James C. Stroud

FOXP3 Controls Regulatory T Cell Function through Cooperation with NFAT

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.

Crystal structure of the extracellular domain of nAChR alpha1 bound to alpha-bungarotoxin at 1.94 A resolution.

We determined the crystal structure of the extracellular domain of the mouse nicotinic acetylcholine receptor (nAChR) α1 subunit bound to α-bungarotoxin at 1.94 Å 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 α1 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.

Docking Motif Interactions in MAP Kinases Revealed by Hydrogen Exchange Mass Spectrometry

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.

Toxic fibrillar oligomers of amyloid-β have cross-β structure.

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-β1–42 (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.

Structure of a TonEBP-DNA complex reveals DNA encircled by a transcription factor.

Tonicity-responsive enhancer binding protein (TonEBP), also known as NFAT5, is a unique member of the NFAT family of transcription factors that regulates gene expression induced by osmotic stress in mammalian cells. Unlike monomeric members of the NFAT family, TonEBP exists as a homodimer and binds asymmetric TonE DNA sites; furthermore, the affinity of TonEBP for DNA is much lower than that of other NFAT proteins. How TonEBP recognizes the TonE site and regulates the activation of hypertonicity response genes has not been clear. Here we show that TonEBP adopts a NF-κB-like structure upon binding to DNA, providing a direct structural link between the NFAT and NF-κB family of transcription factors. We also show that TonEBP completely encircles its DNA target and present biochemical evidence that the DNA encirclement may lead to increased kinetic stability of the TonEBP–DNA complex. Thus, the list of proteins that bind DNA by encirclement is now expanded to include sequence-specific transcription factors.

Structure of NFAT1 bound as a dimer to the HIV-1 LTR kappa B element.

DNA binding by NFAT1 as a dimer has been implicated in the activation of host and viral genes. Here we report a crystal structure of NFAT1 bound cooperatively as a dimer to the highly conserved κB site from the human immunodeficiency virus 1 (HIV-1) long terminal repeat (LTR). This structure reveals a new mode of dimerization and protein-DNA recognition by the Rel homology region (RHR) of NFAT1. The two NFAT1 monomers form a complete circle around the κB DNA through protein-protein interactions mediated by both their N- and C-terminal subdomains. The major dimer interface, formed by the C-terminal domain, is asymmetric and substantially different from the symmetric dimer interface seen in other Rel family proteins. Comparison to other NFAT structures, including NFAT5 and the NFAT1–Fos-Jun–ARRE2 complex, reveals that NFAT1 adopts different conformations and its protein surfaces mediate distinct protein-protein interactions in the context of different DNA sites.

The structured core domain of αB-crystallin can prevent amyloid fibrillation and associated toxicity

Significance We find that the core domain of the human molecular chaperone αB-crystallin can function effectively in preventing protein aggregation and amyloid toxicity. The core domain represents only half the total sequence of the protein, but it is one of the most potent known inhibitors of the aggregation of amyloid-β, a process implicated in Alzheimer’s disease. We have determined high-resolution structures of this core domain and investigated its biophysical properties in solution. We find that the excised domain efficiently prevents amyloid aggregation and thereby reduces the toxicity of the resulting aggregates to cells. The structures of these domains that we present should represent useful scaffolds for the design of novel amyloid inhibitors. 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.

Sequence-specific recruitment of transcriptional co-repressor Cabin1 by myocyte enhancer factor-2

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 Å resolution. The crystal structure reveals a stably folded MEF2S domain on the surface of the MADS box. Cabin1 adopts an amphipathic α-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.

Structure of a domain-swapped FOXP3 dimer on DNA and its function in regulatory T cells

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 the transcription factor 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.

Structural Basis of HIV-1 Activation by NF-κB—A Higher-Order Complex of p50:RelA Bound to the HIV-1 LTR

The activation and latency of human immunodeficiency virus type 1 (HIV-1) are tightly controlled by the transcriptional activity of its long terminal repeat (LTR) region. The LTR is regulated by viral proteins as well as host factors, including the nuclear factor kappaB (NF-kappaB) that becomes activated in virus-infected cells. The two tandem NF-kappaB sites of the LTR are among the most highly conserved sequence elements of the HIV-1 genome. Puzzlingly, these sites are arranged in a manner that seems to preclude simultaneous binding of both sites by NF-kappaB, although previous biochemical work suggests otherwise. Here, we have determined the crystal structure of p50:RelA bound to the tandem kappaB element of the HIV-1 LTR as a dimeric dimer, providing direct structural evidence that NF-kappaB can occupy both sites simultaneously. The two p50:RelA dimers bind the adjacent kappaB sites and interact through a protein contact that is accommodated by DNA bending. The two dimers clamp DNA from opposite faces of the double helix and form a topological trap of the bound DNA. Consistent with these structural features, our biochemical analyses indicate that p50:RelA binds the HIV-1 LTR tandem kappaB sites with an apparent anti-cooperativity but enhanced kinetic stability. The slow on and off rates we observe may be relevant to viral latency because viral activation requires sustained NF-kappaB activation. Furthermore, our work demonstrates that the specific arrangement of the two kappaB sites on the HIV-1 LTR can modulate the assembly kinetics of the higher-order NF-kappaB complex on the viral promoter. This phenomenon is unlikely restricted to the HIV-1 LTR but probably represents a general mechanism for the function of composite DNA elements in transcription.