Exploiting Protein Engineering and Crystal Polymorphism for Successful X-ray Structure Determination

Bonnefond, L.; Schellenberger, Pascale; Basquin, J.; Demangeat, Gérard; Ritzenthaler, Christophe; Chênevert, Robert; Balg, Christian; Frugier, Magali; Rudinger-Thirion, Joëlle; Giegé, Richard; Lorber, Bernard; Sauter, Claude

doi:10.1021/cg101468p

Cited by 12 publications

(14 citation statements)

References 52 publications

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“…Our previous analyses of human WT mt-AspRS had revealed that the synthetic analog of the aminoacylation intermediate aspartyl-adenylate, Asp-AMS, binds cooperatively 28 and increases the thermal stability of the protein by as much as 6 °C 26 . This family of compounds is commonly used in crystallographic studies to rigidify the region around the catalytic site 29 30 . Here, mutants R58G, T136S, L613F and L626Q were stabilized to the same extent as the WT protein, whereas mutants Q184K and R263Q were not stabilized at all by Asp-AMS.…”

Section: Discussionmentioning

confidence: 99%

Neurodegenerative disease-associated mutants of a human mitochondrial aminoacyl-tRNA synthetase present individual molecular signatures

Sauter

Lorber

Gaudry

et al. 2015

Sci Rep

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Mutations in human mitochondrial aminoacyl-tRNA synthetases are associated with a variety of neurodegenerative disorders. The effects of these mutations on the structure and function of the enzymes remain to be established. Here, we investigate six mutants of the aspartyl-tRNA synthetase correlated with leukoencephalopathies. Our integrated strategy, combining an ensemble of biochemical and biophysical approaches, reveals that mutants are diversely affected with respect to their solubility in cellular extracts and stability in solution, but not in architecture. Mutations with mild effects on solubility occur in patients as allelic combinations whereas those with strong effects on solubility or on aminoacylation are necessarily associated with a partially functional allele. The fact that all mutations show individual molecular and cellular signatures and affect amino acids only conserved in mammals, points towards an alternative function besides aminoacylation.

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Section: Discussionmentioning

confidence: 99%

Neurodegenerative disease-associated mutants of a human mitochondrial aminoacyl-tRNA synthetase present individual molecular signatures

Sauter

Lorber

Gaudry

et al. 2015

Sci Rep

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“…The mutation of proteins and protein complexes so that they will consistently yield diffraction-quality crystals is an approach that has been in increasingly wide use (Dale et al, 2003;Bonnefond et al, 2011). One method, which was first suggested by Derewenda, is the engineering of protein surfaces (Derewenda, 2004).…”

Section: Introductionmentioning

confidence: 99%

The structure of an MDM2–Nutlin-3a complex solved by the use of a validated MDM2 surface-entropy reduction mutant

Anil

Riedinger

Endicott

et al. 2013

Acta Crystallogr D Biol Crystallogr

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The p53-binding site of MDM2 holds great promise as a target for therapeutic intervention in MDM2-amplified p53 wild-type forms of cancer. Despite the extensive validation of this strategy, there are relatively few crystallographically determined co-complex structures for small-molecular inhibitors of the MDM2-p53 interaction available in the PDB. Here, a surface-entropy reduction mutant of the N-terminal domain of MDM2 that has been designed to enhance crystallogenesis is presented. This mutant has been validated by comparative ligand-binding studies using differential scanning fluorimetry and fluorescence polarization anisotropy and by cocrystallization with a peptide derived from p53. Using this mutant, the cocrystal structure of MDM2 with the benchmark inhibitor Nutlin-3a has been determined, revealing subtle differences from the previously described co-complex of MDM2 with Nutlin-2.

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“…Biophysical approaches to understand the effect of these mutations on enzyme properties have proven that they alter the stability and solubility of the enzyme. The aminoacylation-intermediate complex has been shown to have a stabilizing effect on structure (32)(33)(34) and is also used frequently for structural studies to stabilize the catalytic region (35,36). The aggregation kinetics data of the mutants at 37 C in the presence of L-Phe and ATP by both static light scattering (SLS) and DLS experiments (Figures 6a-d and 7) suggested that binding of the Phe-AMP complex does not have as pronounced an effect on stability in the case of the pathogenic variants as was seen for the WT HsmitPheRS.…”

Section: Discussionmentioning

confidence: 99%

Biophysical characterization Of Alpers encephalopathy associated mutants of human mitochondrial phenylalanyl‐tRNA synthetase

2019

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Mutations in nucleus‐encoded mitochondrial aminoacyl‐tRNA synthetases (mitaaRSs) lead to defects in mitochondrial translation affecting the expression and function of 13 subunits of the respiratory chain complex leading to diverse pathological conditions. Mutations in the FARS2 gene encoding human mitochondrial phenylalanyl‐tRNA synthetase (HsmitPheRS) have been found to be associated with two different clinical representations, infantile Alpers encephalopathy and spastic paraplegia. Here we have studied three pathogenic mutants (Tyr144Cys, Ile329Thr, and Asp391Val) associated with Alpers encephalopathy to understand how these variants affect the biophysical properties of the enzyme. These mutants have already been reported to have reduced aminoacylation activity. Our study established that the mutants are significantly more thermolabile compared to the wild‐type enzyme with reduced solubility in vitro. The presence of aggregation‐prone insoluble HsmitPheRS variants could have a detrimental impact on organellar translation, and potentially impact normal mitochondrial function. © 2019 IUBMB Life, 71(8): 1141–1149, 2019 © 2019 IUBMB Life, 71(8):1141–1149, 2019

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Exploiting Protein Engineering and Crystal Polymorphism for Successful X-ray Structure Determination

Cited by 12 publications

References 52 publications

Neurodegenerative disease-associated mutants of a human mitochondrial aminoacyl-tRNA synthetase present individual molecular signatures

Neurodegenerative disease-associated mutants of a human mitochondrial aminoacyl-tRNA synthetase present individual molecular signatures

The structure of an MDM2–Nutlin-3a complex solved by the use of a validated MDM2 surface-entropy reduction mutant

Biophysical characterization Of Alpers encephalopathy associated mutants of human mitochondrial phenylalanyl‐tRNA synthetase

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