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Soluble single-domain fragments derived from the unique variable region of camelid heavy-chain antibodies (VHHs) against enzymes may behave as potent inhibitors. The immunization of alpacas with the CMY-2 β-lactamase led to the isolation of three VHHs that specifically recognized and inhibited CMY-2. The structure of the complex VHH cAbCMY-2(254)/CMY-2 was determined by X-ray crystallography. We showed that the epitope is close to the active site and that the CDR3 of the VHH protrudes in the catalytic site. The β-lactamase inhibition was found to follow a mixed profile with a predominant non-competitive component. The three isolated VHHs recognized overlapping epitopes since they behaved as competitive binder. Our study identified a binding site that can be targeted by a new class of β-lactamase’s inhibitors designed with the help of a peptidomimetic approach. Furthermore, the use of mono or bivalent VHH and rabbit polyclonal anti-CMY-2 antibodies enable the development of the first generation of ELISA test for the detection of CMY-2 produced by resistant bacteria.IMPORTANCEThe still increasing antimicrobial resistance in human clinic or veterinary medicine is a major threat for modern chemotherapy. Beside the major caution in the use of current antibiotics, it is important to develop new classes of antibiotics. This work was focused on β-lactamases that are the enzymes involved in the hydrolysis of the major class of antibiotics, the β-lactam compounds. We selected camelid antibodies that inhibit CMY-2, a class C β-lactamase produced by bacteria isolated from the veterinary and human settings. We characterized the conformational epitope present in CMY-2 in order to create a new family of inhibitors based on the paratope of the antibody. Finally, we designed a primary version of a detection system based on an ELISA using VHH and polyclonal antibodies.

As solid organ recipients are at high risk of severe COVID-19 and respond poorly to primary SARS-CoV-2 mRNA vaccination, they have been prioritized for booster vaccination. However, an immunological correlate of protection has not been identified in this vulnerable population. We conducted a prospective monocentric cohort study of 65 kidney transplant recipients who received three doses of SARS-CoV-2 BNT162b2 mRNA vaccination. Associations between symptomatic breakthrough infection (BTI) and vaccine responses, patient demographic and clinical characteristics were explored. Symptomatic COVID-19 was diagnosed in 32% of kidney transplant recipients during a period of six months after the administration of the third vaccine dose. During this period, SARS-CoV-2 delta and omicron were the dominant variants in the general population. Univariate analyzes identified avidity of SARS-CoV-2 receptor binding domain (RBD) binding IgG, neutralizing antibodies and SARS-CoV-2 S2 domain-specific IFN-γ responses as correlates of protection against BTI. Some demographic and clinical parameters correlated with vaccine responses, but none correlated with the risk of BTI. In multivariate analysis, the risk of BTI was best predicted by neutralizing antibody and S2-specific IFN-γ responses, adjusting for age, graft function and mycophenolate mofetil use. In conclusion, both antibody and T cell responses predict the risk of BTI in kidney transplant recipients who received three doses of SARS-CoV-2 mRNA vaccine. T cell responses may help compensate for the suboptimal antibody response to vaccination in this vulnerable population.One Sentence SummaryAntibody and T cell responses to booster SARS-CoV-2 vaccination predict the risk of symptomatic breakthrough infection in kidney transplant recipients

Uncontrolled growth of solid tumors will result in a hallmark hypoxic condition, whereby the key transcriptional regulator of hypoxia inducible factor-1α (HIF-1α) will be stabilized to activate the transcription of target genes that are responsible for the metabolism, proliferation, and metastasis of tumor cells. Targeting and inhibiting the transcriptional activity of HIF-1 may provide an interesting strategy for cancer therapy. In the present study, an immune library and a synthetic library were constructed for the phage display selection of Nbs against recombinant PAS B domain protein (rPasB) of HIF-1α. After panning and screening, seven different nanobodies (Nbs) were selected, of which five were confirmed via immunoprecipitation to target the native HIF-1α subunit. The inhibitory effect of the selected Nbs on HIF-1 induced activation of target genes has been evaluated after intracellular expression of these Nbs in HeLa cells. The dramatic inhibition of both intrabody formats on the expression of HIF-1-related target genes has been confirmed, which indicated the inhibitory efficacy of selected Nbs on the transcriptional activity of HIF-1.

Among various factors, the direct environment (e.g. pH, buffer components, salts, additives, etc.…) is known to have a crucial effect on both the stability and activity of proteins. In particular, proper buffer and pH conditions can improve their stability and function significantly during purification, storage and handling, which is highly relevant for both academic and industrial applications. It can also promote data reproducibility, support the interpretation of experimental results and, finally, contribute to our general understanding of the biophysical properties of proteins. In this study, we have developed a high throughput screen of 158 different buffers/pH conditions in which we evaluated: (i) the protein stability, using differential scanning fluorimetry and (ii) the protein function, using either enzymatic assays or binding activity measurements, both in an automated manner. The modular setup of the screen allows for easy implementation of other characterization methods and parameters, as well as additional test conditions. The buffer/pH screen was validated with five different proteins used as models, i.e. two active-site serine β-lactamases, two metallo-β-lactamases (one of which is only active as a tetramer) and a single-domain dromedary antibody fragment (VHH or nanobody). The formulation screen allowed automated and fast determination of optimum buffer and pH profiles for the tested proteins. Besides the determination of the optimum buffer and pH, the collection of pH profiles of many different proteins may also allow to delineate general concepts to understand and predict the relationship between pH and protein properties.

Carbohydrate-binding modules (CBM) play important roles in targeting and increasing the concentration of carbohydrate active enzymes on their substrates. Using NMR to get the solution structure of CBM14, we can gain insight into secondary structure elements and intramolecular interactions with our assigned nuclear overhauser effect peaks. This reveals that two conserved aromatic residues (Phe437 and Phe456) make up the hydrophobic core of the CBM. These residues are also responsible for connecting the two β-sheets together, by being part of β2 and β4, respectively, and together with disulfide bridges, they create CBM14’s characteristic “hevein-like” fold. Most CBMs rely on aromatic residues for substrate binding; however, CBM14 contains just a single tryptophan (Trp465) that together with Asn466 enables substrate binding. Interestingly, an alanine mutation of a single residue (Leu454) located behind Trp465 renders the CBM incapable of binding. Fluorescence spectroscopy performed on this mutant reveals a significant blue shift, as well as a minor blue shift for its neighbor Val455. The reduction in steric hindrance causes the tryptophan to be buried into the hydrophobic core of the structure and therefore suggests a preorganized binding site for this CBM. Our results show that both Trp465 and Asn466 are affected when CBM14 interacts with both (GlcNAc)3 and β-chitin, that the binding interactions are weak, and that CBM14 displays a slightly higher affinity toward β-chitin. This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.

The heterodimeric transcription elongation factor Spt4/Spt5 (Spt4/5) tightly associates with RNAPII to regulate both transcriptional elongation and co-transcriptional pre-mRNA processing; however, the mechanisms by which Spt4/5 acts are poorly understood. Recent studies of the human and Drosophila Spt4/5 complexes indicate that they can bind nucleic acids in vitro. We demonstrate here that yeast Spt4/5 can bind in a sequence-specific manner to single stranded RNA containing AAN repeats. Furthermore, we show that the major protein determinants for RNA-binding are Spt4 together with the NGN domain of Spt5 and that the KOW domains are not required for RNA recognition. These findings attribute a new function to a domain of Spt4/5 that associates directly with RNAPII, making significant steps towards elucidating the mechanism behind transcriptional control by Spt4/5.

Deciphering the function of the nonprotein-coding portion of genomes represents one of the major challenges that molecular biology is facing today. Numerous classes of RNAs have been discovered over the last past decade and appear to play important regulatory roles in gene expression and disease. The ability to study and manipulate these RNAs relies on the development of programmable RNA-binding molecules such as RNA-binding proteins. Most RNA-binding proteins have modular architectures and combine different RNA-binding domains that provide binding affinity toward a specific RNA sequence and/or structure. Herein, we describe a general strategy to design single-stranded RNA-binding proteins using RanBP2-type zinc-finger (ZF) domains that can recognize a given RNA sequence with high affinity and specificity.

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental monitoring, and food safety control. In this context, we used β-lactamases as efficient reporter enzymes in several protein-protein interaction studies. Furthermore, their ability to accept insertions of peptides or structured proteins/domains strongly encourages the use of these enzymes to generate chimeric proteins. In a recent study, we inserted a single-domain antibody fragment into the Bacillus licheniformis BlaP β-lactamase. These small domains, also called nanobodies, are defined as the antigen-binding domains of single chain antibodies from camelids. Like common double chain antibodies, they show high affinities and specificities for their targets. The resulting chimeric protein exhibited a high affinity against its target while retaining the β-lactamase activity. This suggests that the nanobody and β-lactamase moieties remain functional. In the present work, we report a detailed protocol that combines our hybrid β-lactamase system to the biosensor technology. The specific binding of the nanobody to its target can be detected thanks to a conductimetric measurement of the protons released by the catalytic activity of the enzyme.

Recent advances in transcriptome sequencing and analysis have revealed the complexity of the human genome. The majority (≈ 98%) of cellular transcripts is not translated into proteins and represents a vast, unchartered world of functional non-coding RNAs. Most of them adopt a well-defined three-dimensional structure to achieve their biological functions. However, only very few RNA structures are currently available which reflects the challenges associated with RNA crystallization. Nevertheless, these structures would represent a critical step in understanding functions of non-coding RNAs and their molecular mechanisms in the cell. The overall goal of this study is to develop an innovative and versatile tool to facilitate the functional study and crystallization of structured RNAs (stRNAs). In this work, we have engineered an antibody fragment from camelid heavy-chain antibody (nanobody) able to specifically bind with low nanomolar affinity to stRNA, while no binding could be detected for single-stranded DNA/RNA, double-stranded DNA/RNA or a negatively charged protein. However, this nanobody recognizes different and non-related stRNAs, this observation suggests that it binds to an epitope shared by these stRNAs. Finally, our data also show that the binding of the nanobody does not alter the secondary structure content of the stRNA as well as its unfolding/refolding processes during heat treatment. This work constitutes a successful proof of concept demonstrating that nanobodies can be engineered to recognize RNA-related epitopes.

Deciphering the function of the nonprotein-coding portion of genomes represents one of the major challenges that molecular biology is facing today. Numerous classes of RNAs have been discovered over the last past decade and appear to play important regulatory roles in gene expression and disease. The ability to study and manipulate these RNAs relies on the development of programmable RNA-binding molecules such as RNA-binding proteins. Most RNA-binding proteins have modular architectures and combine different RNA-binding domains that provide binding affinity toward a specific RNA sequence and/or structure. Herein, we describe a general strategy to design single-stranded RNA-binding proteins using RanBP2-type zinc-finger (ZF) domains that can recognize a given RNA sequence with high affinity and specificity.

Antibody-based products constitute one of the most attractive biological molecules for diagnostic, medical imagery and therapeutic purposes with very few side effects. Their development has become a major priority of biotech and pharmaceutical industries. Recently, a growing number of modified antibody-based products have emerged including fragments, multi-specific and conjugate antibodies. In this study, using protein engineering, we have functionalized the anti-hen egg-white lysozyme (HEWL) camelid VHH antibody fragment (cAb-Lys3), by insertion into a solvent-exposed loop of the Bacillus licheniformis β-lactamase BlaP. We showed that the generated hybrid protein conserved its enzymatic activity while the displayed nanobody retains its ability to inhibit HEWL with a nanomolar affinity range. Then, we successfully implemented the functionalized cAb-Lys3 in enzyme-linked immunosorbent assay, potentiometric biosensor and drug screening assays. The hybrid protein was also expressed on the surface of phage particles and, in this context, was able to interact specifically with HEWL while the β-lactamase activity was used to monitor phage interactions. Finally, using thrombin-cleavage sites surrounding the permissive insertion site in the β-lactamase, we reported an expression system in which the nanobody can be easily separated from its carrier protein. Altogether, our study shows that insertion into the BlaP β-lactamase constitutes a suitable technology to functionalize nanobodies and allows the creation of versatile tools that can be used in innovative biotechnological assays.

Nitroxide labels are useful probes of biomolecule structure that can be detected by NMR and EPR spectroscopy. Although many methods exist for labeling oligonucleotides with nitroxides, most require reagents that are expensive or laborious to prepare. A simpler approach is described herein using commercially available phosphorothioate oligonucleotides and 2-(3-iodoacetamidomethyl)-PROXYL. We describe semi-optimized conditions for labeling DNA and RNA oligonucleotides and methodology for purifying and identifying these reagents by MALDI-MS. The nitroxide label showed some propensity to hydrolyze at high temperature and over prolonged periods at room temperature. Nitroxide-labeled DNA oligonucleotides gave characteristic EPR spectra and caused the disappearance of bound protein signals in 15N HSQC spectra consistent with the paramagnetic relaxation enhancement (PRE) effect.

International audience; The controlled delivery of proteins within calcium carbonate (CaCO 3) particles is currently widely investigated. The success of these carriers is driven by ionic interactions between the encapsulated proteins and the particles. This poses a great limitation on the successful loading of proteins that have no ionic affinity to CaCO 3. In this study, we explored the use of polysaccharide–protein interactions to strongly enhance the encapsulation of proteins in CaCO 3 microparticles. Previously, Vandevenne and colleagues inserted a human chitin binding domain (ChBD) that has intrinsic affinity for hyaluronic acid (HA) into a b-lactamase (BlaP). This generated chimeric protein, named BlaPChBD, was shown to be fully bifunctional. In this study we showed that this hybrid protein can associate with HA and be successfully loaded into vaterite CaCO 3 microparticles using supercritical CO 2 (ScCO 2) technology aided by the templating effect of HA on CaCO 3. The presence of ChBD inserted into BlaP increased the encapsulation of the protein by 6-fold when complexed with HA. Furthermore, thrombin cleavage sites were engineered on both sides of the inserted ChBD in the chimeric BlaP to achieve release of the protein from the micro-particles by protease cleavage. Our results showed that thrombin cleavage increased the release of the protein from the microparticles within 36 hours from o20% to 87%. In conclusion, the presence of ChBD successfully improved the encapsulation yield of the protein while retaining up to 82% of its activity and efficient release of the protein from the microparticles was achieved by protease cleavage.

Chitin is an important structural component of numerous fungal pathogens and parasitic nematodes. The human macrophage chitotriosidase (HCHT) is a chitinase that hydrolyses glycosidic bonds between the N-acetyl-D-glucosamine units of this biopolymer. HCHT belongs to the Glycoside Hydrolase (GH) superfamily and contains a well-characterized catalytic domain appended to a chitin-binding domain (ChBDCHIT1). Although its precise biological function remains unclear, HCHT has been described to be involved in innate immunity. In this study, the molecular basis for interaction with insoluble chitin as well as with soluble chito-oligosaccharides has been determined. The results suggest a new mechanism as a common binding mode for many Carbohydrate Binding Modules (CBMs). Furthermore, using a phylogenetic approach, we have analysed the modularity of HCHT and investigated the evolutionary paths of its catalytic and chitin binding domains. The phylogenetic analyses indicate that the ChBDCHIT1 domain dictates the biological function of HCHT and not its appended catalytic domain. This observation may also be a general feature of GHs. Altogether, our data have led us to postulate and discuss that HCHT acts as an immune catalyser. © 2017 The Authors. Published by Nature Publishing Group. This is an open access article licensed under a Creative Commons Attribution 4.0 International License

The realization that gene transcription is much more pervasive than previously thought and that many diverse RNA species exist in simple as well as complex organisms has triggered efforts to develop functionalized RNA-binding proteins (RBPs) that have the ability to probe and manipulate RNA function. Previously, we showed that the RanBP2-type zinc finger (ZF) domain is a good candidate for an addressable single-stranded-RNA (ssRNA) binding domain that can recognize ssRNA in a modular and specific manner. In the present study, we successfully engineered a sequence specificity change onto this ZF scaffold by using a combinatorial approach based on phage display. This work constitutes a foundation from which a set of RanBP2 ZFs might be developed that is able to recognize any given RNA sequence.

Single-stranded DNA (ssDNA)-binding protein (SSB) protects ssDNA from degradation and recruits other proteins for DNA replication and repair. Escherichia coli SSB is the prototypical eubacterial SSB in a family of tetrameric SSBs. It consists of a structurally well-defined ssDNA binding domain (OB-domain) and a disordered C-terminal domain (C-domain). The eight-residue C-terminal segment of SSB (C-peptide) mediates the binding of SSB to many different SSB-binding proteins. Previously published nuclear magnetic resonance (NMR) data of the monomeric state at pH 3.4 showed that the C-peptide binds to the OB-domain at a site that overlaps with the ssDNA binding site, but investigating the protein at neutral pH is difficult because of the high molecular mass and limited solubility of the tetramer. Here we show that the C-domain is highly mobile in the SSB tetramer at neutral pH and that binding of the C-peptide to the OB-domain is so weak that most of the C-peptides are unbound even in the absence of ssDNA. We address the problem of determining intramolecular binding affinities in the situation of fast exchange between two states, one of which cannot be observed by NMR and cannot be fully populated. The results were confirmed by electron paramagnetic resonance spectroscopy and microscale thermophoresis. The C-peptide-OB-domain interaction is shown to be driven primarily by electrostatic interactions, so that binding of 1 equiv of (dT)35 releases practically all C-peptides from the OB-domain tetramer. The interaction is much more sensitive to NaCl than to potassium glutamate, which is the usual osmolyte in E. coli. As the C-peptide is predominantly in the unbound state irrespective of the presence of ssDNA, long-range electrostatic effects from the C-peptide may contribute more to regulating the activity of SSB than any engagement of the C-peptide by the OB-domain.

Classical zinc fingers (ZFs) are one of the most abundant and best characterized DNA-binding domains. Typically, tandem arrays of three or more ZFs bind DNA target sequences with high affinity and specificity, and the mode of DNA recognition is sufficiently well understood that tailor-made ZF-based DNA-binding proteins can be engineered. We have shown previously that a two-zinc finger unit found in the transcriptional coregulator ZNF217 recognizes DNA but with an affinity and specificity that is lower than other ZF arrays. To investigate the basis for these differences, we determined the structure of a ZNF217-DNA complex. We show that although the overall position of the ZFs on the DNA closely resembles that observed for other ZFs, the side-chain interaction pattern differs substantially from the canonical model. The structure also reveals the presence of two methyl-π interactions, each featuring a tyrosine contacting a thymine methyl group. To our knowledge, interactions of this type have not previously been described in classical ZF-DNA complexes. Finally, we investigated the sequence specificity of this two-ZF unit and discuss how ZNF217 might discriminate its target DNA sites in the cell.

International audience; Despite impressive successes in protein design, designing a well-folded protein of more 100 amino acids de novo remains a formidable challenge. Exploiting the promising biophysical features of the artificial protein Octarellin V, we improved this protein by directed evolution, thus creating a more stable and soluble protein: Octarellin V.1. Next, we obtained crystals of Octarellin V.1 in complex with crystallization chaperons and determined the tertiary structure. The experimental structure of Octarellin V.1 differs from its in silico design: the (αβα) sandwich architecture bears some resemblance to a Rossman-like fold instead of the intended TIM-barrel fold. This surprising result gave us a unique and attractive opportunity to test the state of the art in protein structure prediction, using this artificial protein free of any natural selection. We tested 13 automated webservers for protein structure prediction and found none of them to predict the actual structure. More than 50% of them predicted a TIM-barrel fold, i.e. the structure we set out to design more than 10years ago. In addition, local software runs that are human operated can sample a structure similar to the experimental one but fail in selecting it, suggesting that the scoring and ranking functions should be improved. We propose that artificial proteins could be used as tools to test the accuracy of protein structure prediction algorithms, because their lack of evolutionary pressure and unique sequences features.

Irreversible accumulation of protein aggregates represents an important problem both in vivo and in vitro. The aggregation of proteins is of critical importance in a wide variety of biomedical situations, ranging from diseases (such as Alzheimer's and Parkinson's diseases) to the production (e.g. inclusion bodies), stability, storage and delivery of protein drugs. β-Cyclodextrin (β-CD) is a circular heptasaccharide characterized by a hydrophilic exterior and a hydrophobic interior ring structure. In this research, we studied the effects of a chemically modified β-CD (BCD07056), on the aggregating and refolding properties of BlaPChBD, a hybrid protein obtained by inserting the chitin binding domain of the human macrophage chitotriosidase into the class A β-lactamase BlaP from Bacillus licheniformis 749/I during its thermal denaturation. The results show that BCD07056 strongly increases the refolding yield of BlaPChBD after thermal denaturation and constitutes an excellent additive to stabilize the protein over time at room temperature. Our data suggest that BCD07056 acts early in the denaturation process by preventing the formation of an intermediate which leads to an aggregated state. Finally, the role of β-CD derivatives on the stability of proteins is discussed.

This work analyses the chitin-binding and catalytic domains of the human macrophage chitotriosidase and investigates the physiological role of this glycoside hydrolase in a complex mechanism such as the innate immune system, especially its antifungal activity. Accordingly, we first analyzed the ability of its chitin-binding domain to interact with chitin embedded in fungal cell walls using the β-lactamase activity reporter system described in our previous work. The data showed that the chitin-binding activity was related to the cell wall composition of the fungi strains and that their peptide-N-glycosidase/zymolyase treatments increased binding to fungal by increasing protein permeability. We also investigated the antifungal activity of the enzyme against Candida albicans. The antifungal properties of the complete chitotriosidase were analyzed and compared with those of the isolated chitin-binding and catalytic domains. The isolated catalytic domain but not the chitin-binding domain was sufficient to provide antifungal activity. Furthermore, to explain the lack of obvious pathologic phenotypes in humans homozygous for a widespread mutation that renders chitotriosidase inactive, we postulated that the absence of an active chitotriosidase might be compensated by the expression of another human hydrolytic enzyme such as lysozyme. The comparison of the antifungal properties of chitotriosidase and lysozyme indicated that surprisingly, both enzymes have similar in vitro antifungal properties. Furthermore, despite its more efficient hydrolytic activity on chitin, the observed antifungal activity of chitotriosidase was lower than that of lysozyme. Finally, this antifungal duality between chitotriosidase and lysozyme is discussed in the context of innate immunity.

Using genetic engineering technologies, the chitin-binding domain (ChBD) of the human macrophage chitotriosidase has been inserted into the host protein BlaP, a class A beta-lactamase produced by Bacillus licheniformis. The product of this construction behaved as a soluble chimeric protein that conserves both the capacity to bind chitin and to hydrolyze beta-lactam moiety. Here we describe the biochemical and biophysical properties of this protein (BlaPChBD). This work contributes to a better understanding of the reciprocal structural and functional effects of the insertion on the host protein scaffold and the heterologous structured protein fragments. The use of BlaP as a protein carrier represents an efficient approach to the functional study of heterologous protein fragments.

Chromodomain Helicase DNA-binding protein 4 (CHD4) is a chromatin-remodeling enzyme that has been reported to regulate DNA-damage responses through its N-terminal region in a poly(ADP-ribose) polymerase-dependent manner. We have identified and determined the structure of a stable domain (CHD4-N) in this N-terminal region. The-fold consists of a four-α-helix bundle with structural similarity to the high mobility group box, a domain that is well known as a DNA binding module. We show that the CHD4-N domain binds with higher affinity to poly(ADP-ribose) than to DNA. We also show that the N-terminal region of CHD4, although not CHD4-N alone, is essential for full nucleosome remodeling activity and is important for localizing CHD4 to sites of DNA damage. Overall, these data build on our understanding of how CHD4-NuRD acts to regulate gene expression and participates in the DNA-damage response.

Myelin transcription factor 1 (MyT1/NZF2), a member of the neural zinc-finger (NZF) protein family, is a transcription factor that plays a central role in the developing central nervous system. It has also recently been shown that, in combination with two other transcription factors, the highly similar paralog MyT1L is able to direct the differentiation of murine and human stem cells into functional neurons. MyT1 contains seven zinc fingers (ZFs) that are highly conserved throughout the protein and throughout the NZF family. We recently presented a model for the interaction of the fifth ZF of MyT1 with a DNA sequence derived from the promoter of the retinoic acid receptor (RARE) gene. Here, we have used NMR spectroscopy, in combination with surface plasmon resonance and data-driven molecular docking, to delineate the mechanism of DNA binding for double ZF polypeptides derived from MyT1. Our data indicate that a two-ZF unit interacts with the major groove of the entire RARE motif and that both fingers bind in an identical manner and with overall two-fold rotational symmetry, consistent with the palindromic nature of the target DNA. Several key residues located in one of the irregular loops of the ZFs are utilized to achieve specific binding. Analysis of the human and mouse genomes based on our structural data reveals three putative MyT1 target genes involved in neuronal development.

Classical C2H2 zinc finger proteins are among the most abundant transcription factors found in eukaryotes, and the mechanisms through which they recognize their target genes have been extensively investigated. In general, a tandem array of three fingers separated by characteristic TGERP links is required for sequence-specific DNA recognition. Nevertheless, a significant number of zinc finger proteins do not contain a hallmark three-finger array of this type, raising the question of whether and how they contact DNA. We have examined the multi-finger protein ZNF217, which contains eight classical zinc fingers. ZNF217 is implicated as an oncogene and in repressing the E-cadherin gene. We show that two of its zinc fingers, 6 and 7, can mediate contacts with DNA. We examine its putative recognition site in the E-cadherin promoter and demonstrate that this is a suboptimal site. NMR analysis and mutagenesis is used to define the DNA binding surface of ZNF217, and we examine the specificity of the DNA binding activity using fluorescence anisotropy titrations. Finally, sequence analysis reveals that a variety of multi-finger proteins also contain two-finger units, and our data support the idea that these may constitute a distinct subclass of DNA recognition motif.

The system described here allows the expression of protein fragments into a solvent-exposed loop of a carrier protein, the beta-lactamase BlaP. When using Escherichia coli constitutive expression vectors, a positive selection of antibioresistant bacteria expressing functional hybrid beta-lactamases is achieved in the presence of beta-lactams making further screening of correctly folded and secreted hybrid beta-lactamases easier. Protease-specific recognition sites have been engineered on both sides of the beta-lactamase permissive loop in order to cleave off the exogenous protein fragment from the carrier protein by an original two-step procedure. According to our data, this approach constitutes a suitable alternative for production of difficult to express protein domains. This work demonstrates that the use of BlaP as a carrier protein does not alter the biochemical activity and the native disulphide bridge formation of the inserted chitin binding domain of the human macrophage chitotriosidase. We also report that the beta-lactamase activity of the hybrid protein can be used to monitor interactions between the inserted protein fragments and its ligands and to screen neutralizing molecules.