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Noda Institute for Scientiﬁc Research GRANT
2011 Research Grant

Regulation of gene expression by a heme as a signaling
molecule in Lactococcus lactis
Shigetoshi AONO
National Institutes of Natural Sciences, Okazaki Institute for Integrative Bioscience

Research aims

Results & Discussion

Lactococcus lactis undergoes a metabolic shift from fermentation to respiration in an aerobic environment whenheme is supplied1). Acquired heme is used as a source ofheme, rather than of iron, to activate cytochrome oxidase(cytochrome bd) for oxygen respiration, because this bacterium cannot biosynthesize heme. As free heme molecules
are cytotoxic, some regulatory system is required to avoid
this toxicity. In L. lactis, a heme-efﬂux system is reported
to be responsible for heme tolerance and hemeostasis.
Though a transcriptional regulator HrtR will regulate theexpression of the heme-efﬂux system, the detailed mechanisms remain to be elucidated. In this work, I have determined the crystal structures of HrtR in heme-free (apo-),heme-bound (holo-), and DNA-bound forms with biochemical and spectroscopic characterization, based on which I
propose a heme-responsive regulatory mechanism of HrtR
responsible for transcriptional regulation of heme homeostasis.

To avoid structural distortions that could be produced as
the consequence of the presence of tags, intact HrtR without any tag sequence was expressed and puriﬁed in thiswork. Titration of apo-HrtR with heme revealed that apoHrtR takes up 1 mol equivalent of heme with respect toeach HrtR protomer to form a 1 : 1 complex that shows a
Soret peak, and a and b bands, at 413, 562, and 536 nm,
respectively, in the ferric form (Fig. 1). Reduction of holoHrtR changed the Soret, a and b peaks to 425, 560, and530 nm, respectively2). These spectra are similar to those ofheme proteins with bis-His axial ligands, suggesting thatthe heme in holo-HrtR is coordinated by two histidines.
The crystal structure of holo-HrtR described below showedthat this is the case.
Electrophoretic mobility shift assays (EMSAs) revealed
that apo-HrtR can directly interact with the putative operator-promoter region of the hrtRBA operon (Fig. 2)2). The
HrtR-binding sequence (5⬘-ATGACACAGTGTCAT-3⬘) islocated at the position of -6 to -20 relative to the putativetranscriptional start site of the hrtRBA operon, which isoverlapped with the putative -10 region of the hrtRBA promoter. Apo-HrtR bound to the target DNA, which gave aband-shift due to the formation of the apo-HrtR/DNA complex (Fig. 2). The addition of a 10–50 mol equivalent excessof unlabeled target DNA relative to labeled target DNA tothe reaction mixture resulted in the disappearance of the retarded band concomitantly with the appearance of a banddue to free DNA, while 50 mol equivalent excess of unlabeled non-target DNA did not show any competition. Thesecompetition assays revealed that the binding of HrtR to tar-Methods
The hrtR gene synthesized by GenScript was inserted between the NdeI and BamHI sites of pET3a to construct anexpression vector for HrtR, by which an intact HrtR is expressed without any tag sequences. The codons in the synthesized hrtR gene were optimized for protein expression inEscherichia coli. X-ray diffraction data were collected on
BL26B1, BL26B2, and BL41XU at the SPring-8.

Figure 1(A) Heme titration into apo-HrtR (B) Electronic absorption spectra of holo-HrtR

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Figure 2 EMSA of HrtR
Lane 1: free DNA
Lane 2: apo-HrtR, Lane3: holo-HrtR
Lane 4: apo-HrtR + 0.2 eq. hemin
Lane 5: apo-HrtR + 0.5 eq. hemin
Lane 6: apo-HrtR + 1.0 eq. hemin

get DNA is speciﬁc.
Next the regulatory role of heme for DNA-binding wasstudied by EMSAs and ﬂuorescence polarization assays.
The addition of 1 mol equivalent of heme relative to HrtR
protomer was sufﬁcient to fully dissociate HrtR from DNA(Fig. 2). Consistent with these results, holo-HrtR showedneither a band shift on EMSAs nor a change in ﬂuorescencepolarization when reacting with the target DNA. These results indicate that heme acts as an effector molecule bywhich the DNA-binding activity of HrtR is regulated toderepress the hrtRBA operon upon sensing free heme molecules in L. lactis.
To obtain structural insights into the heme-responsive
regulatory mechanisms of HrtR function, the crystal structures of apo-HrtR, holo-HrtR, and the apo-HrtR/DNA complex have been determined at resolutions of 3.1, 1.9, and2.0 Å for apo-HrtR, holo-HrtR, and the apo-HrtR/DNAcomplex, respectively2). The global fold of HrtR is similar
to that of TetR family transcriptional regulators that posses
nine conserved helices, within which helices 1 to 3 comprise the DNA-binding domain, and helices 4 to 9 comprisethe ligand-binding domain. In addition to the nine conserved helices, HrtR possesses extra short helices at the Nand C-termini, though it is not clear due to disorder if there
is an a C helix in holo-HrtR. The DNA-binding domain andthe heme-sensing (heme-binding) domain consist of a N,a 1, a 2, and a 3 helices, and a 4, a 5, a 6, a 7, a 8, a 9, anda C helices, respectively. The interface between the DNAbinding and heme-binding domains is formed through interactions among a 1, a 4, and a 6 helices (see below). Thea 8 and a 9 helices in each subunit were found to form afour-helix bundle that constitutes a dimer interface.

The crystal structure of holo-HrtR revealed that the hemeis accommodated in a large cavity that is open to solvent,with His72 and His149 as the axial ligands to form a 6-coordinated heme. The crystal structures of HrtR determinedin this study reveal that heme-binding to apo-HrtR triggers
a conformational change that regulates the DNA-binding
activity of HrtR. The change in relative orientation of theDNA-binding domain is induced upon heme-binding witha rigid-body motion of the DNA-binding domain. Thechange in relative orientation of the DNA-binding domain
upon heme-binding resulted in a change in the center-tocenter distance between the recognition helices in the helixturn-helix DNA-binding motif of each subunit. The distance between the Ca atoms of Tyr50 in the a 3 helix ofeach subunit increased upon heme-binding from 35 to 47 Å.
As the distance between two consecutive major grooves in
B-DNA is 34 Å, the inter-helical distance between therecognition helices in holo-HrtR (47 Å between the C aatom of Tyr50 in the a 3 helix) is too wide for the recognition helices to ﬁt into two consecutive major grooves and tobind to the target DNA. Thus, the coil-to-helix transition induced by the coordination of His72 and His149 to the heme
shifts HrtR from a DNA-binding competent conformation
to one that is not.
Conclusion
In this work, it has been reported that the transcriptional
regulator HrtR senses and binds a heme molecule as its
physiological effector to regulate the expression of theheme-efﬂux system responsible for heme homeostasis in L.
lactis. To elucidate the molecular mechanisms of how HrtR
senses a heme molecule and regulates gene expression forthe heme efﬂux system, the crystal structures of the apoHrtR/DNA complex, apo-HrtR, and holo-HrtR have been
determined.
References1)

2)

Pedersen, M. B. et al. (2008) Impact of aeration and heme-activatedrespiration on Lactococcus lactis gene expression: identiﬁcation of aheme-responsive operon. J. Bacteriol. 190: 4903–4911.
Sawai, H., Yamanaka, M., Sugimoto, H., Shiro, Y., and Aono, S.
(2012) Structural basis for the transcriptional regulation of heme
homeostasis in Lactococcus lactis. J. Biol. Chem. 287: 30755-30768.