Predictive approach for protein aggregation: Correlation of protein surface characteristics and conformational flexibility to protein aggregation propensity

Galm, Lara; Amrhein, Sven; Hubbuch, Jürgen

doi:10.1002/bit.25949

Cited by 25 publications

(5 citation statements)

References 46 publications

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“…There was precipitate visible by the end of each ITC titration, and the stoichiometry of inhibitor binding indicated that only 10% of the protein was able to bind ligands by the end of the titration. This was perhaps not surprising considering the conformational flexibility of the unliganded protein observed by HDX (see below) and the known connection between highly flexible peptides and susceptibility to aggregation, − as well as the fact that even freshly purified DAHPS(Phe) was previously found to be only 30% active . Protein inactivation during the titration might be expected to alter the apparent K d values; however, the fitted K d values were close to those determined by other methods, with K d,Mn = 5.3 ± 1.7 μM and K d,DAHP oxime = 3.1 ± 1.2 μM.…”

Section: Resultssupporting

confidence: 56%

“…Previously, spatially resolved hydrogen/deuterium exchange (HDX) measurements showed that DAHPS G had unusually large amounts of conformational flexibility, including stretches that are α-helices in crystal structures . This lack of well-defined structure at the surface tends to be correlated with the propensity for aggregation and denaturation, − as was observed in the ITC experiments.…”

Section: Resultsmentioning

confidence: 89%

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Potent Inhibition of 3-Deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) Synthase by DAHP Oxime, a Phosphate Group Mimic

Balachandran¹,

Heimhalt²,

Liuni

et al. 2016

Biochemistry

147

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3-Deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) synthase catalyzes the first step in the shikimate pathway. It catalyzes an aldol-like reaction of phosphoenolpyruvate (PEP) with erythrose 4-phosphate (E4P) to form DAHP. The kinetic mechanism was rapid equilibrium sequential ordered ter ter, with the essential divalent metal ion, Mn, binding first, followed by PEP and E4P. DAHP oxime, in which an oxime group replaces the keto oxygen, was a potent inhibitor, with K = 1.5 ± 0.4 μM, though with residual activity at high inhibitor concentrations. It displayed slow-binding inhibition with a residence time, t, of 83 min. The crystal structure revealed that the oxime functional group, combined with two crystallographic waters, bound at the same location in the catalytic center as the phosphate group of the tetrahedral intermediate. DAHP synthase has a dimer-of-dimers homotetrameric structure, and DAHP oxime bound to only one subunit of each tight dimer. Inhibitor binding was competitive with respect to all three substrates in the subunits to which it bound. DAHP oxime did not overlap with the metal binding site, so the cause of their mutually exclusive binding was not clear. Similarly, there was no obvious structural reason for inhibitor binding in only two subunits; however, changes in global hydrogen/deuterium exchange showed large scale changes in protein dynamics upon inhibitor binding. The k value for the residual activity at high inhibitor concentrations was 3-fold lower, and the apparent K value decreased at least 10-fold. This positive cooperativity of binding between DAHP oxime in subunits B and C, and E4P in subunits A and D appears to be the dominant cause for incomplete inhibition at high inhibitor concentrations. In spite of its lack of obvious structural similarity to phosphate, the oxime and crystallographic waters acted as a small, neutral phosphate mimic.

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Section: Resultssupporting

confidence: 56%

Section: Resultsmentioning

confidence: 89%

Potent Inhibition of 3-Deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) Synthase by DAHP Oxime, a Phosphate Group Mimic

Balachandran¹,

Heimhalt²,

Liuni

et al. 2016

Biochemistry

147

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“…There are a lot of studies on interrelationships of protein flexibility, aggregation, and chemical stability. [244][245][246] Therefore, the method development for modeling protein flexibility is another important area in computeraided antibody design.…”

Section: Perspectivesmentioning

confidence: 99%

Engineering Stability, Viscosity, and Immunogenicity of Antibodies by Computational Design

Kuroda

Tsumoto

2020

Journal of Pharmaceutical Sciences

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In recent years, computational methods have garnered much attention in protein engineering. A large number of computational methods have been developed to analyze the sequences and structures of proteins and have been used to predict the various properties. Antibodies are one of the emergent protein therapeutics, and thus, methods to control their physicochemical properties are highly desirable. However, despite the tremendous efforts of past decades, computational methods to predict the physicochemical properties of antibodies are still in their infancy. Experimental validations are certainly required for real-world applications, and the results should be interpreted with caution. Among the various properties of antibodies, we focus in this review on stability, viscosity, and immunogenicity, and we present the current status of computational methods to engineer such properties.

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“…Aggregation propensity is directly related with protein surface hydrophobicity ( Galm et al, 2017 ). We propose that reducing the hydrophobicity of CH2 may attenuate antibody aggregation under low pH conditions.…”

Section: Discussionmentioning

confidence: 99%

Decreasing hydrophobicity or shielding hydrophobic areas of CH2 attenuates low pH-induced IgG4 aggregation

Wu,

Cao,

Wei

et al. 2023

Front. Bioeng. Biotechnol.

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Protein aggregation is a major challenge in the development of therapeutic monoclonal antibodies (mAbs). Several stressors can cause protein aggregation, including temperature shifts, mechanical forces, freezing-thawing cycles, oxidants, reductants, and extreme pH. When antibodies are exposed to low pH conditions, aggregation increases dramatically. However, low pH treatment is widely used in protein A affinity chromatography and low pH viral inactivation procedures. In the development of an IgG4 subclass antibody, mAb1-IgG4 showed a strong tendency to aggregate when temporarily exposed to low pH conditions. Our findings showed that the aggregation of mAb1-IgG4 under low pH conditions is determined by the stability of the Fc. The CH2 domain is the least stable domain in mAb1-IgG4. The L309E, Q311D, and Q311E mutations in the CH2 domain significantly reduced the aggregation propensity, which could be attributed to a reduction in the hydrophobicity of the CH2 domain. Protein stabilizers, such as sucrose and mannose, could also attenuate low pH-induced mAb1-IgG4 aggregation by shielding hydrophobic areas and increasing protein stability. Our findings provide valuable strategies for managing the aggregation of protein therapeutics with a human IgG4 backbone.

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Predictive approach for protein aggregation: Correlation of protein surface characteristics and conformational flexibility to protein aggregation propensity

Cited by 25 publications

References 46 publications

Potent Inhibition of 3-Deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) Synthase by DAHP Oxime, a Phosphate Group Mimic

Potent Inhibition of 3-Deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) Synthase by DAHP Oxime, a Phosphate Group Mimic

Engineering Stability, Viscosity, and Immunogenicity of Antibodies by Computational Design

Decreasing hydrophobicity or shielding hydrophobic areas of CH2 attenuates low pH-induced IgG4 aggregation

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