期刊
NUCLEIC ACIDS RESEARCH
卷 50, 期 12, 页码 7002-7012出版社
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkac529
关键词
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资金
- National Natural Science Foundation of China [12090051, 11834018, 91753104, U1930402, 12090050, 12022409]
- Strategic Priority Research Program of the Chinese Academy of Sciences [XDB37000000]
- National Key Research and Development Program of China [2019YFA0709304]
- CAS Key Research Program of Frontier Sciences [ZDBS-LY-SLH015]
- Youth Innovation Promotion Association of CAS [Y2021003]
- Tianhe-2JK computing time award at the Beijing Computational Research Center (CSRC)
In this study, the authors used single-molecule techniques and computational modeling to investigate the dynamic conformations and their impact on structure-function relationships in two Pif1 helicases. They found that solvent-exposed nucleotides interact dynamically with the helicase surfaces and these interactions play a crucial role in the enzymatic activities of the helicases.
Flexible regions in biomolecular complexes, although crucial to understanding structure-function relationships, are often unclear in high-resolution crystal structures. In this study, we showed that single-molecule techniques, in combination with computational modeling, can characterize dynamic conformations not resolved by high-resolution structure determination methods. Taking two Pif1 helicases (ScPif1 and BsPif1) as model systems, we found that, besides a few tightly bound nucleotides, adjacent solvent-exposed nucleotides interact dynamically with the helicase surfaces. The whole nucleotide segment possessed curved conformations and covered the two RecA-like domains of the helicases, which are essential for the inch-worm mechanism. The synergetic approach reveals that the interactions between the exposed nucleotides and the helicases could be reduced by large stretching forces or electrostatically shielded with high-concentration salt, subsequently resulting in reduced translocation rates of the helicases. The dynamic interactions between the exposed nucleotides and the helicases underlay the force- and salt-dependences of their enzymatic activities. The present single-molecule based approach complements high-resolution structural methods in deciphering the molecular mechanisms of the helicases.
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