NrdR is a bacterial transcriptional repressor consisting of a zinc (Zn)-ribbon domain followed by an ATP-cone domain. Understanding its mechanism of action could aid the design of novel antibacterials. NrdR binds specifically to two “NrdR boxes” upstream of ribonucleotide reductase operons, of which Escherichia coli has three: nrdHIEF, nrdDG and nrdAB, in the last of which we identified a new box. We show that E. coli NrdR (EcoNrdR) has similar binding strength to all three sites when loaded with ATP plus deoxyadenosine triphosphate (dATP) or equivalent diphosphate combinations. No other combination of adenine nucleotides promotes binding to DNA. We present crystal structures of EcoNrdR–ATP–dATP and EcoNrdR–ADP–dATP, which are the first high-resolution crystal structures of an NrdR. We have also determined cryo-electron microscopy structures of DNA-bound EcoNrdR–ATP–dATP and novel filaments of EcoNrdR–ATP. Tetrameric forms of EcoNrdR involve alternating interactions between pairs of Zn-ribbon domains and ATP-cones. The structures reveal considerable flexibility in relative orientation of ATP-cones vs Zn-ribbon domains. The structure of DNA-bound EcoNrdR–ATP–dATP shows that significant conformational rearrangements between ATP-cones and Zn-ribbons accompany DNA binding while the ATP-cones retain the same relative orientation. In contrast, ATP-loaded EcoNrdR filaments show rearrangements of the ATP-cone pairs and sequester the DNA-binding residues of NrdR such that they are unable to bind to DNA. Our results, in combination with a previous structural and biochemical study, point to highly flexible EcoNrdR structures that, when loaded with the correct nucleotides, adapt to an optimal promoter-binding conformation.

Galactolipids are characteristic lipids of the photosynthesis membranes of higher plants and cyanobacteria. Due to their close relationship to the stability of the photosystem protein complexes, the biogenesis of galactolipids has been intensively studied on the genetic and molecular levels. There are two major types of galactolipids in chloroplastic membranes: monogalactosyldiacylglycerol and digalactosyldiacylglycerol (DGDG). Under phosphate-limiting conditions, the amount of DGDG increases dramatically to allow for phosphate salvage from phospholipids. In Arabidopsis thaliana, the membrane-associated glycosyltransferase digalactosyldiacylglycerol synthase 2 (atDGD2) is highly responsive to phosphate starvation and is significantly upregulated during such conditions. The lipid galactosylation reactions are also fundamentally interesting as they require a catalyst that is capable of bringing a hydrophilic and lipophilic substrate together at the solution-membrane phase border. Here, we present the X-ray crystal structure of atDGD2, which is the first reported DGDG synthase structure. AtDGD2 is most structurally similar to functionally unrelated GT-B enzymes. Interestingly, in spite of significant donor substrate binding differences, we identified four amino acids (Gly22, His151, Lys243, and Glu321, atDGD2 numbering) which were entirely conserved between the structurally similar enzymes. We also investigated the membrane interaction kinetics and membrane anchoring mechanism of atDGD2. This demonstrated that atDGD2 is membrane-bound but also showed that membrane binding is highly dynamic. Furthermore, our structural information in context of previous biophysical studies highlights regions of the enzyme exhibiting a high degree of structural plasticity, which we propose to be important for allowing atDGD2 to quickly adapt its activity based on the membrane lipid environment.

Botulinum neurotoxins (BoNTs) are the most potent toxins known and are used to treat an increasing number of medical disorders. All BoNTs are naturally co-expressed with a protective partner protein (NTNH) with which they form a 300 kDa complex, to resist acidic and proteolytic attack from the digestive tract. We have previously identified a new botulinum neurotoxin serotype, BoNT/X, that has unique and therapeutically attractive properties. We present the cryo-EM structure of the BoNT/X-NTNH/X complex and the crystal structure of the isolated NTNH protein. Unexpectedly, the BoNT/X complex is stable and protease-resistant at both neutral and acidic pH and disassembles only in alkaline conditions. Using the stabilizing effect of NTNH, we isolated BoNT/X and showed that it has very low potency both in vitro and in vivo. Given the high catalytic activity and translocation efficacy of BoNT/X, low activity of the full toxin is likely due to the receptor-binding domain, which presents very weak ganglioside binding and exposed hydrophobic surfaces.

Imaging of living synapses has relied for over two decades on the overexpression of synaptic proteins fused to fluorescent reporters. This strategy alters the stoichiometry of synaptic components and ultimately affects synapse physiology. To overcome these limitations, here a nanobody is presented that binds the calcium sensor synaptotagmin-1 (NbSyt1). This nanobody functions as an intrabody (iNbSyt1) in living neurons and is minimally invasive, leaving synaptic transmission almost unaffected, as suggested by the crystal structure of the NbSyt1 bound to Synaptotagmin-1 and by the physiological data. Its single-domain nature enables the generation of protein-based fluorescent reporters, as showcased here by measuring spatially localized presynaptic Ca²⁺ with a NbSyt1- jGCaMP8 chimera. Moreover, the small size of NbSyt1 makes it ideal for various super-resolution imaging methods. Overall, NbSyt1 is a versatile binder that will enable imaging in cellular and molecular neuroscience with unprecedented precision across multiple spatiotemporal scales. 

Ribonucleotide reductase (RNR) is an essential enzyme that catalyzes the synthesis of DNA building blocks in virtually all living cells. NrdR, an RNR-specific repressor, controls the transcription of RNR genes and, often, its own, in most bacteria and some archaea. NrdR senses the concentration of nucleotides through its ATP-cone, an evolutionarily mobile domain that also regulates the enzymatic activity of many RNRs, while a Zn-ribbon domain mediates binding to NrdR boxes upstream of and overlapping the transcription start site of RNR genes. Here, we combine biochemical and cryo-EM studies of NrdR from Streptomyces coelicolor to show, at atomic resolution, how NrdR binds to DNA. The suggested mechanism involves an initial dodecamer loaded with two ATP molecules that cannot bind to DNA. When dATP concentrations increase, an octamer forms that is loaded with one molecule each of dATP and ATP per monomer. A tetramer derived from this octamer then binds to DNA and represses transcription of RNR. In many bacteria — including well-known pathogens such as Mycobacterium tuberculosis — NrdR simultaneously controls multiple RNRs and hence DNA synthesis, making it an excellent target for novel antibiotics development.

Botulinum neurotoxins (BoNTs) are the causative agents of a potentially lethal paralytic disease targeting cholinergic nerve terminals. Multiple BoNT serotypes exist, with types A, B and E being the main cause of human botulism. Their extreme toxicity has been exploited for cosmetic and therapeutic uses to treat a wide range of neuromuscular disorders. Although naturally occurring BoNT types share a common end effect, their activity varies significantly based on the neuronal cell-surface receptors and intracellular SNARE substrates they target. These properties are the result of structural variations that have traditionally been studied using biophysical methods such as X-ray crystallography. Here, we determined the first structures of botulinum neurotoxins using single-particle cryogenic electron microscopy. The maps obtained at 3.6 and 3.7 Å for BoNT/B and /E, respectively, highlight the subtle structural dynamism between domains, and of the binding domain in particular. This study demonstrates how the recent advances made in the field of single-particle electron microscopy can be applied to bacterial toxins of clinical relevance and the botulinum neurotoxin family in particular.

Ribonucleotide reductase (RNR) is a central enzyme for the synthesis of DNA building blocks. Most aerobic organisms, including nearly all eukaryotes, have class I RNRs consisting of R1 and R2 subunits. The catalytic R1 subunit contains an overall activity site that can allosterically turn the enzyme on or off by the binding of ATP or dATP, respectively. The mechanism behind the ability to turn the enzyme off via the R1 subunit involves the formation of different types of R1 oligomers in most studied species and R1–R2 octamers in Escherichia coli. To better understand the distribution of different oligomerization mechanisms, we characterized the enzyme from Clostridium botulinum, which belongs to a subclass of class I RNRs not studied before. The recombinantly expressed enzyme was analyzed by size-exclusion chromatography, gas-phase electrophoretic mobility macromolecular analysis, EM, X-ray crystallography, and enzyme assays. Interestingly, it shares the ability of the E. coli RNR to form inhibited R1–R2 octamers in the presence of dATP but, unlike the E. coli enzyme, cannot be turned off by combinations of ATP and dGTP/dTTP. A phylogenetic analysis of class I RNRs suggests that activity regulation is not ancestral but was gained after the first subclasses diverged and that RNR subclasses with inhibition mechanisms involving R1 oligomerization belong to a clade separated from the two subclasses forming R1–R2 octamers. These results give further insight into activity regulation in class I RNRs as an evolutionarily dynamic process.

This thesis is divided into two sections; the first part describes our work in the field of botulinum neurotoxins (presented in papers I, II, III, and manuscript IV) and the second part summarizes our work involving the design of a new biochemical tool (presented in paper V).

Botulinum neurotoxins (BoNTs) produced by the anaerobic bacterium Clostridium botulinum are the most poisonous substances known to date. They have a conserved structure that consists of three domains (receptor-binding, translocation, and catalytic domain), each of which has a distinct function. The receptor-binding domain binds to neuronal receptors, and after endocytosis the translocation domain shuttles the catalytic domain into the cytosol, where it cleaves neuronal proteins of the SNARE family, which are part of the vesicle-membrane fusion machinery.

In paper I, we studied proteins of unknown function (OrfX1, OrfX2, OrfX3, and P47), which are co-expressed with certain BoNTs. We solved the crystal structures of OrfX2 and P47, and their structural resemblance to tubular lipid binding proteins (TULIP) together with lipid binding studies, led us to conclude that OrfX1 and P47 are able to bind phosphatidyl inositol phosphates (PIPs) in vitro.

In paper II, we studied the binding of BoNT/B, /DC and /G to their protein receptor synaptotagmin (Syt). We determined their affinities to synaptotagmins from different species, and concluded that residue F50 in bovine Syt-II is responsible for its increased affinity towards BoNT/DC. In addition, we studied the interaction between BoNT/G and Syt-II via STD-NMR. Our results showed the binding to be similar to BoNT/B and Syt-II, and that the N-terminal region of the Syt peptide is important for the binding of BoNTs to synaptotagmin, even though it is not part of the binding interface.

In paper III and manuscript IV, we present the identification of a novel BoNT serotype named BoNT/X. We showed that BoNT/X cleaves the non-canonical substrates VAMP4, VAMP5 and Ykt6, as well as the canonical substrate VAMP1-3 at a new cleavage site, distinct from other BoNTs. In addition, we present the cryo-EM structure of BoNT/X in complex with its non-toxic interaction partner NTNH. Our pH stability experiments revealed that BoNT/X-NTNH remain bound at neutral to moderately high pH, in contrast with what is observed for BoNT/A-NTNH.

In paper V we present the design of a novel epitope tag named the ALFA system. The ALFA tag is a short α-helical protein tag that is highly stable and electroneutral. The ALFA nanobody has a very high affinity for the tag and is small enough to allow for high performance in high-resolution microscopy. The crystal structure of the ALFA nanobody in complex with the tag led to a modified version of the ALFA nanobody that can release the tag via competitive elution with free ALFA peptide. Our results showed that this system outperforms several commercially available systems in protein purification and high-resolution microscopy.

Botulinum neurotoxins (BoNTs) are exceptionally toxic proteins that cause paralysis but are also extensively used as treatment for various medical conditions. Most BoNTs bind two receptors on neuronal cells, namely, a ganglioside and a protein receptor. Differences in the sequence between the protein receptors from different species can impact the binding affinity and toxicity of the BoNTs. Here we have investigated how BoNT/B, /DC, and /G, all three toxins that utilize synaptotagmin I and II (Syt-I and Syt-II, respectively) as their protein receptors, bind to Syt-I and -II of mouse/rat, bovine, and human origin by isothermal titration calorimetry analysis. BoNT/G had the highest affinity for human Syt-I, and BoNT/DC had the highest affinity for bovine Syt-II. As expected, BoNT/B, /DC, and /G showed very low levels of binding to human Syt-II. Furthermore, we carried out saturation transfer difference (STD) and STD-TOCSY NMR experiments that revealed the region of the Syt peptide in direct contact with BoNT/G, which demonstrate that BoNT/G recognizes the Syt peptide in a model similar to that in the established BoNT/B-Syt-II complex. Our analyses also revealed that regions outside the Syt peptide’s toxin-binding region are important for the helicity of the peptide and, therefore, the binding affinity.

Specialized epitope tags are widely used for detecting, manipulating or purifying proteins, but often their versatility is limited. Here, we introduce the ALFA-tag, a rationally designed epitope tag that serves a remarkably broad spectrum of applications in life sciences while outperforming established tags like the HA-, FLAG (R)- or myc-tag. The ALFA-tag forms a small and stable a-helix that is functional irrespective of its position on the target protein in prokaryotic and eukaryotic hosts. We characterize a nanobody (NbALFA) binding ALFA-tagged proteins from native or fixed specimen with low picomolar affinity. It is ideally suited for super-resolution microscopy, immunoprecipitations and Western blotting, and also allows in vivo detection of proteins. We show the crystal structure of the complex that enabled us to design a nanobody mutant (NbALFA(PE)) that permits efficient one-step purifications of native ALFA-tagged proteins, complexes and even entire living cells using peptide elution under physiological conditions.