Full-Length Structure and Heme Binding in the Transcriptional Regulator HcpR

HcpR is a heme-dependent transcriptional regulator present in many Gram-negative anaerobic bacteria. In the perio-pathogen <i>Porphyromonas gingivalis</i>, HcpR is crucial for the response to reactive nitrogen species such as nitric oxide (NO). Binding of NO to the heme group of HcpR leads to transcription of redox enzyme Hcp. However, the molecular mechanisms of binding of heme to HcpR remain unknown. In this study, we present the 2.3 Å structure of the <i>P. gingivalis</i> HcpR. Interdomain interactions present in the structure help to form a hydrophobic pocket in the N-terminal sensing domain. A comparison analysis with other CRP/FNR-family members reveals that the molecular mechanisms of HcpR-mediated regulation may be distinct from those of other family members. Using docking studies, we identified a putative heme-binding site in the sensing domain pocket. <i>In vivo</i> complementation and mutagenesis studies verify one pocket residue, Met68, as an important reagent in HcpR-mediated transcriptional activation. Finally, heme-binding studies with purified forms of recombinant HcpR support Met68 as a crucial residue for heme binding as well as coordination.