PrP C -deficient mice expressing prion protein variants with large amino-proximal deletions (termed PrP DF ) suffer from neurodegeneration, which is rescued by full-length PrP C . We now report that expression of PrP DCD , a PrP variant lacking 40 central residues (94-134), induces a rapidly progressive, lethal phenotype with extensive central and peripheral myelin degeneration. This phenotype was rescued dose-dependently by coexpression of full-length PrP C or PrP C lacking all octarepeats. Expression of a PrP C variant lacking eight residues (114-121) was innocuous in the presence or absence of full-length PrP C , yet enhanced the toxicity of PrP DCD and diminished that of PrP DF . Therefore, deletion of the entire central domain generates a strong recessive-negative mutant of PrP C , whereas removal of residues 114-121 creates a partial agonist with context-dependent action. These findings suggest that myelin integrity is maintained by a constitutively active neurotrophic protein complex involving PrP C , whose effector domain encompasses residues 94-134.

The tyrosine kinase Met, the product of the c-met proto-oncogene and the receptor for hepatocyte growth factor/scatter factor (HGF/SF), mediates signals critical for cell survival and migration. The human pathogen Listeria monocytogenes exploits Met signaling for invasion of host cells via its surface protein InlB. We present the crystal structure of the complex between a large fragment of the human Met ectodomain and the Met-binding domain of InlB. The concave face of the InlB leucine-rich repeat region interacts tightly with the first immunoglobulin-like domain of the Met stalk, a domain which does not bind HGF/SF. A second contact between InlB and the Met Sema domain locks the otherwise flexible receptor in a rigid, signaling competent conformation. Full Met activation requires the additional C-terminal domains of InlB which induce heparin-mediated receptor clustering and potent signaling. Thus, although it elicits a similar cellular response, InlB is not a structural mimic of HGF/SF.

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Flavin-dependent halogenases catalyse halogenation of aromatic compounds. In most cases, this reaction proceeds with high regioselectivity and requires only the presence of FADH2, oxygen, and halide salts. Since marine habitats contain high concentrations of halides, organisms populating the oceans might be valuable sources of yet undiscovered halogenases. A new Hidden-Markov-Model (HMM) based on the PFAM tryptophan halogenase model was used for the analysis of marine metagenomes. Eleven metagenomes were screened leading to the identification of 254 complete or partial putative flavin-dependent halogenase genes. One predicted halogenase gene (brvH) was selected, codon optimised for E. coli, and overexpressed. Substrate screening revealed that this enzyme represents an active flavin-dependent halogenase able to convert indole to 3-bromoindole. Remarkably, bromination prevails also in a large excess of chloride. The BrvH crystal structure is very similar to that of tryptophan halogenases but reveals a substrate binding site that is open to the solvent instead of being covered by a loop.

Edited by Ruma BanerjeeFlavin-dependent halogenases increasingly attract attention as biocatalysts in organic synthesis, facilitating environmentally friendly halogenation strategies that require only FADH 2 , oxygen, and halide salts. Different flavin-dependent tryptophan halogenases regioselectively chlorinate or brominate tryptophan's indole moiety at C5, C6, or C7. Here, we present the first substrate-bound structure of a tryptophan 6-halogenase, namely Thal, also known as ThdH, from the bacterium Streptomyces albogriseolus at 2.55 Å resolution. The structure revealed that the C6 of tryptophan is positioned next to the ⑀-amino group of a conserved lysine, confirming the hypothesis that proximity to the catalytic residue determines the site of electrophilic aromatic substitution. Although Thal is more similar in sequence and structure to the tryptophan 7-halogenase RebH than to the tryptophan 5-halogenase PyrH, the indole binding pose in the Thal active site more closely resembled that of PyrH than that of RebH. The difference in indole orientation between Thal and RebH appeared to be largely governed by residues positioning the Trp backbone atoms. The sequences of Thal and RebH lining the substrate binding site differ in only few residues. Therefore, we exchanged five amino acids in the Thal active site with the corresponding counterparts in RebH, generating the quintuple variant Thal-RebH5. Overall conversion of L-Trp by the Thal-RebH5 variant resembled that of WT Thal, but its regioselectivity of chlorination and bromination was almost completely switched from C6 to C7 as in RebH. We conclude that structure-based protein engineering with targeted substitution of a few residues is an efficient approach to tailoring flavin-dependent halogenases.The authors declare that they have no conflicts of interest with the contents of this article. This article contains Tables S1 and S2 and Figs. S1-S17. The atomic coordinates and structure factors (codes 6H43, 6H44, and 6IB5) have been deposited in the Protein Data Bank (http://wwpdb.org/). . 3 The abbreviations used are: FDH, flavin-dependent halogenase; aa, amino acid(s); ESI, electrospray ionization; HSQC, heteronuclear single-quantum coherence; r.m.s.d., root mean square deviation; ROESY, rotating frame nuclear Overhauser effect spectroscopy; Bicine, N,N-bis(2-hydroxyethyl)-glycine; H-bond, hydrogen bond; Ni-NTA, nickel-nitrilotriacetic acid; RR-ADH, Rhodococcus ruber alcohol dehydrogenase; PDB, Protein Data Bank.Figure 8. Steric conflicts upon placing L-Trp from Trp-RebH in Trp-Thal and vice versa. Minor clashes are indicated as small green hexagons, and more severe clashes are shown as larger red hexagons. a, L-Trp from Trp-RebH (PDB code 2E4G) was placed in Trp-Thal. Clashes mainly occur between the L-Trp indole and the side chains of conserved residues Phe-112 and Trp-466. b, L-Trp from Trp-Thal was placed in Trp-RebH (PDB code 2E4G). Clashes mainly occur between the L-Trp indole and the side chain of Asn-470 and between the L-Trp carboxylate and the side chain...