STRUCTURAL AND EVOLUTIONARY ANALYSIS OF THE RHOMBOID SUPERFAMILY’S MOLECULAR MECHANISM

Rhomboid proteins form a large superfamily of intramembrane polytopic proteins present across all domains of life. They are classified into four groups: one of catalytically active serine proteases and three others, including iRhoms, which lack catalytic activity. Although their phylogeny and classification remain under debate, it is widely accepted that all groups evolved from an active ancestral protease. Rhomboid serine proteases are distinctive for containing a Ser-His catalytic dyad instead of the classical Ser-His-Asp triad, providing insight into how hydrolytic activity occurs within membranes. Structural studies with peptide-like inhibitors and molecular dynamics simulations have deepened our understanding of their mechanisms. This study focuses on the structural analysis of the GlpG, RHDF2, and Pcp1 rhomboids. As most functional and structural data derive from E. coli, investigating eukaryotic rhomboids and the inactive iRhoms enables comparative analyses of function, structure, and evolution across taxa. We conducted conservation and correlation analysis on a large dataset of rhomboid sequences from Bacteria, Archaea, and Eukarya. The Decomposition of Residue Coevolution Networks (DRCN) method, as implemented in PFstats, was applied to detect coevolving residues likely linked to key structural or functional roles. Additionally, sequences from selected taxa were analyzed using the Maximum Likelihood method to reconstruct rhomboid evolutionary relationships. Graphics from DRCN were used to annotate the phylogenetic tree. Conserved and coevolving residues were mapped onto the available 3D structures of GlpG (PDB: 6PJA) and RHDF2 (PDB: 9O58). As no experimental structure exists for mitochondrial rhomboid Pcp1, comparative modeling was performed using Modeller, with GlpG as a template. All visualizations were generated in PyMOL. Conservation analysis identified 12 highly conserved positions, some previously validated by site-directed mutagenesis, suggesting similar importance for uncharacterized ones. Three sets of correlated residues were found: two related to protein stability and structural core, and a third involving residues linked to ligand specificity and water retention. The phylogenetic reconstruction reflects these patterns, supporting clade-specific residue conservation. This study provides new insights into rhomboid evolution and their structure-function relationships.

This work was financially supported by the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) through a master's degree fellowship.