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Selective targeting of germline B cells with specifically designed germline-targeting HIV-1 envelope immunogens (GT-Env) is considered a feasible vaccination strategy to elicit broadly neutralizing antibodies (bnAbs). BnAbs are extremely valuable because they neutralize genetically distant viral strains at the same time. To overcome its inherently low affinity to germline B cells, the aim of the study was to present GT-Env via different immobilization strategies densely arrayed on the surface of nanoparticles. We engineered a prefusion-stabilized GT-Env trimer with affinity to VRC01 germline B cells using a bioinformatics-supported design approach. Distinct glycan modifications and amino acid substitutions yielded a GT-Env trimer which bound to the receptor with a K

Chromatin remodelers use the energy of ATP hydrolysis to regulate chromatin dynamics. Their impact for development and disease requires strict enzymatic control. Here, we address the differential regulability of the ATPase domain of hSNF2H and hCHD3, exhibiting similar substrate affinities and enzymatic activities. Both enzymes are comparably strongly inhibited in their ATP hydrolysis activity by the competitive ATPase inhibitor ADP. However, the nucleosome remodeling activity of SNF2H is more strongly affected than that of CHD3. Beside ADP, also IP6 inhibits the nucleosome translocation of both enzymes to varying degrees, following a competitive inhibition mode at CHD3, but not at SNF2H. Our observations are further substantiated by mutating conserved Q- and K-residues of ATPase domain motifs. The variants still bind both substrates and exhibit a wild-type similar, basal ATP hydrolysis. Apart from three CHD3 variants, none of the variants can translocate nucleosomes, suggesting for the first time that the basal ATPase activity of the CHD3 is sufficient for nucleosome remodeling. Together with the ADP data our results propose a more efficient coupling of ATP hydrolysis and remodeling in CHD3. This aspect correlates with findings that CHD3 nucleosome translocation is visible at much lower ATP concentrations than SNF2H. We propose sequence differences between the ATPase domains of both enzymes as an explanation for the functional differences and suggest that aa interactions, including the conserved Q- and K-residues distinctly regulate ATPase-dependent functions of both proteins. Our data emphasize the benefits of remodeler ATPase domains for selective drugability and/or regulability of chromatin dynamics.

Tryptophan synthase (TS) is a heterotetrameric αββα complex. It is characterized by the channeling of the reaction intermediate indole and the mutual activation of the α-subunit TrpA and the β-subunit TrpB via a complex allosteric network. We have analyzed this allosteric network by means of ancestral sequence reconstruction (ASR), which is an in silico method to resurrect extinct ancestors of modern proteins. Previously, the sequences of TrpA and TrpB from the last bacterial common ancestor (LBCA) have been computed by means of ASR and characterized. LBCA-TS is similar to modern TS by forming a αββα complex with indole channeling taking place. However, LBCA-TrpA allosterically decreases the activity of LBCA-TrpB, whereas, for example, the modern ncTrpA from Neptuniibacter caesariensis allosterically increases the activity of ncTrpB. To identify amino acid residues that are responsible for this inversion of the allosteric effect, all 6 evolutionary TrpA and TrpB intermediates that stepwise link LBCA-TS with ncTS were characterized. Remarkably, the switching from TrpB inhibition to TrpB activation by TrpA occurred between 2 successive TS intermediates. Sequence comparison of these 2 intermediates and iterative rounds of site-directed mutagenesis allowed us to identify 4 of 413 residues from TrpB that are crucial for its allosteric activation by TrpA. The effect of our mutational studies was rationalized by a community analysis based on molecular dynamics simulations. Our findings demonstrate that ancestral sequence reconstruction can efficiently identify residues contributing to allosteric signal propagation in multienzyme complexes.

Allosteric communication between different subunits in metabolic enzyme complexes is of utmost physiological importance but only understood for few systems. We analyzed the structural basis of allostery in aminodeoxychorismate synthase (ADCS), which is a member of the family of glutamine amidotransferases and catalyzes the committed step of the folate biosynthetic pathway. ADCS consists of the synthase subunit PabB and the glutaminase subunit PabA, which is allosterically stimulated by the presence of the PabB substrate chorismate. We first solved the crystal structure of a PabA subunit at 1.9-Å resolution. Based on this structure and the known structure of PabB, we computed an atomic model for the ADCS complex. We then used alanine scanning to test the functional role of 59 conserved residues located between the active sites of PabB and PabA. Steady-state kinetic characterization revealed four branches of a conserved network of mainly charged residues that propagate the signal from chorismate at the PabB active site to the PabA active site. The branches eventually lead to activity-inducing transformations at (i) the oxyanion hole motif, (ii) the catalytic Cys-His-Glu triad, and (iii) glutamine binding residues at the PabA active site. We compare our findings with previously postulated activation mechanisms of different glutamine amidotransferases and propose a unifying regulation mechanism for this ubiquitous family of enzymes.

It is an important goal of computational biology to correctly predict the association state of a protein based on its amino acid sequence and the structures of known homologues. We have pursued this goal on the example of anthranilate phosphoribosyltransferase (AnPRT), an enzyme that is involved in the biosynthesis of the amino acid tryptophan. Firstly, known crystal structures of naturally occurring homodimeric AnPRTs were analyzed using the Protein Interfaces, Surfaces, and Assemblies (PISA) service of the European Bioinformatics Institute (EBI). This led to the identification of two hydrophobic "hot spot" amino acids in the protein-protein interface that were predicted to be essential for self-association. Next, in a comprehensive multiple sequence alignment (MSA), naturally occurring AnPRT variants with hydrophilic or charged amino acids in place of hydrophobic residues in the two hot spot positions were identified. Representative variants were characterized in terms of thermal stability, enzymatic activity, and quaternary structure. We found that AnPRT variants with charged residues in both hot spot positions exist exclusively as monomers in solution. Variants with hydrophilic amino acids in one hot spot position occur in both forms, monomer and dimer. The results of the present study provide a detailed characterization of the determinants of the AnPRT monomer-dimer equilibrium and show that analysis of hot spots in combination with MSAs can be a valuable tool in prediction of protein quaternary structures.

For evolutionary studies, but also for protein engineering, ancestral sequence reconstruction (ASR) has become an indispensable tool. The first step of every ASR protocol is the preparation of a representative sequence set containing at most a few hundred recent homologs whose composition determines decisively the outcome of a reconstruction. A common approach for sequence selection consists of several rounds of manual recompilation that is driven by embedded phylogenetic analyses of the varied sequence sets. For ASR of a geranylgeranylglyceryl phosphate synthase, we additionally utilized FitSS4ASR, which replaces this time-consuming protocol with an efficient and more rational approach. FitSS4ASR applies orthogonal filters to a set of homologs to eliminate outlier sequences and those bearing only a weak phylogenetic signal. To demonstrate the usefulness of FitSS4ASR, we determined experimentally the oligomerization state of eight predecessors, which is a delicate and taxon-specific property. Corresponding ancestors deduced in a manual approach and by means of FitSS4ASR had the same dimeric or hexameric conformation; this concordance testifies to the efficiency of FitSS4ASR for sequence selection. FitSS4ASR-based results of two other ASR experiments were added to the Supporting Information. Program and documentation are available at https://gitlab.bioinf.ur.de/hek61586/FitSS4ASR.

Computational protein design has the ambitious goal of crafting novel proteins that address challenges in biology and medicine. To overcome these challenges, the computational protein modeling suite Rosetta has been tailored to address various protein design tasks. Recently, statistical methods have been developed that identify correlated mutations between residues in a multiple sequence alignment of homologous proteins. These subtle inter-dependencies in the occupancy of residue positions throughout evolution are crucial for protein function, but we found that three current Rosetta design approaches fail to recover these co-evolutionary couplings. Thus, we developed the Rosetta method ResCue (residue-coupling enhanced) that leverages co-evolutionary information to favor sequences which recapitulate correlated mutations, as observed in nature. To assess the protocols via recapitulation designs, we compiled a benchmark of ten proteins each represented by two, structurally diverse states. We could demonstrate that ResCue designed sequences with an average sequence recovery rate of 70%, whereas three other protocols reached not more than 50%, on average. Our approach had higher recovery rates also for functionally important residues, which were studied in detail. This improvement has only a minor negative effect on the fitness of the designed sequences as assessed by Rosetta energy. In conclusion, our findings support the idea that informing protocols with co-evolutionary signals helps to design stable and native-like proteins that are compatible with the different conformational states required for a complex function., Author summary In homologous proteins, functionally or structurally important residues are strongly conserved. Thus, the consideration of conservation signals during protein design protocols can help to create sequences that are more native-like. However, the number of conserved residues is small in many proteins and not all important residues can be captured by conservation analysis. Residues are forming networks whose composition is dictated by protein structure function and thus is visible through the co-evolutionary analysis. Nowadays, advanced methods allow us to deduce these networks from multiple sequence alignments. Thus, we have implemented the novel Rosetta method termed ‘ResCue’ that informs the design protocol with co-evolutionary signals. Recapitulation designs based on ten difficult benchmarks made clear that this protocol creates sequences that are more native-like than three other, state-of-the-art design protocols.

A comparison of protein backbones makes clear that not more than approximately 1400 different folds exist, each specifying the three-dimensional topology of a protein domain. Large proteins are composed of specific domain combinations and many domains can accommodate different functions. These findings confirm that the reuse of domains is key for the evolution of multi-domain proteins. If reuse was also the driving force for domain evolution, ancestral fragments of sub-domain size exist that are shared between domains possessing significantly different topologies. For the fully automated detection of putatively ancestral motifs, we developed the algorithm Fragstatt that compares proteins pairwise to identify fragments, that is, instantiations of the same motif. To reach maximal sensitivity, Fragstatt compares sequences by means of cascaded alignments of profile Hidden Markov Models. If the fragment sequences are sufficiently similar, the program determines and scores the structural concordance of the fragments. By analyzing a comprehensive set of proteins from the CATH database, Fragstatt identified 12 532 partially overlapping and structurally similar motifs that clustered to 134 unique motifs. The dissemination of these motifs is limited: We found only two domain topologies that contain two different motifs and generally, these motifs occur in not more than 18% of the CATH topologies. Interestingly, motifs are enriched in topologies that are considered ancestral. Thus, our findings suggest that the reuse of sub-domain sized fragments was relevant in early phases of protein evolution and became less important later on.

Light regulation of drug molecules has gained growing interest in biochemical and pharmacological research in recent years. In addition, a serious need for novel molecular targets of antibiotics has emerged presently. Herein, the development of a photocontrollable, azobenzene‐based antibiotic precursor towards tryptophan synthase (TS), an essential metabolic multienzyme complex in bacteria, is presented. The compound exhibited moderately strong inhibition of TS in its E configuration and five times lower inhibition strength in its Z configuration. A combination of biochemical, crystallographic, and computational analyses was used to characterize the inhibition mode of this compound. Remarkably, binding of the inhibitor to a hitherto‐unconsidered cavity results in an unproductive conformation of TS leading to noncompetitive inhibition of tryptophan production. In conclusion, we created a promising lead compound for combatting bacterial diseases, which targets an essential metabolic enzyme, and whose inhibition strength can be controlled with light., Photocontrollable antibiotic: There is growing interest in the spatiotemporal control of drugs for research and medical applications. To this end, a photocontrollable azobenzene‐based inhibitor of tryptophan synthase, an essential metabolic enzyme complex in bacteria, was designed. Thorough biochemical, crystallographic, and computational analyses elucidated the noncompetitive mode of action of this promising antibiotic lead compound.

Summary The formation of specific protein complexes in a cell is a non-trivial problem given the co-existence of thousands of different polypeptide chains. A particularly difficult case are two glutamine amidotransferase complexes (anthranilate synthase [AS] and aminodeoxychorismate synthase [ADCS]), which are composed of homologous pairs of synthase and glutaminase subunits. We have attempted to identify discriminating interface residues of the glutaminase subunit TrpG from AS, which are responsible for its specific interaction with the synthase subunit TrpEx and prevent binding to the closely related synthase subunit PabB from ADCS. For this purpose, TrpG-specific interface residues were grafted into the glutaminase subunit PabA from ADCS by two different approaches, namely a computational and a data-driven one. Both approaches resulted in PabA variants that bound TrpEx with higher affinity than PabB. Hence, we have accomplished a reprogramming of protein-protein interaction specificity that provides insights into the evolutionary adaptation of protein interfaces.

Mutations in the RS1 gene encoding retinoschisin cause X-linked juvenile retinoschisis (XLRS), a hereditary retinal dystrophy in males. While most of the XLRS associated mutations strongly interfere with cellular secretion, this is not true for mutants RS1-F108C, -R141G, -R141H, -R182C, -H207Q and -R209H. Native retinoschisin builds double-octamers and binds to retinal membranes, interacting with the retinal Na/K-ATPase. Functionally, it regulates MAP kinase signaling and Na/K-ATPase localization, and hampers photoreceptor degeneration. In this study, we investigated the capacity of the retinoschisin mutants still secreted extracellularly to fulfil these tasks. We addressed secretion and oligomerization of the heterologously expressed mutants as well as their binding to recombinant retinal Na/K-ATPases and murine retinoschisin-deficient (Rs1h-/Y) retinal and non-retinal explants. This has refined the categorization of secreted retinoschisin mutants: (i) no octamerization, unspecific membrane binding (RS1-F108C and -R182C), (ii) double-octamerization but no membrane binding (RS1-R141H), and (iii) double-octamerization and unspecific membrane binding (RS1-R141G, -H207Q, and -R209H). Notably, selected mutants of all categories (RS1-F108C, -R141H, and -R209H) failed to regulate retinal MAP kinase signaling and Na/K-ATPase localization in Rs1h-/Y retinal explants, and could not attenuate photoreceptor degeneration. Bioinformatic modeling of the secreted mutants depicted prominent alterations in the spatial and temporal conformation of a substructure called "spike 3" and its vicinity, implying a crucial role of this substructure for binding capacity and specificity. Taken together, our data point to a pathomechanism for secreted retinoschisin mutants, specifically to disturbances of the retinoschisin interface accompanied by unphysiological membrane interactions and impaired regulatory functions.

The artificial regulation of proteins by light is an emerging subdiscipline of synthetic biology. Here, we used this concept to photocontrol both catalysis and allostery within the heterodimeric enzyme complex imidazole glycerol phosphate synthase (ImGP-S). ImGP-S consists of the cyclase subunit HisF and the glutaminase subunit HisH, which is allosterically stimulated by substrate binding to HisF. We show that a light-sensitive diarylethene (1,2-dithienylethene, DTE)-based competitive inhibitor in its ring-open state binds with low micromolar affinity to the cyclase subunit and displaces its substrate from the active site. As a consequence, catalysis by HisF and allosteric stimulation of HisH are impaired. Following UV-light irradiation, the DTE ligand adopts its ring-closed state and loses affinity for HisF, restoring activity and allostery. Our approach allows for the switching of ImGP-S activity and allostery during catalysis and appears to be generally applicable for the light regulation of other multienzyme complexes.

The cell membranes of all archaea contain ether lipids, and a number of archaea are hyperthermophilic. Consequently, the enzymes that catalyze the synthesis of membrane ether lipids had to adopt to these rough conditions. Interestingly, the enzyme that establishes the first ether bond in these lipids, the geranylgeranylglyceryl phosphate synthase (GGGPS), forms hexamers in many hyperthermophilic archaea, while also dimeric variants of this enzyme exist in other species. We used Methanothermobacter thermautotrophicus GGGPS (mtGGGPS) as a model to elucidate the benefit of hexamerization. We studied the oligomerization interfaces in detail by introducing disturbing mutations and subsequently compared the stability and activity of generated dimeric and monomeric variants with the wild-type enzyme. Differential scanning calorimetry revealed a biphasic denaturation of mtGGGPS. The temperature of the first transition varies and rises with increasing oligomerization state. This first phase of denaturation leads to catalytic inactivation, but CD spectroscopy indicated only minor changes on the secondary structure level. The residual part of the fold is extremely thermostable and denatures in a second phase at temperatures120 °C. The analysis of another distant native GGGPS enzyme affirms these observations. Molecular dynamics simulations revealed three structural elements close to the substrate binding sites with elevated flexibility. We assume that hexamerization might stabilize these structures, and kinetic studies support this hypothesis for the binding pocket of the substrate glycerol 1-phosphate. Oligomerization might thus positively affect the thermostability-flexibility trade-off in GGGPS by allowing a higher intrinsic flexibility of the individual protomers.

It is important to understand how the catalytic activity of enzymes is related to their conformational flexibility. We have studied this activity–flexibility correlation using the example of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (ssIGPS), which catalyzes the fifth step in the biosynthesis of tryptophan. ssIGPS is a thermostable representative of enzymes with the frequently encountered and catalytically versatile (βα)8-barrel fold. Four variants of ssIGPS with increased catalytic turnover numbers were analyzed by transient kinetics at 25 °C, and wild-type ssIGPS was likewise analyzed both at 25 °C and at 60 °C. Global fitting with a minimal three-step model provided the individual rate constants for substrate binding, chemical transformation, and product release. The results showed that in both cases, namely, the application of activating mutations and temperature increase, the net increase in the catalytic turnover number is afforded by acceleration of the product release rate ...

Azobenzenes are of particular interest as a photochromic scaffold for biological applications because of their high fatigue resistance, their large geometrical change between extended (trans) and bent (cis) isomer, and their diverse synthetic accessibility. Despite their wide-spread use, there is no reported photochromic inhibitor of the well-investigated enzyme β-galactosidase, which plays an important role for biochemistry and single molecule studies. Herein, we report the synthesis of photochromic competitive β-galactosidase inhibitors based on the molecular structure of 2-phenylethyl β-d-thiogalactoside (PETG) and 1-amino-1-deoxy-β-d-galactose (β-d-galactosylamine). The thermally highly stable PETG-based azobenzenes show excellent photochromic properties in polar solvents and moderate to high photostationary states (PSS). The optimized compound 37 is a strong competitive inhibitior of β-galactosidase from Escherichia coli and its inhibition constant (Ki) changes between 60 nM and 290 nM upon irradiation with light. Additional docking experiments supported the observed structure-activity relationship.

Rational protein design aims at the targeted modification of existing proteins. To reach this goal, software suites like Rosetta propose sequences to introduce the desired properties. Challenging design problems necessitate the representation of a protein by means of a structural ensemble. Thus, Rosetta multi-state design (MSD) protocols have been developed wherein each state represents one protein conformation. Computational demands of MSD protocols are high, because for each of the candidate sequences a costly three-dimensional (3D) model has to be created and assessed for all states. Each of these scores contributes one data point to a complex, design-specific energy landscape. As neural networks (NN) proved well-suited to learn such solution spaces, we integrated one into the frameworkRosetta:MSFinstead of the so far used genetic algorithm with the aim to reduce computational costs. As its predecessor,Rosetta:MSF:NNadministers a set of candidate sequences and their scores and scans sequence space iteratively. During each iteration, the union of all candidate sequences and their Rosetta scores are used to re-train NNs that possess a design-specific architecture. The enormous speed of the NNs allows an extensive assessment of alternative sequences, which are ranked on the scores predicted by the NN. Costly 3D models are computed only for a small fraction of best-scoring sequences; these and the corresponding 3D-based scores replace half of the candidate sequences during each iteration. The analysis of two sets of candidate sequences generated for a specific design problem by means of a genetic algorithm confirmed that the NN predicted 3D-based scores quite well; the Pearson correlation coefficient was at least 0.95. ApplyingRosetta:MSF:NN:enzdesto a benchmark consisting of 16 ligand-binding problems showed that this protocol converges ten-times faster than the genetic algorithm and finds sequences with comparable scores.

It is important to identify hotspot residues that determine protein-protein interactions in interfaces of macromolecular complexes. We have applied a combination of ancestral sequence reconstruction and protein design to identify hotspots within imidazole glycerol phosphate synthase (ImGPS). ImGPS is a key metabolic enzyme complex, which links histidine and de novo purine biosynthesis and consists of the cyclase subunit HisF and the glutaminase subunit HisH. Initial fluorescence titration experiments showed that HisH from Zymomonas mobilis (zmHisH) binds with high affinity to the reconstructed HisF from the last universal common ancestor (LUCA-HisF) but not to HisF from Pyrobaculum arsenaticum (paHisF), which differ by 103 residues. Subsequent titration experiments with a reconstructed evolutionary intermediate linking LUCA-HisF and paHisF and inspection of the subunit interface of a contemporary ImGPS allowed us to narrow down the differences crucial for zmHisH binding to nine amino acids of HisF. Homology modeling and in silico mutagenesis studies suggested that at most two of these nine HisF residues are crucial for zmHisH binding. These computational results were verified by experimental site-directed mutagenesis, which finally enabled us to pinpoint a single amino acid residue in HisF that is decisive for high-affinity binding of zmHisH. Our work shows that the identification of protein interface hotspots can be very efficient when reconstructed proteins with different binding properties are included in the analysis. This article is protected by copyright. All rights reserved.

The members of the glutamine amidotransferase (GATase) family catalyze the incorporation of ammonia within numerous metabolic pathways and can be categorized in two classes. Here, we concentrated on class I GATases, which are heteromeric enzyme complexes consisting of synthase subunits and glutaminase subunits with a catalytic Cys-His-Glu triad. Glutamine hydrolysis at the glutaminase subunit is (i) dependent on the formation of tight synthase-glutaminase complexes and (ii) allosterically coupled to the presence of the substrate at the synthase subunit. The structural basis of both complex formation and allostery is poorly understood. However, previous work on 4-amino-4-deoxychorismate synthase and imidazole glycerol phosphate synthase suggested that a conserved aspartate residue in their synthase subunits, which is located at the subunit interface close to the glutaminase catalytic triad, might be important for both features. We performed a computational screen of class I GATases from the Protein Data Bank and identified conserved and similarly located aspartate residues. We then generated alanine and glutamate mutants of these residues and characterized them by analytical gel filtration and steady-state enzyme kinetics. The results confirmed the important role of the wild-type aspartate residues for the formation of stable synthase-glutaminase complexes (in three of four cases) and the stimulation of glutaminase activity in the analyzed GATases (in all four cases). We present a model for rationalizing the dual role of the conserved aspartate residue toward a unifying regulation mechanism in the entire class I GATase family.

Several long non-coding RNAs (lncRNAs) act as regulators of cellular homeostasis; however, few of these molecules were functionally characterized in a mature human tissue environment. Here, we report that the lncRNA LINC00941 is a crucial regulator of human epidermal homeostasis. LINC00941 is enriched in progenitor keratinocytes and acts as a repressor of keratinocyte differentiation. Furthermore, LINC00941 represses SPRR5, a previously uncharacterized molecule, which functions as an essential positive regulator of keratinocyte differentiation. Interestingly, 54.8% of genes repressed in SPRR5-deficient epidermal tissue are induced in LINC00941-depleted organotypic epidermis, suggesting a common mode of action for both molecules.

Insect pheromones are often derived from fatty acid metabolism. Fatty acid desaturases, enzymes introducing double bonds into fatty acids, are crucial for the biosynthesis of these chemical signals. Δ12-desaturases catalyse the biosynthesis of linoleic acid by introducing a second double bond into oleic acid, but have been identified in only a few animal species. Here, we report the functional characterisation of two Δ12-desaturases, Nvit_D12a and Nvit_D12b, from the parasitic wasp Nasonia vitripennis. We demonstrate that Nvit_D12a is expressed in the rectal vesicle of males where they produce a linoleic acid-derived sex pheromone to attract virgin females. C-labelling experiments with Urolepis rufipes, a closely related species belonging to the ‘Nasonia group’, revealed that females, but not males, are able to synthesise linoleic acid. U. rufipes males produce an isoprenoid sex pheromone in the same gland and do not depend on linoleic acid for pheromone production. This suggests that Δ12-desaturases are common in the ‘Nasonia group’, but acquired a specialised function in chemical communication of those species that use linoleic acid as a pheromone precursor. Phylogenetic analysis suggests that insect Δ12-desaturases have evolved repeatedly from Δ9-desaturases in different insect taxa. Hence, insects have developed a way to produce linoleic acid independent of the omega desaturase subfamily which harbours all of the eukaryotic Δ12-desaturases known so far., F.S. was supported by a doctoral fellowship from the Konrad-Adenauer-Stiftung; J.E. and M.M. were supported by the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NOW; VICI grant 865.12.003).

Summary The amino acid sequences of primordial enzymes from extinct organisms can be determined by an in silico approach termed ancestral sequence reconstruction (ASR). In the first step of an ASR, a multiple sequence alignment (MSA) comprising extant homologous enzymes is being composed. On the basis of this MSA and a stochastic model of sequence evolution, a phylogenetic tree is calculated by means of a maximum likelihood approach. Finally, the sequences of the ancestral proteins at all internal nodes including the root of the tree are deduced. We present several examples of ASR and the subsequent experimental characterization of enzymes as old as four billion years. The results show that most ancestral enzymes were highly thermostable and catalytically active. Moreover, they adopted three-dimensional structures similar to those of extant enzymes. These findings suggest that sophisticated enzymes were invented at a very early stage of biological evolution.

Modern enzyme complexes are characterized by a high catalytic efficiency and allosteric communication between the constituting protein subunits. We were interested in whether primordial enzyme complexes from extinct species displayed a similar degree of functional sophistication. To this end, we used ancestral sequence reconstruction to resurrect the α and β subunits of the tryptophan synthase (TS) complex from the last bacterial common ancestor (LBCA), which presumably existed more than 3.4 billion years ago. We show that the LBCA TS subunits are thermostable and exhibit high catalytic activity. Moreover, they form a complex with αββα stoichiometry whose crystal structure is similar to that of modern TS. Kinetic analysis revealed that the reaction intermediate indole is channeled from the α to the β subunits and suggests that allosteric communication already occurred in LBCA TS.

The La-related protein 7 (LARP7) forms a complex with the nuclear 7SK RNA to regulate RNA polymerase II transcription. It has been implicated in cancer and the Alazami syndrome, a severe developmental disorder. Here, we report a so far unknown role of this protein in RNA modification. We show that LARP7 physically connects the spliceosomal U6 small nuclear RNA (snRNA) with a distinct subset of box C/D small nucleolar RNAs (snoRNAs) guiding U6 2'-O-methylation. Consistently, these modifications are severely compromised in the absence of LARP7. Although general splicing remains largely unaffected, transcriptome-wide analysis revealed perturbations in alternative splicing in LARP7-depleted cells. Importantly, we identified defects in 2'-O-methylation of the U6 snRNA in Alazami syndrome siblings carrying a LARP7 mutation. Our data identify LARP7 as a bridging factor for snoRNA-guided modification of the U6 snRNA and suggest that alterations in splicing fidelity contribute to the etiology of the Alazami syndrome.

Packaging of DNA into chromatin regulates DNA accessibility and consequently all DNA-dependent processes. The nucleosome is the basic packaging unit of DNA forming arrays that are suggested, by biochemical studies, to fold hierarchically into ordered higher-order structures of chromatin. This organization has been recently questioned using microscopy techniques, proposing an irregular structure. To address the principles of chromatin organization, we applied an in situ differential MNase-seq strategy and analyzed in silico the results of complete and partial digestions of human chromatin. We investigated whether different levels of chromatin packaging exist in the cell. We assessed the accessibility of chromatin within distinct domains of kb to Mb genomic regions, performed statistical analyses and computer modelling. We found no difference in MNase accessibility, suggesting no difference in fiber folding between domains of euchromatin and heterochromatin or between other sequence and epigenomic features of chromatin. Thus, our data suggests the absence of differentially organized domains of higher-order structures of chromatin. Moreover, we identified only local structural changes, with individual hyper-accessible nucleosomes surrounding regulatory elements, such as enhancers and transcription start sites. The regulatory sites per se are occupied with structurally altered nucleosomes, exhibiting increased MNase sensitivity. Our findings provide biochemical evidence that supports an irregular model of large-scale chromatin organization.

Ancestral sequence reconstruction (ASR) is a powerful tool to infer primordial sequences from contemporary, i.e., extant ones. An essential element of ASR is the computation of a phylogenetic tree whose leaves are the chosen extant sequences. Most often, the reconstructed sequence related to the root of this tree is of greatest interest: It represents the common ancestor (CA) of the sequences under study. If this sequence encodes a protein, one can "resurrect" the CA by means of gene synthesis technology and study biochemical properties of this extinct predecessor with the help of wet-lab experiments.However, ASR deduces also sequences for all internal nodes of the tree, and the well-considered analysis of these "intermediates" can help to elucidate evolutionary processes. Moreover, one can identify key mutations that alter proteins or protein complexes and are responsible for the differing properties of extant proteins. As an illustrative example, we describe the protocol for the rapid identification of hotspots determining the binding of the two subunits within the heteromeric complex imidazole glycerol phosphate synthase.

Ancestral sequence reconstruction (ASR) is a powerful tool to infer primordial sequences from contemporary, i.e., extant ones. An essential element of ASR is the computation of a phylogenetic tree whose leaves are the chosen extant sequences. Most often, the reconstructed sequence related to the root of this tree is of greatest interest: It represents the common ancestor (CA) of the sequences under study. If this sequence encodes a protein, one can "resurrect" the CA by means of gene synthesis technology and study biochemical properties of this extinct predecessor with the help of wet-lab experiments.However, ASR deduces also sequences for all internal nodes of the tree, and the well-considered analysis of these "intermediates" can help to elucidate evolutionary processes. Moreover, one can identify key mutations that alter proteins or protein complexes and are responsible for the differing properties of extant proteins. As an illustrative example, we describe the protocol for the rapid identification of hotspots determining the binding of the two subunits within the heteromeric complex imidazole glycerol phosphate synthase.

Packaging of DNA into chromatin regulates DNA accessibility and, consequently, all DNA-dependent processes, such as transcription, recombination, repair, and replication. The nucleosome is the basic packaging unit of DNA forming arrays that are suggested, by biochemical studies, to fold hierarchically into ordered higher-order structures of chromatin. This defined organization of chromatin has been recently questioned using microscopy techniques, proposing a rather irregular structure. To gain more insight into the principles of chromatin organization, we applied an in situ differential MNase-seq strategy and analyzed in silico the results of complete and partial digestions of human chromatin. We investigated whether different levels of chromatin packaging exist in the cell. Thus, we assessed the accessibility of chromatin within distinct domains of kb to Mb genomic regions by utilizing statistical data analyses and computer modelling. We found no difference in the degree of compaction between domains of euchromatin and heterochromatin or between other sequence and epigenomic features of chromatin. Thus, our data suggests the absence of differentially compacted domains of higher-order structures of chromatin. Moreover, we identified only local structural changes, with individual hyper-accessible nucleosomes surrounding regulatory elements, such as enhancers and transcription start sites. The regulatory sites per se are occupied with structurally altered nucleosomes, exhibiting increased MNase sensitivity. Our findings provide biochemical evidence that supports an irregular model of large-scale chromatin organization.

The conserved nuclear protein ENY2 (Sus1 in yeast) is involved in coupling transcription and mRNA export in yeast and metazoa, as it is a component both of the transcriptional co-activator complex SAGA and of the mRNA export complex TREX-2. Arabidopsis thaliana ENY2 is widely expressed in the plant and it localizes to the nucleoplasm, but unlike its yeast/metazoan orthologs, it is not enriched in the nuclear envelope. Affinity purification of ENY2 in combination with mass spectrometry revealed that it co-purified with SAGA components, but not with the nuclear pore-associated TREX-2. In addition, further targeted proteomics analyses by reciprocal tagging established the composition of the Arabidopsis SAGA complex consisting of the four modules HATm, SPTm, TAFm and DUBm, and that several SAGA subunits occur in alternative variants. While the HATm, SPTm and TAFm robustly co-purified with each other, the deubiquitination module (DUBm) appears to associate with the other SAGA modules more weakly/dynamically. Consistent with a homology model of the Arabidopsis DUBm, the SGF11 protein interacts directly with ENY2 and UBP22. Plants depleted in the DUBm components, SGF11 or ENY2, are phenotypically only mildly affected, but they contain increased levels of ubiquitinated histone H2B, indicating that the SAGA-DUBm has histone deubiquitination activity in plants. In addition to transcription-related proteins (i.e., transcript elongation factors, Mediator), many splicing factors were found to associate with SAGA, linking the SAGA complex and ongoing transcription with mRNA processing.

Chorismat-umsetzende Enzyme spielen in Pflanzen sowie frei lebenden und infektiosen Mikroorganismen eine entscheidende Rolle bei der Biosynthese zahlreicher Metabolite. Zu diesen Enzymen zahlen die primarmetabolische Anthranilatsynthase (AS) und die homologe, sekundarmetabolische Isochorismatsynthase (ICS). Beide Enzyme katalysieren mechanistisch ahnliche Reaktionen, wobei sie Ammoniak bzw. Wasser als Nukleophil nutzen. Wir zeigen hier, dass die Nukleophilspezifitat der AS durch nur zwei Aminosaureaustausche im Zugangskanal zum aktiven Zentrum von Ammoniak auf Wasser erweitert werden kann. Die resultierende Bifunktionalitat dieser AS/ICS-Variante verdeutlicht, dass die Evolution eines sekundarmetabolischen Enzyms von einem primarmetabolischen aus durch wenige Mutationen erfolgen kann und nicht zwingend eine initiale Genduplikation voraussetzt. Allgemein betrachtet, tragen unsere Erkenntnisse zum Verstandnis bei, wie in der Natur innerhalb von Enzym-Superfamilien neue Reaktionen evolvieren.

Highlight EMBRYO SAC family peptides show multiple activities in mediating pollen tube burst and fungal growth inhibition. They interact differently with cell surface receptors of pollen tube and fungal cells., EMBRYO SAC1-4 (ES1-4) peptides belong to the defensin subgroup of cysteine-rich peptides known to mediate pollen tube burst in Zea mays (maize). ES1-4 are reported here to also be capable of inhibiting germination and growth of the maize fungal pathogens Fusarium graminearum and Ustilago maydis at higher concentrations. Dividing the peptides into smaller pieces showed that a 15-amino-acid peptide located in a highly variable loop region lacking similarity to other defensins or defensin-like peptides binds to maize pollen tube surfaces, causing swelling prior to burst. This peptide fragment and a second conserved neighbouring fragment showed suppression of fungal germination and growth. The two peptides caused swelling of fungal cells, production of reactive oxygen species, and finally the formation of big vacuoles prior to burst at high peptide concentration. Furthermore, peptide fragments were found to bind differently to fungal cells. In necrotrophic F. graminearum, a peptide fragment named ES-d bound only at cell surfaces whereas the peptide ES-c bound at cell surfaces and also accumulated inside cells. Conversely, in biotrophic U. maydis, both peptide fragments accumulated inside cells, but, if applied at higher concentration, ES-c but not ES-d accumulated mainly in vacuoles. Mapping of peptide interaction sites identified amino acids differing in pollen tube burst and fungal response reactions. In summary, these findings indicate that residues targeting pollen tube burst in maize are specific to the ES family, while residues targeting fungal growth are conserved within defensins and defensin-like peptides.