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Georgoulia, P. S., Todde, G., Bjelic, S. & Friedman, R. (2019). The catalytic activity of Abl1 single and compound mutations: Implications for the mechanism of drug resistance mutations in chronic myeloid leukaemia. Biochimica et Biophysica Acta - General Subjects, 1863(4), 732-741
Open this publication in new window or tab >>The catalytic activity of Abl1 single and compound mutations: Implications for the mechanism of drug resistance mutations in chronic myeloid leukaemia
2019 (English)In: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1863, no 4, p. 732-741Article in journal (Refereed) Published
Abstract [en]

Background

Abl1 is a protein tyrosine kinase whose aberrant activation due to mutations is the culprit of several cancers, most notably chronic myeloid leukaemia. Several Abl1 inhibitors are used as anti-cancer drugs. Unfortunately, drug resistance limits their effectiveness. The main cause for drug resistance is mutations in the kinase domain (KD) of Abl1 that evolve in patients. The T315I mutation confers resistance against all clinically-available inhibitors except ponatinib. Resistance to ponatinib can develop by compound (double) mutations.

Methods

Kinetic measurements of the KD of Abl1 and its mutants were carried out to examine their catalytic activity. Specifically, mutants that lead to drug resistance against ponatinib were considered. Molecular dynamics simulations and multiple sequence analysis were used for explanation of the experimental findings.

Results

The catalytic efficiency of the T315I pan-resistance mutant is more than two times lower than that of the native KD. All ponatinib resistant mutations restore the catalytic efficiency of the enzyme. Two of them (G250E/T315I and Y253H/E255V) have a catalytic efficiency that is more than five times that of the native KD.

Conclusions

The measurements and analysis suggest that resistance is at least partially due to the development of a highly efficient kinase through subsequent mutations. The simulations highlight modifications in two structurally important regions of Abl1, the activation and phosphate binding loops, upon mutations.

General significance

Experimental and computational methods were used together to explain how mutations in the kinase domain of Abl1 lead to resistance against the most advanced drug currently in use to treat chronic myeloid leukaemia.

Place, publisher, year, edition, pages
Elsevier, 2019
National Category
Biophysics Theoretical Chemistry Biochemistry and Molecular Biology
Research subject
Natural Science, Biomedical Sciences; Natural Science, Chemistry; Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-80308 (URN)10.1016/j.bbagen.2019.01.011 (DOI)000460853200009 ()30684523 (PubMedID)
Funder
Swedish Cancer Society, CAN 2015/387
Available from: 2019-02-07 Created: 2019-02-07 Last updated: 2019-03-29Bibliographically approved
Andre, I. & Bjelic, S. (2018). Computational assessment of folding energy landscapes in heterodimeric coiled coils. Proteins: Structure, Function, and Bioinformatics, 86(7), 790-801
Open this publication in new window or tab >>Computational assessment of folding energy landscapes in heterodimeric coiled coils
2018 (English)In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 86, no 7, p. 790-801Article in journal (Refereed) Published
Abstract [en]

The coiled coil structural motif consists of alpha helices supercoiling around each other to form staggered knobs-into-holes packing. Such structures are deceptively simple, especially as they often can be described with parametric equations, but are known to exist in various conformations. Even the simplest systems, consisting of 2 monomers, can assemble into a wide range of states. They can form canonical as well as noncanonical coiled coils, be parallel or antiparallel, where helices associate with different degrees of shift, tilt, and rotation. Here, we investigate the energy landscape of heterodimeric coiled coils by carrying out de novo folding simulations starting from amino acid sequence. We folded a diverse set of 22 heterodimers and demonstrate that the approach is capable of identifying the atomic details in the experimental structure in the majority of cases. Our methodology also enables exploration of alternative states that can be accessible in solution beyond the experimentally determined structure. For many systems, we observe folding energy landscapes with multiple energy minima and several isoenergetic states. By comparing coiled coils from single domains and those extracted from larger proteins, we find that standalone coiled coils have deeper energy wells at the experimentally determined conformation. By folding the competing homodimeric states in addition to the heterodimers, we observe that the structural specificity towards the heteromeric state is often small. Taken together, our results demonstrate that de novo folding simulations can be a powerful tool to characterize structural specificity of coiled coils when coupled to assessment of energy landscapes.

Place, publisher, year, edition, pages
Wiley-Blackwell, 2018
Keywords
coiled coil, de novo folding, energy landscapes, Rosetta, structural specificity
National Category
Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-77009 (URN)10.1002/prot.25516 (DOI)000435812700009 ()29675909 (PubMedID)
Available from: 2018-07-27 Created: 2018-07-27 Last updated: 2018-07-27Bibliographically approved
Wijma, H. J., Floor, R. J., Bjelic, S., Marrink, S. J., Baker, D. & Janssen, D. B. (2015). Enantioselective enzymes by computational design and in silico screening. Angewandte Chemie International Edition, 54(12), 3726-3730
Open this publication in new window or tab >>Enantioselective enzymes by computational design and in silico screening
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2015 (English)In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 54, no 12, p. 3726-3730Article in journal (Refereed) Published
Abstract [en]

Computational enzyme design holds great promise for providing new biocatalysts for synthetic chemistry. A strategy to design small mutant libraries of complementary enantioselective epoxide hydrolase variants for the production of highly enantioenriched (S,S)-diols and (R,R)-diols is developed. Key features of this strategy (CASCO, catalytic selectivity by computational design) are the design of mutations that favor binding of the substrate in a predefined orientation, the introduction of steric hindrance to prevent unwanted substrate binding modes, and ranking of designs by high-throughput molecular dynamics simulations. Using this strategy we obtained highly stereoselective mutants of limonene epoxide hydrolase after experimental screening of only 37 variants. The results indicate that computational methods can replace a substantial amount of laboratory work when developing enantioselective enzymes.

National Category
Chemical Sciences Biochemistry and Molecular Biology
Research subject
Natural Science, Chemistry
Identifiers
urn:nbn:se:lnu:diva-50605 (URN)10.1002/anie.201411415 (DOI)25651000 (PubMedID)
Available from: 2016-03-11 Created: 2016-03-11 Last updated: 2019-02-26Bibliographically approved
Bjelic, S. (2015). Protein carriers for passage of the Blood-Brain Barrier. Paper presented at 29th Annual Symposium of the Protein-Society, JUL 22-25, 2015, Barcelona, SPAIN. Protein Science, 24, 177-177
Open this publication in new window or tab >>Protein carriers for passage of the Blood-Brain Barrier
2015 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 24, p. 177-177Article in journal, Meeting abstract (Other academic) Published
National Category
Biochemistry and Molecular Biology
Research subject
Natural Science, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-47383 (URN)000363658100294 ()
Conference
29th Annual Symposium of the Protein-Society, JUL 22-25, 2015, Barcelona, SPAIN
Available from: 2015-11-24 Created: 2015-11-24 Last updated: 2019-02-26Bibliographically approved
Nivón, L. G., Bjelic, S., King, C. & Baker, D. (2014). Automating human intuition for protein design. Proteins: Structure, Function, and Bioinformatics, 82(5), 858-866
Open this publication in new window or tab >>Automating human intuition for protein design
2014 (English)In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 82, no 5, p. 858-866Article in journal (Refereed) Published
Abstract [en]

In the design of new enzymes and binding proteins, human intuition is often used to modify computationally designed amino acid sequences prior to experimental characterization. The manual sequence changes involve both reversions of amino acid mutations back to the identity present in the parent scaffold and the introduction of residues making additional interactions with the binding partner or backing up first shell interactions. Automation of this manual sequence refinement process would allow more systematic evaluation and considerably reduce the amount of human designer effort involved. Here we introduce a benchmark for evaluating the ability of automated methods to recapitulate the sequence changes made to computer-generated models by human designers, and use it to assess alternative computational methods. We find the best performance for a greedy one-position-at-a-time optimization protocol that utilizes metrics (such as shape complementarity) and local refinement methods too computationally expensive for global Monte Carlo (MC) sequence optimization. This protocol should be broadly useful for improving the stability and function of designed binding proteins.

National Category
Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-50608 (URN)10.1002/prot.24463 (DOI)24265170 (PubMedID)
Available from: 2016-03-11 Created: 2016-03-11 Last updated: 2019-02-26Bibliographically approved
Liu, Y., Zhang, X., Tan, Y. L., Bhabha, G., Ekiert, D. C., Kipnis, Y., . . . Kelly, J. W. (2014). De Novo-Designed Enzymes as Small-Molecule-Regulated Fluorescence Imaging Tags and Fluorescent Reporters. Journal of the American Chemical Society, 136(38), 13102-13105
Open this publication in new window or tab >>De Novo-Designed Enzymes as Small-Molecule-Regulated Fluorescence Imaging Tags and Fluorescent Reporters
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2014 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 136, no 38, p. 13102-13105Article in journal (Refereed) Published
Abstract [en]

Enzyme-based tags attached to a protein-of-interest (POI) that react with a small molecule, rendering the conjugate fluorescent, are very useful for studying the POI in living cells. These tags are typically based on endogenous enzymes, so protein engineering is required to ensure that the small-molecule probe does not react with the endogenous enzyme in the cell of interest. Here we demonstrate that de novo-designed enzymes can be used as tags to attach to POIs. The inherent bioorthogonality of the de novo-designed enzyme-small-molecule probe reaction circumvents the need for protein engineering, since these enzyme activities are not present in living organisms. Herein, we transform a family of de novo-designed retroaldolases into variable-molecular-weight tags exhibiting fluorescence imaging, reporter, and electrophoresis applications that are regulated by tailored, reactive small-molecule fluorophores.

National Category
Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-50606 (URN)10.1021/ja5056356 (DOI)25209927 (PubMedID)
Available from: 2016-03-11 Created: 2016-03-11 Last updated: 2019-02-26Bibliographically approved
Bjelic, S., Kipnis, Y., Wang, L., Pianowski, Z., Vorobiev, S., Su, M., . . . Baker, D. (2014). Exploration of Alternate Catalytic Mechanisms and Optimization Strategies for Retroaldolase Design. Journal of Molecular Biology, 426(1), 256-271
Open this publication in new window or tab >>Exploration of Alternate Catalytic Mechanisms and Optimization Strategies for Retroaldolase Design
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2014 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 426, no 1, p. 256-271Article in journal (Refereed) Published
Abstract [en]

Designed retroaldolases have utilized a nucleophilic lysine to promote carbon-carbon bond cleavage of β-hydroxy-ketones via a covalent Schiff base intermediate. Previous computational designs have incorporated a water molecule to facilitate formation and breakdown of the carbinolamine intermediate to give the Schiff base and to function as a general acid/base. Here we investigate an alternative active-site design in which the catalytic water molecule was replaced by the side chain of a glutamic acid. Five out of seven designs expressed solubly and exhibited catalytic efficiencies similar to previously designed retroaldolases for the conversion of 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone to 6-methoxy-2-naphthaldehyde and acetone. After one round of site-directed saturation mutagenesis, improved variants of the two best designs, RA114 and RA117, exhibited among the highest kcat (>10(-3)s(-1)) and kcat/KM (11-25M(-1)s(-1)) values observed for retroaldolase designs prior to comprehensive directed evolution. In both cases, the >10(5)-fold rate accelerations that were achieved are within 1-3 orders of magnitude of the rate enhancements reported for the best catalysts for related reactions, including catalytic antibodies (kcat/kuncat=10(6) to 10(8)) and an extensively evolved computational design (kcat/kuncat>10(7)). The catalytic sites, revealed by X-ray structures of optimized versions of the two active designs, are in close agreement with the design models except for the catalytic lysine in RA114. We further improved the variants by computational remodeling of the loops and yeast display selection for reactivity of the catalytic lysine with a diketone probe, obtaining an additional order of magnitude enhancement in activity with both approaches.

Keywords
retroaldolase, enzyme design, protein engineering, enzyme optimization
National Category
Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-50609 (URN)10.1016/j.jmb.2013.10.012 (DOI)24161950 (PubMedID)
Available from: 2016-03-11 Created: 2016-03-11 Last updated: 2019-02-26Bibliographically approved
Liu, Y., Tan, Y. L., Zhang, X., Bhabha, G., Ekiert, D. C., Genereux, J. C., . . . Kelly, J. W. (2014). Small molecule probes to quantify the functional fraction of a specific protein in a cell with minimal folding equilibrium shifts.. Proceedings of the National Academy of Sciences of the United States of America, 111(12), 4449-4454
Open this publication in new window or tab >>Small molecule probes to quantify the functional fraction of a specific protein in a cell with minimal folding equilibrium shifts.
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2014 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 111, no 12, p. 4449-4454Article in journal (Refereed) Published
Abstract [en]

Although much is known about protein folding in buffers, it remains unclear how the cellular protein homeostasis network functions as a system to partition client proteins between folded and functional, soluble and misfolded, and aggregated conformations. Herein, we develop small molecule folding probes that specifically react with the folded and functional fraction of the protein of interest, enabling fluorescence-based quantification of this fraction in cell lysate at a time point of interest. Importantly, these probes minimally perturb a protein's folding equilibria within cells during and after cell lysis, because sufficient cellular chaperone/chaperonin holdase activity is created by rapid ATP depletion during cell lysis. The folding probe strategy and the faithful quantification of a particular protein's functional fraction are exemplified with retroaldolase, a de novo designed enzyme, and transthyretin, a nonenzyme protein. Our findings challenge the often invoked assumption that the soluble fraction of a client protein is fully folded in the cell. Moreover, our results reveal that the partitioning of destabilized retroaldolase and transthyretin mutants between the aforementioned conformational states is strongly influenced by cytosolic proteostasis network perturbations. Overall, our results suggest that applying a chemical folding probe strategy to other client proteins offers opportunities to reveal how the proteostasis network functions as a system to regulate the folding and function of individual client proteins in vivo.

Keywords
chemical probes, pharmacologic chaperone, fluorescence labeling
National Category
Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-50607 (URN)10.1073/pnas.1323268111 (DOI)24591605 (PubMedID)
Available from: 2016-03-11 Created: 2016-03-11 Last updated: 2019-02-26Bibliographically approved
Bjelic, S., Nivón, L. G., Çelebi-Ölçüm, N., Kiss, G., Rosewall, C. F., Lovick, H. M., . . . Baker, D. (2013). Computational design of enone-binding proteins with catalytic activity for the Morita-Baylis-Hillman reaction. ACS Chemical Biology, 8(4), 749-757
Open this publication in new window or tab >>Computational design of enone-binding proteins with catalytic activity for the Morita-Baylis-Hillman reaction
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2013 (English)In: ACS Chemical Biology, ISSN 1554-8929, E-ISSN 1554-8937, Vol. 8, no 4, p. 749-757Article in journal (Refereed) Published
Abstract [en]

The Morita-Baylis-Hillman reaction forms a carbon-carbon bond between the α-carbon of a conjugated carbonyl compound and a carbon electrophile. The reaction mechanism involves Michael addition of a nucleophile catalyst at the carbonyl β-carbon, followed by bond formation with the electrophile and catalyst disassociation to release the product. We used Rosetta to design 48 proteins containing active sites predicted to carry out this mechanism, of which two show catalytic activity by mass spectrometry (MS). Substrate labeling measured by MS and site-directed mutagenesis experiments show that the designed active-site residues are responsible for activity, although rate acceleration over background is modest. To characterize the designed proteins, we developed a fluorescence-based screen for intermediate formation in cell lysates, carried out microsecond molecular dynamics simulations, and solved X-ray crystal structures. These data indicate a partially formed active site and suggest several clear avenues for designing more active catalysts.

National Category
Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-50610 (URN)10.1021/cb3006227 (DOI)23330600 (PubMedID)
Available from: 2016-03-11 Created: 2016-03-11 Last updated: 2019-02-26Bibliographically approved
Richter, F., Leaver-Fay, A., Khare, S. D., Bjelic, S. & Baker, D. (2011). De novo enzyme design using Rosetta3. PLoS ONE, 6(5)
Open this publication in new window or tab >>De novo enzyme design using Rosetta3
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2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 5Article in journal (Refereed) Published
Abstract [en]

The Rosetta de novo enzyme design protocol has been used to design enzyme catalysts for a variety of chemical reactions, and in principle can be applied to any arbitrary chemical reaction of interest. The process has four stages: 1) choice of a catalytic mechanism and corresponding minimal model active site, 2) identification of sites in a set of scaffold proteins where this minimal active site can be realized, 3) optimization of the identities of the surrounding residues for stabilizing interactions with the transition state and primary catalytic residues, and 4) evaluation and ranking the resulting designed sequences. Stages two through four of this process can be carried out with the Rosetta package, while stage one needs to be done externally. Here, we demonstrate how to carry out the Rosetta enzyme design protocol from start to end in detail using for illustration the triosephosphate isomerase reaction.

National Category
Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-50611 (URN)10.1371/journal.pone.0019230 (DOI)21603656 (PubMedID)
Available from: 2016-03-11 Created: 2016-03-11 Last updated: 2019-02-26Bibliographically approved
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Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-9300-614X

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