Botulinum Neurotoxin F Subtypes Cleaving the VAMP-2 Q58⁻K59 Peptide Bond Exhibit Unique Catalytic Properties and Substrate Specificities

In the recent past, about 40 botulinum neurotoxin (BoNT) subtypes belonging to serotypes A, B, E, and F pathogenic to humans were identified among hundreds of independent isolates. BoNTs are the etiological factors of botulism and represent potential bioweapons; however, they are also recognized pharmaceuticals for the efficient counteraction of hyperactive nerve terminals in a variety of human diseases. The detailed biochemical characterization of subtypes as the basis for development of suitable countermeasures and possible novel therapeutic applications is lagging behind the increase in new subtypes. Here, we report the primary structure of a ninth subtype of BoNT/F. Its amino-acid sequence diverges by at least 8.4% at the holotoxin and 13.4% at the enzymatic domain level from all other known BoNT/F subtypes. We found that BoNT/F9 shares the scissile Q58/K59 bond in its substrate vesicle associated membrane protein 2 with the prototype BoNT/F1. Comparative biochemical analyses of four BoNT/F enzymatic domains showed that the catalytic efficiencies decrease in the order F1 > F7 > F9 > F6, and vary by up to a factor of eight. KM values increase in the order F1 > F9 > F6 ≈ F7, whereas kcat decreases in the order F7 > F1 > F9 > F6. Comparative substrate scanning mutagenesis studies revealed a unique pattern of crucial substrate residues for each subtype. Based upon structural coordinates of F1 bound to an inhibitor polypeptide, the mutational analyses suggest different substrate interactions in the substrate binding channel of each subtype

Assigning Backbone NMR Resonances for Full Length Tau Isoforms: Efficient Compromise between Manual Assignments and Reduced Dimensionality

Tau protein is the longest disordered protein for which nearly complete backbone NMR resonance assignments have been reported. Full-length tau protein was initially assigned using a laborious combination of bootstrapping assignments from shorter tau fragments and conventional triple resonance NMR experiments. Subsequently it was reported that assignments of comparable quality could be obtained in a fully automated fashion from data obtained using reduced dimensionality NMR (RDNMR) experiments employing a large number of indirect dimensions. Although the latter strategy offers many advantages, it presents some difficulties if manual intervention, confirmation, or correction of the assignments is desirable, as may often be the case for long disordered and degenerate polypeptide sequences. Here we demonstrate that nearly complete backbone resonance assignments for full-length tau isoforms can be obtained without resorting either to bootstrapping from smaller fragments or to very high dimensionality experiments and automation. Instead, a set of RDNMR triple resonance experiments of modest dimensionality lend themselves readily to efficient and unambiguous manual assignments. An analysis of the backbone chemical shifts obtained in this fashion indicates several regions in full length tau with a notable propensity for helical or strand-like structure that are in good agreement with previous observations

Separation of Recombination and SOS Response in Escherichia coli RecA Suggests LexA Interaction Sites

RecA plays a key role in homologous recombination, the induction of the DNA damage response through LexA cleavage and the activity of error-prone polymerase in Escherichia coli. RecA interacts with multiple partners to achieve this pleiotropic role, but the structural location and sequence determinants involved in these multiple interactions remain mostly unknown. Here, in a first application to prokaryotes, Evolutionary Trace (ET) analysis identifies clusters of evolutionarily important surface amino acids involved in RecA functions. Some of these clusters match the known ATP binding, DNA binding, and RecA-RecA homo-dimerization sites, but others are novel. Mutation analysis at these sites disrupted either recombination or LexA cleavage. This highlights distinct functional sites specific for recombination and DNA damage response induction. Finally, our analysis reveals a composite site for LexA binding and cleavage, which is formed only on the active RecA filament. These new sites can provide new drug targets to modulate one or more RecA functions, with the potential to address the problem of evolution of antibiotic resistance at its root

DETERMINATION OF (3)J(H-I(N),C-I(')) COUPLING-CONSTANTS IN PROTEINS WITH THE C'-FIDS METHOD

We introduce the C'-FIDS-H-1, N-15-HSQC experiment: a new method for the determination of (3)J(H-i(N), C-i) coupling constants in proteins, yielding information about the torsional angle phi. It relies on the H-1, N-15-HSQC or HNCO experiment, two of the the most sensitive heteronuclear correlation experiments for isotopically labeled proteins. A set of three H-1, N-15-HSQC or HNCO spectra are recorded: a reference experiment in which the carbonyl spins are decoupled during t(1) and t(2), a second experiment in which they are decoupled exclusively during t(1) and a third one in which they are coupled in t(1), as well as t(2). The last experiment yields an E.COSY-type pattern from which the (2)J(HNiN, C-i-1) and (1)J(N-i, C-i-1) coupling constants can be extracted. By comparison of the coupled multiplet (obtained from the second experiment) with the decoupled multiplet (obtained from the first experiment) convoluted with the (2)J(H-i(N), C-i-1) coupling, the (3)J(HN:CI) coupling can be found in a one-parameter fitting procedure. The method is demonstrated for the protein rhodniin, containing 103 amino acids. Systematic errors due to differential relaxation are small for (n)J(H-N, C-n) couplings in biomacromolecules of the size currently under NMR spectroscopic investigation

Determination of 3J(H infi supN ,C infi sup? ) coupling constants in proteins with the C?-FIDS method

We introduce the C′-FIDS-1H,15N-HSQC experiment, a new method for the determination of 3J(H infi supN ,C infi sup′ ) coupling constants in proteins, yielding information about the torsional angle ϕ. It relies on the 1H,15N-HSQC or HNCO experiment, two of the the most sensitive heteronuclear correlation experiments for isotopically labeled proteins. A set of three 1H,15N-HSQC or HNCO spectra are recorded: a reference experiment in which the carbonyl spins are decoupled during t1 and t2, a second experiment in which they are decoupled exclusively during t1 and a third one in which they are coupled in t1 as well as t2. The last experiment yields an E.COSY-type pattern from which the 2J(H infi supN ,C infi-1 sup′ ) and 1J(Ni,C infi-1 sup′ ) coupling constants can be extracted. By comparison of the coupled multiplet (obtained from the second experiment) with the decoupled multiplet (obtained from the first experiment) convoluted with the 2J(H infi supN ,C infi-1 sup′ ) coupling, the 3J(H infi supN ,C infi sup′ ) coupling can be found in a one-parameter fitting procedure. The method is demonstrated for the protein rhodniin, containing 103 amino acids. Systematic errors due to differential relaxation are small for nJ(HN,C′) couplings in biomacromolecules of the size currently under NMR spectroscopic investigation