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bioRxiv · 10.1101/2021.08.18.456811

Plasticity in AAA+ proteases reveals ATP-dependent substrate specificity principles

Mahmoud, S. A.·Aldikacti, B.·Chien, P.
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Abstract

In bacteria, AAA+ proteases such as Lon and ClpXP degrade substrates with exquisite specificity. These machines capture the energy of ATP hydrolysis to power unfolding and degradation of target substrates. Here, we show that a mutation in the ATP binding site of ClpX shifts protease specificity to promote degradation of normally Lon-restricted substrates. However, this ClpX mutant is worse at degrading ClpXP targets, suggesting an optimal balance in substrate preference for a given protease that is surprisingly easy to alter. In vitro, wildtype ClpXP also degrades Lon-restricted substrates more readily when ATP levels are reduced, similar to the shifted specificity of mutant ClpXP, which has altered ATP hydrolysis kinetics. Based on these results, we suggest that rates of ATP hydrolysis not only power substrate unfolding and degradation, but also tune protease specificity. We consider various models for this effect based on emerging structures of AAA+ machines showing conformationally distinct states. O_FIG O_LINKSMALLFIG WIDTH=197 HEIGHT=200 SRC="FIGDIR/small/456811v2_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@1b72081org.highwire.dtl.DTLVardef@1b713c7org.highwire.dtl.DTLVardef@73a92aorg.highwire.dtl.DTLVardef@167422d_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG eTOCAAA+ proteases, such as Lon and ClpXP, select distinct targets for degradation to maintain proteostasis. Mahmoud et al. show that ATP hydrolysis can tune substrate specificity of ClpX, allowing ClpX to degrade Lon-restricted substrates under limiting ATP conditions or in the presence of a ClpX mutant. HighlightsO_LIA Walker B mutation of the AAA+ protease ClpX alters substrate specificity C_LIO_LIClpX mutant degrades new substrates but degrades canonical substrates less well C_LIO_LIDecreasing ATP levels enhances ClpXP mediated degradation of some classes of substrates C_LIO_LIATP-induced changes in conformational states accompany alterations in ClpX specificity C_LI

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Mahmoud, S. A., Aldikacti, B., Chien, P.. 2021-08-18. Plasticity in AAA+ proteases reveals ATP-dependent substrate specificity principles. https://doi.org/10.1101/2021.08.18.456811

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