Roger Benoit

Structural basis for recognition of synaptic vesicle protein 2C by botulinum neurotoxin A

Botulinum neurotoxin A (BoNT/A) belongs to the most dangerous class of bioweapons. Despite this, BoNT/A is used to treat a wide range of common medical conditions such as migraines and a variety of ocular motility and movement disorders. BoNT/A is probably best known for its use as an antiwrinkle agent in cosmetic applications (including Botox and Dysport). BoNT/A application causes long-lasting flaccid paralysis of muscles through inhibiting the release of the neurotransmitter acetylcholine by cleaving synaptosomal-associated protein 25 (SNAP-25) within presynaptic nerve terminals. Two types of BoNT/A receptor have been identified, both of which are required for BoNT/A toxicity and are therefore likely to cooperate with each other: gangliosides and members of the synaptic vesicle glycoprotein 2 (SV2) family, which are putative transporter proteins that are predicted to have 12 transmembrane domains, associate with the receptor-binding domain of the toxin. Recently, fibroblast growth factor receptor 3 (FGFR3) has also been reported to be a potential BoNT/A receptor. In SV2 proteins, the BoNT/A-binding site has been mapped to the luminal domain, but the molecular details of the interaction between BoNT/A and SV2 are unknown. Here we determined the high-resolution crystal structure of the BoNT/A receptor-binding domain (BoNT/A-RBD) in complex with the SV2C luminal domain (SV2C-LD). SV2C-LD consists of a right-handed, quadrilateral β-helix that associates with BoNT/A-RBD mainly through backbone-to-backbone interactions at open β-strand edges, in a manner that resembles the inter-strand interactions in amyloid structures. Competition experiments identified a peptide that inhibits the formation of the complex. Our findings provide a strong platform for the development of novel antitoxin agents and for the rational design of BoNT/A variants with improved therapeutic properties.

Botulinum neurotoxins: new questions arising from structural biology.

Botulinum neurotoxins (BoNTs) are the most toxic substances known and cause botulism in vertebrates. They have also emerged as effective and powerful reagents for cosmetic and medical applications. One important prerequisite for understanding BoNT function in disease, and the further development of the toxins for cosmetic and medical applications, is a detailed knowledge of BoNT interactions with non-toxic neurotoxin-associated proteins and cell surface receptors. Based on the substantial recent progress in obtaining high-resolution crystal structures of key BoNT complexes, we summarize the major advances in understanding BoNT interactions and discuss the resulting potential implications, in particular those relating to BoNT serotype A.

Crystal structure of the BoNT/A2 receptor-binding domain in complex with the luminal domain of its neuronal receptor SV2C

A detailed molecular understanding of botulinum neurotoxin (BoNT)/host-cell-receptor interactions is fundamental both for developing strategies against botulism and for generating improved BoNT variants for medical applications. The X-ray crystal structure of the receptor-binding domain (HC) of BoNT/A1 in complex with the luminal domain (LD) of its neuronal receptor SV2C revealed only few specific side-chain – side-chain interactions that are important for binding. Notably, two BoNT/A1 residues, Arg 1156 and Arg 1294, that are crucial for the interaction with SV2, are not conserved among subtypes. Because it has been suggested that differential receptor binding of subtypes might explain their differences in biological activity, we determined the crystal structure of BoNT/A2-HC in complex with SV2C-LD. Although only few side-chain interactions are conserved between the two BoNT/A subtypes, the overall binding mode of subtypes A1 and A2 is virtually identical. In the BoNT/A2-HC – SV2C complex structure, a missing cation-π stacking is compensated for by an additional salt bridge and an anion-π stacking interaction, which explains why the binding of BoNT/A subtypes to SV2C tolerates variable side chains. These findings suggest that motif extensions and a shallow binding cleft in BoNT/A-HC contribute to binding specificity.

Structural basis for misregulation of kinesin KIF21A autoinhibition by CFEOM1 disease mutations

Tight regulation of kinesin activity is crucial and malfunction is linked to neurological diseases. Point mutations in the KIF21A gene cause congenital fibrosis of the extraocular muscles type 1 (CFEOM1) by disrupting the autoinhibitory interaction between the motor domain and a regulatory region in the stalk. However, the molecular mechanism underlying the misregulation of KIF21A activity in CFEOM1 is not understood. Here, we show that the KIF21A regulatory domain containing all disease-associated substitutions in the stalk forms an intramolecular antiparallel coiled coil that inhibits the kinesin. CFEOM1 mutations lead to KIF21A hyperactivation by affecting either the structural integrity of the antiparallel coiled coil or the autoinhibitory binding interface, thereby reducing its affinity for the motor domain. Interaction of the KIF21A regulatory domain with the KIF21B motor domain and sequence similarities to KIF7 and KIF27 strongly suggest a conservation of this regulatory mechanism in other kinesin-4 family members.

High-throughput mutagenesis using a two-fragment PCR approach

Site-directed scanning mutagenesis is a powerful protein engineering technique which allows studies of protein functionality at single amino acid resolution and design of stabilized proteins for structural and biophysical work. However, creating libraries of hundreds of mutants remains a challenging, expensive and time-consuming process. The efficiency of the mutagenesis step is the key for fast and economical generation of such libraries. PCR artefacts such as misannealing and tandem primer repeats are often observed in mutagenesis cloning and reduce the efficiency of mutagenesis. Here we present a high-throughput mutagenesis pipeline based on established methods that significantly reduces PCR artefacts. We combined a two-fragment PCR approach, in which mutagenesis primers are used in two separate PCR reactions, with an in vitro assembly of resulting fragments. We show that this approach, despite being more laborious, is a very efficient pipeline for the creation of large libraries of mutants.

Structure of the BoNT/A1--receptor complex.

Botulinum neurotoxin A causes botulism but is also used for medical and cosmetic applications. A detailed molecular understanding of BoNT/A--host receptor interactions is therefore fundamental for improving current clinical applications and for developing new medical strategies targeting human disorders. Towards this end, we recently solved an X-ray crystal structure of BoNT/A1 in complex with its neuronal protein receptor SV2C. Based on our findings, we discuss the potential implications for BoNT/A function.

Trennverfahren und allgemeine Analysemethoden

Zur Analyse biochemischer Vorgange mussen oft Zellbestandteile voneinander getrennt werden. Die Biomolekule unterscheiden sich in Struktur, Grose, Ladung und Affinitat zu Bindungspartnern (Liganden) und ermoglichen damit ihre Auftrennung. Ein besonderes Problem besteht darin, dass viele Biomolekule Polymere aus einer sehr beschrankten Anzahl von Bausteinen sind: Zwei DNA-Stucke von 30 bp, deren Sequenzen sich in einem einzigen Nucleotid unterscheiden, sind schwierig voneinander zu trennen. Zwei Molekule von gleicher Grose und Ladung konnen sich sogar nur in der Sequenz der Bausteine unterscheiden. Auch die niedrige Konzentration von Biomolekulen kann ein Problem darstellen: Meist kommen nur eine oder zwei Kopien eines DNA-Molekuls in einer Zelle vor. Erst in den letzten Jahrzehnten sind effiziente Amplifizierungs- und Trennmethoden fur Makromolekule entwickelt worden. Dieses Kapitel stellt die wichtigsten Trenn- und allgemeinen Analyseverfahren vor.

Gluconeogenese, Glykogen, Disaccharide und Pentosephosphatweg

Woher kommt die Glucose, welche der Zelle uber Glykolyse, Citratzyklus und oxidative Phosphorylierung die Synthese von ATP ermoglicht? Glucose wird bei Mensch und Tier entweder mit der Nahrung in Form von Starke und Disacchariden zugefuhrt oder durch Abbau des Reservekohlenhydrats Glykogen erhalten. Sind diese Quellen erschopft, wird Glucose aus Nichtkohlenhydrat-Vorlaufern synthetisiert (Gluconeogenese).

Biochemische Aspekte der menschlichen Ernährung

Unterernahrung (ungenugende Zufuhr von Brennstoffen), Fehlernahrung (ungenugende Zufuhr von Proteinen und essenziellen Nahrungsbestandteilen) und Uberernahrung (ubermasige Zufuhr von Brennstoffen) grassieren in verschiedenen Regionen der Erde und verursachen etliche der medizinischen Probleme unserer Zeit. Es gibt keine gesundheitsfordernden Masnahmen, die wirkungsvoller und kostengunstiger sind als eine adaquate Ernahrungsweise.

Biochemie und Molekularbiologie

Studienleitung Biochemie und Molekularbiologie: Sie zeigt, dass vieles, was im Leben auf den ersten Blick unerklärbar und geheimnisvoll scheint, mit gewissen Grundlagen aus der Chemie und der Physik eben doch wahrgenommen und erklärt werden kann. Man erkennt dadurch auch Zusammenhänge zwischen den Leitwissenschaften des vorigen Jahrhunderts – also zwischen Chemie und Physik – mit der gegenwärtigen Leitwissenschaft, der Biologie.