| 1. |
- Andre, Ingemar, et al.
(författare)
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Computational assessment of folding energy landscapes in heterodimeric coiled coils
- 2018
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Ingår i: Proteins. - : Wiley-Blackwell. - 0887-3585 .- 1097-0134. ; 86:7, s. 790-801
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
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| 3. |
- Bjelic, Sinisa, et al.
(författare)
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Cold adaptation of enzyme reaction rates
- 2008
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 47:38, s. 10049-10057
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Tidskriftsartikel (refereegranskat)abstract
- A major issue for organisms living at extreme temperatures is to preserve both stability and activity of their enzymes. Cold-adapted enzymes generally have a reduced thermal stability, to counteract freezing, and show a lower enthalpy and a more negative entropy of activation compared to mesophilic and thermophilic homologues. Such a balance of thermodynamic activation parameters can make the reaction rate decrease more linearly, rather than exponentially, as the temperature is lowered, but the structural basis for rate optimization toward low working temperatures remains unclear. In order to computationally address this problem, it is clear that reaction simulations rather than standard molecular dynamics calculations are needed. We have thus carried out extensive computer simulations of the keto-enol(ate) isomerization steps in differently adapted citrate synthases to explore the structure-function relationships behind catalytic rate adaptation to different temperatures. The calculations reproduce the absolute rates of the psychrophilic and mesophilic enzymes at 300 K, as well as the lower enthalpy and more negative entropy of activation of the cold-adapted enzyme, where the latter simulation result is obtained from high-precision Arrhenius plots. The overall catalytic effect originates from electrostatic stabilization of the transition state and enolate and the reduction of reorganization free energy. The simulations, however, show psychrophilic, mesophilic, and hyperthermophilic citrate synthases to have increasingly stronger electrostatic stabilization of the transition state, while the energetic penalty in terms of internal protein interactions follows the reverse order with the cold-adapted enzyme having the most favorable energy term. The lower activation enthalpy and more negative activation entropy observed for cold-adapted enzymes are found to be associated with a decreased protein stiffness. The origin of this effect is, however, not localized to the active site but to other regions of the protein structure.
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| 4. |
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| 5. |
- Bjelic, Sinisa, et al.
(författare)
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Computational design of enone-binding proteins with catalytic activity for the Morita-Baylis-Hillman reaction
- 2013
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Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 8:4, s. 749-757
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Bjelic, Sinisa, et al.
(författare)
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Computational inhibitor design against malaria plasmepsins
- 2007
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Ingår i: Cellular and Molecular Life Sciences (CMLS). - : Springer Science and Business Media LLC. - 1420-682X .- 1420-9071. ; 64:17, s. 2285-2305
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Tidskriftsartikel (refereegranskat)abstract
- Plasmepsins are aspartic proteases involved in the degradation of the host cell hemoglobin that is used as a food source by the malaria parasite. Plasmepsins are highly promising as drug targets, especially when combined with the inhibition of falcipains that are also involved in hemoglobin catabolism. In this review, we discuss the mechanism of plasmepsins I-IV in view of the interest in transition state mimetics as potential compounds for lead development. Inhibitor development against plasmepsin II as well as relevant crystal structures are summarized in order to give an overview of the field. Application of computational techniques, especially binding affinity prediction by the linear interaction energy method, in the development of malarial plasmepsin inhibitors has been highly successful and is discussed in detail. Homology modeling and molecular docking have been useful in the current inhibitor design project, and the combination of such methods with binding free energy calculations is analyzed.
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| 7. |
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| 8. |
- Bjelic, Sinisa, et al.
(författare)
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Exploration of Alternate Catalytic Mechanisms and Optimization Strategies for Retroaldolase Design
- 2014
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 426:1, s. 256-271
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 9. |
- Bjelic, Sinisa, 1977-
(författare)
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Molecular Simulation of Enzyme Catalysis and Inhibition
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The reaction mechanisms for the hemoglobin degrading enzymes in the Plasmodium falciparum malaria parasite, plasmepsin II (Plm II) and histo-aspartic protease (HAP), have been analyzed by molecular simulations. The reaction free energy profiles, calculated by the empirical valence bond (EVB) method in combination with molecular dynamics (MD) and free energy perturbation (FEP) simulations are in good agreement with experimental data. Additional computational methods, such as homology modelling and automated substrate docking, were necessary to generate a 3D model and a reactive substrate conformation before the reaction mechanism in HAP could be investigated. HAP is found to be an aspartic protease with a peptide cleaving mechanism similar to plasmepsin II. The major difference between these enzymes is that the negatively charged tetrahedral intermediate is stabilized by the charged histidine in HAP while in Plm II it is a neutral aspartic acid. Also the reaction mechanism for two other aspartic proteases, cathepsin D and HIV-1 protease, was simulated. These enzymes are relevant both for the inhibitor selectivity and for obtaining a general picture of catalysis in aspartic proteases. Another project involves inhibitor design towards plasmepsins. In particular, Plm II directed inhibitors based on the dihydroxyethylene scaffold have been characterized computationally. Molecular dynamics (MD) simulations were used to propagate the investigated system through time and to generate ensembles used for the calculation of free energies. The ligand binding affinities were calculated with the linear interaction energy (LIE) method. The most potent inhibitor had a Ki value of 6 nM and showed 78 % parasite inhibition when tested on red blood cells infected by malaria parasite P. falciparum. Citrate synthase is part of the citric acid cycle and is present in organisms that live in cold sea water as well as hot springs. The temperature adaptation of citrate synthase to cold and heat was investigated in terms of the difference in transition state stabilization between the psychrophilic, mesophilic and hyperthermophilic homologues. The EVB, FEP and MD methods were used to generate reaction free energy profiles. The investigated energetics points toward the electrostatic stabilization during the reaction as the major difference between the different citrate synthase homologues. The electrostatic stabilization of the transition state is most effective in the following order of the citrate synthase homologues: hyperthermophile, mesophile, psycrophile. This could be a general rule for temperature adaptation of enzyme catalysis.
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