Structural basis of regioselective tryptophan dibromination by the single-component flavin-dependent halogenase AetF

The flavin-dependent halogenase (FDH) AetF successively brominates tryptophan at C5 and C7 to generate 5,7-dibromotryptophan. In contrast to the well studied two-component tryptophan halogenases, AetF is a single-component flavoprotein monooxygenase. Here, crystal structures of AetF alone and in complex with various substrates are presented, representing the first experimental structures of a single-component FDH. Rotational pseudo­symmetry and pseudomerohedral twinning complicated the phasing of one structure. AetF is structurally related to flavin-dependent monooxygenases. It contains two dinucleotide-binding domains for binding the ADP moiety with unusual sequences that deviate from the consensus sequences GXGXXG and GXGXXA. A large domain tightly binds the cofactor flavin adenine dinucleotide (FAD), while the small domain responsible for binding the nicotinamide adenine dinucleotide (NADP) is unoccupied. About half of the protein forms additional structural elements containing the tryptophan binding site. FAD and tryptophan are about 16 Å apart. A tunnel between them presumably allows diffusion of the active halogenating agent hypohalous acid from FAD to the substrate. Tryptophan and 5-bromotryptophan bind to the same site but with a different binding pose. A flip of the indole moiety identically positions C5 of tryptophan and C7 of 5-bromotryptophan next to the tunnel and to catalytic residues, providing a simple explanation for the regioselectivity of the two successive halogenations. AetF can also bind 7-bromotryptophan in the same orientation as tryptophan. This opens the way for the biocatalytic production of differentially dihalogenated tryptophan derivatives. The structural conservation of a catalytic lysine suggests a way to identify novel single-component FDHs

Barnase fusion as a tool to determine the crystal structure of the small disulfide-rich protein McoEeTI

Niemann H, Schmoldt HU, Wentzel A, Kolmar H, Heinz DW. Barnase fusion as a tool to determine the crystal structure of the small disulfide-rich protein McoEeTI. J Mol Biol. 2006;356(1):1-8

Crystal structure of a dynamin GTPase domain in both nucleotide-free and GDP-bound forms

Dynamins form a family of multidomain GTPases involved in endocytosis, vesicle trafficking and maintenance of mitochondrial morphology. In contrast to the classical switch GTPases, a force-generating function has been suggested for dynamins. Here we report the 2.3 Å crystal structure of the nucleotide-free and GDP-bound GTPase domain of Dictyostelium discoideum dynamin A. The GTPase domain is the most highly conserved region among dynamins. The globular structure contains the G-protein core fold, which is extended from a six-stranded β-sheet to an eight-stranded one by a 55 amino acid insertion. This topologically unique insertion distinguishes dynamins from other subfamilies of GTP-binding proteins. An additional N-terminal helix interacts with the C-terminal helix of the GTPase domain, forming a hydrophobic groove, which could be occupied by C-terminal parts of dynamin not present in our construct. The lack of major conformational changes between the nucleotide-free and the GDP-bound state suggests that mechanochemical rearrangements in dynamin occur during GTP binding, GTP hydrolysis or phosphate release and are not linked to loss of GDP

The formation of new nucleoli during macronuclear development of the hypotrichous ciliate Stylonychia lemnae visualized by in situ hybridization

Maercker C, Harjes P, Neben M, Niemann H, Sianidis G, Lipps HJ. The formation of new nucleoli during macronuclear development of the hypotrichous ciliate Stylonychia lemnae visualized by in situ hybridization. Chromosome Res. 1997;5(5):333-335

Fold and function of the InlB B-repeat

Host cell invasion by the facultative intracellular pathogen Listeria monocytogenes requires the invasion protein InlB in many cell types. InlB consists of an N-terminal internalin domain that binds the host cell receptor tyrosine kinase Met and C-terminal GW domains that bind to glycosaminoglycans (GAGs). Met binding and activation is required for host cell invasion, while the interaction between GW domains and GAGs enhances this effect. Soluble InlB elicits the same cellular phenotypes as the natural Met ligand hepatocyte growth factor/scatter factor (HGF/SF), e.g. cell scatter. So far, little is known about the central part of InlB, the B-repeat. Here we present a structural and functional characterization of the InlB B-repeat. The crystal structure reveals a variation of the β-grasp fold that is most similar to small ubiquitin-like modifiers (SUMOs). However, structural similarity also suggests a potential evolutionary relation to bacterial mucin-binding proteins. The B-repeat defines the prototype structure of a hitherto uncharacterized domain present in over a thousand bacterial proteins. Generally, this domain probably acts as a spacer or a receptor-binding domain in extracellular multi-domain proteins. In cellular assays the B-repeat acts synergistically with the internalin domain conferring to it the ability to stimulate cell motility. Thus, the B-repeat probably binds a further host cell receptor and thereby enhances signaling downstream of Met

INORGANIC CLUSTER COMPOUNDS AS MODELS FOR THE STRUCTURE OF ACTIVE-SITES IN PROMOTED HYDRODESULFURIZATION CATALYSTS

TOPSOE H, CLAUSEN BS, TOPSOE NY, et al. INORGANIC CLUSTER COMPOUNDS AS MODELS FOR THE STRUCTURE OF ACTIVE-SITES IN PROMOTED HYDRODESULFURIZATION CATALYSTS. JOURNAL OF THE CHEMICAL SOCIETY-FARADAY TRANSACTIONS I. 1987;83(7):2157-2167

Crystal structure of a dynamin GTPase domain

Dynamins form a family of multidomain GTPases involved in endocytosis, vesicle trafficking and maintenance of mitochondrial morphology. In contrast to the classical switch GTPases, a force-generating function has been suggested for dynamins. Here we report the 2.3 Å crystal structure of the nucleotide-free and GDP-bound GTPase domain of Dictyostelium discoideum dynamin A. The GTPase domain is the most highly conserved region among dynamins. The globular structure contains the G-protein core fold, which is extended from a six-stranded ß-sheet to an eight-stranded one by a 55 amino acid insertion. This topologically unique insertion distinguishes dynamins from other subfamilies of GTP-binding proteins. An additional N-terminal helix interacts with the C-terminal helix of the GTPase domain, forming a hydrophobic groove, which could be occupied by C-terminal parts of dynamin not present in our construct. The lack of major conformational changes between the nucleotide-free and the GDP-bound state suggests that mechanochemical rearrangements in dynamin occur during GTP binding, GTP hydrolysis or phosphate release and are not linked to loss of GDP

Crystal structure of a dynamin GTPase domain

Dynamins form a family of multidomain GTPases involved in endocytosis, vesicle trafficking and maintenance of mitochondrial morphology. In contrast to the classical switch GTPases, a force-generating function has been suggested for dynamins. Here we report the 2.3 Å crystal structure of the nucleotide-free and GDP-bound GTPase domain of Dictyostelium discoideum dynamin A. The GTPase domain is the most highly conserved region among dynamins. The globular structure contains the G-protein core fold, which is extended from a six-stranded ß-sheet to an eight-stranded one by a 55 amino acid insertion. This topologically unique insertion distinguishes dynamins from other subfamilies of GTP-binding proteins. An additional N-terminal helix interacts with the C-terminal helix of the GTPase domain, forming a hydrophobic groove, which could be occupied by C-terminal parts of dynamin not present in our construct. The lack of major conformational changes between the nucleotide-free and the GDP-bound state suggests that mechanochemical rearrangements in dynamin occur during GTP binding, GTP hydrolysis or phosphate release and are not linked to loss of GDP

The dynamin A ring complex: molecular organisation and nucleotide-dependent conformational changes

Here we show that Dictyostelium discoideum dynamin A is a fast GTPase, binds to negatively charged lipids, and self-assembles into rings and helices in a nucleotide-dependent manner, similar to human dynamin-1. Chemical modification of two cysteine residues, positioned in the middle domain and GTPase effector domain (GED), leads to altered assembly properties and the stabilization of a highly regular ring complex. Single particle analysis of this dynamin A* ring complex led to a three-dimensional map, which shows that the nucleotide-free complex consists of two layers with 11-fold symmetry. Our results reveal the molecular organization of the complex and indicate the importance of the middle domain and GED for the assembly of dynamin family proteins. Nucleotide-dependent changes observed with the unmodified and modified protein support a mechanochemical action of dynamin, in which tightening and stretching of a helix contribute to membrane fission