ABSTRACT
Advances in sequencing technologies have led to a rapid growth of public protein sequence databases, whereby the fraction of proteins with experimentally verified function continuously decreases. This problem is currently addressed by automated functional annotations with computational tools, which however lack the accuracy of experimental approaches and are susceptible to error propagation. Here, we present an approach that combines the efficiency of functional annotation by in silico methods with the rigor of enzyme characterization in vitro. First, a thorough experimental analysis of a representative enzyme of a group of homologues is performed which includes a focused alanine scan of the active site to determine a fingerprint of function-determining residues. In a second step, this fingerprint is used in combination with a sequence similarity network to identify putative isofunctional enzymes among the homologues. Using this approach in a proof-of-principle study, homologues of the histidinol phosphate phosphatase (HolPase) from Pseudomonas aeruginosa, many of which were annotated as phosphoserine phosphatases, were predicted to be HolPases. This functional annotation of the homologues was verified by in vitro testing of several representatives and an analysis of the occurrence of annotated HolPases in the corresponding phylogenetic groups. Moreover, the application of the same approach to the homologues of the HolPase from the archaeon Nitrosopumilus maritimus, which is not related to the HolPase from P. aeruginosa and was newly discovered in the course of this work, led to the annotation of the putative HolPase from various archaeal species.
Subject(s)
Bacterial Proteins , Histidinol-Phosphatase , Histidinol-Phosphatase/chemistry , Amino Acid Sequence , Phylogeny , Bacterial Proteins/chemistryABSTRACT
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.
Subject(s)
HIV-1 , Silicon DioxideABSTRACT
The artificial regulation of enzymatic activity by light is an important goal of synthetic biology that can be achieved by the incorporation of light-responsive noncanonical amino acids via genetic code expansion. Here, we apply this concept to anthranilate synthase from Salmonella typhimurium (stTrpE). This enzyme catalyzes the first step of tryptophan biosynthesis, and its activity is feedback-inhibited by the binding of the end-product of the pathway to an allosteric site. To put this feedback inhibition of stTrpE by tryptophan under the control of light, we individually replaced 15 different amino acid residues with the photosensitive noncanonical amino acid o-nitrobenzyl-O-tyrosine (ONBY). ONBY contains a sterically demanding caging group that was meant to cover the allosteric site. Steady-state enzyme kinetics showed that the negative effect of tryptophan on the catalytic activity of the two variants stTrpE-K50ONBY and stTrpE-Y455ONBY was diminished compared to the wild-type enzyme by 1 to 2 orders of magnitude. Upon light-induced decaging of ONBY to the less space-consuming tyrosine residue, tryptophan binding to the allosteric site was restored and catalytic activity was inhibited almost as efficiently as observed for wild-type stTrpE. Based on these results, direct photocontrol of feedback inhibition of stTrpE-K50ONBY and stTrpE-Y455ONBY could be achieved by irradiation during the reaction. Molecular modeling studies allowed us to rationalize the observed functional conversion from the noninhibited caged to the tryptophan-inhibited decaged states. Our study shows that feedback inhibition, which is an important mechanism to regulate key metabolic enzymes, can be efficiently controlled by the purposeful use of light-responsive noncanonical amino acids.
Subject(s)
Anthranilate Synthase , Tryptophan , Amino Acids , Anthranilate Synthase/genetics , Anthranilate Synthase/metabolism , Feedback , Kinetics , Tryptophan/metabolism , TyrosineABSTRACT
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.
Subject(s)
Algorithms , Computational Biology/methods , Neural Networks, Computer , Proteins/chemistry , Amino Acid Sequence , Databases, Protein , Epistasis, Genetic , Mutation/genetics , Proteins/genetics , ThermodynamicsABSTRACT
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.
Subject(s)
Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA Helicases/metabolism , Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism , Adenosine Diphosphate/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Amino Acid Sequence , Binding, Competitive , Chromatin/genetics , Chromatin/metabolism , Chromatin Assembly and Disassembly , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/genetics , DNA Helicases/chemistry , DNA Helicases/genetics , Humans , Hydrolysis , Mi-2 Nucleosome Remodeling and Deacetylase Complex/chemistry , Mi-2 Nucleosome Remodeling and Deacetylase Complex/genetics , Mutation , Nucleosomes/genetics , Nucleosomes/metabolism , Protein Binding , Protein Domains , Sequence Homology, Amino Acid , Substrate SpecificityABSTRACT
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.
Subject(s)
Azo Compounds/pharmacology , Enzyme Inhibitors/pharmacology , Tryptophan Synthase/antagonists & inhibitors , Enzyme Inhibitors/radiation effectsABSTRACT
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.
Subject(s)
Anthranilate Synthase/chemistry , Carbon-Carbon Lyases/chemistry , Escherichia coli Proteins/chemistry , Transaminases/chemistry , Amino Acid Substitution , Anthranilate Synthase/genetics , Anthranilate Synthase/metabolism , Binding Sites , Carbon-Carbon Lyases/genetics , Carbon-Carbon Lyases/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Molecular Docking Simulation/methods , Protein Binding , Protein Interaction Mapping/methods , Transaminases/genetics , Transaminases/metabolismABSTRACT
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.
Subject(s)
Carbon-Carbon Lyases/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Transaminases/metabolism , Allosteric Regulation , Allosteric Site , Carbon-Carbon Lyases/chemistry , Catalytic Domain , Crystallography, X-Ray , Escherichia coli/chemistry , Escherichia coli Proteins/chemistry , Models, Molecular , Protein Conformation , Protein Subunits/chemistry , Protein Subunits/metabolism , Transaminases/chemistryABSTRACT
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.
Subject(s)
Chromatin Assembly and Disassembly , DNA Helicases/chemistry , DNA Helicases/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Motifs , Amino Acid Sequence , Cell Line , DNA Helicases/genetics , Humans , Models, Molecular , Mutation , Nuclear Proteins/genetics , Nucleosomes/metabolism , Transcription Factors/geneticsABSTRACT
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 ß-galactosidase, which plays an important role for biochemistry and single molecule studies. Herein, we report the synthesis of photochromic competitive ß-galactosidase inhibitors based on the molecular structure of 2-phenylethyl ß-d-thiogalactoside (PETG) and 1-amino-1-deoxy-ß-d-galactose (ß-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 ß-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.
Subject(s)
Drug Design , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Light , beta-Galactosidase/antagonists & inhibitors , Azo Compounds/chemistry , Enzyme Inhibitors/metabolism , Escherichia coli/enzymology , Galactose/chemistry , Molecular Docking Simulation , Protein Conformation , Temperature , Thiogalactosides/chemistry , beta-Galactosidase/chemistry , beta-Galactosidase/metabolismABSTRACT
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.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/growth & development , Nuclear Pore/metabolism , Trans-Activators/metabolism , Transcription Factors/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Cell Nucleus/metabolism , Histones/metabolism , Mass Spectrometry , Nuclear Pore/genetics , Proteomics , Trans-Activators/genetics , UbiquitinationABSTRACT
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 (ßα)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 relative to the chemical transformation steps. Measurements of the solvent viscosity effect at 25 °C versus 60 °C confirmed this change in the rate-determining step with temperature, which is in accordance with a kink in the Arrhenius diagram of ssIGPS at â¼40 °C. When rotational diffusion rates of electron paramagnetic spin-labels attached to active site loop ß1α1 are plotted in the form of an Arrhenius diagram, kinks are observed at the same temperature. These findings, together with molecular dynamics simulations, demonstrate that a different degree of loop mobility correlates with different rate-limiting steps in the catalytic mechanism of ssIGPS.