# Exploring Metalloprotease from _Dunaliella_ sp.: Production, Regulation, and Structural Insight
## 3 Results and Discussion
### Structural Analysis
#### 3.6.2 Quaternary Organization  
Many studies have shown that FtsH enzymes are self-compartmentalizing proteases with six active sites located inside a universally conserved hexameric molecule, fulfilling key functions in various cellular activities and signaling. FtsH enzymes transform ATP chemical energy into mechanical energy exploited for substrate unfolding and translocation into the proteolytic site [129].  
Previous crystal structures of the bacterial FtsH in nucleotide-bound or free conformations have been shown to be organized into hexameric rings with two-, three-, or six-fold symmetry [130; 131]. But recently, the solved structure of mitochondrial FtsH YME1 from _Saccharomyces cerevisiae_ has revealed a distinctly different quaternary organization where the YME1 AAA+ domains assemble into a spiral staircase, with the ATPases progressively rotated and translated with respect to one another. This non-symmetric organization has also been observed for other AAA+ unfoldases, like the 26S proteasome ATPase [127].  
Here, the best stability indexes checked by ERRAT [132] and Verify 3D [133] were obtained with models having C2 and C6 symmetric folds using the apo-FtsH crystal structure from _Thermotoga maritima_ as the template.  
The 3D model of the apo form presented in Figure 6A,C showed a perfect six-fold symmetry with the important aromatic pore residues like PHE 158 at the top of the open circular entrance. A comparison between the apo- and the ADP-bound model (Figure 6B,D) showed movement in the aromatic pore residues, triggered by translocations of AAA protease domains, inducing pore closure and a more compact appearance. The same observations were described by [PERSON] et al. [129]. The authors suggested that the protease ring is the stationary component, while the nucleotide-determined translocations in the AAA domains are responsible for substrate unfolding and pulling through the central pore. They do not describe a completely closed pore. These movements are guided by the successive nucleotide fixation in AAA domains since ATPs are bound with different affinities and not simultaneously. More recently, [PERSON] et al. [127] observed three distinct nucleotide states in the same molecule in such a way that four subunits contained an ATP in the nucleotide-binding pocket, one subunit contained an ADP, and one contained an apo-like subunit. These three distinct states correlate with substrate engagement and hydrolysis, with an allosterically regulated pore opening mechanism.  
As shown in Figure 6E, the ATPase and the protease subdomains enclosed a negatively charged entrance and exit from the proteolytic chamber in the apo-FtsH model. In the ADP-bound model, two distinct sites are observed in the asymmetric unit (Figure 6F). The first one is positively charged and completely closed, which probably corresponds to the hydrolysis progress, and the second one is open from the top of the molecule, probably for product release from the proteolytic chamber [134]. These very important rearrangements enhanced by ATP fixation offer the necessary mechanical force and vectorial movement for substrate translocation and hydrolysis. Recent crystal structures showed that the ADP-bound state could be two-fold and three-fold, and even each of the six monomers could have a different conformation [127; 135].