Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase required for signaling from immunoreceptors in various hematopoietic cells. Phosphorylation of two tyrosine residues in the activation ...loop of the Syk kinase catalytic domain is necessary for signaling, a phenomenon typical of tyrosine kinase family members. Syk in vitro enzyme activity, however, does not depend on phosphorylation (activation loop tyrosine → phenylalanine mutants retain catalytic activity). We have determined the x-ray structure of the unphosphorylated form of the kinase catalytic domain of Syk. The enzyme adopts a conformation of the activation loop typically seen only in activated, phosphorylated tyrosine kinases, explaining why Syk does not require phosphorylation for activation. We also demonstrate that Gleevec (STI-571, Imatinib) inhibits the isolated kinase domains of both unphosphorylated Syk and phosphorylated Abl with comparable potency. Gleevec binds Syk in a novel, compact cis-conformation that differs dramatically from the binding mode observed with unphosphorylated Abl, the more Gleevec-sensitive form of Abl. This finding suggests the existence of two distinct Gleevec binding modes: an extended, trans-conformation characteristic of tight binding to the inactive conformation of a protein kinase and a second compact, cis-conformation characteristic of weaker binding to the active conformation. Finally, the Syk-bound cis-conformation of Gleevec bears a striking resemblance to the rigid structure of the nonspecific, natural product kinase inhibitor staurosporine.
Background: In
Arabidopsis thaliana, ethylene perception and signal transduction into the cell are carried out by a family of membrane-bound receptors, one of which is ethylene resistant 1 (ETR1). ...The large cytoplasmic domain of the receptor showed significant sequence homology to the proteins of a common bacterial regulatory pathway, the two-component system. This system consists of a transmitter histidine kinase and a response regulator (or signal receiver). We present the crystal structures of the first plant receiver domain ETR
RD (residues 604–738) of ETR1 in two conformations.
Results: The monomeric form of ETR
RD resembles the known structure of the bacterial receiver domain. ETR
RD forms a homodimer in solution and in the crystal, an interaction that has not been described previously. Dimerization is mediated by the C terminus, which forms an extended
β sheet with the dimer-related
β-strand core. Furthermore, the loop immediately following the active site adopts an exceptional conformation.
Conclusions: The three-dimensional structure of ETR
RD shows the expected conformational conservation to prokaryotic receiver proteins, such as CheY and CheB, both of which are part of the chemotaxis signaling pathway. ETR
RD provides the first detailed example of a dimerized receiver domain. Given that the dimer interface of ETR
RD coincides with the phosphorylation-dependent interfaces of CheY and CheB, we suggest that the monomerization of ETR
RD is phosphorylation-dependent too. In the Mg
2+-free form of ETR
RD, the
γ-loop conformation does not allow a comparable interaction as observed in the active-site architectures of Mg
2+-bound CheY from
Escherichia coli and
Salmonella typhimurium.
Lipid A modification with 4-amino-4-deoxy-L-arabinose confers on certain pathogenic bacteria, such as
Salmonella, resistance to cationic antimicrobial peptides, including those derived from the ...innate immune system. ArnB catalysis of amino group transfer from glutamic acid to the 4″-position of a UDP-linked ketopyranose molecule to form UDP-4-amino-4-deoxy-L-arabinose represents a key step in the lipid A modification pathway. Structural and functional studies of the ArnB aminotransferase were undertaken by combining X-ray crystallography with biochemical analyses. High-resolution crystal structures were solved for two native forms and one covalently inhibited form of
S. typhimurium ArnB. These structures permitted identification of key residues involved in substrate binding and catalysis, including a rarely observed nonprolyl
cis peptide bond in the active site.
Uridylate kinase from Saccharomyces cerevisiae is a member of the nucleoside monophosphate (NMP) kinase family and catalyzes the reaction ATP+NMP<==>ADP+NDP with moderate specificity for UMP. The ...recombinant enzyme crystallized together with two substrate molecules. The structure was solved, by multiple isomorphous replacement and solvent flattening, at 3.0 A and then refined at 2.13 A resolution. The present R-factor is 19%. Superposition onto the structure of a substrate-free adenylate kinase revealed the motions induced by substrate binding. A further superposition onto an adenylate kinase with bound P1,P5-bis(5'-adenosyl)pentaphosphate (Ap5A), a two-substrate-mimicking inhibitor, failed to explain the UMP preference of the uridylate kinase, but superimposed the nucleosides and in particular the non-transferred phosphates at the ATP- and NMP-site rather well. The coincidence of the phosphates indicate strongly that these groups assume their final positions during catalysis. This locates the transition state, which can be modeled with reasonable geometry in agreement with an in-line associative SN2 mechanism.
Two crystal structures of ligated uridylate kinase fromSaccharomyces cerevisiaewere determined by X-ray analyses. The ligands were ADP and AMP. Cocrystallization with ATP yielded crystals with ADP at ...the ATP site and a mixture of AMp and ADP at the NMP site. Cocrystallization with ADP gave rise to a distinct crystal type with ADP at the ATP site, but only AMP at the NMP site. In both cases, the substrates are kept in place by favorable crystal contacts. The structures have been refined toR-factors of 17.8% and 19.6% at resolutions of 2.1 Å and 1.9 Å, respectively. A comparison with the related cytosolic adenylate kinase from pig disclosed large induced-fit movements on substrate binding and the disassembly of the catalytic center in the absence of substrates. The relatively high side-activity of uridylate kinase for AMP is explained by the finding that the binding pocket is sized for an AMP, but constructed to bind UMP together with a water molecule.
The signal receiver domain of ETR1, an ethylene receptor from Arabidopsis thaliana, has been subcloned and expressed in E. coli and purified by affinity chromatography. Crystals of both native and a ...selenomethionine‐substituted form of the receiver domain have been obtained. Native crystals grew in 1.6 M Li2SO4 and 0.1 M HEPES pH 7.5 and once flash‐frozen diffract to 2.1 Å resolution. They belong to space group P41212 with unit‐cell dimensions a = b = 48.4, c = 112.3 Å.
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