Cotranslocational processing of the protein substrate calmodulin by an AAA plus unfoldase occurs via unfolding and refolding intermediates

Augustyniak Rafal; Kay Lewis E.

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Cotranslocational processing of the protein substrate calmodulin by an AAA plus unfoldase occurs via unfolding and refolding intermediates

机译：Cotranslocational processing of the protein substrate calmodulin by an AAA plus unfoldase occurs via unfolding and refolding intermediates

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摘要

Protein remodeling by AAA+ enzymes is central for maintaining proteostasis in a living cell. However, a detailed structural description of how this is accomplished at the level of the substrate molecules that are acted upon is lacking. Here, we combine chemical cross-linking and methyl transverse relaxation-optimized NMR spectroscopy to study, at atomic resolution, the stepwise unfolding and subsequent refolding of the two-domain substrate calmodulin by the VAT AAA+ unfoldase from Thermoplasma acidophilum. By engineering intermolecular disulphide bridges between the substrate and VAT we trap the substrate at different stages of translocation, allowing structural studies throughout the translocation process. Our results show that VAT initiates substrate translocation by pulling on intrinsically unstructured N or C termini of substrate molecules without showing specificity for a particular amino acid sequence. Although the B1 domain of protein G is shown to unfold cooperatively, translocation of calmodulin leads to the formation of intermediates, and these differ on an individual domain level in a manner that depends on whether pulling is from the N or C terminus. The approach presented generates an atomic resolution picture of substrate unfolding and subsequent refolding by unfoldases that can be quite different from results obtained via in vitro denaturation experiments.

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《Proceedings of the National Academy of Sciences of the United States of America.》 |2018年第21期|E4786-E4795|共10页
作者
Augustyniak Rafal; Kay Lewis E.;
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作者单位

Univ Toronto, Dept Mol Genet, Toronto, ON M5S 1A8, Canada;

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收录信息美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
原文格式 PDF
正文语种英语
中图分类自然科学总论;
关键词
VAT; protein unfolding; protein translocation; AAA plus ATPase; methyl-TROSY NMR;

Cotranslocational processing of the protein substrate calmodulin by an AAA plus unfoldase occurs via unfolding and refolding intermediates

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