Abstract
Escherichia coli FtsH is an ATP-dependent protease that belongs to the AAA protein family. The second region of homology (SRH) is a highly conserved motif among AAA family members and distinguishes these proteins in part from the wider family of Walker-type ATPases. Despite its conservation across the AAA family of proteins, very little is known concerning the function of the SRH. To address this question, we introduced point mutations systematically into the SRH of FtsH and studied the activities of the mutant proteins. Highly conserved amino acid residues within the SRH were found to be critical for the function of FtsH, with mutations at these positions leading to decreased or abolished ATPase activity. The effects of the mutations on the protease activity of FtsH correlated strikingly with their effects on the ATPase activity. The ATPase-deficient SRH mutants underwent an ATP-induced conformational change similar to wild type FtsH, suggesting an important role for the SRH in ATP hydrolysis but not ATP binding. Analysis of the data in the light of the crystal structure of the hexamerization domain of N-ethylmaleimide-sensitive fusion protein suggests a plausible mechanism of ATP hydrolysis by the AAA ATPases, which invokes an intermolecular catalytic role for the SRH.
Highlights
Escherichia coli FtsH is an ATP-dependent protease that belongs to the AAA protein family
The AAA protein family is a distinct subfamily of the Walker-type ATPases that has been defined on the basis of amino acid sequence homology
To determine whether or not the second region of homology (SRH) is important for FtsH function and, if so, which amino acid residues in the SRH are important, we introduced Ala substitutions into some conserved and less conserved residues in the SRH
Summary
Strains and Media—AR3289 (sfhC21 zad220::Tn10) [10, 20], AR423 (met gal supE hsdR sfiC ⌬(srl-recA)306::Tn10 ⌬ftsH3::kan [pAR171, ftsHϩ repts camR]) [19], AR754 (thr-1 leu-6 thi-1 supE44 lacY1 tonA21 zha-6::Tn10 ftsH1) [24], and BL21(DE3) (FϪ ompT hsdS gal dcm [DE3]) [25] have been described. PSTD401 carries the wild type ftsH gene with its own Shine-Dalgarno sequence under the control of the lac promoter [19]. The mutants are as follows: 1) K201N (AAA to AAC): the KpnI-PstI segment of pIFH108 was replaced with the corresponding segment of pSTD41. Purification of FtsH—AR5088 cells carrying pIFH108 were grown in 2 liters of L medium containing ampicillin (100 g/ml) at 37 °C to a cell density corresponding to 50 Klett units. At this point, expression of FtsH was induced by the addition of IPTG (1 mM) followed by growth for a further 3 h. Pelleted cells were suspended in 100 l of the SDS loading buffer. 20 l of the samples were analyzed by SDS-PAGE followed by immunoblotting using anti-FtsH or anti-32 serum [29] and detected by ECL
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