Mechanism and Structural Insights Into a Novel Esterase, E53, Isolated From Erythrobacter longus

Esterases are a class of enzymes that split esters into an acid and an alcohol in a chemical reaction with water, having high potential in pharmaceutical, food and biofuel industrial applications. To advance the understanding of esterases, we have identified and characterized E53, an alkalophilic esterase from a marine bacterium Erythrobacter longus. The crystal structures of wild type E53 and three variants were solved successfully using the X-ray diffraction method. Phylogenetic analysis classified E53 as a member of the family IV esterase. The enzyme showed highest activity against p-nitrophenyl butyrate substrate at pH 8.5–9.5 and 40°C. Based on the structural feature, the catalytic pocket was defined as R1 (catalytic center), R2 (pocket entrance), and R3 (end area of pocket) regions. Nine variants were generated spanning R1–R3 and thorough functional studies were performed. Detailed structural analysis and the results obtained from the mutagenesis study revealed that mutations in the R1 region could regulate the catalytic reaction in both positive and negative directions; expanding the bottleneck in R2 region has improved the enzymatic activity; and R3 region was associated with the determination of the pH pattern of E53. N166A in R3 region showed reduced activity only under alkaline conditions, and structural analysis indicated the role of N166 in stabilizing the loop by forming a hydrogen bond with L193 and G233. In summary, the systematic studies on E53 performed in this work provide structural and functional insights into alkaliphilic esterases and further our knowledge of these enzymes.

Halo-Tolerant And Thermostable Mechanisms of An Endoglucanase From Marine Aspergillus Niger

The cellulase cocktail of marine Aspergillus niger exhibited halo-tolerant and thermostable properties, which is of great potential in industrial application. In order to excavate the single tolerant cellulase components from complex cellulase cocktail, constitutive homologous expression was employed for direct obtainment of the endoglucanase (AnEGL). Enzymatic property study revealed that AnEGL exhibited a property of halo-tolerance and an outstanding thermostability in high salinity environment. Significantly, its activity increased by 29% and the half-life at 65 °C increased by 26.7-fold with the presence of 4.5 M NaCl. Molecular dynamics simulation revealed that Na+ and Cl－ could form salt bridges with charged residues, and then influenced the activity of loops and the stability of substrate binding pocket, which accounted for the halo-tolerance and thermostability. Further, site-specific mutagenesis study proved that the residues Asp95 and Asp99 in the pocket were of great concern for the tolerant properties. The halo-tolerant and thermostable AnEGL was of great value in lignocellulosic utilization and the conjectural mechanisms were of referential significance for other tolerant enzymes.

Tandem WW/PPxY motif interactions in WWOX: the multifaceted role of the second WW domain

Class I WW domains mediate protein interactions by binding short linear PPxY motifs. They occur predominantly as tandem repeats, and their target proteins often contain multiple PPxY motifs, but the interplay of WW/peptide interactions is not always intuitive. WW domain-containing oxidoreductase (WWOX) protein harbors two WW domains: unstable WW1 capable of PPxY binding, and well-folded but mutated WW2 that cannot bind such motifs. WW2 is considered to act as a WW1 chaperone, but the underlying mechanism remains to be revealed. Here we combine NMR, ITC and structural modeling to elucidate the role of both WW domains in WWOX binding to single and double motif peptides derived from its substrate ErbB4. Using NMR we identified an interaction surface between the two domains that supports a WWOX conformation that is compatible with peptide substrate binding. ITC and NMR measurements reveal that while binding affinity to a single motif is marginally increased in the presence of WW2, affinity to a dual motif peptide increases tenfold, and that WW2 can directly bind double motif-peptides using its canonical binding site. Finally, differential binding of peptides in a mutagenesis study is consistent with a parallel orientation binding to the WW1-WW2 tandem domain, agreeing with structural models of the interaction. Our results reveal the complex nature of tandem WW domain organization and substrate binding, highlighting the contribution of WWOX WW2 to both stability and binding. This opens the way to assess how evolution can utilize the multivariate nature of binding to fine-tune interactions for specific biological functions.

An increase in surface hydrophobicity mediates chaperone activity in N-chlorinated proteins

Under physiological conditions, Escherichia coli RidA is an enamine/imine deaminase, which promotes the release of ammonia from reactive enamine/imine intermediates. However, when modified by hypochlorous acid (HOCl), as produced by the host defense, RidAHOCl turns into a potent chaperone-like holdase that can effectively protect the proteome of E. coli during oxidative stress. We previously reported that the activation of RidA's chaperone-like function coincides with the addition of at least seven and up to ten chlorine atoms. These atoms are reversibly added to basic amino acids in RidAHOCl and removal by reducing agents leads to inactivation. Nevertheless, it remains unclear, which residues in particular need to be chlorinated for activation. Here, we employ a combination of LC-MS/MS analysis, a chemo-proteomic approach, and a mutagenesis study to identify residues responsible for RidA's chaperone-like function. Through LC-MS/MS of digested RidAHOCl, we obtained direct evidence of the chlorination of one arginine residue (and, coincidentally, two tyrosine residues), while other N-chlorinated residues could not be detected, presumably due to the instability of the modification and its potential interference with a proteolytic digest. Therefore, we established a chemoproteomic approach using 5-(dimethylamino) naphthalene-1-sulfinic acid (DANSO2H) as a probe to label N-chlorinated lysines. Using this probe, we were able to detect the N-chlorination of six additional lysine residues. Moreover, using a mutagenesis study to genetically probe the role of single arginine and lysine residues, we found that the removal of arginines R105 and R128 leads to a substantial reduction of RidAHOCl's chaperone activity. These results, together with structural analysis, confirm that the chaperone activity of RidA is concomitant with the loss of positive charges on the protein surface, leading to an increased overall protein hydrophobicity. Molecular modelling of RidAHOCl and the rational design of a RidA variant that shows chaperone activity even in the absence of HOCl further supports our hypothesis. Our data provide a molecular mechanism for HOCl-mediated chaperone activity found in RidA and a growing number of other HOCl-activated chaperones.

Assessment of Phenotype Relevant Amino Acid Residues in TEM-β-Lactamases by Mathematical Modelling and Experimental Approval

Single substitutions or combinations of them alter the hydrolytic activity towards specific β-lactam-antibiotics and β-lactamase inhibitors of TEM-β-lactamases. The sequences and phenotypic classification of allelic TEM variants, as provided by the NCBI National Database of Antibiotic Resistant Organisms, does not attribute phenotypes to all variants. Some entries are doubtful as the data assessment differs strongly between the studies or no data on the methodology are provided at all. This complicates mathematical and bioinformatic predictions of phenotypes that rely on the database. The present work aimed to prove the role of specific substitutions on the resistance phenotype of TEM variants in, to our knowledge, the most extensive mutagenesis study. In parallel, the predictive power of extrapolation algorithms was assessed. Most well-known substitutions with direct impact on the phenotype could be reproduced, both mathematically and experimentally. Most discrepancies were found for supportive substitutions, where some resulted in antagonistic effects in contrast to previously described synergism. The mathematical modelling proved to predict the strongest phenotype-relevant substitutions accurately but showed difficulties in identifying less prevalent but still phenotype transforming ones. In general, mutations increasing cephalosporin resistance resulted in increased sensitivity to β-lactamase inhibitors and vice versa. Combining substitutions related to cephalosporin and β-lactamase inhibitor resistance in almost all cases increased BLI susceptibility, indicating the rarity of the combined phenotype.

The structurally conserved TELR region on shelterin protein TPP1 is essential for telomerase processivity but not recruitment

The shelterin protein TPP1 is involved in both recruiting telomerase and stimulating telomerase processivity in human cells. Assessing the in vivo significance of the latter role of TPP1 has been difficult, because TPP1 mutations that perturb telomerase function tend to abolish both telomerase recruitment and processivity. The Saccharomyces cerevisiae telomerase-associated Est3 protein adopts a protein fold similar to the N-terminal region of TPP1. Interestingly, a previous structure-guided mutagenesis study of Est3 revealed a TELR surface region that regulates telomerase function via an unknown mechanism without affecting the interaction between Est3 and telomerase [T. Rao et al., Proc. Natl. Acad. Sci. U.S.A. 111, 214–218 (2014)]. Here, we show that mutations within the structurally conserved TELR region on human TPP1 impaired telomerase processivity while leaving telomerase recruitment unperturbed, hence uncoupling the two roles of TPP1 in regulating telomerase. Telomeres in cell lines containing homozygous TELR mutations progressively shortened to a critical length that caused cellular senescence, despite the presence of abundant telomerase in these cells. Our findings not only demonstrate that telomerase processivity can be regulated by TPP1 in a process separable from its role in recruiting telomerase, but also establish that the in vivo stimulation of telomerase processivity by TPP1 is critical for telomere length homeostasis and long-term viability of human cells.

Circ_0008542 in osteoblast exosomes promotes osteoclast-induced bone resorption through m6A methylation

With an increasing aging society, China is the world’s fastest growing markets for oral implants. Compared with traditional oral implants, immediate implants cause marginal bone resorption and increase the failure rate of osseointegration, but the mechanism is still unknown. Therefore, it is important to further study mechanisms of tension stimulus on osteoblasts and osteoclasts at the early stage of osseointegration to promote rapid osseointegration around oral implants. The results showed that exosomes containing circ_0008542 from MC3T3-E1 cells with prolonged tensile stimulation promoted osteoclast differentiation and bone resorption. Circ_0008542 upregulated Tnfrsf11a (RANK) gene expression by acting as a miR-185-5p sponge. Meanwhile, the circ_0008542 1916-1992 bp segment exhibited increased m6A methylation levels. Inhibiting the RNA methyltransferase METTL3 or overexpressing the RNA demethylase ALKBH5 reversed osteoclast differentiation and bone resorption induced by circ_0008542. Injection of circ_0008542 + ALKBH5 into the tail vein of mice reversed the same effects in vivo. Site-directed mutagenesis study demonstrated that 1956 bp on circ_0008542 is the m6A functional site with the abovementioned biological functions. In conclusion, the RNA methylase METTL3 acts on the m6A functional site of 1956 bp in circ_0008542, promoting competitive binding of miRNA-185-5p by circ_0008542, and leading to an increase in the target gene RANK and the initiation of osteoclast bone absorption. In contrast, the RNA demethylase ALKBH5 inhibits the binding of circ_0008542 with miRNA-185-5p to correct the bone resorption process. The potential value of this study provides methods to enhance the resistance of immediate implants through use of exosomes releasing ALKBH5.

Crystal structure of schizorhodopsin reveals mechanism of inward proton pumping

Schizorhodopsins (SzRs), a new rhodopsin family identified in Asgard archaea, are phylogenetically located at an intermediate position between type-1 microbial rhodopsins and heliorhodopsins. SzRs work as light-driven inward H+ pumps as xenorhodopsins in bacteria. Although E81 plays an essential role in inward H+ release, the H+ is not metastably trapped in such a putative H+ acceptor, unlike the other H+ pumps. It remains elusive why SzR exhibits different kinetic behaviors in H+ release. Here, we report the crystal structure of SzR AM_5_00977 at 2.1 Å resolution. The SzR structure superimposes well on that of bacteriorhodopsin rather than heliorhodopsin, suggesting that SzRs are classified with type-1 rhodopsins. The structure-based mutagenesis study demonstrated that the residues N100 and V103 around the β-ionone ring are essential for color tuning in SzRs. The cytoplasmic parts of transmembrane helices 2, 6, and 7 are shorter than those in the other microbial rhodopsins, and thus E81 is located near the cytosol and easily exposed to the solvent by light-induced structural change. We propose a model of untrapped inward H+ release; H+ is released through the water-mediated transport network from the retinal Schiff base to the cytosol by the side of E81. Moreover, most residues on the H+ transport pathway are not conserved between SzRs and xenorhodopsins, suggesting that they have entirely different inward H+ release mechanisms.

The dodecin of Mycobacterium tuberculosis : biological function and biotechnical applications

Biological Function of Bacterial Dodecins In this thesis, the dodecins of Mycobacterium tuberculosis (MtDod), Streptomyces coelicolor (ScDod) and Streptomyces davaonensis (SdDod) were studied. Kinetic measurements of the flavin binding of MtDod revealed that the dodecin binding pocket is filled in two distinct steps, for which a kinetic model then was established and verified by experimental data. The analysis with the two-step model showed that the unique binding pocket of dodecins allows them to bind excessive amounts of flavins, while at low flavin concentrations, flavin is released and only weakly bound. This function of flavin buffering prevents accumulation of free oxidised flavins and therefore helps to keep the redox balance of the cell and prevents potential cell damage caused by excessive free flavins. To further gain insights into the role of bacterial dodecins, the effect of knocking out the dodecin encoding gene in S. davaonensis was analysed. The knockout strain showed increased concentrations of various stress related metabolites, indicating that without dodecin the cellular balance is disrupted, which supports the role of dodecins as a flavin homeostasis factor. With a self-designed affinity measurement method based on the temperature dependent dissociation of the dodecin:flavin complex, which allowed parallel screening of multiple conditions, it was shown that MtDod, ScDod and SdDod have much higher affinities towards FMN and FAD under acidic conditions. Under these conditions, the three dodecins might function as a FMN storage. M. tuberculosis encounters multiple acidic environments during its infection cycle of humans and can adopt a state of dormancy. During recovery from the dormant state, a flavin storage might be beneficial. For some Streptomyces species it was reported that the formed spores are slightly acidic and therefore ScDod and SdDod could function as flavin storages for the spores. Further details on the flavin binding mechanism of MtDod were revealed by a mutagenesis study, identifying the importance of a histidine residue at the fourth position of the protein sequence for flavin binding, but contrary to expectations, this residue seems only to be partly involved in the pH related affinity shift. The data, reported in this thesis, demonstrates that bacterial dodecins likely function as flavin homeostasis factors, which allow overall higher flavin pools in the cell without disrupting the cellular balance. Further, the reported acid-dependent increase in binding affinity suggests that under certain conditions bacterial dodecins can also function as a flavin storage system. Application of the Dodecin of M. tuberculosis In this thesis, the stability of MtDod, ScDod SdDod and HsDod was analysed to find a suitable dodecin for the use as a carrier/scaffold. Therefore, a method to easily measure the stability of dodecins was designed, which measures the ability of the dodecamer to rebind flavins after a heating phase with stepwise increasing temperatures. Using this assay and testing the stability against detergents by SDS PAGE, showed that the dodecamer of MtDod possesses an excellent stability against a vast array of conditions, like temperatures above 95 °C, low pH and about 2% SDS. By solving the crystal structure of ScDod and SdDod, the latter forming a less stable dodecamer, combined with a mutagenesis study, the importance of a specific salt bridge for dodecamer stability was revealed and might be helpful to find further highly stable dodecins. In addition to the intrinsic high stability of the MtDod dodecamer, also the robustness of the fold was tested by creating diverse MtDod fusion constructs and producing them in Escherichia coli. Here it was shown that MtDod easily tolerates the attachment of proteins up to 4-times of its own size and that both termini can be modified without affecting the dodecamer noticeably. Further, it was shown that MtDod and many MtDod fusion constructs could be purified in high yields via a protocol based on the removal of E. coli proteins through heat denaturation and subsequent centrifugation. In a case study, by fusing diverse antigens from mostly human proteins to MtDod and using these constructs to produce antibodies in rabbits, it was demonstrated that MtDod is immunogenic and presents the attached antigens to the immune system. The here reported properties of MtDod and to a lesser degree of other bacterial dodecins, show that bacterial dodecins are a valuable addition to the pool of scaffold and carrier proteins and have great potential as antigen carriers.

The structurally conserved TELR region on shelterin protein TPP1 is essential for telomerase processivity but not recruitment

In addition to mediating telomerase recruitment, shelterin protein TPP1 also stimulates telomerase processivity. Assessing the in vivo significance of the latter role of TPP1 has been difficult, as TPP1 mutations that perturb telomerase function tend to abolish both telomerase recruitment and processivity. We sought to separate the two activities of TPP1 in regulating telomerase by considering a structure-guided mutagenesis study on the S. cerevisiae telomerase-associated Est3 protein, which revealed a TELR surface region on Est3 that regulates telomerase function via an unknown mechanism without affecting the interaction between Est3 and telomerase (1). Here, we show that mutations within the structurally conserved TELR region on TPP1 impaired telomerase processivity while leaving telomerase recruitment unperturbed, hence uncoupling the two roles of TPP1 in regulating telomerase. Telomeres in cell lines containing homozygous TELR mutations progressively shortened to a critical length that caused cellular senescence, despite the presence of abundant telomerase in these cells. Our findings not only demonstrate that telomerase processivity can be regulated by TPP1, in a process separable from its role in recruiting telomerase to telomeres, but also establish that the in vivo stimulation of telomerase processivity by TPP1 is critical for telomere length homeostasis and long-term cell viability.SignificanceTelomerase directs the synthesis of new telomeric repeats at chromosome ends, enabling cells to overcome the end replication problem and continue to divide. The shelterin protein TPP1 interacts with telomerase, promoting both telomerase recruitment and processivity (the addition of multiple telomeric repeats after a single substrate binding event). Here we show the identification of separation-of-function mutants of TPP1 that eliminate telomerase processivity but leave the telomerase recruitment function intact. When introduced into human cells in a homozygous manner, these mutations can induce critical telomere shortening and cellular senescence. Our observations therefore provide the first demonstration that telomerase processivity, in addition to telomerase recruitment, is a key regulatory step in vivo for continued human cell proliferation.