Essentiality of core hydrophobicity to the structure and function of archaeal chromatin protein Cren7.

Lei Tian, Niannian Ding, Xuehui Liu, Yuanyuan Chen, Zhenfeng Zhang

International journal of biological macromolecules 2022 Aug 01

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Studies on the structure-function relationship of protein greatly help to understand not only the principles of protein folding but also the rationales of protein engineering. Crenarchaeal chromatin protein Cren7 provides an excellent research model for this issue. The small protein adopts a 'β-barrel' fold, formed by the double-stranded antiparallel β-sheet 1 tightly packing with the triple-stranded antiparallel β-sheet 2. The simple structure of Cren7 is stabilized by the hydrophobic core between the β-sheets, consisting of the side chains of V8, V10, L20, V25, F41 and F50. In the present work, mutation analyses by alanine substitution of each of the residues in the hydrophobic core were performed. Circular dichroism spectra and nuclear magnetic resonance analyses showed that mutation of F41 led to a significant misfolding of Cren7 through disruption of the β-sheets. Meanwhile, the mutant F41A showed a reduced thermostatility (Tm of 53.2 °C), as compared with the wild-type Cren7 (Tm > 80 °C). Biolayer interferometry and nick-closure assays showed the largely unchanged activities in DNA binding and supercoiling of F41A, indicating the DNA interface of Cren7 was generally retained in F41A. However, F41A was unable to mediate DNA bridging, probably due to the impairment in forming oligomers/polymers on DNA. Atomic force microscopic images of the F41A-DNA complexes also revealed that F41A nearly completely lost the ability to compact DNA into highly condensed structures. Our results not only reveal the critical role of F41 in protein folding of Cren7 but also provide new insights into the structure-function relationships of thermostable proteins. Copyright © 2022. Published by Elsevier B.V.

Citation

Lei Tian, Niannian Ding, Xuehui Liu, Yuanyuan Chen, Zhenfeng Zhang. Essentiality of core hydrophobicity to the structure and function of archaeal chromatin protein Cren7. International journal of biological macromolecules. 2022 Aug 01;214:381-390