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Kulkarni, Yashraj
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Publications (7 of 7) Show all publications
Parkash, V., Kulkarni, Y., ter Beek, J., Shcherbakova, P. V., Kamerlin, S. C. L. & Johansson, E. (2019). Structural consequence of the most frequently recurring cancer-associated substitution in DNA polymerase epsilon. Nature Communications, 10, Article ID 373.
Open this publication in new window or tab >>Structural consequence of the most frequently recurring cancer-associated substitution in DNA polymerase epsilon
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2019 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 10, article id 373Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2019
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-376827 (URN)10.1038/s41467-018-08114-9 (DOI)000456286400001 ()30670696 (PubMedID)
Funder
Knut and Alice Wallenberg FoundationSwedish Research CouncilSwedish Cancer SocietyNIH (National Institute of Health), ES015869
Available from: 2019-02-11 Created: 2019-02-11 Last updated: 2019-02-11Bibliographically approved
Osterlund, N., Kulkarni, Y. S., Misiaszek, A. D., Wallin, C., Krueger, D. M., Liao, Q., . . . Graslund, A. (2018). Amyloid-beta Peptide Interactions with Amphiphilic Surfactants: Electrostatic and Hydrophobic Effects. ACS Chemical Neuroscience, 9(7), 1680-1692
Open this publication in new window or tab >>Amyloid-beta Peptide Interactions with Amphiphilic Surfactants: Electrostatic and Hydrophobic Effects
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2018 (English)In: ACS Chemical Neuroscience, ISSN 1948-7193, E-ISSN 1948-7193, Vol. 9, no 7, p. 1680-1692Article in journal (Refereed) Published
Abstract [en]

The amphiphilic nature of the amyloid-beta (A beta) peptide associated with Alzheimer's disease facilitates various interactions with biomolecules such as lipids and proteins, with effects on both structure and toxicity of the peptide. Here, we investigate these peptide-amphiphile interactions by experimental and computational studies of A beta(1-40) in the presence of surfactants with varying physicochemical properties. Our findings indicate that electrostatic peptide-surfactant interactions are required for coclustering and structure induction in the peptide and that the strength of the interaction depends on the surfactant net charge. Both aggregation-prone peptide-rich coclusters and stable surfactant-rich coclusters can form. Only A beta(1-40) monomers, but not oligomers, are inserted into surfactant micelles in this surfactant-rich state. Surfactant headgroup charge is suggested to be important as electrostatic peptide-surfactant interactions on the micellar surface seems to be an initiating step toward insertion. Thus, no peptide insertion or change in peptide secondary structure is observed using a nonionic surfactant. The hydrophobic peptide-surfactant interactions instead stabilize the A beta monomer, possibly by preventing self-interaction between the peptide core and C terminus, thereby effectively inhibiting the peptide aggregation process. These findings give increased understanding regarding the molecular driving forces for A beta aggregation and the peptide interaction with amphiphilic biomolecules.

Place, publisher, year, edition, pages
AMER CHEMICAL SOC, 2018
Keywords
Alzheimer's disease, A beta aggregation, surfactant interactions, optical and NMR spectroscopy, mass spectrometry, molecular dynamics simulations
National Category
Neurosciences
Identifiers
urn:nbn:se:uu:diva-386225 (URN)10.1021/acschemneuro.8b00065 (DOI)000439531400017 ()29683649 (PubMedID)
Funder
Swedish Research Council
Available from: 2019-06-19 Created: 2019-06-19 Last updated: 2019-06-19Bibliographically approved
Liao, Q., Kulkarni, Y., Sengupta, U., Petrovic, D., Mulholland, A. J., van der Kamp, M. W., . . . Kamerlin, S. C. L. (2018). Loop Motion in Triosephosphate Isomerase Is Not a Simple Open and Shut Case. Journal of the American Chemical Society, 140(46), 15889-15903
Open this publication in new window or tab >>Loop Motion in Triosephosphate Isomerase Is Not a Simple Open and Shut Case
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2018 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 140, no 46, p. 15889-15903Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-367313 (URN)10.1021/jacs.8b09378 (DOI)000451496800048 ()30362343 (PubMedID)
Funder
Swedish Research CouncilSwedish National Infrastructure for Computing (SNIC), 2016/1-293Swedish National Infrastructure for Computing (SNIC), 2017/12-11
Available from: 2018-11-29 Created: 2018-11-29 Last updated: 2019-01-24Bibliographically approved
Kulkarni, Y. S., Liao, Q., Bylehn, F., Amyes, T. L., Richard, J. P. & Kamerlin, S. C. L. (2018). Role of Ligand-Driven Conformational Changes in Enzyme Catalysis: Modeling the Reactivity of the Catalytic Cage of Triosephosphate Isomerase. Journal of the American Chemical Society, 140(11), 3854-3857
Open this publication in new window or tab >>Role of Ligand-Driven Conformational Changes in Enzyme Catalysis: Modeling the Reactivity of the Catalytic Cage of Triosephosphate Isomerase
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2018 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 140, no 11, p. 3854-3857Article in journal (Refereed) Published
Abstract [en]

We have previously performed empirical valence bond calculations of the kinetic activation barriers, Delta G(calc) double dagger, for the deprotonation of complexes between TIM and the whole substrate glyceraldehyde-3-phosphate (GAP, Kulkarni et al. J. Am. Chem. Soc. 2017, 139, 10514-10525). We now extend this work to also study the deprotonation of the substrate pieces glycolaldehyde (GA) and GA.HPi [HPi = phosphite dianion]. Our combined calculations provide activation barriers, Delta G(calc)(double dagger) for the TIM-catalyzed deprotonation of GAP (12.9 +/- 0.8 kcal.mol(-1)), of the substrate piece GA (15.0 +/- 2.4 kcal.mol(-1)), and of the pieces GA.HP, (15.5 +/- 3.5 kcal.mol(-1)). The effect of bound dianion on Delta G(calc) double dagger is small (<= 2.6 kcal.mol(-1)), in comparison to the much larger 12.0 and 5.8 kcal.mol(-1) intrinsic phosphodianion and phosphite dianion binding energy utilized to stabilize the transition states for TIM-catalyzed deprotonation of GAP and GA. HP, respectively. This shows that the dianion binding energy is essentially fully expressed at our protein model for the Michaelis complex, where it is utilized to drive an activating change in enzyme conformation. The results represent an example of the synergistic use of results from experiments and calculations to advance our understanding of enzymatic reaction mechanisms.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2018
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-354362 (URN)10.1021/jacs.8b00251 (DOI)000428356000010 ()29516737 (PubMedID)
Funder
Swedish Research Council, 2015-04928
Available from: 2018-06-25 Created: 2018-06-25 Last updated: 2018-06-25Bibliographically approved
Amrein, B. A., Steffen-Munsberg, F., Szeler, I., Purg, M., Kulkarni, Y. & Kamerlin, S. C. (2017). CADEE: Computer-Aided Directed Evolution of Enzymes. IUCrJ, 4(1), 50-64
Open this publication in new window or tab >>CADEE: Computer-Aided Directed Evolution of Enzymes
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2017 (English)In: IUCrJ, ISSN 0972-6918, E-ISSN 2052-2525, Vol. 4, no 1, p. 50-64Article in journal (Refereed) Published
Abstract [en]

The tremendous interest in enzymes as biocatalysts has led to extensive work in enzyme engineering, as well as associated methodology development. Here, a new framework for computer-aided directed evolution of enzymes (CADEE) is presented which allows a drastic reduction in the time necessary to prepare and analyze in silico semi-automated directed evolution of enzymes. A pedagogical example of the application of CADEE to a real biological system is also presented in order to illustrate the CADEE workflow.

Keywords
computational directed evolution, computational enzyme design, distributed computing, empirical valence bond, triosephosphate isomerase
National Category
Structural Biology Bioinformatics (Computational Biology) Theoretical Chemistry
Identifiers
urn:nbn:se:uu:diva-314218 (URN)10.1107/S2052252516018017 (DOI)000392925800007 ()
Funder
EU, FP7, Seventh Framework Programme, 306474Knut and Alice Wallenberg FoundationThe Royal Swedish Academy of SciencesSwedish Research Council, 2015-04928Swedish National Infrastructure for Computing (SNIC), 2015/16-12
Available from: 2017-01-31 Created: 2017-01-31 Last updated: 2018-01-13Bibliographically approved
Kulkarni, Y. S., Liao, Q., Petrovic, D., Krüger, D. M., Strodel, B., Amyes, T. L., . . . Kamerlin, S. C. L. (2017). Enzyme Architecture: Modeling the Operation of a Hydrophobic Clamp in Catalysis by Triosephosphate Isomerase. Journal of the American Chemical Society, 139(30), 10514-10525
Open this publication in new window or tab >>Enzyme Architecture: Modeling the Operation of a Hydrophobic Clamp in Catalysis by Triosephosphate Isomerase
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2017 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 139, no 30, p. 10514-10525Article in journal (Refereed) Published
Abstract [en]

Triosephosphate isomerase (TIM) is a proficient catalyst of the reversible isomerization of dihydroxyacetone phosphate (DHAP) to D-glyceraldehyde phosphate (GAP), via general base catalysis by E165. Historically, this enzyme has been an extremely important model system for understanding the fundamentals of biological catalysis. TIM is activated through an energetically demanding conformational change, which helps position the side chains of two key hydrophobic residues (1170 and L230), over the carboxylate side chain of E165. This is critical both for creating a hydrophobic pocket for the catalytic base and for maintaining correct active site architecture. Truncation of these residues to alanine causes significant falloffs in TIM's catalytic activity, but experiments have failed to provide a full description of the action of this clamp in promoting substrate deprotonation. We perform here detailed empirical valence bond calculations of the TIM-catalyzed deprotonation of DHAP and GAP by both wild type TIM and its 1170A, L230A, and 1170A/L230A mutants, obtaining exceptional quantitative agreement with experiment. Our calculations provide a linear free energy relationship, with slope 0.8, between the activation barriers and Gibbs free energies for these TIM-catalyzed reactions. We conclude that these clamping side chains minimize the Gibbs free energy for substrate deprotonation, and that the effects on reaction driving force are largely expressed at the transition state for proton transfer. Our combined analysis of previous experimental and current computational results allows us to provide an overview of the breakdown of ground-state and transition state effects in enzyme catalysis in unprecedented detail, providing a molecular description of the operation of a hydrophobic clamp in triosephosphate isomerase.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2017
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-334051 (URN)10.1021/jacs.7b05576 (DOI)000407089500046 ()28683550 (PubMedID)
Available from: 2017-11-21 Created: 2017-11-21 Last updated: 2017-11-21Bibliographically approved
Wallin, C., Kulkarni, Y. S., Abelein, A., Jarvet, J., Liao, Q., Strodel, B., . . . Wärmländer, S. K. T. (2016). Characterization of Mn(II) ion binding to the amyloid-beta peptide in Alzheimer's disease. Journal of Trace Elements in Medicine and Biology, 38, 183-193
Open this publication in new window or tab >>Characterization of Mn(II) ion binding to the amyloid-beta peptide in Alzheimer's disease
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2016 (English)In: Journal of Trace Elements in Medicine and Biology, ISSN 0946-672X, E-ISSN 1878-3252, Vol. 38, p. 183-193Article in journal (Refereed) Published
Abstract [en]

Growing evidence links neurodegenerative diseases to metal exposure. Aberrant metal ion concentrations have been noted in Alzheimer's disease (AD) brains, yet the role of metals in AD pathogenesis remains unresolved. A major factor in AD pathogenesis is considered to be aggregation of and amyloid formation by amyloid-beta (A beta) peptides. Previous studies have shown that A beta displays specific binding to Cu(II) and Zn(II) ions, and such binding has been shown to modulate A beta aggregation. Here, we use nuclear magnetic resonance (NMR) spectroscopy to show that Mn(II) ions also bind to the N-terminal part of the A beta(1-40) peptide, with a weak binding affinity in the milli- to micromolar range. Circular dichroism (CD) spectroscopy, solid state atomic force microscopy (AFM), fluorescence spectroscopy, and molecular modeling suggest that the weak binding of Mn(II) to A beta may not have a large effect on the peptide's aggregation into amyloid fibrils. However, identification of an additional metal ion displaying A beta binding reveals more complex AD metal chemistry than has been previously considered in the literature.

Keywords
Manganese, Neurodegeneration, Metal-protein binding, Spectroscopy, Molecular dynamics
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-308063 (URN)10.1016/j.jtemb.2016.03.009 (DOI)000385473600023 ()27085215 (PubMedID)
Available from: 2016-11-30 Created: 2016-11-23 Last updated: 2018-12-04Bibliographically approved
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