A conformational change in the helicase core is necessary but not sufficient for RNA unwinding by the DEAD box helicase YxiN

Cooperative binding of ATP and RNA to DEAD-box helicases induces the closed conformation of their helicase core, with extensive interactions across the domain interface. The bound RNA is bent, and its distortion may constitute the first step towards RNA unwinding. To dissect the role of the conformational change in the helicase core for RNA unwinding, we characterized the RNA-stimulated ATPase activity, RNA unwinding and the propensity to form the closed conformer for mutants of the DEAD box helicase YxiN. The ATPase-deficient K52Q mutant forms a closed conformer upon binding of ATP and RNA, but is deficient in RNA unwinding. A mutation in motif III slows down the catalytic cycle, but neither affects the propensity for the closed conformer nor its global conformation. Hence, the closure of the cleft in the helicase core is necessary but not sufficient for RNA unwinding. In contrast, the G303A mutation in motif V prevents a complete closure of the inter-domain cleft, affecting ATP binding and hydrolysis and is detrimental to unwinding. Possibly, the K52Q and motif III mutants still introduce a kink into the backbone of bound RNA, whereas G303A fails to kink the RNA substrat

A conformational change in the helicase core is necessary but not sufficient for RNA unwinding by the DEAD box helicase YxiN

Cooperative binding of ATP and RNA to DEAD-box helicases induces the closed conformation of their helicase core, with extensive interactions across the domain interface. The bound RNA is bent, and its distortion may constitute the first step towards RNA unwinding. To dissect the role of the conformational change in the helicase core for RNA unwinding, we characterized the RNA-stimulated ATPase activity, RNA unwinding and the propensity to form the closed conformer for mutants of the DEAD box helicase YxiN. The ATPase-deficient K52Q mutant forms a closed conformer upon binding of ATP and RNA, but is deficient in RNA unwinding. A mutation in motif III slows down the catalytic cycle, but neither affects the propensity for the closed conformer nor its global conformation. Hence, the closure of the cleft in the helicase core is necessary but not sufficient for RNA unwinding. In contrast, the G303A mutation in motif V prevents a complete closure of the inter-domain cleft, affecting ATP binding and hydrolysis and is detrimental to unwinding. Possibly, the K52Q and motif III mutants still introduce a kink into the backbone of bound RNA, whereas G303A fails to kink the RNA substrate

Domain orientation in the RNA helicase YxiN and the role of conformational changes for RNA unwinding

The RNA helicase YxiN from Bacillus subtilis is a member of the family of DEAD box proteins. YxiN is able to unwind RNA double strands in an ATP-dependent manner. The ability to catalyse RNA rearrangement is in vivo presumably necessary for the bacterial ribosome biogenesis. YxiN comprises a two-domain helicase core region and a C-terminal RNA binding domain. While crystal structures of the C-terminal core domain and the RNA binding domain separately have been determined before, the structure of full-length YxiN is not known. In the current project the orientation of these three domains to each other was determined employing fluorescence resonance energy transfer (FRET) experiments at the single-molecule level. Therefore the approximate architecture of the full-length enzyme in solution can now be described. The two core domains exhibit a conformation similar to the crystal structure of the DEAD box protein MjDeaD. The RNA binding domain is adjacent to the C-terminal core domain. Presumably the central -sheet of the RNA binding domain faces towards a patch of the core domain that is formed by loops. During catalysis YxiN undergoes a conformational change. The conformation of the core domains mentioned above is adopted in the absence of substrates and in the presence of RNA, ADP, ATP or ADPNP. In the presence of both RNA and ATP (or ADPNP) the core domains approach each other constituting a closed conformation. During the catalytic cycle this conformational change takes place initially after binding of RNA and ATP. The conformational change is necessary for RNA unwinding. But it is not sufficient since the YxiN_K52Q mutant adopts the closed conformation upon binding of RNA and ATP (or ADPNP) but is unwinding deficient. Transitions between the open and the closed conformation could only rarely been detected in the FRET experiments on a confocal microscope due to a limited observation time. To be able to monitor the conformation of YxiN on longer timescale the protein was engineered for FRET experiments on a total internal reflection microscope. A protocol was developed that comprises fluorophore double labelling of YxiN and the attachment of a biotin at the protein’s C-terminus. The biotinylation procedure is based on the reaction type of expressed protein ligation. The labelled and biotinylated YxiN construct could be specifically immobilized on a streptavidin coated surface for total internal reflection microscopy. Subsequently, YxiN could be monitored for up to a few seconds. Expressed protein ligation was furthermore employed to develop a specific fluorophore double labelling strategy for FRET experiments. Employing this strategy a YxiN construct could be generated that carries one certain fluorophore exclusively at one position in the protein. A different fluorophore can attach to the same position or to one further site within the protein. The procedure was therefore termed semi-site-specific double labelling. In comparison with statistic labelling procedures the semi-site-specific double labelling allows for decreasing the sample heterogeneity in FRET experiments. Taken together, this study revealed the conformation of the three-domain RNA helicase YxiN, its conformational change during catalysis which is essential for the activity of the helicase and the study established protein preparation techniques that provide the basis for further studies on the helicase mechanism

Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domains

RNA helicases mediate structural rearrangements of RNA or RNA–protein complexes at the expense of ATP hydrolysis. Members of the DEAD box helicase family consist of two flexibly connected helicase domains. They share nine conserved sequence motifs that are involved in nucleotide binding and hydrolysis, RNA binding, and helicase activity. Most of these motifs line the cleft between the two helicase domains, and extensive communication between them is required for RNA unwinding. The two helicase domains of the Bacillus subtilis RNA helicase YxiN were produced separately as intein fusions, and a functional RNA helicase was generated by expressed protein ligation. The ligated helicase binds adenine nucleotides with very similar affinities to the wild-type protein. Importantly, its intrinsically low ATPase activity is stimulated by RNA, and the Michaelis–Menten parameters are similar to those of the wild-type. Finally, ligated YxiN unwinds a minimal RNA substrate to an extent comparable to that of the wild-type helicase, confirming authentic interdomain communication

Adaption of human antibody lambda and kappa light chain architectures to CDR repertoires

Monoclonal antibodies bind with high specificity to a wide range of diverse antigens, primarily mediated by their hypervariable complementarity determining regions (CDRs). The defined antigen binding loops are supported by the structurally conserved β-sandwich framework of the light chain (LC) and heavy chain (HC) variable regions. The LC genes are encoded by two separate loci, subdividing the entity of antibodies into kappa (LCκ) and lambda (LCλ) isotypes that exhibit distinct sequence and conformational preferences. In this work, a diverse set of techniques were employed including machine learning, force field analysis, statistical coupling analysis and mutual information analysis of a non-redundant antibody structure collection. Thereby, it was revealed how subtle changes between the structures of LCκ and LCλ isotypes increase the diversity of antibodies, extending the predetermined restrictions of the general antibody fold and expanding the diversity of antigen binding. Interestingly, it was found that the characteristic framework scaffolds of κ and λ are stabilized by diverse amino acid clusters that determine the interplay between the respective fold and the embedded CDR loops. In conclusion, this work reveals how antibodies use the remarkable plasticity of the beta-sandwich Ig fold to incorporate a large diversity of CDR loops.status: publishe

Improved Solution-State Properties of Monoclonal Antibodies by Targeted Mutations

Monoclonal antibody (mAb)-based therapeutics often require high-concentration formulations. Unfortunately, highly concentrated antibody solutions often have biophysical properties that are disadvantageous for therapeutic development, such as high viscosity, solubility limitations, precipitation issues, or liquid–liquid phase separation. In this work, we present a computational rational design principle for improving the thermodynamic stability of mAb solutions through targeted point mutations. Two publicly available IgG1 monoclonal antibodies that exhibit high viscosity at high concentrations were used as model systems. Guided by a computationally efficient approach that combines molecular dynamics simulations with three-dimensional reference interaction site model theory, point mutations of charged residues were introduced in the variable Fv regions in such a manner that the hydration free energy was optimized. Two selected point mutants were then produced by transient expression and characterized experimentally. Both engineered mAbs have reduced viscosity at high concentration, less negative second virial coefficient, and improved solubility compared to the respective wild-types. The results obtained with the suggested straightforward design principle underline the relevance of solvation effects for understanding, and ultimately optimizing, the properties of highly concentrated mAb solutions, with possible implications also for other biomolecular systems

Monocyte transcriptomes from patients with axial spondyloarthritis reveal dysregulated monocytopoiesis and a distinct inflammatory imprint

International audienceBackground: In patients with axial spondyloarthritis (axSpA), monocytes show a pre-activated phenotype. Gut inflammation is a trigger of monocyte activation and may also affect their development in the bone marrow (BM). As gut inflammation is commonly observed in axSpA patients, we performed a detailed analysis of monocyte transcriptomes of axSpA patients in two cohorts and searched for signs of activation and developmental adaptations as putative imprints of gut inflammation. Methods: Transcriptomes of blood CD14+ monocytes of HLA-B27+ axSpA patients and healthy controls (HC) were generated by microarrays from cohort 1 and by RNA-sequencing from cohort 2. Differentially expressed genes from both analyses were subjected to gene set enrichment analysis (GSEA) and to co-expression analysis in reference transcriptomes from BM cells, blood cells and activated monocytes. As serological markers of translocation, 1,3 beta-glycan, intestinal fatty acid binding protein, and lipopolysaccharide binding protein (LBP) were determined by LAL and ELISA. Results: Transcriptome analysis identified axSpA-specific monocyte signatures showing an imprint of LPS/cytokine-activated monocytes, late granulopoietic BM cells, blood neutrophils, and G-CSF-mobilized blood cells, which suggests LPS/TNF activation and more prominent BM adaptation promoting a neutrophil-like phenotype. GSEA mapped axSpA upregulated genes to inflammatory responses and TNFα signaling and downregulated probe-sets to metabolic pathways. Among translocation markers, LBP levels were significantly increased in axSpA patients vs. HC (p < 0.001). Stratified analysis by disease activity and stage identified an “active disease signature” (BASDAI ≥ 4) with an imprint of LPS/cytokine-activated monocytes and CD16+ monocyte subsets. The “AS signature” (vs. non-radiographic axSpA) showed a reinforced neutrophil-like phenotype due to deprivation of dendritic cell transcripts. Conclusions: The neutrophil-like phenotype of axSpA monocytes points towards a biased monocytopoiesis from granulocyte-monocyte progenitors. This shift in monocytopoiesis and the LPS/cytokine imprint as well as the elevated LBP levels are indicators of systemic inflammation, which may result from bacterial translocation. The BM adaptation is most prominent in AS patients while disease activity appears to be linked to activation and trafficking of monocytes

Clinical and Molecular Heterogeneity of RTEL1 Deficiency

Typical features of dyskeratosis congenita (DC) resulting from excessive telomere shortening include bone marrow failure (BMF), mucosal fragility, and pulmonary or liver fibrosis. In more severe cases, immune deficiency and recurring infections can add to disease severity. RTEL1 deficiency has recently been described as a major genetic etiology, but the molecular basis and clinical consequences of RTEL1-associated DC are incompletely characterized. We report our observations in a cohort of six patients: five with novel biallelic RTEL1 mutations p.Trp456Cys, p.Ile425Thr, p.Cys1244ProfsX17, p.Pro884_Gln885ins53X13, and one with novel heterozygous mutation p.Val796AlafsX4. The most unifying features were hypocellular BMF in 6/6 and B-/NK-cell lymphopenia in 5/6 patients. In addition, three patients with homozygous mutations p.Trp456Cys or p.Ile425Thr also suffered from immunodeficiency, cerebellar hypoplasia, and enteropathy, consistent with Hoyeraal-Hreidarsson syndrome. Chromosomal breakage resembling a homologous recombination defect was detected in patient-derived fibroblasts but not in hematopoietic compartment. Notably, in both cellular compartments, differential expression of 1243aa and 1219/1300aa RTEL1 isoforms was observed. In fibroblasts, response to ionizing irradiation and non-homologous end joining were not impaired. Telomeric circles did not accumulate in patient-derived primary cells and lymphoblastoid cell lines, implying alternative pathomechanisms for telomeric loss. Overall, RTEL1-deficient cells exhibited a phenotype of replicative exhaustion, spontaneous apoptosis and senescence. Specifically, CD34(+) cells failed to expand in vitro, B-cell development was compromised, and T-cells did not proliferate in long-term culture. Finally, we report on the natural history and outcome of our patients. While two patients died from infections, hematopoietic stem cell transplantation (HSCT) resulted in sustained engraftment in two patients. Whether chemotherapy negatively impacts on the course and onset of other DC-related symptoms remains open at present. Early-onset lung disease occurred in one of our patients after HSCT. In conclusion, RTEL deficiency can show a heterogeneous clinical picture ranging from mild hypocellular BMF with B/NK cell lymphopenia to early-onset, very severe, and rapidly progressing cellular deficiency