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In plants, MADS domain transcription factors act as central regulators of diverse developmental pathways. In Arabidopsis thaliana, one of the most central members of this family is SEPALLATA3 ( SEP3), which is involved in many aspects of plant reproduction, including floralmeristem and floral organ development. SEP3 has been shown to form homo and heterooligomeric complexes with other MADS domain transcription factors through its intervening ( I) and keratin-like ( K) domains. SEP3 function depends on its ability to form specific protein-protein complexes; however, the atomic level determinants of oligomerization are poorly understood. Here, we report the 2.5 angstrom crystal structure of a small portion of the intervening and the complete keratin-like domain of SEP3. The domains form two amphipathic alpha helices separated by a rigid kink, which prevents intramolecular association and presents separate dimerization and tetramerization interfaces comprising predominantly hydrophobic patches. Mutations to the tetramerization interface demonstrate the importance of highly conserved hydrophobic residues for tetramer stability. Atomic force microscopy was used to show SEP3-DNA interactions and the role of oligomerization in DNA binding and conformation. Based on these data, the oligomerization patterns of the larger family of MADS domain transcription factors can be predicted and manipulated based on the primary sequence.

Brunkard et al. propose that the identification of novel LEAFY sequences contradicts our model of evolution through promiscuous intermediates. Based on the debate surrounding land plant phylogeny and on our analysis of these interesting novel sequences, we explain why there is no solid evidence to disprove our model.

In plants, the AUXIN RESPONSE FACTOR (ARF) transcription factor family regulates gene expression in response to auxin. In the absence of auxin, ARF transcription factors are repressed by interaction with AUXIN/INDOLE 3-ACETIC ACID (Aux/IAA) proteins. Although the C termini of ARF and Aux/IAA proteins facilitate their homo- and heterooligomerization, the molecular basis for this interaction remained undefined. The crystal structure of the C-terminal interaction domain of Arabidopsis ARF7 reveals a Phox and Bem1p (PB1) domain that provides both positive and negative electrostatic interfaces for directional protein interaction. Mutation of interface residues in the ARF7 PB1 domain yields monomeric protein and abolishes interaction with both itself and IAA17. Expression of a stabilized Aux/IAA protein (i.e., IAA16) bearing PB1 mutations in Arabidopsis suggests a multimerization requirement for ARF protein repression, leading to a refined auxin-signaling model.

The plant hormone auxin is a key morphogenetic regulator acting from embryogenesis onwards. Transcriptional events in response to auxin are mediated by the auxin response factor (ARF) transcription factors and the Aux/IAA (IAA) transcriptional repressors. At low auxin concentrations, IAA repressors associate with ARF proteins and recruit corepressors that prevent auxin-induced gene expression. At higher auxin concentrations, IAAs are degraded and ARFs become free to regulate auxin-responsive genes. The interaction between ARFs and IAAs is thus central to auxin signalling and occurs through the highly conserved domain III/IV present in both types of proteins. Here, we report the crystal structure of ARF5 domain III/IV and reveal the molecular determinants of ARF-IAA interactions. We further provide evidence that ARFs have the potential to oligomerize, a property that could be important for gene regulation in response to auxin.

Isolated influenza A virus nucleoprotein exists in an equilibrium between monomers and trimers. Samples containing only monomers or only trimers can be stabilized by respectively low and high salt. The trimers bind RNA with high affinity but remain trimmers, whereas the monomers polymerise onto RNA forming nucleoprotein-RNA complexes. When wild type (wt) nucleoprotein is crystallized, it forms trimers, whether one starts with monomers or trimers. We therefore crystallized the obligate monomeric R416A mutant nucleoprotein and observed how the domain exchange loop that leads over to a neighbouring protomer in the trimer structure interacts with equivalent sites on the mutant monomer surface, avoiding polymerisation. The C-terminus of the monomer is bound to the side of the RNA binding surface, lowering its positive charge. Biophysical characterization of the mutant and wild type monomeric proteins gives the same results, suggesting that the exchange domain is folded in the same way for the wild type protein. In a search for how monomeric wt nucleoprotein may be stabilized in the infected cell we determined the phosphorylation sites on nucleoprotein isolated from virus particles. We found that serine 165 was phosphorylated and conserved in all influenza A and B viruses. The S165D mutant that mimics phosphorylation is monomeric and displays a lowered affinity for RNA compared with wt monomeric NP. This suggests that phosphorylation may regulate the polymerisation state and RNA binding of nucleoprotein in the infected cell. The monomer structure could be used for finding new anti influenza drugs because compounds that stabilize the monomer may slow down viral infection.

Transcription factors (TFs) are key players in evolution. Changes affecting their function can yield novel life forms but may also have deleterious effects. Consequently, gene duplication events that release one gene copy from selective pressure are thought to be the common mechanism by which TFs acquire new activities. Here, we show that LEAFY, a major regulator of flower development and cell division in land plants, underwent changes to its DNA binding specificity, even though plant genomes generally contain a single copy of the LEAFY gene. We examined how these changes occurred at the structural level and identify an intermediate LEAFY form in hornworts that appears to adopt all different specificities. This promiscuous intermediate could have smoothed the evolutionary transitions, thereby allowing LEAFY to evolve new binding specificities while remaining a single-copy gene.

The LEAFY (LFY) protein is a key regulator of flower development in angiosperms. Its gradually increased expression governs the sharp floral transition, and LFY subsequently controls the patterning of flower meristems by inducing the expression of floral homeotic genes. Despite a wealth of genetic data, how LFY functions at the molecular level is poorly understood. Here, we report crystal structures for the DNA-binding domain of Arabidopsis thaliana LFY bound to two target promoter elements. LFY adopts a novel seven-helix fold that binds DNA as a cooperative dimer, forming base-specific contacts in both the major and minor grooves. Cooperativity is mediated by two basic residues and plausibly accounts for LFY's effectiveness in triggering sharp developmental transitions. Our structure reveals an unexpected similarity between LFY and helix-turn-helix proteins, including homeodomain proteins known to regulate morphogenesis in higher eukaryotes. The appearance of flowering plants has been linked to the molecular evolution of LFY. Our study provides a unique framework to elucidate the molecular mechanisms underlying floral development and the evolutionary history of flowering plants.

The nucleoprotein (NP) of influenza virus covers the viral RNA entirely and it is this NP-RNA complex that is the template for transcription and replication by the viral polymerase. Purified NP forms a dynamic equilibrium between monomers and small oligomers, but only the monomers can oligomerize onto RNA. Therefore, drugs that stabilize the monomers or that induce abnormal oligomerization may have an antiviral effect, as would drugs that interfere with RNA binding. Crystal structures have been produced for monomeric and dimeric mutants, and for trimers and tetramers; high-resolution electron microscopy structures have also been calculated for the viral NP-RNA complex. We explain how these structures and the dynamic oligomerization equilibrium of NP can be and have been used for anti-influenza drug development.

The typical bullet shape of Rhabdoviruses is thought to rely on the matrix protein for stabilizing the nucleocapsid coil. Here we scrutinize the morphology of purified and recombinant nucleocapsids of vesicular stomatitis virus in vitro. We elucidate pH and ionic strength conditions for their folding into conical tips and further growth into whole bullets, and provide cryo-electron microscopy reconstructions of the bullet tip and the helical trunk. We address conformational variability of the reconstituted nucleocapsids and the issue of constraints imposed by the binding of matrix protein. Our findings bridge the gap between the isolated nucleoprotein-RNA string in its form of an undulating ribbon, and the tight bullet-shaped virion skeleton.

Human adenovirus serotype 5 (HAdV5)-based vectors administered intravenously accumulate in the liver as the result of their direct binding to blood coagulation factor X (FX) and subsequent interaction of the FX-HAdV5 complex with heparan sulfate proteoglycan (HSPG) at the surface of liver cells. Intriguingly, the serotype 35 fiber-pseudotyped vector HAdV5F35 has liver transduction efficiencies 4-logs lower than HAdV5, even though both vectors carry the same hexon capsomeres. In order to reconcile this apparent paradox, we investigated the possible role of other viral capsid proteins on the FX/HSPG-mediated cellular uptake of HAdV5-based vectors. Using CAR-and CD46-negative CHO cells varying in HSPG expression, we confirmed that FX bound to serotype 5 hexon protein and to HAdV5 and HAdV5F35 virions via its Gla-domain, and enhanced the binding of both vectors to surface-immobilized hypersulfated heparin and cellular HSPG. Using penton mutants, we found that the positive effect of FX on HAdV5 binding to HSPG and cell transduction did not depend on the penton base RGD and fiber shaft KKTK motifs. However, we found that FX had no enhancing effect on the HAdV5F35-mediated cell transduction, but a negative effect which did not involve the cell attachment or endocytic step, but the intracellular trafficking and nuclear import of the FX-HAdV5F35 complex. By cellular imaging, HAdV5F35 particles were observed to accumulate in the late endosomal compartment, and were released in significant amounts into the extracellular medium via exocytosis. We showed that the stability of serotype 5 hexon: FX interaction was higher at low pH compared to neutral pH, which could account for the retention of FX-HAdV5F35 complexes in the late endosomes. Our results suggested that, despite the high affinity interaction of hexon capsomeres to FX and cell surface HSPG, the adenoviral fiber acted as the dominant determinant of the internalization and trafficking pathway of HAdV5-based vectors.