| 1. |
- Szeler, Klaudia
(author)
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Computational Protein Evolution : Modeling the Selectivity and Promiscuity of Engineered Enzymes
- 2020
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Doctoral thesis (other academic/artistic)abstract
- Enzymes are biological catalysts that significantly increase the rate of all biochemical reactions that take place within cells and are essential to maintain life. Many questions regarding their function remain unknown. Experimental techniques, such as kinetic measurements, spectroscopy, and site-directed mutagenesis, can provide relevant information about enzyme structure, key residues, active site conformations, and kinetics. However, they struggle to provide a full picture of enzyme catalysis. Combining experiments with computational techniques gives the possibility to generate a complete explanation with atomistic resolution. Computational modeling offers an incredibly robust toolkit that can provide detailed insight into the reactivity and dynamics of biomolecules.Compounds that contain phosphate and sulfate groups are essential in the living world. They are present as i.e., a biological source of energy (ATP), signaling molecules (GTP), coenzymes, building blocks (DNA, RNA). Furthermore, phosphate esters can be used as insecticides, herbicides, flame retardants, and as chemical weapons. Cleavage of the phosphate bond involves an extremely low rate of spontaneous hydrolysis, nevertheless it is common reaction in living organisms. Phosphatases (enzymes catalysing cleavage of phosphate bond) are crucial in both physiological regulation as well as serious pathological conditions including asthma, immunosuppression, cardiovascular diseases, diabetes.Understanding the basis of phosphoryl and sulfuryl transfer reactions is crucial for medical, biological, and biotechnological industries in order to i.e., create and improve existing drugs, modify enzyme structures, understand the development of some diseases. However, despite decades of both experimental and computational studies, mechanistic details of these reactions remain controversial. These reactions can occur via multiple different mechanisms involving intermediate steps or transition state structures. To solve these puzzles, we performed computational studies to verify the reaction pathway of diaryl sulfate diesters hydrolysis. We suggest that the reaction proceeds through a concerted mechanism with a loose (slightly dissociative) transition state.Serum paraoxonase 1 (PON1) is calcium-dependent lactonase, which is bound to high-density lipoprotein (HDL) with apolipoprotein A-I (ApoA-I). The enzyme is highly promiscuous and catalyzes the hydrolysis of multiple, different types of chemical compounds, such as lactones, aromatic esters, oxons, and organophosphates. We performed several, complex studies on PON1’s reaction mechanism, promiscuity, PON1-HDL interactions, and evolutionary trajectories. One of the most extensively used approaches in this thesis was the empirical valence bond (EVB) method. Our models reproduce essential experimental observables and provide mechanistic insights and a better understanding of the enzymes role and its evolutionary derived promiscuity.
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| 2. |
- Ben-David, Moshe, et al.
(author)
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Enzyme Evolution An Epistatic Ratchet versus a Smooth Reversible Transition
- 2020
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In: Molecular biology and evolution. - : Oxford University Press (OUP). - 0737-4038 .- 1537-1719. ; 37:4, s. 1133-1147
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Journal article (peer-reviewed)abstract
- Evolutionary trajectories are deemed largely irreversible. In a newly diverged protein, reversion of mutations that led to the functional switch typically results in loss of both the new and the ancestral functions. Nonetheless, evolutionary transitions where reversions are viable have also been described. The structural and mechanistic causes of reversion compatibility versus incompatibility therefore remain unclear. We examined two laboratory evolution trajectories of mammalian paraoxonase-1, a lactonase with promiscuous organophosphate hydrolase (OPH) activity. Both trajectories began with the same active-site mutant, His115Trp, which lost the native lactonase activity and acquired higher OPH activity. A neo-functionalization trajectory amplified the promiscuous OPH activity, whereas the re-functionalization trajectory restored the native activity, thus generating a new lactonase that lacks His115. The His115 revertants of these trajectories indicated opposite trends. Revertants of the neo-functionalization trajectory lost both the evolved OPH and the original lactonase activity. Revertants of the trajectory that restored the original lactonase function were, however, fully active. Crystal structures and molecular simulations show that in the newly diverged OPH, the reverted His115 and other catalytic residues are displaced, thus causing loss of both the original and the new activity. In contrast, in the re-functionalization trajectory, reversion compatibility of the original lactonase activity derives from mechanistic versatility whereby multiple residues can fulfill the same task. This versatility enables unique sequence-reversible compositions that are inaccessible when the active site was repurposed toward a new function.
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| 3. |
- Parkash, Vimal, et al.
(author)
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A sensor complements the steric gate when DNA polymerase ε discriminates ribonucleotides
- 2023
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In: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 51:20, s. 11225-11238
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Journal article (peer-reviewed)abstract
- The cellular imbalance between high concentrations of ribonucleotides (NTPs) and low concentrations of deoxyribonucleotides (dNTPs), is challenging for DNA polymerases when building DNA from dNTPs. It is currently believed that DNA polymerases discriminate against NTPs through a steric gate model involving a clash between a tyrosine and the 2 '-hydroxyl of the ribonucleotide in the polymerase active site in B-family DNA polymerases. With the help of crystal structures of a B-family polymerase with a UTP or CTP in the active site, molecular dynamics simulations, biochemical assays and yeast genetics, we have identified a mechanism by which the finger domain of the polymerase sense NTPs in the polymerase active site. In contrast to the previously proposed polar filter, our experiments suggest that the amino acid residue in the finger domain senses ribonucleotides by steric hindrance. Furthermore, our results demonstrate that the steric gate in the palm domain and the sensor in the finger domain are both important when discriminating NTPs. Structural comparisons reveal that the sensor residue is conserved among B-family polymerases and we hypothesize that a sensor in the finger domain should be considered in all types of DNA polymerases.
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| 4. |
- Parkash, Vimal, et al.
(author)
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Structural consequence of the most frequently recurring cancer-associated substitution in DNA polymerase epsilon
- 2019
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In: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 10
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Journal article (peer-reviewed)abstract
- The most frequently recurring cancer-associated DNA polymerase epsilon (Pol epsilon) mutation is a P286R substitution in the exonuclease domain. While originally proposed to increase genome instability by disrupting exonucleolytic proofreading, the P286R variant was later found to be significantly more pathogenic than Pol epsilon proofreading deficiency per se. The mechanisms underlying its stronger impact remained unclear. Here we report the crystal structure of the yeast orthologue, Pol epsilon-P301R, complexed with DNA and an incoming dNTP. Structural changes in the protein are confined to the exonuclease domain, with R301 pointing towards the exonuclease site. Molecular dynamics simulations suggest that R301 interferes with DNA binding to the exonuclease site, an outcome not observed with the exonuclease-inactive Pol epsilon-D290A, E292A variant lacking the catalytic residues. These results reveal a distinct mechanism of exonuclease inactivation by the P301R substitution and a likely basis for its dramatically higher mutagenic and tumorigenic effects.
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| 5. |
- Janfalk Carlsson, Åsa, 1980-, et al.
(author)
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Epoxide Hydrolysis as a Model System for Understanding Flux Through a Branched Reaction Scheme
- 2018
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In: IUCrJ. - 2052-2525. ; 5:3, s. 269-282
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Journal article (peer-reviewed)abstract
- The epoxide hydrolase StEH1 catalyzes the hydrolysis of trans-methylstyrene oxide to 1-phenylpropane-1,2-diol. The (S,S)-epoxide is exclusively transformed into the (1R,2S)-diol, while hydrolysis of the (R,R)-epoxide results in a mixture of product enantiomers. In order to understand the differences in the stereoconfigurations of the products, the reactions were studied kinetically during both the pre-steady-state and steady-state phases. A number of closely related StEH1 variants were analyzed in parallel, and the results were rationalized by structure–activity analysis using the available crystal structures of all tested enzyme variants. Finally, empirical valence-bond simulations were performed in order to provide additional insight into the observed kinetic behaviour and ratios of the diol product enantiomers. These combined data allow us to present a model for the flux through the catalyzed reactions. With the (R,R)-epoxide, ring opening may occur at either C atom and with similar energy barriers for hydrolysis, resulting in a mixture of diol enantiomer products. However, with the (S,S)-epoxide, although either epoxide C atom may react to form the covalent enzyme intermediate, only the pro-(R,S) alkylenzyme is amenable to subsequent hydrolysis. Previously contradictory observations from kinetics experiments as well as product ratios can therefore now be explained for this biocatalytically relevant enzyme.
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| 6. |
- Liao, Qinghua, et al.
(author)
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Loop Motion in Triosephosphate Isomerase Is Not a Simple Open and Shut Case
- 2018
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In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 140:46, s. 15889-15903
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Journal article (peer-reviewed)abstract
- Conformational changes are crucial for the catalytic action of many enzymes. A prototypical and well-studied example is loop opening and closure in triosephosphate isomerase (TIM), which is thought to determine the rate of catalytic turnover in many circumstances. Specifically, TIM loop 6 “grips” the phosphodianion of the substrate and, together with a change in loop 7, sets up the TIM active site for efficient catalysis. Crystal structures of TIM typically show an open or a closed conformation of loop 6, with the tip of the loop moving ∼7 Å between conformations. Many studies have interpreted this motion as a two-state, rigid-body transition. Here, we use extensive molecular dynamics simulations, with both conventional and enhanced sampling techniques, to analyze loop motion in apo and substrate-bound TIM in detail, using five crystal structures of the dimeric TIM from Saccharomyces cerevisiae. We find that loop 6 is highly flexible and samples multiple conformational states. Empirical valence bond simulations of the first reaction step show that slight displacements away from the fully closed-loop conformation can be sufficient to abolish most of the catalytic activity; full closure is required for efficient reaction. The conformational change of the loops in TIM is thus not a simple “open and shut” case and is crucial for its catalytic action. Our detailed analysis of loop motion in a highly efficient enzyme highlights the complexity of loop conformational changes and their role in biological catalysis.
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| 7. |
- Pfeiffer, Martin, et al.
(author)
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Essential Functional Interplay of the Catalytic Groups in Acid Phosphatase
- 2022
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In: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; 12:6, s. 3357-3370
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Journal article (peer-reviewed)abstract
- The cooperative interplay between the functional devices of a preorganized active site is fundamental to enzyme catalysis. An in-depth understanding of this phenomenon is central to elucidating the remarkable efficiency of natural enzymes and provides an essential benchmark for enzyme design and engineering. Here, we study the functional interconnectedness of the catalytic nucleophile (His18) in an acid phosphatase by analyzing the consequences of its replacement with aspartate. We present crystallographic, biochemical, and computational evidence for a conserved mechanistic pathway via a phospho-enzyme intermediate on Asp18. Linear free-energy relationships for phosphoryl transfer from phosphomonoester substrates to His18/Asp18 provide evidence for the cooperative interplay between the nucleophilic and general-acid catalytic groups in the wild-type enzyme, and its substantial loss in the H18D variant. As an isolated factor of phosphatase efficiency, the advantage of a histidine compared to an aspartate nucleophile is similar to 10(4)-fold. Cooperativity with the catalytic acid adds >= 10(2)-fold to that advantage. Empirical valence bond simulations of phosphoryl transfer from glucose 1-phosphate to His and Asp in the enzyme explain the loss of activity of the Asp18 enzyme through a combination of impaired substrate positioning in the Michaelis complex, as well as a shift from early to late protonation of the leaving group in the H18D variant. The evidence presented furthermore suggests that the cooperative nature of catalysis distinguishes the enzymatic reaction from the corresponding reaction in solution and is enabled by the electrostatic preorganization of the active site. Our results reveal sophisticated discrimination in multifunctional catalysis of a highly proficient phosphatase active site.
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| 8. |
- Longo, Liam M., et al.
(author)
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Short and simple sequences favored the emergence of N-helix phospho-ligand binding sites in the first enzymes
- 2020
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In: Proceedings of the National Academy of Sciences of the United States of America. - : NATL ACAD SCIENCES. - 0027-8424 .- 1091-6490. ; 117:10, s. 5310-5318
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Journal article (peer-reviewed)abstract
- The ubiquity of phospho-ligands suggests that phosphate binding emerged at the earliest stage of protein evolution. To evaluate this hypothesis and unravel its details, we identified all phosphate-binding protein lineages in the Evolutionary Classification of Protein Domains database. We found at least 250 independent evolutionary lineages that bind small molecule cofactors and metabolites with phosphate moieties. For many lineages, phosphate binding emerged later as a niche functionality, but for the oldest protein lineages, phosphate binding was the founding function. Across some 4 billion y of protein evolution, side-chain binding, in which the phosphate moiety does not interact with the backbone at all, emerged most frequently. However, in the oldest lineages, and most characteristically in alpha beta alpha sandwich enzyme domains, N-helix binding sites dominate, where the phosphate moiety sits atop the N terminus of an alpha-helix. This discrepancy is explained by the observation that N-helix binding is uniquely realized by short, contiguous sequences with reduced amino acid diversity, foremost Gly, Ser, and Thr. The latter two amino acids preferentially interact with both the backbone amide and the side-chain hydroxyl (bidentate interaction) to promote binding by short sequences. We conclude that the first alpha beta alpha sandwich domains emerged from shorter and simpler polypeptides that bound phospho-ligands via N-helix sites.
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| 9. |
- Crean, Rory M., et al.
(author)
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KIF-Key Interactions Finder : A program to identify the key molecular interactions that regulate protein conformational changes
- 2023
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In: Journal of Chemical Physics. - : American Institute of Physics (AIP). - 0021-9606 .- 1089-7690. ; 158:14
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Journal article (peer-reviewed)abstract
- Simulation datasets of proteins (e.g., those generated by molecular dynamics simulations) are filled with information about how a non-covalent interaction network within a protein regulates the conformation and, thus, function of the said protein. Most proteins contain thousands of non-covalent interactions, with most of these being largely irrelevant to any single conformational change. The ability to automatically process any protein simulation dataset to identify non-covalent interactions that are strongly associated with a single, defined conformational change would be a highly valuable tool for the community. Furthermore, the insights generated from this tool could be applied to basic research, in order to improve understanding of a mechanism of action, or for protein engineering, to identify candidate mutations to improve/alter the functionality of any given protein. The open-source Python package Key Interactions Finder (KIF) enables users to identify those non-covalent interactions that are strongly associated with any conformational change of interest for any protein simulated. KIF gives the user full control to define the conformational change of interest as either a continuous variable or categorical variable, and methods from statistics or machine learning can be applied to identify and rank the interactions and residues distributed throughout the protein, which are relevant to the conformational change. Finally, KIF has been applied to three diverse model systems (protein tyrosine phosphatase 1B, the PDZ3 domain, and the KE07 series of Kemp eliminases) in order to illustrate its power to identify key features that regulate functionally important conformational dynamics.
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| 10. |
- Baier, Florian, et al.
(author)
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Cryptic genetic variation shapes the adaptive evolutionary potential of enzymes
- 2019
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In: eLIFE. - : ELIFE SCIENCES PUBLICATIONS LTD. - 2050-084X. ; 8
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Journal article (peer-reviewed)abstract
- Genetic variation among orthologous proteins can cause cryptic phenotypic properties that only manifest in changing environments. Such variation may impact the evolvability of proteins, but the underlying molecular basis remains unclear. Here, we performed comparative directed evolution of four orthologous metallo-beta-lactamases toward a new function and found that different starting genotypes evolved to distinct evolutionary outcomes. Despite a low initial fitness, one ortholog reached a significantly higher fitness plateau than its counterparts, via increasing catalytic activity. By contrast, the ortholog with the highest initial activity evolved to a less-optimal and phenotypically distinct outcome through changes in expression, oligomerization and activity. We show how cryptic molecular properties and conformational variation of active site residues in the initial genotypes cause epistasis, that could lead to distinct evolutionary outcomes. Our work highlights the importance of understanding the molecular details that connect genetic variation to protein function to improve the prediction of protein evolution.
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