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Resolving the Dynamic Interplay of the Conformational States in the TPR Domain of Human AMPylase FicD

Runge, S.; Bilgen, E.; Itzen, A.; Lamb, D. C.

2024-12-20 biophysics
10.1101/2024.12.18.629078 bioRxiv
Show abstract

The human Fic-enzyme FicD plays an important role in regulating the Hsp70 homolog BiP in the endoplasmic reticulum (ER): FicD reversibly modulates BiPs activity through attaching an adenosine monophosphate (AMP) to the substrate binding domain. This reduces BIPs chaperone activity by shifting it into a conformation with reduced substrate affinity. Crystal structures of FicD in the apo, ATP-bound, and BiP-bound states revealed significant conformational variability in the tetratricopeptide repeat (TPR) motifs. In this study, we investigate the conformational dynamics of FicDs TPR motifs using two-color single-molecule Forster resonance energy transfer (smFRET). We demonstrate that the TPR motifs exhibit conformational dynamics between the TPR-out and TPR-in conformations on timescales ranging from milliseconds to microseconds. In addition, we extend our investigation on multiple labeling positions within FicD, revealing how conformational dynamics vary depending on the location within the TPR motif. We quantify the motions with dynamic photon distribution analysis (PDA) for the FRET constructs and propose a conformational landscape model for FicD where the TPR-in/out states exist in an equilibrium that is altered due to the presence of ATP and BiP.

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