Isolation and Functional Characterization of the Novel Clostridium botulinum Neurotoxin A8 Subtype

Botulism is a severe neurological disease caused by the complex family of botulinum neurotoxins (BoNT). Based on the different serotypes known today, a classification of serotype variants termed subtypes has been proposed according to sequence diversity and immunological properties. However, the relevance of BoNT subtypes is currently not well understood. Here we describe the isolation of a novel Clostridium botulinum strain from a food-borne botulism outbreak near Chemnitz, Germany. Comparison of its botulinum neurotoxin gene sequence with published sequences identified it to be a novel subtype within the BoNT/A serotype designated BoNT/A8. The neurotoxin gene is located within an ha-orfX+ cluster and showed highest homology to BoNT/A1, A2, A5, and A6. Unexpectedly, we found an arginine insertion located in the HC domain of the heavy chain, which is unique compared to all other BoNT/A subtypes known so far. Functional characterization revealed that the binding characteristics to its main neuronal protein receptor SV2C seemed unaffected, whereas binding to membrane-incorporated gangliosides was reduced in comparison to BoNT/A1. Moreover, we found significantly lower enzymatic activity of the natural, full-length neurotoxin and the recombinant light chain of BoNT/A8 compared to BoNT/A1 in different endopeptidase assays. Both reduced ganglioside binding and enzymatic activity may contribute to the considerably lower biological activity of BoNT/A8 as measured in a mouse phrenic nerve hemidiaphragm assay. Despite its reduced activity the novel BoNT/A8 subtype caused severe botulism in a 63-year-old male. To our knowledge, this is the first description and a comprehensive characterization of a novel BoNT/A subtype which combines genetic information on the neurotoxin gene cluster with an in-depth functional analysis using different technical approaches. Our results show that subtyping of BoNT is highly relevant and that understanding of the detailed toxin function might pave the way for the development of novel therapeutics and tailor-made antitoxins

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

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 HN,Hα and HN,C′ coupling constants in 13C, 15N-labeled proteins

Sensitive three-dimensional NMR experiments, based on the E.COSY principle, are presented for the measurement of the 3J(HN,Hα) and 3J(HN,C′) coupling constants in uniformly 13C- and 15N-labeled proteins. They employ gradient coherence selection in combination with the sensitivity enhancement method in HSQC-type spectra (Cavanagh et al., 1991; Palmer et al., 1991). In most cases, the two measured coupling constants unambiguously define the ϕ-angle for protein structure determination. The method is applied to uniformly 13C, 15N-labeled ribonuclease T1

DETERMINATION OF H(N),H-ALPHA AND H(N), C' COUPLING-CONSTANTS IN C-13,N-15-LABELED PROTEINS

Sensitive three-dimensional NMR experiments, based on the E.COSY principle, are presented for the measurement of the 3J(H(N),H(alpha) and 3J(H(N),C') coupling constants in uniformly C-13- and N-15-labeled proteins. They employ gradient coherence selection in combination with the sensitivity enhancement method in HSQC-type spectra (Cavanagh et al., 1991; Palmer et al., 1991). In most cases, the two measured coupling constants unambiguously define the phi-angle for protein structure determination. The method is applied to uniformly C-13,N-15-labeled ribonuclease T1