Transition metals are essential to many biological systems for the proper function of enzymes. However, excess levels of free transition metal ions are toxic. Therefore, tight regulation offered by a metal specific trafficking system is often employed to maintain metal homoeostasis. RcnR is a Ni(II) and Co(II) responsive transcriptional regulator in the <i>E. coli</i> Ni-trafficking system and controls the expression of the Ni(II)/Co(II) exporter proteins, RcnAB. The mechanism of DNA binding by RcnR and how cognate metals induce protein allosteric regulation has not been fully elucidated. Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) was used to probe the RcnR structure in the presence of DNA, cognate metals (Ni and Co), and the non-cognate metal, Zn. The result reveals that apo-RcnR, RcnR-DNA site 1, RcnR-DNA site 1+2 complexes and Zn(II)-RcnR complex have similar deuterium uptake kinetics with hot spots that are located near the N-terminus (residues 14-24). This deuterium uptake pattern is not observed in the Ni(II)-RcnR and Co(II)-RcnR complex, and the largest deuterium incorporation difference is located near the N-terminus, which has also been shown to be involved in metal binding. This suggests that cognate metals binding to the N-terminal region results in the decrease of helix 1 flexibility or induces conformational changes that bury positively charged residues, such as R14 and K17, that are important for DNA binding, and that these changes decrease the protein-DNA binding affinity.

HDX-MS reveals metal-specific structural changes important to DNA binding by the cobalt and nickel responsive transcriptional regulator, RcnR

253rd National Meeting (2017)

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