution-sensitive Arg33 and Cys353 mutants could also be considered as IVM-sensitive hits. The pattern of these 12 residues was consistent with helical topology of both TMs, with every third or fourth amino acid affected by substitution. These predominantly hydrophobic-nonpolar residues are also present in the IVM-sensitive Schistosoma mansoni P2X subunit. They lie on the same side of their helices and could face lipids in the open conformation state and provide the binding pocket for IVM. In contrast, the IVM-independent hits Met31, Tyr42, Gly45, Val49, Gly340, Leu343, Ala344, Gly347, Thr350, Asp354, and Val357 map on the opposite side of their helices, probably facing the pore of receptor or protein and playing important roles in gating.

Structural basis for DNA recognition by FOXO proteins.

The FOXO forkhead transcription factors are involved in metabolism control, cell survival, cellular proliferation, DNA damage repair response, and stress resistance. Their transcriptional activity is regulated through a number of posttranslational modifications, including phosphorylation, acetylation and ubiquitination. The recently determined three-dimensional structures of FOXO forkhead domains bound to DNA enable to explain the structural basis for DNA recognition by FOXO proteins and its regulation. The aim of this review is to summarize the recent structural characterization of FOXO proteins, the mechanisms of DNA recognition and the role of posttranslational modifications in the regulation of FOXO DNA-binding properties. This article is part of a Special Issue entitled: PI3K-AKT-FOXO axis in cancer and aging.