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Figure 2.
Insights from crystal structures of FIH-D201A and FIH-D201G.
a, stereo view of the iron binding site of the
FIH-D201A·Fe(II)·2OG·HIF-1α[786–826]
complex. Experimental electron density OMIT map (F[o] - F[c])
contoured to 5σ represented as blue mesh (electron density is
carved out around the residues and ligands displayed for
clarity). The unanticipated electron density adjacent to the
iron was provisionally modeled as (bi)carbonate (see
“Results” for discussion). b, comparison of the wild-type
FIH·Fe(II)·2OG·HIF-1α[786–826] complex
(PDB ID 1H2L) with the
FIH-D201A·Fe(II)·2OG·HIF-1α[786–826]
complex (wild-type FIH (blue) in complex with HIF substrate
(cyan) and FIH-D201A (yellow) in complex with HIF substrate
(magenta)). This figure emphasizes several important
interactions between wild-type FIH and HIF-1α that are lost in
the FIH-D201A complex: wild-type FIH Asp-201 and HIF-1α Asn-803
main chain nitrogen (yellow dash), wild-type FIH Gln-239 and
HIF-1α Asn-803 side chain, and wild-type FIH Trp-296 and
HIF-1α Val-802. Note the presence of the assigned sulfate ion
(orange and red) in the FIH-D201A structure apparently replacing
the carboxylate of the HIF-1α Glu-801 in the wild-type
FIH·Fe(II)·2OG·HIF-1α[786–826] complex,
which is also observed in uncomplexed wild-type FIH structure
(PDB 1H2N, not shown). c, stereo views from the crystal
structures of 2OG-dependent halogenase, SyrB2 (pink)
superimposed on the FIH-D201A variant (yellow). The FIH-D201A
variant shares the same HXA... H motif as SyrB2 (PDB ID 2FCT);
however, the FIH-D201A apparently does not provide enough space
for a chloride ion to complete octahedral coordination to the
Fe(II), which could explain why FIH-D201A does not have
halogenase activity toward HIF-1α under our assay conditions.
Distances between the FIH-D201A Ala-201 Cβ methyl group and
Fe(II) in each of the structures are shown as black dashed lines
to emphasize this point. d, stereo view ball-and-stick
representation of the
FIH-D201G·Zn(II)·2OG·HIF-1α[786–826]
complex metal binding site. Experimental electron density 2F[o]
- F[c] contoured to 1.0σ represented as blue mesh (electron
density is carved out around the residues and ligands displayed
for clarity). FIH and 2OG are colored green, HIF-1α[786–826]
is colored yellow, Zn(II) is colored as a gray sphere, and the
Zn(II) bound water is colored as a red sphere. Wat, water.
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