Computational Analysis of Periplasmic Protein-Mediated Resistance to Membrane Extraction of a Trimeric...

Gram-negative bacteria display large surface proteins exposed to substantial mechanical stresses during surface attachment, fluid flow, and physical perturbations. AtaA, a trimeric autotransporter adhesin from Acinetobacter sp. Tol 5, forms long homotrimeric fibers (~260 nm) that mediate robust adhesion. Its C-terminal transmembrane (TM) domain anchors the fiber to the outer membrane and also associates with the N-terminal domain of the periplasmic auxiliary protein TpgA (TpgA-N). However, the mechanical importance of this interaction remains elusive. Here, we quantified TpgA-mediated stabilization of AtaA-TM anchoring using all-atom steered molecular dynamics and free energy calculations in an asymmetric Acinetobacter outer membrane model. TpgA-N binding significantly enhanced resistance to membrane extraction, approximately doubling the peak force required compared with AtaA-TM alone. Structural analyses revealed that multiple electrostatic contacts at the AtaA-TM-TpgA-N interface collectively reinforce the complex under tensile loading. Furthermore, thermodynamic integration yielded a favorable restrained separation free energy, supporting stabilization of the complex. Because TpgA-N is a hydrophilic periplasmic protein, its translocation through the hydrophobic membrane core is energetically unfavorable. Thus, membrane extraction of AtaA-TM necessitates the prior dissociation of the stable AtaA-TM-TpgA-N interaction. Together, our results demonstrate that TpgA mechanically reinforces the membrane anchor of AtaA through both thermodynamic stabilization and increased resistance to tensile forces. This study highlights a specialized strategy where a periplasmic protein strengthens the membrane anchoring of giant bacterial adhesins to withstand environmental loads.