Crystallographic analysis of a putative FAD-dependent oxidoreductase identifies a potentially misfolded apo structure...

Misfolded proteins, if not refolded by molecular chaperones, are typically targeted for degradation by cellular quality control systems or tend to aggregate. In the present study, we report a rarely observed crystal structure of a misfolded structure that exists as a stable monomer in solution. The CT375 protein from Chlamydia trachomatis, annotated as a putative d-amino acid dehydrogenase (DAADH), was confirmed to contain the flavin adenine dinucleotide (FAD) through ultraviolet-visible absorption spectroscopy and liquid chromatography-mass spectrometry analyses. However, high-resolution electron density maps excluded the presence of an FAD cofactor bound within the crystallized CT375. This abnormal apo conformation cannot be explained by the loss of FAD during crystallization, as the loop aberrantly occupying the active pocket cannot transition between its current conformation and the FAD-bound conformation observed in homologous oxidoreductases without passing through a two-stranded β-sheet. It is likely that the interactions between this misfolded loop, instead of FAD, and the active pocket residues contribute to stabilizing the overall fold of CT375 in this misfolded state, and that the misfolded protein, present within the heterogeneous CT375 sample, was fortuitously crystallized. The structure of misfolded CT375 underscores the critical role of the cofactor in correct protein folding and provides a valuable model for advancing the understanding of protein misfolding and its potential mechanisms underlying protein dysfunction.