Desensitization is a universal feature of ligand-gated ion channels. Using the crystal structure of the GluR2 L483Y mutant channel as a guide, we attempted to build non-desensitizing kainate-subtype glutamate receptors. Success was achieved for GluR5, GluR6 and GluR7 with intermolecular disulfide cross-links but not by engineering the dimer interface. Crystallographic analysis of the GluR6 Y490C L752C dimer revealed relaxation from the active conformation, which functional studies reveal is not sufficient to trigger desensitization. The equivalent non-desensitizing cross-linked GluR2 mutant retained weak sensitivity to a positive allosteric modulator, which had no effect on GluR2 L483Y. These results establish that the active conformation of AMPA and kainate receptors is conserved and further show that their desensitization requires dimer rearrangements, that subtle structural differences account for their diverse functional properties and that the ligand-binding core dimer is a powerful regulator of ion-channel activity.

Glutamate receptor (GluR) ion channels mediate fast synaptic transmission in the mammalian CNS. Numerous crystallographic studies, the majority on the GluR2-subtype AMPA receptor, have revealed the structural basis for binding of subtype-specific agonists. In contrast, because there are far fewer antagonist-bound structures, the mechanisms for antagonist binding are much less well understood, particularly for kainate receptors that exist as multiple subtypes with a distinct biology encoded by the GluR5-7, KA1, and KA2 genes. We describe here high-resolution crystal structures for the GluR5 ligand-binding core complex with UBP302 and UBP310, novel GluR5-selective antagonists. The crystal structures reveal the structural basis for the high selectivity for GluR5 observed in radiolabel displacement assays for the isolated ligand binding cores of the GluR2, GluR5, and GluR6 subunits and during inhibition of glutamate-activated currents in studies on full-length ion channels. The antagonists bind via a novel mechanism and do not form direct contacts with the E723 side chain as occurs in all previously solved AMPA and kainate receptor agonist and antagonist complexes. This results from a hyperextension of the ligand binding core compared with previously solved structures. As a result, in dimer assemblies, there is a 22 Å extension of the ion channel linkers in the transition from antagonist-to glutamate-bound forms. This large conformational change is substantially different from that described for AMPA receptors, was not possible to predict from previous work, and suggests that glutamate receptors are capable of much larger movements than previously thought.

No abstract

The role of the beta-ketoacyl synthase domains in dimerization of the 2505 residue subunits of the multifunctional animal FAS has been evaluated by a combination of crosslinking and characterization of several truncated forms of the protein. Polypeptides containing only the N-terminal 971 residues can form dimers, but polypeptides lacking only the N-terminal 422 residue beta-ketoacyl synthase domain cannot. FAS subunits can be crosslinked with spacer lengths as short as 6 A, via cysteine residues engineered near the N terminus of the full-length polypeptides. The proximity of the N-terminal beta-ketoacyl synthase domains and their essential role in dimerization is consistent with a revised model for the FAS in which a head-to-head arrangement of two coiled subunits facilitates functional interactions between the dimeric beta-ketoacyl synthase and the acyl carrier protein domains of either subunit.

Resistance to apoptosis is a hallmark of leukemia, representing a major therapeutic obstacle. We previously showed that CCAAT/enhancer binding protein α (C/EBPα) and its AML mutants inhibit apoptosis in hematopoietic cells dependent upon induction of bcl-2 in cooperation with NF-κB p50. We now identify the genes encoding mcl-1, an anti-apoptotic bcl-2 member and FLIP, an inhibitor of the extrinsic apoptosis pathway, as additional direct genetic targets of the C/EBPα:NF-κB complex. In particular, C/EBPα binds to the BCL2, FLIP or MCL1 promoters, as detected by ChIP assay in both Ba/F3 cells and normal murine marrow or spleen cells. In contrast, C/EBPα does not bind to the promoters of these genes in marrow or spleen cells obtained from nfkb1(−/−) mice that lack NF-κB p50. In addition, induction of C/EBPα from the zinc-responsive MT promoter in Ba/F3 cells leads to induction of BCL2, FLIP, and MCL1 mRNAs. Furthermore, C/EBPα(ΔK312), an AML derived leucine zipper (LZ) mutant that does not bind DNA but can interact with NF-κB p50, retains the ability to localize to the BCL2, FLIP, and MCL1 promoters and to induce their corresponding mRNAs. The promoters for each of these genes also bind endogenous NF-κB p50 as assessed by ChIP assay. We previously demonstrated that the ability of C/EBPα to block the intrinsic apoptotic pathway and protect cells from radiation- or cytokine withdrawal-induced apoptosis is dependent upon induction of bcl-2. We now show that in C/EBPα transgenic splenocytes bcl-2 protein is induced only in the presence of NF-κB p50. As expected from the induction of FLIP by C/EBPα, we now also find that transgenic C/EBPα protects wild-type but not nfkb1(−/−) splenocytes from apoptosis induced by FasL activation of the extrinsic apoptotic pathway. In addition, we now demonstrate direct interaction of C/EBPα with NF-κB p50 using highly purified proteins isolated from E. coli. Together these data indicate that BCL2, MCL1, or FLIP are induced cooperatively by C/EBPα or C/EBPα(ΔK312) proteins that tether to the corresponding promoters via bound NF-κB p50. Additionally, we found that C/EBPα or C/EBPα(ΔK312) bind to the nfkb1 promoter and induce its mRNA expression. Conversely, we also demonstrate that immature, lineage-negative marrow cells isolated from nfkb1(−/−) mice have an approximately 3-fold reduced level of C/EBPα and that NF-κB p50 binds the endogenous CEBPA promoter in normal murine marrow cells in the ChIP assay, indicating cross-regulation and a potential positive feedback loop between C/EBPα and NF-κB p50. Indeed, induction of exogenous C/EBPα in Ba/F3 cells increases nuclear localization of NF-κB as assessed by gel shift assay. In summary, C/EBPα and its AML mutants interact directly with NF-κB p50 via their basic regions; through binding to a DNA κB site the complex activates several anti-apoptotic genes including BCL2, MCL1 and FLIP, thereby inhibiting both the extrinsic and intrinsic apoptotic pathways. In addition, through positive feedback NF-κB p50 induces C/EBPα expression, and C/EBPα induces NF-κB p50. These effects are retained by the C/EBPα AML mutants and do not depend on DNA binding, contributing to NF-κB deregulation in malignant blasts. As C/EBPα or mutant C/EBPα variants and activated NF-κB are commonly expressed together in AML stem cells, targeting the C/EBPα:NF-κB p50 interaction with small molecules may provide a novel therapeutic approach to this and other malignancies.