Enhancing the promiscuity of a member of the Caspase protease family by rational design

Öhlknecht, Christoph; Petrov, Dražen; Engele, Petra; Kröß, Christina; Sprenger, Bernhard; Fischer, Andreas; Lingg, Nico; Schneider, Rainer; Oostenbrink, Chris

doi:10.1002/prot.25950

Cited by 9 publications

(17 citation statements)

References 90 publications

(102 reference statements)

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“…Ile and Thr are both β-branched and weak binders of the S1′ site ( K M of Ile/Thr in cp-Casp2: 71 ± 25/75 ± 25 μM). 7 As a consequence, the data for these two amino acids will be compared within the following analysis. While it is unfortunate that an exact comparison cannot be made, we chose not to adjust our simulations after the validation experiments were performed but to compare how relevant the predictions are in a realistic experimental setting where the exact relevant experiments may not be accessible.…”

Section: Resultsmentioning

confidence: 99%

“…We recently provided a successful example of such a rational design procedure. 7 In this work, statistical and computational methods were jointly applied to engineer human Caspase-2 (Casp-2). The task was to create a biochemical scissor that is fit to cleave fusion tags from a wide variety of proteins.…”

Section: Introductionmentioning

confidence: 99%

“…24 To develop a versatile protease that can cleave fusion tags from the N-terminus of any protein, the general aim was to identify mutations that enhance the promiscuity of the S1′ binding pocket. 7 To achieve this, we calculated changes in the binding free energy of a model substrate upon mutation of both sites while keeping the respective other site unmutated. All calculations were performed twice using two different P1′ amino acids in the substrate, Ile and Pro.…”

Section: Introductionmentioning

confidence: 99%

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Efficient In Silico Saturation Mutagenesis of a Member of the Caspase Protease Family

Öhlknecht

Katz

Kröß

et al. 2021

J. Chem. Inf. Model.

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Rational-design methods have proven to be a valuable toolkit in the field of protein design. Numerical approaches such as free-energy calculations or QM/MM methods are fit to widen the understanding of a protein-sequence space but require large amounts of computational time and power. Here, we apply an efficient method for free-energy calculations that combines the one-step perturbation (OSP) with the third-power-fitting (TPF) approach. It is fit to calculate full free energies of binding from three different end states only. The nonpolar contribution to the free energies are calculated for a set of chosen amino acids from a single simulation of a judiciously chosen reference state. The electrostatic contributions, on the other hand, are predicted from simulations of the neutral and charged end states of the individual amino acids. We used this method to perform in silico saturation mutagenesis of two sites in human Caspase-2. We calculated relative binding free energies toward two different substrates that differ in their P1′ site and in their affinity toward the unmutated protease. Although being approximate, our approach showed very good agreement upon validation against experimental data. 76% of the predicted relative free energies of amino acid mutations was found to be true positives or true negatives. We observed that this method is fit to discriminate amino acid mutations because the rate of false negatives is very low (<1.5%). The approach works better for a substrate with medium/low affinity with a Matthews correlation coefficient (MCC) of 0.63, whereas for a substrate with very low affinity, the MCC was 0.38. In all cases, the combined TPF + OSP approach outperformed the linear interaction energy method.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Efficient In Silico Saturation Mutagenesis of a Member of the Caspase Protease Family

Öhlknecht

Katz

Kröß

et al. 2021

J. Chem. Inf. Model.

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“…For an application of X-TI and corrections of netcharge changes in current research see e. g. Ref. [21].…”

Section: Tutorial 2: Double Decoupling Methods and Corrections For Net-cmentioning

confidence: 99%

A Suite of Advanced Tutorials for the GROMOS Biomolecular Simulation Software [Article v1.0]

et al. 2020

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“…Öhlknecht et al. ( 26 ) have used an in silico approach to optimize cp caspase-2. Mutations that improve the P1′ tolerance were predicted by structural comparison of different caspases and confirmed in in vitro experiments.…”

Section: Directed Evolution Screening and Selectionmentioning

confidence: 99%

PROFICS: A bacterial selection system for directed evolution of proteases

Kröß

Engele

Sprenger

et al. 2021

Journal of Biological Chemistry

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Proteases serve as important tools in biotechnology and as valuable drugs or drug targets. Efficient protein engineering methods to study and modulate protease properties are thus of great interest for a plethora of applications. We established PROFICS (PRotease Optimization via Fusion-Inhibited Carbamoyltransferase-based Selection), a bacterial selection system, which enables the optimization of proteases for biotechnology, therapeutics or diagnosis in a simple overnight process. During the PROFICS process, proteases are selected for their ability to specifically cut a tag from a reporter enzyme and leave a native N-terminus. Precise and efficient cleavage after the recognition sequence reverses the phenotype of an Escherichia coli knockout strain deficient in an essential enzyme of pyrimidine synthesis. A toolbox was generated to select for proteases with different preferences for P1′ residues (the residue immediately following the cleavage site). The functionality of PROFICS is demonstrated with viral proteases and human caspase-2. PROFICS improved caspase-2 activity up to 25-fold after only one round of mutation and selection. Additionally, we found a significantly improved tolerance for all P1′ residues caused by a mutation in a substrate interaction site. We showed that this improved activity enables cells containing the new variant to outgrow cells containing all other mutants, facilitating its straightforward selection. Apart from optimizing enzymatic activity and P1′ tolerance, PROFICS can be used to reprogram specificities, erase off-target activity, optimize expression via tags/codon usage, or even to screen for potential drug-resistance-conferring mutations in therapeutic targets such as viral proteases in an unbiased manner.

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Enhancing the promiscuity of a member of the Caspase protease family by rational design

Cited by 9 publications

References 90 publications

Efficient In Silico Saturation Mutagenesis of a Member of the Caspase Protease Family

Efficient In Silico Saturation Mutagenesis of a Member of the Caspase Protease Family

A Suite of Advanced Tutorials for the GROMOS Biomolecular Simulation Software [Article v1.0]

PROFICS: A bacterial selection system for directed evolution of proteases

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