Genome assembly and protein structure modeling reveal key molecular features of divergent <i>wmk</i> homologs in <i>Wolbachia</i>

<i>Wolbachia</i> induces female-biased sex ratios in host populations through male-specific killing, thereby enhancing its spread via maternal transmission. The prophage-associated gene <i>wmk</i> has been proposed as a key effector underlying this male-killing (MK) phenotype. Interestingly, <i>wmk</i> homologs are found across diverse <i>Wolbachia</i> strains, regardless of the presence of male killing, and show extensive sequence divergence. However, the functional implications of this sequence variation-particularly among distant homologs-remain poorly understood. Here, we analyzed <i>wmk</i> homologs from 18 <i>Wolbachia</i> genomes, including 17 publicly available genomes and one de novo assembled genome from the parthenium beetle. We identified a highly divergent <i>wmk</i> homolog in the latter and predicted its protein structure using AlphaFold2, followed by molecular dynamics simulations to characterize its molecular features. Our analyses reveal that <i>wmk</i>-encoded protein exhibits a modular architecture comprising two helix-turn-helix (HTH) domains and an additional accessory domain not previously described. Modeling of inter-domain interactions further supports the functional relevance of this modular organization. Comparative analyses across all homologs distinguish divergent <i>wmk</i> from other canonical variants based on sequence composition, structural organization, and phylogenetic clustering. Notably, the structural features that differentiate these homologs also distinguish <i>wmk</i> from other known HTH-type regulators. Together, our findings provide new molecular insights into the architecture and evolution of <i>wmk</i>, offering a framework to understand the mechanistic basis of the MK phenotype in <i>Wolbachia</i>.IMPORTANCE<i>Wolbachia</i>-induced male killing presents a promising strategy for the biocontrol of vector and pest populations. The <i>wmk</i> gene has been identified as a candidate underlying this phenotype. Yet, the significance of its sequence variation-particularly between highly divergent homologs-remains unclear. Here, we characterize a divergent <i>wmk</i> homolog from a novel <i>Wolbachia</i> strain. Then, we compare sequence and structural features of <i>wmk</i> homologs across a total of 18 <i>Wolbachia</i> strains using AlphaFold2 and molecular dynamics simulations. Our results highlight key molecular features in divergent variants and provide new insights into <i>wmk</i> evolution, laying a basis for exploring its functional diversity across <i>Wolbachia</i> lineages.