Antigen stimulation of immune cells activates the transcription factor NFAT, a key regulator of T cell activation and anergy. NFAT forms cooperative complexes with the AP-1 family of transcription factors and regulates T cell activation-associated genes. Here we show that regulatory T cell (Treg) function is mediated by an analogous cooperative complex of NFAT with the forkhead transcription factor FOXP3, a lineage specification factor for Tregs. The crystal structure of an NFAT:FOXP2:DNA complex reveals an extensive protein-protein interaction interface between NFAT and FOXP2. Structure-guided mutations of FOXP3, predicted to progressively disrupt its interaction with NFAT, interfere in a graded manner with the ability of FOXP3 to repress expression of the cytokine IL2, upregulate expression of the Treg markers CTLA4 and CD25, and confer suppressor function in a murine model of autoimmune diabetes. Thus by switching transcriptional partners, NFAT converts the acute T cell activation program into the suppressor program of Tregs.

We determined the crystal structure of the extracellular domain of the mouse nicotinic acetylcholine receptor (nAChR) alpha1 subunit bound to alpha-bungarotoxin at 1.94 A resolution. This structure is the first atomic-resolution view of a nAChR subunit extracellular domain, revealing receptor-specific features such as the main immunogenic region (MIR), the signature Cys-loop and the N-linked carbohydrate chain. The toxin binds to the receptor through extensive protein-protein and protein-sugar interactions. To our surprise, the structure showed a well-ordered water molecule and two hydrophilic residues deep in the core of the alpha1 subunit. The two hydrophilic core residues are highly conserved in nAChRs, but correspond to hydrophobic residues in the nonchannel homolog acetylcholine-binding proteins. We carried out site-directed mutagenesis and electrophysiology analyses to assess the functional role of the glycosylation and the hydrophilic core residues. Our structural and functional studies show essential features of the nAChR and provide new insights into the gating mechanism.

Protein interactions between MAP kinases and substrates, activators, and scaffolding proteins are regulated by docking site motifs, one containing basic residues proximal to Leu-X-Leu (DEJL) and a second containing Phe-X-Phe (DEF). Hydrogen exchange mass spectrometry was used to identify regions in MAP kinases protected from solvent by docking motif interactions. Protection by DEJL peptide binding was observed in loops spanning beta7-beta8 and alphaD-alphaE in p38alpha and ERK2. In contrast, protection by DEF binding to ERK2 revealed a distinct hydrophobic pocket for Phe-X-Phe binding formed between the P+1 site, alphaF helix, and the MAP kinase insert. In inactive ERK2, this pocket is occluded by intramolecular interactions with residues in the activation lip. In vitro assays confirm the dependence of Elk1 and nucleoporin binding on ERK2 phosphorylation, and provide a structural basis for preferential involvement of active ERK in substrate binding and nuclear pore protein interactions.

Although amyloid fibers are found in neurodegenerative diseases, evidence points to soluble oligomers of amyloid-forming proteins as the cytotoxic species. Here, we establish that our preparation of toxic amyloid-β 1-42 (Abeta42) fibrillar oligomers (TABFOs) shares with mature amyloid fibrils the cross-β structure, in which adjacent β-sheets adhere by interpenetration of protein side chains. We study the structure and properties of TABFOs by powder X-ray diffraction, EM, circular dichroism, FTIR spectroscopy, chromatography, conformational antibodies, and celluar toxicity. In TABFOs, Abeta42 molecules stack into short protofilaments consisting of pairs of helical β-sheets that wrap around each other to form a superhelix. Wrapping results in a hole along the superhelix axis, providing insight into how Abeta may form pathogenic amyloid pores. Our model is consistent with numerous properties of Abeta42 fibrillar oligomers, including heterogenous size, ability to seed new populations of fibrillar oligomers, and fiber-like morphology.Abeta oligomers | toxic oligomers | Alzheimer's disease | domain swapping | protein aggregation S everal neurodegenerative diseases are correlated with amyloid fibrillar deposits (1). For a number of these diseases, it has been postulated that amyloid fibers may not play the primary causative role (2). Rather, soluble aggregates of the amyloidogenic proteins are likely the relevant etiological agents (2, 3). The most prevalent of these neurodegenerative diseases, Alzheimer's disease (4), is strongly linked to the presence of soluble aggregates of amyloid-β (Abeta) (5). Abeta aggregates have been shown to impair neurite function (6), synaptic morphology (7), cognitive function (8), and cell viability (9). In the prion conditions, also classed as amyloid diseases (10), small oligomers have also been identified as the toxic species (11). Recently, the availability of structure-specific antibodies has provided a means to group oligomers into two broad antigenic categories known as prefibrillar and fibrillar oligomers (12). Fibrillar oligomers are recognized by the OC antibody isolated from rabbits immunized with Abeta fibers (13), suggesting that Abeta fibrillar oligomers share surface features with Abeta fibers. In addition to fiber-like morphology, fibrillar oligomers are similar to fibers in that fibrillar oligomers can seed new populations of fibrillar oligomers (14). The ability to seed suggests that, like fibers, fibrillar oligomers are organized into a repeating array or lattice of monomers, wherein the monomers have identical structures. Fibrillar oligomers likely have a distinct lattice from fibers, because Abeta fibrillar oligomers do not seed Abeta fiber formation (14). Here, we characterize a particular preparation of fibrillar oligomers that we term toxic Abeta 1-42 (Abeta42) fibrillar oligomers (TABFOs).The structure of amyloid fibers may provide insight into the structure of fibrillar oligomers. Fiber diffraction studies of chemically pure amyloid display a cross-β diffraction...

FOXP (FOXP1-4) is a newly defined subfamily of the forkhead box (FOX) transcription factors. A mutation in the FOXP2 forkhead domain cosegregates with a severe speech disorder, whereas several mutations in the FOXP3 forkhead domain are linked to the IPEX syndrome in human and a similar autoimmune phenotype in mice. Here we report a 1.9 A crystal structure of the forkhead domain of human FOXP2 bound to DNA. This structure allows us to revise the previously proposed DNA recognition mechanism and provide a unifying model of DNA binding for the FOX family of proteins. Our studies also reveal that the FOXP2 forkhead domain can form a domain-swapped dimer, made possible by a strategic substitution of a highly conserved proline in conventional FOX proteins with alanine in the P subfamily. Disease-causing mutations in FOXP2 and FOXP3 map either to the DNA binding surface or the domain-swapping dimer interface, functionally corroborating the crystal structure.

Mammalian small heat-shock proteins (sHSPs) are molecular chaperones that form polydisperse and dynamic complexes with target proteins, serving as a first line of defense in preventing their aggregation into either amorphous deposits or amyloid fibrils. Their apparently broad target specificity makes sHSPs attractive for investigating ways to tackle disorders of protein aggregation. The two most abundant sHSPs in human tissue are αB-crystallin (ABC) and HSP27; here we present high-resolution structures of their core domains (cABC, cHSP27), each in complex with a segment of their respective C-terminal regions. We find that both truncated proteins dimerize, and although this interface is labile in the case of cABC, in cHSP27 the dimer can be cross-linked by an intermonomer disulfide linkage. Using cHSP27 as a template, we have designed an equivalently locked cABC to enable us to investigate the functional role played by oligomerization, disordered N and C termini, subunit exchange, and variable dimer interfaces in ABC. We have assayed the ability of the different forms of ABC to prevent protein aggregation in vitro. Remarkably, we find that cABC has chaperone activity comparable to that of the full-length protein, even when monomer dissociation is restricted through disulfide linkage. Furthermore, cABC is a potent inhibitor of amyloid fibril formation and, by slowing the rate of its aggregation, effectively reduces the toxicity of amyloid-β peptide to cells. Overall we present a small chaperone unit together with its atomic coordinates that potentially enables the rational design of more effective chaperones and amyloid inhibitors.X-ray crystallography | ion mobility mass spectrometry | nuclear magnetic resonance spectroscopy

The transcription factor FOXP3 is essential for the suppressive function of regulatory T cells that are required for maintaining self-tolerance. We have solved the crystal structure of the FOXP3 forkhead domain, as a ternary complex with the DNA-binding domain of nuclear factor of activated T cells-1 (NFAT1) and a DNA oligonucleotide from the interleukin-2 promoter. A striking feature of this structure is that FOXP3 forms a domain-swapped dimer that bridges two molecules of DNA. Structure-guided or autoimmune disease (IPEX)-associated mutations in the domain-swap interface diminished dimer formation by the FOXP3 forkhead domain without compromising FOXP3 DNA binding. These mutations also eliminated T cell suppressive activity conferred by FOXP3, both in vitro and in a murine model of autoimmune diabetes in vivo. We conclude that FOXP3-mediated suppressor function requires dimerization through the forkhead domain, and that mutations in the dimer interface can lead to the systemic autoimmunity observed in IPEX patients.

The myocyte enhancer factor-2 (MEF2) family of transcription factors has important roles in the development and function of T cells, neuronal cells and muscle cells. MEF2 is capable of repressing or activating transcription by association with a variety of co-repressors or co-activators in a calcium-dependent manner. Transcriptional repression by MEF2 has attracted particular attention because of its potential role in hypertrophic responses of cardiomyocytes. Several MEF2 co-repressors, such as Cabin1/Cain and class II histone deacetylases (HDACs), have been identified. However, the molecular mechanism of their recruitment to specific promoters by MEF2 remains largely unknown. Here we report a crystal structure of the MADS-box/MEF2S domain of human MEF2B bound to a motif of the transcriptional co-repressor Cabin1 and DNA at 2.2 A resolution. The crystal structure reveals a stably folded MEF2S domain on the surface of the MADS box. Cabin1 adopts an amphipathic alpha-helix to bind a hydrophobic groove on the MEF2S domain, forming a triple-helical interaction. Our studies of the ternary Cabin1/MEF2/DNA complex show a general mechanism by which MEF2 recruits transcriptional co-repressor Cabin1 and class II HDACs to specific DNA sites.