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oai:figshare.com:article/33971328 2026-09-23T06:04:58Z category_1 category_4 category_7 category_12 category_24 category_873 category_45 category_931 category_135 portal_63 item_type_2 month_year_09_2026

Conserved Hinge Motif Modulates Conformational Mobility of a Heme Trafficking Protein from Fusobacterium nucleatum Stella I. Widjaja (25094703) M. Hossein Khalilian (20940157) Alexandra K. McGregor (25094706) Kirsten R. Wolthers (1759165) Biophysics Biochemistry Medicine Cell Biology Evolutionary Biology Chemical Sciences not elsewhere classified Sociology Information Systems not elsewhere classified Computational Biology variants exhibiting different 5 ms ). tyr residues located conserved hinge fine bound hmuf paralog 19 </ sup molecular dynamics simulations indicating faster exchange 1d nmr reveals helical cap appended heme trafficking protein vitro </ cis </ slowly exchange nmr spectra hinge bisecting heme trafficking helical cap tyr128 peak tyr probe two resonances two peaks strict prerequisite results indicate remains closed protein structure p151g substitution open complex one conformer md simulation greater perturbation flavodoxin fold flavodoxin domain electron reduction closed complex chemical shifts bind tightly >- pro151 Fusobacterium nucleatum encodes HmuF, which has been shown <i>in vitro</i> to bind tightly to heme, traffic the cofactor to anaerobilin synthase, and catalyze the four-electron reduction of anaerobilin. The structure of the heme-bound HmuF paralog, FldH, reveals an α-helical cap appended to the flavodoxin domain. Molecular dynamics simulations of FldH without heme reveal that the α-helical cap pivots away from the flavodoxin domain to form an open complex, but then reforms the closed complex, which remains closed for the duration of the MD simulation. Transition to the open conformation disrupts noncovalent interactions formed by three conserved residues (<i>cis</i>-Pro151, His150, and Asp155) that form a hinge bisecting the cap and flavodoxin domain. ¹⁹F NMR was used to probe the dynamics of the cap and the role of the hinge motif. 1D NMR reveals that a single ¹⁹F-Tyr probe in the α-helical cap has two resonances that slowly exchange (τ = 1.5 ms). A H150A and D155N substitution caused coalescence of these two peaks, indicating faster exchange or stabilization of one conformer. A P151G substitution not only led to a shift in the ¹⁹F-Tyr128 peak(s), but also to the chemical shifts of the ¹⁹F-Tyr residues located in the flavodoxin fold, indicating a greater perturbation in protein structure. Despite the variants exhibiting different ¹⁹F-NMR spectra, all engage in heme trafficking and anaerobilin reduction. Together, these results indicate that while the conserved hinge fine-tunes the dynamics of the α-helical cap, this motif is not a strict prerequisite for HmuF/FldH function. 2026-09-23T00:00:00Z Dataset Media 10.1021/acs.biochem.6c00314.s002 https://figshare.com/articles/media/Conserved_Hinge_Motif_Modulates_Conformational_Mobility_of_a_Heme_Trafficking_Protein_from_Fusobacterium_nucleatum/33971328 CC BY-NC 4.0