Botulinum neurotoxins (BoNTs) are among the most poisonous biological substances known. They assemble with non-toxic non-hemagglutinin (NTNHA) protein to form the minimally functional progenitor toxin complexes (M-PTC), which protects BoNT in the gastrointestinal tract and releases it upon entry into the circulation. Here we provide molecular insight into the assembly between BoNT/A and NTNHA-A using small-angle X-ray scattering. We found that the free form BoNT/A maintains a pH-independent conformation with limited domain flexibility. Intriguingly, the free form NTNHA-A adopts pH-dependent conformational changes due to a torsional motion of its C-terminal domain. Once forming a complex at acidic pH, they each adopt a stable conformation that is similar to that observed in the crystal structure of the M-PTC. Our results suggest that assembly of the M-PTC depends on the environmental pH and that the complex form of BoNT/A is induced by interacting with NTNHA-A at acidic pH.

Aberrant self-assembly, induced by structural misfolding of the prion proteins, leads to a number of neurodegenerative disorders. In particular, misfolding of the mostly α-helical cellular prion protein (PrP(C)) into a β-sheet-rich disease-causing isoform (PrP(Sc)) is the key molecular event in the formation of PrP(Sc) aggregates. The molecular mechanisms underlying the PrP(C)-to-PrP(Sc) conversion and subsequent aggregation remain to be elucidated. However, in persistently prion-infected cell-culture models, it was shown that treatment with monoclonal antibodies against defined regions of the prion protein (PrP) led to the clearing of PrP(Sc) in cultured cells. To gain more insight into this process, we characterized PrP-antibody complexes in solution using a fast protein liquid chromatography coupled with small-angle x-ray scattering (FPLC-SAXS) procedure. High-quality SAXS data were collected for full-length recombinant mouse PrP [denoted recPrP(23-230)] and N-terminally truncated recPrP(89-230), as well as their complexes with each of two Fab fragments (HuM-P and HuM-R1), which recognize N- and C-terminal epitopes of PrP, respectively. In-line measurements by fast protein liquid chromatography coupled with SAXS minimized data artifacts caused by a non-monodispersed sample, allowing structural analysis of PrP alone and in complex with Fab antibodies. The resulting structural models suggest two mechanisms for how these Fabs may prevent the conversion of PrP(C) into PrP(Sc).

The arginine methyltransferase PRMT5-MEP50 is required for embryogenesis and is misregulated in many cancers. PRMT5 targets a wide variety of substrates, including histone proteins involved in specifying an epigenetic code. However, the mechanism by which PRMT5 utilizes MEP50 to discriminate substrates and to specifically methylate target arginines is unclear. To test a model in which MEP50 is critical for substrate recognition and orientation, we determined the crystal structure of Xenopus laevis PRMT5-MEP50 complexed with S-adenosylhomocysteine (SAH). PRMT5-MEP50 forms an unusual tetramer of heterodimers with substantial surface negative charge. MEP50 is required for PRMT5-catalyzed histone H2A and H4 methyltransferase activity and binds substrates independently. The PRMT5 catalytic site is oriented towards the cross-dimer paired MEP50. Histone peptide arrays and solution assays demonstrate that PRMT5-MEP50 activity is inhibited by substrate phosphorylation and enhanced by substrate acetylation. Electron microscopy and reconstruction showed substrate centered on MEP50. These data support a mechanism in which MEP50 binds substrate and stimulates PRMT5 activity modulated by substrate post-translational modifications.