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We have recently identified BEN1 as a protein interactor of seryl-tRNA synthetase (SerRS) from model plant Arabidopsis thaliana. BEN1 contains an NADP + binding domain and possesses acidic N-terminal extension essential for interaction with A. thaliana SerRS. This extension, specific for BEN1 homologues from Brassicaceae family, is solvent-exposed and distant to the nucleotide-binding site. We prepared a truncated BEN1 variant ΔN17BEN1 lacking the first 17 amino acid of this N-terminal extension as well as full-length BEN1 to investigate how the truncation affects the binding affinity towards coenzyme NADP + . By performing microscale thermophoresis (MST) experiments we have shown that both BEN1 variants bind the NADP + cofactor, however, truncated BEN1 showed 34-fold higher affinity towards NADP + indicating that its core protein structure is not just preserved but it binds NADP + even stronger. To further corroborate the obtained results, we opted for a computational approach based on classical molecular dynamics simulations of both complexes. Our results have shown that both truncated and intact BEN1 variants form the same number of interactions with the NADP + cofactor; however, it was the interaction occupancy that was affected. Namely, three independent MD simulations showed that the ΔN17BEN1 variant in complex with NADP + has significantly higher interaction occupancy thus binds NADP + with more than one order of magnitude higher affinity. Contrary to our expectations, the truncation of this distant region that does not communicate with the nucleotide-binding site didn't result in the gain of interaction but affected the intrinsic conformational dynamics which in turn fine-tuned the binding affinity by increasing the interaction occupancy and strength of the key conserved cation-π interaction between Arg69 and adenine of NADP + and hydrogen bond between Ser244 and phosphate of NADP + ., (Copyright © 2021 Elsevier B.V. All rights reserved.)