Experimental and computational analysis of the ancestry of an evolutionary young enzyme from histidine biosynthesis.

The conservation of fold and chemistry of the enzymes associated with histidine biosynthesis suggests that this pathway evolved prior to the diversification of Bacteria, Archaea, and Eukaryotes. The only exception is the histidinol phosphate phosphatase (HolPase). So far, non-homologous HolPases that possess distinct folds and belong to three different protein superfamilies have been identified in various phylogenetic clades. However, their evolution has remained unknown to date. Here, we analyzed the evolutionary history of the HolPase from γ-Proteobacteria (HisB-N). It has been argued that HisB-N and its closest homologue d-glycero-d-manno-heptose-1,7-bisphosphate 7-phosphatase (GmhB) have emerged from the same promiscuous ancestral phosphatase. GmhB variants catalyze the hydrolysis of the anomeric d-glycero-d-manno-heptose-1,7-bisphosphate (αHBP or ßHBP) with a strong preference for one anomer (αGmhB or ßGmhB). We found that HisB-N from Escherichia coli shows promiscuous activity for ßHBP but not αHBP, while ßGmhB from Crassaminicella sp. shows promiscuous activity for HolP. Accordingly, a combined phylogenetic tree of αGmhBs, ßGmhBs, and HisB-N sequences revealed that HisB-Ns form a compact subcluster derived from ßGmhBs. Ancestral sequence reconstruction and in vitro analysis revealed a promiscuous HolPase activity in the resurrected enzymes prior to functional divergence of the successors. The following increase in catalytic efficiency of the HolP turnover is reflected in the shape and electrostatics of the active site predicted by AlphaFold. An analysis of the phylogenetic tree led to a revised evolutionary model that proposes the horizontal gene transfer of a promiscuous ßGmhB from δ- to γ-Proteobacteria where it evolved to the modern HisB-N.

Multivalent display of engineered HIV-1 envelope trimers on silica nanoparticles for targeting and in vitro activation of germline VRC01 B cells.

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 KD of 11.5 µM. Silica nanoparticles with 200 nm diameter (SiNPs) were used for the multivalent display of the novel GT-Env with a 15 nm mean centre-to-centre spacing either by site-specific, covalent conjugation or at random, non-specific adsorption. Oriented, covalent GT-Env conjugation revealed better binding of structure dependent bnAbs as compared to non-specifically adsorbed GT-Env. In addition, GT-Env covalently attached activated a B cell line expressing the germline VRC01 receptor at an EC50 value in the nanomolar range (4 nM), while soluble GT-Env required 1,000-fold higher concentrations to induce signalling. The significantly lower GT-Env concentration was likely required due to avidity effects, which were in the picomolar range. Thus, low affinity antigens may particularly benefit from a particulate and multivalent delivery. In future, SiNPs are ideal to be modified in a modular design with various GT-Env variants that target different stages of germline and bnAb precursor B cells.

Rosetta:MSF:NN: Boosting performance of multi-state computational protein design with a neural network.

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 framework Rosetta:MSF instead of the so far used genetic algorithm with the aim to reduce computational costs. As its predecessor, Rosetta:MSF:NN administers 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. Applying Rosetta:MSF:NN:enzdes to 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.

Evidence for the preferential reuse of sub-domain motifs in primordial protein folds.

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.

Rosetta design with co-evolutionary information retains protein function.

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.

Sequence and functional differences in the ATPase domains of CHD3 and SNF2H promise potential for selective regulability and drugability.

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 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.

Reprogramming the Specificity of a Protein Interface by Computational and Data-Driven Design.

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.

Towards Photochromic Azobenzene-Based Inhibitors for Tryptophan Synthase.

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.

The Alazami Syndrome-Associated Protein LARP7 Guides U6 Small Nuclear RNA Modification and Contributes to Splicing Robustness.

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.

Conserved genomic neighborhood is a strong but no perfect indicator for a direct interaction of microbial gene products.

BACKGROUND: The order of genes in bacterial genomes is not random; for example, the products of genes belonging to an operon work together in the same pathway. The cotranslational assembly of protein complexes is deemed to conserve genomic neighborhoods even stronger than a common function. This is why a conserved genomic neighborhood can be utilized to predict, whether gene products form protein complexes. RESULTS: We were interested to assess the performance of a neighborhood-based classifier that analyzes a large number of genomes. Thus, we determined for the genes encoding the subunits of 494 experimentally verified hetero-dimers their local genomic context. In order to generate phylogenetically comprehensive genomic neighborhoods, we utilized the tools offered by the Enzyme Function Initiative. For each subunit, a sequence similarity network was generated and the corresponding genome neighborhood network was analyzed to deduce the most frequent gene product. This was predicted as interaction partner, if its abundance exceeded a threshold, which was the frequency giving rise to the maximal Matthews correlation coefficient. For the threshold of 16%, the true positive rate was 45%, the false positive rate 0.06%, and the precision 55%. For approximately 20% of the subunits, the interaction partner was not found in a neighborhood of ± 10 genes. CONCLUSIONS: Our phylogenetically comprehensive analysis confirmed that complex formation is a strong evolutionary factor that conserves genome neighborhoods. On the other hand, for 55% of the cases analyzed here, classification failed. Either, the interaction partner was not present in a ± 10 gene window or was not the most frequent gene product.

Analysis of allosteric communication in a multienzyme complex by ancestral sequence reconstruction.

Tryptophan synthase (TS) is a heterotetrameric αßßα complex. It is characterized by the channeling of the reaction intermediate indole and the mutual activation of the α-subunit TrpA and the ß-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 αßßα 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.

Light-Regulation of Tryptophan Synthase by Combining Protein Design and Enzymology.

The spatiotemporal control of enzymes by light is of growing importance for industrial biocatalysis. Within this context, the photo-control of allosteric interactions in enzyme complexes, common to practically all metabolic pathways, is particularly relevant. A prominent example of a metabolic complex with a high application potential is tryptophan synthase from Salmonella typhimurium (TS), in which the constituting TrpA and TrpB subunits mutually stimulate each other via a sophisticated allosteric network. To control TS allostery with light, we incorporated the unnatural amino acid o-nitrobenzyl-O-tyrosine (ONBY) at seven strategic positions of TrpA and TrpB. Initial screening experiments showed that ONBY in position 58 of TrpA (aL58ONBY) inhibits TS activity most effectively. Upon UV irradiation, ONBY decages to tyrosine, largely restoring the capacity of TS. Biochemical characterization, extensive steady-state enzyme kinetics, and titration studies uncovered the impact of aL58ONBY on the activities of TrpA and TrpB and identified reaction conditions under which the influence of ONBY decaging on allostery reaches its full potential. By applying those optimal conditions, we succeeded to directly light-activate TS(aL58ONBY) by a factor of ~100. Our findings show that rational protein design with a photo-sensitive unnatural amino acid combined with extensive enzymology is a powerful tool to fine-tune allosteric light-activation of a central metabolic enzyme complex.

Light Regulation of Enzyme Allostery through Photo-responsive Unnatural Amino Acids.

Imidazole glycerol phosphate synthase (ImGPS) is an allosteric bienzyme complex in which substrate binding to the synthase subunit HisF stimulates the glutaminase subunit HisH. To control this stimulation with light, we have incorporated the photo-responsive unnatural amino acids phenylalanine-4'-azobenzene (AzoF), o-nitropiperonyl-O-tyrosine (NPY), and methyl-o-nitropiperonyllysine (mNPK) at strategic positions of HisF. The light-mediated isomerization of AzoF at position 55 (fS55AzoFE ↔ fS55AzoFZ) resulted in a reversible 10-fold regulation of HisH activity. The light-mediated decaging of NPY at position 39 (fY39NPY → fY39) and of mNPK at position 99 (fK99mNPK → fK99) led to a 4- to 6-fold increase of HisH activity. Molecular dynamics simulations explained how the unnatural amino acids interfere with the allosteric machinery of ImGPS and revealed additional aspects of HisH stimulation in wild-type ImGPS. Our findings show that unnatural amino acids can be used as a powerful tool for the spatiotemporal control of a central metabolic enzyme complex by light.

Identification of the retinoschisin-binding site on the retinal Na/K-ATPase.

X-linked juvenile retinoschisis (XLRS) is a hereditary retinal dystrophy, caused by mutations in the RS1 gene which encodes the secreted protein retinoschisin. In recent years, several molecules have been proposed to interact with retinoschisin, including the retinal Na/K-ATPase, L-voltage gated Ca2+ channels, and specific sugars. We recently showed that the retinal Na/K-ATPase consisting of subunits ATP1A3 and ATP1B2 is essential for anchoring retinoschisin to plasma membranes and identified the glycosylated ATP1B2 subunit as the direct interaction partner for retinoschisin. We now aimed to precisely map the retinoschisin binding domain(s) in ATP1B2. In general, retinoschisin binding was not affected after selective elimination of individual glycosylation sites via site-directed mutagenesis as well as after full enzymatic deglycosylation of ATP1B2. Applying the interface prediction tool PresCont, two putative protein-protein interaction patches ("patch I" and "patch II") consisting each of four hydrophobic amino acid stretches on the ATP1B2 surface were identified. These were consecutively altered by site-directed mutagenesis. Functional assays with the ATP1B2 patch mutants identified patch II and, specifically, the associated amino acid at position 240 (harboring a threonine in ATP1B2) as crucial for retinoschisin binding to ATP1B2. These and previous results led us to suggest an induced-fit binding mechanism for the interaction between retinoschisin and the Na/K-ATPase, which is dependent on threonine 240 in ATP1B2 allowing the accommodation of hyperflexible retinoschisin spikes by the associated protein-protein interaction patch on ATP1B2.

A Fold-Independent Interface Residue Is Crucial for Complex Formation and Allosteric Signaling in Class I Glutamine Amidotransferases.

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.

Mapping the Allosteric Communication Network of Aminodeoxychorismate Synthase.

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.

Prediction of quaternary structure by analysis of hot spot residues in protein-protein interfaces: the case of anthranilate phosphoribosyltransferases.

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.

Functional characterisation of two Δ12-desaturases demonstrates targeted production of linoleic acid as pheromone precursor in Nasonia.

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. 13C-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.

Sequence selection by FitSS4ASR alleviates ancestral sequence reconstruction as exemplified for geranylgeranylglyceryl phosphate synthase.

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.

Elucidation of the functional roles of the Q and I motifs in the human chromatin-remodeling enzyme BRG1.

The Snf2 proteins, comprising 53 different enzymes in humans, belong to the SF2 family. Many Snf2 enzymes possess chromatin-remodeling activity, requiring a functional ATPase domain consisting of conserved motifs named Q and I-VII. These motifs form two recA-like domains, creating an ATP-binding pocket. Little is known about the function of the conserved motifs in chromatin-remodeling enzymes. Here, we characterized the function of the Q and I (Walker I) motifs in hBRG1 (SMARCA4). The motifs are in close proximity to the bound ATP, suggesting a role in nucleotide binding and/or hydrolysis. Unexpectedly, when substituting the conserved residues Gln758 (Q motif) or Lys785 (I motif) of both motifs, all variants still bound ATP and exhibited basal ATPase activity similar to that of wildtype BRG1 (wtBRG1). However, all mutants lost the nucleosome-dependent stimulation of the ATPase domain. Their chromatin-remodeling rates were impaired accordingly, but nucleosome binding was retained and still comparable with that of wtBRG1. Interestingly, a cancer-relevant substitution, L754F (Q motif), displayed defects similar to the Gln758 variant(s), arguing for a comparable loss of function. Because we excluded a mutual interference of ATP and nucleosome binding, we postulate that both motifs stimulate the ATPase and chromatin-remodeling activities upon binding of BRG1 to nucleosomes, probably via allosteric mechanisms. Furthermore, mutations of both motifs similarly affect the enzymatic functionality of BRG1 in vitro and in living cells. Of note, in BRG1-deficient H1299 cells, exogenously expressed wtBRG1, but not BRG1 Q758A and BRG1 K785R, exhibited a tumor suppressor-like function.