The sulfonylureas and imidazolinones are potent commercial herbicide families. They are among the most popular choices for farmers worldwide, because they are nontoxic to animals and highly selective. These herbicides inhibit branched-chain amino acid biosynthesis in plants by targeting acetohydroxyacid synthase (AHAS, EC 2.2.1.6). This report describes the 3D structure of Arabidopsis thaliana AHAS in complex with five sulfonylureas (to 2.5 Å resolution) and with the imidazolinone, imazaquin (IQ; 2.8 Å). Neither class of molecule has a structure that mimics the substrates for the enzyme, but both inhibit by blocking a channel through which access to the active site is gained. The sulfonylureas approach within 5 Å of the catalytic center, which is the C2 atom of the cofactor thiamin diphosphate, whereas IQ is at least 7 Å from this atom. Ten of the amino acid residues that bind the sulfonylureas also bind IQ. Six additional residues interact only with the sulfonylureas, whereas there are two residues that bind IQ but not the sulfonylureas. Thus, the two classes of inhibitor occupy partially overlapping sites but adopt different modes of binding. The increasing emergence of resistant weeds due to the appearance of mutations that interfere with the inhibition of AHAS is now a worldwide problem. The structures described here provide a rational molecular basis for understanding these mutations, thus allowing more sophisticated AHAS inhibitors to be developed. There is no previously described structure for any plant protein in complex with a commercial herbicide.inhibition ͉ sulfonylurea ͉ x-ray crystallography ͉ imidazolinone ͉ thiamin diphosphate T he sulfonylurea and imidazolinone herbicides are an essential part of the multibillion-dollar weed-control market. There are now Ͼ30 herbicides from these families registered for worldwide use. A major advantage of these compounds is that they are nontoxic to animals, highly selective, and very potent, thereby allowing low application rates. These herbicides act by inhibiting acetohydroxyacid synthase [AHAS; also known as acetolactate synthase; EC 2.2.1.6 (1)], the first common enzyme in the biosynthetic pathway of the branched-chain amino acids. The reaction carried out by this enzyme is the synthesis of either (S)-2-acetolactate from two molecules of pyruvate or (S)-2-aceto-2-hydroxybutyrate from a molecule each of pyruvate and 2-ketobutyrate.AHAS belongs to a superfamily of thiamin diphosphate (ThDP)-dependent enzymes that are capable of catalyzing a variety of reactions, including both the oxidative and nonoxidative decarboxylation of 2-ketoacids. This cofactor is bound by a divalent metal ion such as Mg 2ϩ , which coordinates to the diphosphate group of ThDP and to two highly conserved residues (2) in these proteins. AHAS also binds a molecule of FAD, although this cofactor does not participate in the principal reactions. To date, most AHAS enzymes that have been characterized have both a catalytic subunit (Ϸ65 kDa) and a smaller regulatory subunit, which varies in s...

Already 20 years have passed since the cloning of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor ␣-chain, the first member of the GM-CSF/ interleukin (IL)-3/IL-5 family of hemopoietic cytokine receptors to be molecularly characterized. The intervening 2 decades have uncovered a plethora of biologic functions transduced by the GM-CSF receptor (pleiotropy) and revealed distinct signaling networks that couple the receptor to biologic outcomes. Unlike other hemopoietin receptors, the GM-CSF receptor has a significant nonredundant role in myeloid hematologic malignancies, macrophage-mediated acute and chronic inflammation, pulmonary homeostasis, and allergic disease. The molecular mechanisms underlying GM-CSF receptor activation have recently been revealed by the crystal structure of the GM-CSF receptor complexed to GM-CSF, which shows an unexpected higher order assembly. Emerging evidence also suggests the existence of intracellular signosomes that are recruited in a concentration-dependent fashion to selectively control cell survival, proliferation, and differentiation by GM-CSF. These findings begin to unravel the mystery of cytokine receptor pleiotropy and are likely to also apply to the related IL IntroductionGranulocyte-macrophage colony-stimulating factor (GM-CSF) and the related cytokines interleukin (IL)-3 and IL-5 regulate the production and functional activation of hemopoietic cells, with GM-CSF acting on monocyte/macrophages and all granulocytes. 1 GM-CSF also controls dendritic cell and T-cell function, 2 thus linking innate and acquired immunity. Because of the widespread expression of the GM-CSF receptor in hematopoietic cells, it was assumed that both GM-CSF and its receptor were key players in the regulation of steady-state functions. Whereas this turned out to be true in terms of lung physiology, 3-7 deletion of either the GM-CSF gene or its receptor showed no obvious deficiency in myeloid cell numbers or production. 4,8,9 Rather, a growing body of evidence now suggests that GM-CSF plays a key role in signaling emergency hemopoiesis (predominantly myelopoiesis) in response to infection, including the production of granulocytes and macrophages in the bone marrow and their maintenance, survival, and functional activation at sites of injury or insult. [10][11][12][13] The role of GM-CSF and its receptor in pathology, on the other hand, arises largely as a result of abnormal signaling leading to deregulated myelopoiesis with enhanced proliferation and survival of myeloid precursors, a common feature of myeloproliferative disorders and myeloid leukemias. For example, juvenile myelomonocytic leukemia (JMML), a rare, but potentially fatal myeloproliferative disorder of children clinically presenting with monocytosis, thrombocytopenia, and malignant infiltration of nonhematologic organs by clonal macrophages, 14 exhibits hypersensitivity to 15,16 and a mouse model of JMML shows an absolute requirement for GM-CSF and its receptor in establishment and maintenance of the disease. 17,18...

The GM-CSF, IL-3, and IL-5 receptors constitute the βc family, playing important roles in inflammation, autoimmunity, and cancer. Typical of heterodimeric type I cytokine receptors, signaling requires recruitment of the shared subunit to the initial cytokine:α subunit binary complex through an affinity conversion mechanism. This critical process is poorly understood due to the paucity of crystal structures of both binary and ternary receptor complexes for the same cytokine. We have now solved the structure of the binary GM-CSF:GMRα complex at 2.8-Å resolution and compared it with the structure of the ternary complex, revealing distinct conformational changes. Guided by these differences we performed mutational and functional studies that, importantly, show GMRα interactions playing a major role in receptor signaling while βc interactions control high-affinity binding. These results support the notion that conformational changes underlie the mechanism of GM-CSF receptor activation and also suggest how related type I cytokine receptors signal.

The full-length, two-domain response regulator RegX3 from Mycobacterium tuberculosis is a dimer stabilized by three-dimensional domain swapping. Dimerization is known to occur in the OmpR/PhoB subfamily of response regulators upon activation but has previously only been structurally characterized for isolated receiver domains. The RegX3 dimer has a bipartite intermolecular interface, which buries 2357 Å 2 per monomer. The two parts of the interface are between the two receiver domains (dimerization interface) and between a composite receiver domain and the effector domain of the second molecule (interdomain interface). The structure provides support for the importance of threonine and tyrosine residues in the signal transduction mechanism. These residues occur in an active-like conformation stabilized by lanthanum ions. In solution, RegX3 exists as both a monomer and a dimer in a concentration-dependent equilibrium. The dimer in solution differs from the active form observed in the crystal, resembling instead the model of the inactive full-length response regulator PhoB.Tuberculosis is a global threat to human health, with onethird of the world's population infected with the causative agent of tuberculosis, Mycobacterium tuberculosis. With the emergence of multidrug-resistant strains of M. tuberculosis, novel therapeutic agents are urgently required. This will be best achieved by developing a better understanding of the molecular biology of this pathogen. In this context, signaling systems play an important role in the continued survival of the organism and are central to the way M. tuberculosis responds to environmental stress, especially that generated by the host immune system.The predominant signaling system for adaptive gene expression changes in bacteria is called a two-component system (TCS).3 TCS are found in eubacteria, archaea, and eukarya; however, they are rare in eukarya. As the name implies, the system typically consists of two proteins: a sensor histidine kinase (SK) and a response regulator (RR) (1). SKs are usually membrane-anchored proteins with a characteristic core consisting of a histidine-containing dimerization domain and a catalytic domain with ATPase activity. In response to extracellular conditions, and in some cases intracellular conditions, the SK autophosphorylates at a histidine residue within the dimerization domain. This autophosphorylation occurs upon dimerization through a phosphate transfer from the catalytic domain (annotated as a HATPase_c domain by the SMART server (2, 3)) of the adjacent protomer to the conserved histidine in the dimerization domain (HisKA domain). The activated SK may then function as a phosphate donor to a universally conserved aspartic acid residue in the RR. RRs typically consist of two domains, a receiver and an effector domain, although a large family of 54 activators also contain a central AAAϩ domain. The N-terminal receiver domain acts as the phosphoacceptor, whereas the C-terminal effector domain is usually a DNA-binding domain involved in the tra...

The open reading frame rv1364c of Mycobacterium tuberculosis, which regulates the stress-dependent σ factor, σ(F), has been analyzed structurally and functionally. Rv1364c contains domains with sequence similarity to the RsbP/RsbW/RsbV regulatory system of the stress-response σ factor of Bacillus subtilis. Rv1364c contains, sequentially, a PAS domain (which shows sequence similarity to the PAS domain of the B. subtilis RsbP protein), an active phosphatase domain, a kinase (anti-σ(F) like) domain and a C-terminal anti-σ(F) antagonist like domain. The crystal structures of two PAS domain constructs (at 2.3 and 1.6 Å) and a phosphatase/kinase dual domain construct (at 2.6 Å) are described. The PAS domain is shown to bind palmitic acid but to have 100 times greater affinity for palmitoleic acid. The full-length protein can exist in solution as both monomer and dimer. We speculate that a switch between monomer and dimer, possibly resulting from fatty acid binding, affects the accessibility of the serine of the C-terminal, anti-σ(F) antagonist domain for dephosphorylation by the phosphatase domain thus indirectly altering the availability of σ(F).