Decoding the intricate network of molecular interactions of a hyperstable engineered biocatalyst

Marková, Klára; Chmelova, Klaudia; Marques, Sérgio M.; Carpentier, Philippe; Bednář, David; Damborský, Jiřı́; Marek, Martin

doi:10.1039/d0sc03367g

Cited by 15 publications

(36 citation statements)

References 77 publications

(108 reference statements)

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“…A recent structural study carried out on the monomer of DhaA115 revealed an intricate network of molecular interactions that reinforce the engineered αβα sandwich architecture. 26 Mutations to bulky aromatic amino acids at the protein surface trigger long-distance backbone changes through multiple cooperative interactions. These interactions produce an unprecedented double-lock system that closes the molecular gates to the active site and reduces the volumes of the access tunnels 26 .…”

Section: Discussionmentioning

confidence: 99%

“…In this study, we show that the computationally-driven stabilization of a monomeric haloalkane dehalogenase DhaA115 (Tm app = 73.5°C; ΔTm app > 23°C) 5,26 unintentionally altered the protein folding landscape, resulting in the formation of stable domain-swapped intermediates. Our structural findings reveal that the intended stabilizing mutations were frequently found in the cryptic hinge regions and introduced secondary interfaces where they made new non-covalent interactions between the misfolded, intertwined polypeptide chains.…”

Section: Introductionmentioning

confidence: 89%

“…We have already reported that our previous computer-aided engineering of a haloalkane dehalogenase DhaA from Rhodococcus rhodochrous was unexpectedly accompanied by de novo formation of enzyme oligomers. 26,27 While the DhaA wild-type enzyme (Tm app = ~50.4°C) exists solely in a monomeric form, the most stabilized 11-point mutant DhaA115 (Tm app = ~73.4°C) exists in monomeric (~75.7%), dimeric (~20.3%) and higher oligomeric (~4.0%) forms (Figure 1 and Table S1). A single band of ~35 kDa can be detected using electrophoresis in denaturing conditions for both wild-type DhaA and DhaA115, while native non-denaturing electrophoretic analysis revealed multiple distinct oligomeric states of hyperstable DhaA115 (Figure 1A).…”

Section: De Novo Oligomerization Due To Computer-aided Stabilizationmentioning

confidence: 99%

“…Interestingly, the two domain-swapped dimers deviated from their respective crystal structures considerably with RMSD fluctuations above 2-3 Å, in contrast to the relatively rigid structures of monomeric DhaA115 and wild-type DhaA (RMSD below 1 Å). 26 To investigate the main reason for such large deviations, we clustered those trajectories by the RMSD and analyzed the centroid structures (Figures S3 and S4). We found that the structural changes consisted mainly of the repositioning of the two globular units with respect to each other, while each catalytic unit remained very stable and superimposable with itself (Figures S3C and S4C).…”

Section: Dhaa115 Oligomerizes Through Domain-swapping Mechanismmentioning

confidence: 99%

“…Due to the reduction of enzyme access tunnels 26 , the catalytic activity of DhaA115 at ambient temperature was diminished compared to the wild-type DhaA. 27 To find out how the domain swapping affects catalysis, the dehalogenase activities of the monomeric and dimeric DhaA115 forms were assayed with five halogenated substrates.…”

Section: Domain-swapped Dimers Exhibit Enhanced Catalytic Efficiencymentioning

confidence: 99%

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Computational Protein Stabilization Can Affect Folding Energy Landscapes and Lead to Domain-Swapped Dimers

Marková¹,

Kunka²,

Chmelova³

et al. 2021

Preprint

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The functionality of a protein depends on its unique three-dimensional structure, which is a result of the folding process when the nascent polypeptide follows a funnel-like energy landscape to reach a global energy minimum. Computer-encoded algorithms are increasingly employed to stabilize native proteins for use in research and biotechnology applications. Here, we reveal a unique example where the computational stabilization of a monomeric α/β-hydrolase enzyme (Tm = 73.5°C; ΔTm > 23°C) affected the protein folding energy landscape. Introduction of eleven single-point stabilizing mutations based on force field calculations and evolutionary analysis yielded catalytically active domain-swapped intermediates trapped in local energy minima. Crystallographic structures revealed that these stabilizing mutations target cryptic hinge regions and newly introduced secondary interfaces, where they make extensive non-covalent interactions between the intertwined misfolded protomers. The existence of domain-swapped dimers in a solution is further confirmed experimentally by data obtained from SAXS and crosslinking mass spectrometry. Unfolding experiments showed that the domain-swapped dimers can be irreversibly converted into native-like monomers, suggesting that the domain-swapping occurs exclusively in vivo. Our findings uncovered hidden protein-folding consequences of computational protein design, which need to be taken into account when applying a rational stabilization to proteins of biological and pharmaceutical interest.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 89%

Section: De Novo Oligomerization Due To Computer-aided Stabilizationmentioning

confidence: 99%

Section: Dhaa115 Oligomerizes Through Domain-swapping Mechanismmentioning

confidence: 99%

Section: Domain-swapped Dimers Exhibit Enhanced Catalytic Efficiencymentioning

confidence: 99%

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Computational Protein Stabilization Can Affect Folding Energy Landscapes and Lead to Domain-Swapped Dimers

Marková¹,

Kunka²,

Chmelova³

et al. 2021

Preprint

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Protein Engineering of Enzyme Robustness Relevant to Organic and Pharmaceutical Chemistry and Applications in Biotechnology

2023

Enzyme Engineering

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Multimeric structure of a subfamily III haloalkane dehalogenase‐like enzyme solved by combination of cryo‐EM and x‐ray crystallography

et al. 2023

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Haloalkane dehalogenase (HLD) enzymes employ an SN2 nucleophilic substitution mechanism to erase halogen substituents in diverse organohalogen compounds. Subfamily I and II HLDs are well‐characterized enzymes, but a mode and purpose of multimerization of subfamily III HLDs are unknown. Here we probe the structural organization of DhmeA, a subfamily III HLD‐like enzyme from the archaeon Haloferax mediterranei, by combining cryo‐electron microscopy (cryo‐EM) and X‐ray crystallography. We show that full‐length wild‐type DhmeA forms diverse quaternary structures, ranging from small oligomers to large supramolecular ring‐like assemblies of various sizes and symmetries. We optimized sample preparation steps, enabling three‐dimensional reconstructions of an oligomeric species by single‐particle cryo‐EM. Moreover, we engineered a crystallizable mutant (DhmeAΔGG) that provided diffraction‐quality crystals. The 3.3 Å crystal structure reveals that DhmeAΔGG forms a ring‐like 20‐mer structure with outer and inner diameter of ~200 Å and ~80 Å, respectively. An enzyme homodimer represents a basic repeating building unit of the crystallographic ring. Three assembly interfaces (dimerization, tetramerization and multimerization) were identified to form the supramolecular ring that displays a negatively charged exterior, while its interior part harboring catalytic sites is positively charged. Localization and exposure of catalytic machineries suggest a possible processing of large negatively charged macromolecular substrates.This article is protected by copyright. All rights reserved.

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Decoding the intricate network of molecular interactions of a hyperstable engineered biocatalyst

Abstract: Computational design of protein catalysts with enhanced stabilities for use in research and enzyme technologies is a challenging task. Using force-field calculations and phylogenetic analysis, we previously designed the haloalkane...

Cited by 15 publications

References 77 publications

Computational Protein Stabilization Can Affect Folding Energy Landscapes and Lead to Domain-Swapped Dimers

Computational Protein Stabilization Can Affect Folding Energy Landscapes and Lead to Domain-Swapped Dimers

Protein Engineering of Enzyme Robustness Relevant to Organic and Pharmaceutical Chemistry and Applications in Biotechnology

Multimeric structure of a subfamily III haloalkane dehalogenase‐like enzyme solved by combination of cryo‐EM and x‐ray crystallography

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