Integrative structural and functional analysis of full-length hibernation-promoting factor and its association with antibiotic susceptibility in Mycobacterium smegmatis

ABSTRACT Ribosome hibernation is a critical adaptive mechanism that enables bacteria to survive stress by stabilizing inactive ribosomes and conserving cellular resources. This process is facilitated by hibernation-promoting factors (HPFs). However, structural and functional insights into the full-length architecture of Mycobacterium smegmatis HPF, particularly its C-terminal region, remain limited. Here, we combined in silico and experimental approaches to characterize HPF ( MSMEG_1878 ) from M. smegmatis and investigate its role in ribosome-associated physiology and antibiotic response. Sequence and phylogenetic analyses revealed strong conservation of HPF among mycobacterial species, including the extended C-terminal region. A full-length 230-amino-acid structural model generated using complementary modeling approaches and molecular dynamics refinement showed close agreement with the experimentally resolved region. Structural mapping positioned HPF at the 70S ribosomal intersubunit interface, with the modeled C-terminal region extending toward the vicinity of the 16S rRNA decoding-center. Docking analyses predicted that paromomycin and neomycin localize within the h44-associated decoding-center region, near the HPF-associated surface, whereas chloramphenicol and tetracycline localize to spatially distinct ribosomal regions. Deletion of hpf impaired bacterial growth and reduced susceptibility to paromomycin and neomycin, while complementation restored susceptibility toward the wild-type phenotype. Furthermore, neomycin exposure significantly increased hpf transcript abundance, whereas paromomycin exposure showed a nonsignificant trend toward increased expression. Collectively, these findings suggest that M. smegmatis HPF contributes to ribosome-associated physiology and may influence the cellular response to selected antibiotics, providing insights into the relationship between ribosome hibernation and antibiotic response in mycobacteria. IMPORTANCE This study provides new structural and functional insights into the full-length architecture and ribosome-associated role of HPF ( MSMEG_1878 ) in Mycobacterium smegmatis , with particular emphasis on its C-terminal region. Using complementary structural modeling approaches, we investigated the predicted architecture of the C-terminal region and its spatial relationship with the ribosomal decoding-center region. Functional analyses further demonstrate that loss of HPF affects bacterial growth and alters susceptibility to selected aminoglycosides. Together, these findings suggest that HPF contributes to ribosome-associated physiology and may influence cellular responses to selected antibiotics. This work provides a potential structural and functional framework for further investigation of how ribosome-associated factors influence antibiotic responses in mycobacteria.