The structure of Aquifex aeolicus FtsH in the ADP-bound state reveals a C ₂-symmetric hexamer

Abstract

The crystal structure of a truncated, soluble quadruple mutant of FtsH from Aquifex aeolicus comprising the AAA and protease domains has been determined at 2.96 Å resolution in space group I222. The protein crystallizes as a hexamer, with the protease domain forming layers in the ab plane. Contacts between these layers are mediated by the AAA domains. These are highly disordered in one crystal form, but are clearly visible in a related form with a shorter c axis. Here, adenosine diphosphate (ADP) is bound to each subunit and the AAA ring exhibits twofold symmetry. The arrangement is different from the ADP-bound state of an analogously truncated, soluble FtsH construct from Thermotoga maritima. The pore is completely closed and the phenylalanine residues in the pore line a contiguous path. The protease hexamer is very similar to those described for other FtsH structures. To resolve certain open issues regarding a conserved glycine in the linker between the AAA and protease domains, as well as the active-site switch β-strand, mutations have been introduced in the full-length membrane-bound protein. Activity analysis of these point mutants reveals the crucial importance of these residues for proteolytic activity and is in accord with previous interpretation of the active-site switch and the importance of the linker glycine residue.

Early emergence of the FtsH proteases involved in photosystem II repair

“…5C), whose structural flexibility is considered crucial for the intradomain movements needed for full functionality of the complex (Bieniossek et al 2009, Suno et al 2012, Vostrukhina et al 2015. Mutations in the lid helix lead to a decrease of not only the protease activity but also the ATPase activity (Suno et al 2012), strongly indicating the lid helix and flexible glycine interact with each other.…”

“…Sites 2 and 3, at positions 400-408 and 445-447, respectively, are close to the flexible glycine [G399 in T. thermophilus (Suno et al 2006, Vostrukhina et al 2015 and G404 in T. maritima (Bieniossek et al 2009)] and lid helix (Bieniossek et al 2009, Suno et al 2012 regions R443-E455 in T. thermophilus (Suno et al 2006, Vostrukhina et al 2015 (Fig. 5C), whose structural flexibility is considered crucial for the intradomain movements needed for full functionality of the complex (Bieniossek et al 2009, Suno et al 2012, Vostrukhina et al 2015.…”

Mini review: ATP‐dependent proteases in bacteria

“…Communication between the AAA + motor domain and the peptidase domain of AAA + proteases is crucial for full proteolytic activity. In the FtsH hexamer both domains are connected via a flexible linker of 12 amino acids containing an essential and universally conserved glycine residue . Substitution of this glycine against leucine abolishes oligomerization, ATPase, and protease activity of a soluble FtsH variant from Thermotoga maritima , Δ‐ Tm FtsH(G404L), and ATPase and proteolytic activity of a soluble FtsH variant from Thermus thermophilus Δ‐ Tt FtsH(G399L) .…”

“…In the FtsH hexamer both domains are connected via a flexible linker of 12 amino acids containing an essential and universally conserved glycine residue . Substitution of this glycine against leucine abolishes oligomerization, ATPase, and protease activity of a soluble FtsH variant from Thermotoga maritima , Δ‐ Tm FtsH(G404L), and ATPase and proteolytic activity of a soluble FtsH variant from Thermus thermophilus Δ‐ Tt FtsH(G399L) . The equivalent mutation in a full length version of Aquifex aeolicus FtsH, FL‐ Aa FtsH(G399L), impeded proteolytic activity but retained ATPase activity at a moderate level .…”

“…17,22 The equivalent mutation in a full length version of Aquifex aeolicus FtsH, FL-AaFtsH(G399L), impeded proteolytic activity but retained ATPase activity at a moderate level. 22 The linker between the ATPase and protease domain of Lon is not as conserved as in FtsH. It is for instance formed by a 16-residue helix in Thermococcus onnurineus NA1Lon and an 11-residue extended coil in E. coli Lon.…”