Predicted structure of the 89-kDa invasion-tip protein of Holospora obtusa, an endonuclear symbiont of Paramecium caudatum, suggests a pH-dependent conformational change

The endonuclear symbiont Holospora obtusa employs an invasion-tip protein, weighing 89-kDa, to penetrate the macronucleus of Paramecium caudatum. This protein, part of a unique Holospora family, aids in the escape from the host's digestive vacuole and invasion into the macronucleus. Sequence-based research positioned this protein within the Holospora-specific family and forecasted an N-terminal membrane-anchoring domain and a lengthy alpha-helical C-terminal segment.

AlphaFold2 modeling, complemented by molecular dynamics simulations, revealed that the C-terminal region forms a spiral structure akin to spectrin, featuring a localized trio of coiled-coil helices. The protein demonstrates a substantial pH-dependent charge variation, becoming 55 units more positively charged at pH 4 compared to pH 7.

Differences in simulation outcomes at these pH levels primarily stem from the relative positioning of the N-terminal core and C-terminal rod, characterized by a near 35-degree rotation about a hinge centered on Gly291. However, none of the proximate glutamate residues (Glu273, Glu275, and Glu302) form a pH-dependent salt bridge, and no specific protonation-dependent interaction that underlies the conformational shift has been identified.

Despite this, the authors propose, as a testable hypothesis, that acidification of the digestive vacuole could facilitate this reorientation, effectively relocating the N-terminal membrane-anchoring module to the bacterial surface—an initial step towards vacuole escape.