An enzyme-based biosensor for monitoring and engineering protein stability in vivo.

Protein stability affects the physiological functions of proteins and is also a desirable trait in many protein engineering tasks, yet improving protein stability is challenging because of limitations in methods for directly monitoring protein stability in cells. Here, we report an in vivo stability biosensor wherein a protein of interest (POI) is inserted into a microbial enzyme (CysGA) that catalyzes the formation of endogenous fluorescent compounds, thereby coupling POI stability to simple fluorescence readouts. We demonstrate the utility of the biosensor in directed evolution to obtain stabilized, less aggregation-prone variants of two POIs (including nonamyloidogenic variants of human islet amyloid polypeptide). Beyond engineering applications, we exploited our biosensor in deep mutational scanning for experimental delineation of the stability-related contributions of all residues throughout the catalytic domain of a histone H3K4 methyltransferase, thereby revealing its scientifically informative stability landscape. Thus, our highly accessible method for in vivo monitoring of the stability of diverse proteins will facilitate both basic research and applied protein engineering efforts.

Molecular Basis of Transcription-Coupled Pre-mRNA Capping.

Capping is the first step in pre-mRNA processing, and the resulting 5'-RNA cap is required for mRNA splicing, export, translation, and stability. Capping is functionally coupled to transcription by RNA polymerase (Pol) II, but the coupling mechanism remains unclear. We show that efficient binding of the capping enzyme (CE) to transcribing, phosphorylated yeast Pol II (Pol IIp) requires nascent RNA with an unprocessed 5'-triphosphate end. The transcribing Pol IIp-CE complex catalyzes the first two steps of capping, and its analysis by mass spectrometry, cryo-electron microscopy, and protein crosslinking revealed the molecular basis for transcription-coupled pre-mRNA capping. CE docks to the Pol II wall and spans the end of the RNA exit tunnel to position the CE active sites for sequential binding of the exiting RNA 5' end. Thus, the RNA 5' end triggers its own capping when it emerges from Pol II, to ensure seamless RNA protection from 5'-exonucleases during early transcription.

New structural insights reveal an expanded reaction cycle for inositol pyrophosphate hydrolysis by human DIPP1.

Nudix hydrolases attract considerable attention for their wide range of specialized activities in all domains of life. One particular group of Nudix phosphohydrolases (DIPPs), through their metabolism of diphosphoinositol polyphosphates (PP-InsPs), regulates the actions of these polyphosphates upon bioenergetic homeostasis. In the current study, we describe, at an atomic level, hitherto unknown properties of human DIPP1.We provide X-ray analysis of the catalytic core of DIPP1 in crystals complexed with either natural PP-InsPs, alternative PP-InsP stereoisomers, or non-hydrolysable methylene bisphosphonate analogs ("PCP-InsPs"). The conclusions that we draw from these data are interrogated by studying the impact upon catalytic activity upon mutagenesis of certain key residues. We present a picture of a V-shaped catalytic furrow with overhanging ridges constructed from flexible positively charged side chains; within this cavity, the labile phosphoanhydride bond is appropriately positioned at the catalytic site by an extensive series of interlocking polar contacts which we analogize as "suspension cables." We demonstrate functionality for a triglycine peptide within a ß-strand which represents a non-canonical addition to the standard Nudix catalytic core structure. We describe pre-reaction enzyme/substrate states which we posit to reflect a role for electrostatic steering in substrate capture. Finally, through time-resolved analysis, we uncover a chronological sequence of DIPP1/product post-reaction states, one of which may rationalize a role for InsP6 as an inhibitor of catalytic activity.

Structural insights into DNA double-strand break signaling.

Genomic integrity is most threatened by double-strand breaks, which, if left unrepaired, lead to carcinogenesis or cell death. The cell generates a network of protein-protein signaling interactions that emanate from the DNA damage which are now recognized as a rich basis for anti-cancer therapy development. Deciphering the structures of signaling proteins has been an uphill task owing to their large size and complex domain organization. Recent advances in mammalian protein expression/purification and cryo-EM-based structure determination have led to significant progress in our understanding of these large multidomain proteins. This review is an overview of the structural principles that underlie some of the key signaling proteins that function at the double-strand break site. We also discuss some plausible ideas that could be considered for future structural approaches to visualize and build a more complete understanding of protein dynamics at the break site.

Principles of RNA and nucleotide discrimination by the RNA processing enzyme RppH.

All enzymes face a challenge of discriminating cognate substrates from similar cellular compounds. Finding a correct substrate is especially difficult for the Escherichia coli Nudix hydrolase RppH, which triggers 5'-end-dependent RNA degradation by removing orthophosphate from the 5'-diphosphorylated transcripts. Here we show that RppH binds and slowly hydrolyzes NTPs, NDPs and (p)ppGpp, which each resemble the 5'-end of RNA. A series of X-ray crystal structures of RppH-nucleotide complexes, trapped in conformations either compatible or incompatible with hydrolysis, explain the low reaction rates of mononucleotides and suggest two distinct mechanisms for their hydrolysis. While RppH adopts the same catalytic arrangement with 5'-diphosphorylated nucleotides as with RNA, the enzyme hydrolyzes 5'-triphosphorylated nucleotides by extending the active site with an additional Mg2+ cation, which coordinates another reactive nucleophile. Although the average intracellular pH minimizes the hydrolysis of nucleotides by slowing their reaction with RppH, they nevertheless compete with RNA for binding and differentially inhibit the reactivity of RppH with triphosphorylated and diphosphorylated RNAs. Thus, E. coli RppH integrates various signals, such as competing non-cognate substrates and a stimulatory protein factor DapF, to achieve the differential degradation of transcripts involved in cellular processes important for the adaptation of bacteria to different growth conditions.

The structure of exopolyphosphatase (PPX) from Porphyromonas gingivalis in complex with substrate analogs and magnesium ions reveals the basis for polyphosphate processivity.

The enzymes exopolyphosphatase/guanosine pentaphosphate phosphohydrolase (PPX/GppA) play important roles in the bacterial stringent response. PPX degrades inorganic polyphosphate (polyP), a polymer composed of a few to hundreds of phosphate residues supporting cell survival in the stationary phase. The crystal structure of PPX from Porphyromonas gingivalis (PgPPX) in complex with catalytic magnesium ions and several sulfate ions was solved. PgPPX contained two domains and represented a "closed" configuration. Four sulfate ions forming a linear dispersed chain were observed in the aqueduct of the PPX dimer, which the long polyP chain most likely occupied. The side chain of R255 stretched into the cavity where polyP could be located, obstructing the entrance of larger substrates such as NTP and NDP. This study provided the first view into the structure of the PPX/GppA homolog in complex with magnesium ions and substrate analogs and explained how PgPPX implemented its functionality.

Functional Characterization of COG1713 (YqeK) as a Novel Diadenosine Tetraphosphate Hydrolase Family.

Diadenosine tetraphosphate (Ap4A) is a dinucleotide found in both prokaryotes and eukaryotes. In bacteria, its cellular levels increase following exposure to various stress signals and stimuli, and its accumulation is generally correlated with increased sensitivity to a stressor(s), decreased pathogenicity, and enhanced antibiotic susceptibility. Ap4A is produced as a by-product of tRNA aminoacylation, and is cleaved to ADP molecules by hydrolases of the ApaH and Nudix families and/or by specific phosphorylases. Here, considering evidence that the recombinant protein YqeK from Staphylococcus aureus copurified with ADP, and aided by thermal shift and kinetic analyses, we identified the YqeK family of proteins (COG1713) as an unprecedented class of symmetrically cleaving Ap4A hydrolases. We validated the functional assignment by confirming the ability of YqeK to affect in vivo levels of Ap4A in B. subtilis YqeK shows a catalytic efficiency toward Ap4A similar to that of the symmetrically cleaving Ap4A hydrolases of the known ApaH family, although it displays a distinct fold that is typical of proteins of the HD domain superfamily harboring a diiron cluster. Analysis of the available 3D structures of three members of the YqeK family provided hints to the mode of substrate binding. Phylogenetic analysis revealed the occurrence of YqeK proteins in a consistent group of Gram-positive bacteria that lack ApaH enzymes. Comparative genomics highlighted that yqeK and apaH genes share a similar genomic context, where they are frequently found in operons involved in integrated responses to stress signals.IMPORTANCE Elevation of Ap4A level in bacteria is associated with increased sensitivity to heat and oxidative stress, reduced antibiotic tolerance, and decreased pathogenicity. ApaH is the major Ap4A hydrolase in gamma- and betaproteobacteria and has been recently proposed as a novel target to weaken the bacterial resistance to antibiotics. Here, we identified the orphan YqeK protein family (COG1713) as a highly efficient Ap4A hydrolase family, with members distributed in a consistent group of bacterial species that lack the ApaH enzyme. Among them are the pathogens Staphylococcus aureus, Streptococcus pneumoniae, and Mycoplasma pneumoniae By identifying the player contributing to Ap4A homeostasis in these bacteria, we disclose a novel target to develop innovative antibacterial strategies.

Protein adaptation to high hydrostatic pressure: Computational analysis of the structural proteome.

Hydrostatic pressure has a vital role in the biological adaptation of the piezophiles, organisms that live under high hydrostatic pressure. However, the mechanisms by which piezophiles are able to adapt their proteins to high hydrostatic pressure is not well understood. One proposed hypothesis is that the volume changes of unfolding (ΔVTot ) for proteins from piezophiles is distinct from those of nonpiezophilic organisms. Since ΔVTot defines pressure dependence of stability, we performed a comprehensive computational analysis of this property for proteins from piezophilic and nonpiezophilic organisms. In addition, we experimentally measured the ΔVTot of acylphosphatases and thioredoxins belonging to piezophilic and nonpiezophilic organisms. Based on this analysis we concluded that there is no difference in ΔVTot for proteins from piezophilic and nonpiezophilic organisms. Finally, we put forward the hypothesis that increased concentrations of osmolytes can provide a systemic increase in pressure stability of proteins from piezophilic organisms and provide experimental thermodynamic evidence in support of this hypothesis.

High resolution structure of Vibrio cholerae acylphosphatase (VcAcP) cage: Identification of drugs, location of its binding site and engineering to facilitate cage formation.

Protein cages have recently emerged as an extraordinary drug-delivery system due to its biocompatibility, biodegradability, low toxicity, ease to manipulate and engineer. We have reported earlier the formation and architecture of a do-decameric cage-like architecture of Vibrio cholerae acylphosphatase (VcAcP) at 3.1 Å. High resolution (2.4 Å) crystal structure of VcAcP cage, reported here, illuminates a potential binding site for sulphate/phosphate containing drugs whereas analysis of its subunit association and interfaces indicates high potential for cage engineering. Tryptophan quenching studies indeed discloses noteworthy binding with various sulphate/phosphate containing nucleotide-based drugs and vitamin B6 (PLP) demonstrating that exterior surface of VcAcP protein cage can be exploited as multifunctional carrier. Moreover, a quadruple mutant L30C/T68C/N40C/L81C-VcAcP (QM-VcAcP) capable to form an intricate disulphide bonded VcAcP cage has been designed. SEC, SDS-PAGE analysis and DLS experiment confirmed cysteine mediated engineered VcAcP cage formation.

Structural and functional study of SaAcP, an acylphosphatase from Staphylococcus aureus.

Acylphosphatase is the smallest enzyme that is widely distributed in many diverse organisms ranging from archaebacteria to higher-eukaryotes including the humans. The enzyme hydrolyzes the carboxyl-phosphate bonds of the acyl phosphates which are important intermediates in glycolysis, membrane pumps, tricarboxylic acid cycle, and urea biosynthesis. Despite its biological importance in critical cellular functions, very limited structural investigations have been conducted on bacterial acylphosphatases. Here, we first unveiled the crystal structure of SaAcP, an acylphosphatase from gram-positive S. aureus at the atomic level. Structural insights on the active site together with mutation study provided greater understanding of the catalytic mechanism of SaAcP as a bacterial acylphosphatase and as a putative apyrase. Furthermore, through NMR titration experiment of SaAcP in its solution state, the dynamics and the alterations of residues affected by the phosphate ion were validated. Our findings elucidate the structure-function relationship of acylphosphatases in gram-positive bacteria and will provide a valuable basis for researchers in the field related to bacterial acylphosphatases.

Structural and kinetic insights into stimulation of RppH-dependent RNA degradation by the metabolic enzyme DapF.

Vitally important for controlling gene expression in eukaryotes and prokaryotes, the deprotection of mRNA 5' termini is governed by enzymes whose activity is modulated by interactions with ancillary factors. In Escherichia coli, 5'-end-dependent mRNA degradation begins with the generation of monophosphorylated 5' termini by the RNA pyrophosphohydrolase RppH, which can be stimulated by DapF, a diaminopimelate epimerase involved in amino acid and cell wall biosynthesis. We have determined crystal structures of RppH-DapF complexes and measured rates of RNA deprotection. These studies show that DapF potentiates RppH activity in two ways, depending on the nature of the substrate. Its stimulatory effect on the reactivity of diphosphorylated RNAs, the predominant natural substrates of RppH, requires a substrate long enough to reach DapF in the complex, while the enhanced reactivity of triphosphorylated RNAs appears to involve DapF-induced changes in RppH itself and likewise increases with substrate length. This study provides a basis for understanding the intricate relationship between cellular metabolism and mRNA decay and reveals striking parallels with the stimulation of decapping activity in eukaryotes.

DapF stabilizes the substrate-favoring conformation of RppH to stimulate its RNA-pyrophosphohydrolase activity in Escherichia coli.

mRNA decay is an important strategy by which bacteria can rapidly adapt to their ever-changing surroundings. The 5'-terminus state of mRNA determines the velocity of decay of many types of RNA. In Escherichia coli, RNA pyrophosphohydrolase (RppH) is responsible for the removal of the 5'-terminal triphosphate from hundreds of mRNAs and triggers its rapid degradation by ribonucleases. A diaminopimelate epimerase, DapF, can directly interact with RppH and stimulate its hydrolysis activity in vivo and in vitro. However, the molecular mechanism remains to be elucidated. Here, we determined the complex structure of DapF-RppH as a heterotetramer in a 2:2 molar ratio. DapF-bound RppH exhibits an RNA-favorable conformation similar to the RNA-bound state, suggesting that association with DapF promotes and stabilizes RppH in a conformation that facilitates substrate RNA binding and thus stimulates the activity of RppH. To our knowledge, this is the first published structure of an RNA-pyrophosphohydrolysis complex in bacteria. Our study provides a framework for further investigation of the potential regulators involved in the RNA-pyrophosphohydrolysis process in prokaryotes.

Inorganic polyphosphate interacts with nucleolar and glycosomal proteins in trypanosomatids.

Inorganic polyphosphate (polyP) is a polymer of three to hundreds of phosphate units bound by high-energy phosphoanhydride bonds and present from bacteria to humans. Most polyP in trypanosomatids is concentrated in acidocalcisomes, acidic calcium stores that possess a number of pumps, exchangers, and channels, and are important for their survival. In this work, using polyP as bait we identified > 25 putative protein targets in cell lysates of both Trypanosoma cruzi and Trypanosoma brucei. Gene ontology analysis of the binding partners found a significant over-representation of nucleolar and glycosomal proteins. Using the polyphosphate-binding domain (PPBD) of Escherichia coli exopolyphosphatase (PPX), we localized long-chain polyP to the nucleoli and glycosomes of trypanosomes. A competitive assay based on the pre-incubation of PPBD with exogenous polyP and subsequent immunofluorescence assay of procyclic forms (PCF) of T. brucei showed polyP concentration-dependent and chain length-dependent decrease in the fluorescence signal. Subcellular fractionation experiments confirmed the presence of polyP in glycosomes of T. brucei PCF. Targeting of yeast PPX to the glycosomes of PCF resulted in polyP hydrolysis, alteration in their glycolytic flux and increase in their susceptibility to oxidative stress.

Ppn2, a novel Zn2+-dependent polyphosphatase in the acidocalcisome-like yeast vacuole.

Acidocalcisome-like organelles are found in all kingdoms of life. Many of their functions, such as the accumulation and storage of metal ions, nitrogen and phosphate, the activation of blood clotting and inflammation, depend on the controlled synthesis and turnover of polyphosphate (polyP), a polymer of inorganic phosphate linked by phosphoric anhydride bonds. The exploration of the role of acidocalcisomes in metabolism and physiology requires the manipulation of polyP turnover, yet the complete set of proteins responsible for this turnover is unknown. Here, we identify a novel type of polyphosphatase operating in the acidocalcisome-like vacuoles of the yeast Saccharomyces cerevisiae, which we called Ppn2. Ppn2 belongs to the PPP-superfamily of metallophosphatases, is activated by Zn2+ ions and exclusively shows endopolyphosphatase activity. It is sorted to vacuoles via the multivesicular body pathway. Together with Ppn1, Ppn2 is responsible for a substantial fraction of polyphosphatase activity that is necessary to mobilize polyP stores, for example in response to phosphate scarcity. This finding opens the way to manipulating polyP metabolism more profoundly and deciphering its roles in phosphate and energy homeostasis, as well as in signaling.

Enzymatic quantification and length determination of polyphosphate down to a chain length of two.

Polyacrylamide gel electrophoresis, being the current method of choice for length determination of inorganic polyphosphate (polyP), requires a sequencing apparatus, relies on commercially not available polyP length standards and yields only a chain length distribution. State of the art polyP quantification involves enzymatic hydrolysis of polyP to orthophosphate with the Saccharomyces cerevisiae exopolyphosphatase 1 (scPpx1p) and subsequent colorimetric orthophosphate detection. Because scPpx1p leaves one pyrophosphate per polyP, short chain polyPs are only partially detected. To overcome this analytical limitation, a method involving both the scPpx1p and the S. cerevisiae inorganic pyrophosphatase (scIpp1p) is proposed. Differential enzymatic hydrolysis of polyP with scPpx1p, and a combination of scIpp1p and scPpx1p allows not only for comprehensive quantification of polyP (excluding cyclic polyP) down to a chain length of two, but also absolute average chain length determination in the range of two to approximately 80. An optimized one-reagent method for rapid (2 min) orthophosphate quantification is part of the assay. Biological phosphorous containing molecules at equimolar phosphorous concentrations regarding polyP do not interfere. The method requires 1.5 µg polyP and calls only for a plate reader. This is the first enzymatic method for simultaneous average polyP chain length determination as well as comprehensive quantification.

Direct Conversion of an Enzyme from Native-like to Amyloid-like Aggregates within Inclusion Bodies.

The acylphosphatase from Sulfolobus solfataricus (Sso AcP) is a globular protein able to aggregate in vitro from a native-like conformational ensemble without the need for a transition across the major unfolding energy barrier. This process leads to the formation of assemblies in which the protein retains its native-like structure, which subsequently convert into amyloid-like aggregates. Here, we investigate the mechanism by which Sso AcP aggregates in vivo to form bacterial inclusion bodies after expression in E. coli. Shortly after the initiation of expression, Sso AcP is incorporated into inclusion bodies as a native-like protein, still exhibiting small but significant enzymatic activity. Additional experiments revealed that this overall process of aggregation is enhanced by the presence of the unfolded N-terminal region of the sequence and by destabilization of the globular segment of the protein. At later times, the Sso AcP molecules in the inclusion bodies lose their native-like properties and convert into ß-sheet-rich amyloid-like structures, as indicated by their ability to bind thioflavin T and Congo red. These results show that the aggregation behavior of this protein is similar in vivo to that observed in vitro, and that, at least for a predominant part of the protein population, the transition from a native to an amyloid-like structure occurs within the aggregate state.

Hydrolysis of diadenosine polyphosphates. Exploration of an additional role of Mycobacterium smegmatis MutT1.

Diadenosine polyphosphates (ApnA, n=2-6), particularly Ap4A, are involved in several important physiological processes. The substantial sequence identity of the Nudix hydrolase domain (domain 1) of Mycobacterium smegmatis MutT1 (MsMutT1) with a known Ap4A hydrolase suggested that MsMutT1 could also hydrolyse diadenosine polyphosphates. Biochemical experiments yielded results in conformity with this suggestion, with Ap4A as the best among the substrates. ATP is a product in all experiments; small amounts of ADP were also observed in the experiments involving Ap4A and Ap6A. Hydrolysis was inhibited by fluoride ions in all cases. The mechanism of action and its inhibition in relation to ApnA were explored through the X-ray analysis of the crystals of the MsMutT1 complexes with Ap5A; Ap5A and MnCl2; Ap4A; ATP; and ATP.NaF.MgCl2. The aggregation pattern of molecules in the first four crystals is similar to that found in a majority of MsMutT1-NTP crystals. Substrate molecules occupy the primary binding site and ATP occupies a site at an intermolecular interface, in the first two. ATP occupies both the sites in the third and fourth crystal. The protein-ligand interactions observed in these crystal structures lead to an explanation of the molecular mechanism of hydrolysis of ApnA by MsMutT1. The fifth crystal exhibits a new packing arrangement. The structure of the complex provides an explanation for the fluoride inhibition of the activity of the enzyme. It would thus appear that MutT1 has a major role involving the hydrolysis of diadenosine polyphosphates, which could be elucidated at the molecular level.

Novel Nucleoside Diphosphatase Contributes to Staphylococcus aureus Virulence.

We identified SA1684 as a Staphylococcus aureus virulence gene using a silkworm infection model. The SA1684 gene product carried the DUF402 domain, which is found in RNA-binding proteins, and had amino acid sequence similarity with a nucleoside diphosphatase, Streptomyces coelicolor SC4828 protein. The SA1684-deletion mutant exhibited drastically decreased virulence, in which the LD50 against silkworms was more than 10 times that of the parent strain. The SA1684-deletion mutant also exhibited decreased exotoxin production and colony-spreading ability. Purified SA1684 protein had Mn(2+)- or Co(2+)-dependent hydrolyzing activity against nucleoside diphosphates. Alanine substitutions of Tyr-88, Asp-106, and Asp-123/Glu-124, which are conserved between SA1684 and SC4828, diminished the nucleoside diphosphatase activity. Introduction of the wild-type SA1684 gene restored the hemolysin production of the SA1684-deletion mutant, whereas none of the alanine-substituted SA1684 mutant genes restored the hemolysin production. RNA sequence analysis revealed that SA1684 is required for the expression of the virulence regulatory genes agr, sarZ, and sarX, as well as metabolic genes involved in glycolysis and fermentation pathways. These findings suggest that the novel nucleoside diphosphatase SA1684 links metabolic pathways and virulence gene expression and plays an important role in S. aureus virulence.

Applications of pressure perturbation calorimetry to study factors contributing to the volume changes upon protein unfolding.

BACKGROUND: Pressure perturbation calorimetry (PPC) is a biophysical method that allows direct determination of the volume changes upon conformational transitions in macromolecules. SCOPE OF THIS REVIEW: This review provides novel details of the use of PPC to analyze unfolding transitions in proteins. The emphasis is made on the data analysis as well as on the validation of different structural factors that define the volume changes upon unfolding. Four case studies are presented that show the application of these concepts to various protein systems. MAJOR CONCLUSIONS: The major conclusions are: 1. Knowledge of the thermodynamic parameters for heat induced unfolding facilitates the analysis of the PPC profiles. 2. The changes in the thermal expansion coefficient upon unfolding appear to be temperature dependent.3.Substitutions on the protein surface have negligible effects on the volume changes upon protein unfolding. 4. Structural plasticity of proteins defines the position dependent effect of amino acid substitutions of the residues buried in the native state. 5. Small proteins have positive volume changes upon unfolding which suggests difference in balance between the cavity/void volume in the native state and the hydration volume changes upon unfolding as compared to the large proteins that have negative volume changes. GENERAL SIGNIFICANCE: The information provided here gives a better understanding and deeper insight into the role played by various factors in defining the volume changes upon protein unfolding.

Cell wall canals formed upon growth of Candida maltosa in the presence of hexadecane are associated with polyphosphates.

Canals are supramolecular complexes observed in the cell wall of Candida maltosa grown in the presence of hexadecane as a sole carbon source. Such structures were not observed in glucose-grown cells. Microscopic observations of cells stained with diaminobenzidine revealed the presence of oxidative enzymes in the canals. 4΄,6΄-diamino-2-phenylindole staining revealed that a substantial part of cellular polyphosphate was present in the cell wall of cells grown on hexadecane in condition of phosphate limitation. The content and chain length of polyphosphates were higher in hexadecane-grown cells than in glucose grown ones. The treatment of cells with yeast polyphosphatase PPX1 resulted in the decrease of the canal size. These data clearly indicated that polyphosphates are constituents of canals; they might play an important role in the canal structure and functioning.