Author:
Pacheco-García Juan Luis,Loginov Dmitry S.,Naganathan Athi N.,Vankova Pavla,Cano-Muñoz Mario,Man Petr,Pey Angel L.
Abstract
AbstractPhosphoglycerate kinase has been a model for the stability, folding cooperativity and catalysis of a two-domain protein. The human isoform 1 (hPGK1) is associated with cancer development and rare genetic diseases that affect several of its features. To investigate how mutations affect hPGK1 folding landscape and interaction networks, we have introduced mutations at a buried site in the N-terminal domain (F25 mutants) that either created cavities (F25L, F25V, F25A), enhanced conformational entropy (F25G) or introduced structural strain (F25W) and evaluated their effects using biophysical experimental and theoretical methods. All F25 mutants folded well, but showed reduced unfolding cooperativity, kinetic stability and altered activation energetics according to the results from thermal and chemical denaturation analyses. These alterations correlated well with the structural perturbation caused by mutations in the N-terminal domain and the destabilization caused in the interdomain interface as revealed by H/D exchange under native conditions. Importantly, experimental and theoretical analyses showed that these effects are significant even when the perturbation is mild and local. Our approach will be useful to establish the molecular basis of hPGK1 genotype–phenotype correlations due to phosphorylation events and single amino acid substitutions associated with disease.
Funder
ERDF/Spanish Ministry of Science, Innovation and Universities—State Research Agency
Consejería de Economía, Conocimiento, Empresas y Universidad, Junta de Andalucía
ERDF/ Counseling of Economic transformation, Industry, Knowledge and Universities, Junta de Andalucía
Horizon 2020 EU_FT-ICR_MS project
EU/MEYS projects BioCeV
EU/MEYS CIISB
the Science and Engineering Research Board, SERB, India
Publisher
Springer Science and Business Media LLC
Cited by
4 articles.
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