# Exploring Metalloprotease from _Dunaliella_ sp.: Production, Regulation, and Structural Insight
## 3 Results and Discussion
### Structural Analysis
#### 3.6.1 Secondary Organization  
The modeled FtsH2 from _Dunaliella salina_ consist on an N-terminal AAA+ (ATPases associated with diverse cellular activities) domain responsible for ATPase activity and a C-terminal protease domain responsible for digesting soluble proteins in order to control some cellular regulation [125]. The AAA+ domain bears the Walker A and B motifs needed for nucleotide binding and hydrolysis, as well as other conserved sequence motifs, namely, SRH motifs that carry conserved arginine residues desired for oligomerization and ATP hydrolysis. The AAA+ domain equally bears the conserved FGV pore motif required for substrate recognition and translocation [126]. The AAA+ domain is composed of a smallsubdomain and a large subdomain that act coordinately with the ones of the adjacent monomer to form a nucleotide-binding pocket at their interface (Figure 5A) [127].  
The analysis of the monomeric 3D model showed that the protease domain carries a zinc ion in the zinc-binding HEXCH finger motif, where X can be any residue (HEVGH in our case). The zinc ion is coordinated by three residues, which are the two histidine residues in the HEXCH motif (H346 and H350) and the aspartate 424. FtsH is a member of the zinc-dependent protease family with similar organization to that observed in other M41 proteases. This ion-coordinated site is commonly involved in fixing the substrate amino acid chain in a stretched configuration around the scissile peptide bond [126].  
A search using PDBeFOLD [128] found known structures with greater than 90% similarity in the total number of Secondary Structure Elements (%SSEs) like mitochondrial ATPase protease YME1 from _Saccharomyces cerevisiae_, 6 az0 (95%), and apo-FtsH crystal structure from _Thermotoga maritima_, 3 kds (92%). In addition, a ConSurf analysis was conducted, in which 29,969 unique HMMER hits, including the query, were analyzed. The calculations were performed using 150 sequence homologs to FtsH that were fitted into  
Figure 5: (**A**) Modeled ribbon presentation of DsftsH2 showing its distinct domain; blue represents the AAA+ large subdomain, orange represents the AAA+ small subdomain, and 1 ilac represents the M41 protease domain. (**B**) A superposition of the constructed model (same colors as A) and the model used as the template (yellow) showing an insertion in the protease domain leading to an extra helix–turn–helix motif (black circle). (**C**) Ribbon representation of the degree of conservation of every amino acid in the FtsH2 sequence using multiple sequence alignment in the ConSurf tool. (**D**) Root Mean Square Fluctuation indexes in the CABSflex2.0 analysis results, the arrow shows high RMSF values.  
a 3D monomer model. These calculations revealed high sequence conservation on the AAA+ domain, while the ATPase domain showed maximal variability among the aligned sequences (Figure 5C).  
A superposition of the constructed model and the model used as the template (Figure 5B) showed an insertion in the protease domain leading to an extra helix-turn-helix motif rich with methionine and arginine residues (454 AQSQDMIMRMMAR) as well as high Root Mean Square Fluctuation (RMSF) indexes in CABSflex2.0 analysis (Figure 5D) with a maximum of 2.995 for the residue I460 [56]. This sequence is conserved in microalgae FtsH; regarding its position within the structure, it could play a role in substrate hydrolysis involved in closed and open conformations.