The metal-responsive transcriptional repressors of the SmtB/ArsR family repress
the expression of their respective operons in the absence of metal and are released from
the operator/promoter region when metal ions bind, thus allowing RNA polymerase to
bind and transcribe the operon, which encodes genes involved in homeostasis and
resistance. To elucidate the determinants of metal ion selectivity, comparative metalbinding
and DNA-binding properties of S. aureus CzrA and M. tuberculosis NmtR were
characterized. The structure of the metal coordination complexes of CzrA and NmtR
reveal that CzrA forms a 4-coordinate, tetrahedral complex with both Zn(II) and Co(II)
potent regulators of czr operator/promoter (O/P) binding in vitro and de-repression in
vivo. In contrast, NmtR adopts 5- or 6-coordinate complexes with Ni(II) and Co(II), the
strongest allosteric regulators of nmt O/P binding in vitro and de-repression in vivo.
Zn(II), a non-inducer in vivo and poor regulator in vitro, binds NmtR with high affinity
and forms a non-native 4-coordinate complex. These studies suggest that metal
coordination geometries (number), not metal binding affinities, are primary determinants
of functionality.
To gain molecular insight into the mechanism of allosteric regulation of O/P
binding by metal ions, NMR and X-ray crystallographic studies of apo- and zinc forms
of CzrA, and another ArsR/SmtB zinc sensor, Synechococcus PCC7942 SmtB, were
performed. These studies showed that formation of the metal chelate drives a quaternary
structural switch mediated by an intersubunit hydrogen-binding network that originates
with the nonliganding Nε2 face of His97 in CzrA (His117 in SmtB) that stabilizes a low
affinity DNA-binding conformation.
Mutagenesis experiments reveal that substitution of D84 and H97 in CzrA,
results in the formation of higher coordination number complexes that are nonfunctional
in driving zinc-mediated allosteric regulation of DNA binding. In contrast, conservative
mutations of H86 and H100 in CzrA bind Co(II) or Zn(II) in a tetrahedral manner, albeit
with greatly reduced affinity, and allosterically regulate O/P binding with significant
lower coupling free energies compared to wild-type CzrA. These findings further
reinforce the notion that metal coordination geometry is the primary determinant for
functional sites in metal-sensing transcriptional repressors.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/2303 |
| Date | 29 August 2005 |
| Creators | Pennella, Mario Antonio |
| Contributors | Giedroc, David P. |
| Publisher | Texas A&M University |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation, text |
| Format | 6702347 bytes, electronic, application/pdf, born digital |
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