Published on Web 11/08/2008
Ligand-Induced Conformational Heterogeneity of Cytochrome P450 CYP119
Identified by 2D NMR Spectroscopy with the Unnatural Amino Acid
13C-p-Methoxyphenylalanine
Jed N. Lampe, Stephen N. Floor, John D. Gross, Clinton R. Nishida, Yongying Jiang,
Michael J. Trnka, and Paul R. Ortiz de Montellano*
Department of Pharmaceutical Chemistry, UniVersity of California, San Francisco, California 94158-2517
Received September 9, 2008; E-mail: Ortiz@cgl.ucsf.edu
It has become increasingly clear that protein dynamics are
required for enzymatic function.1-3 In fact, the native state of an
enzyme can be described as a statistical ensemble of different
conformational substates with similar free energies.4 Modern NMR
techniques have proved successful at connecting conformational
substates and their respective free energies with their individual
contributions to catalysis.5 The cytochrome P450 (CYP) enzyme
superfamily is a ubiquitous set of heme-containing monooxygenases
that typically catalyze the incorporation of one atom of molecular
oxygen into a hydrocarbon substrate.6 P450 enzymes are known
to exhibit a wide range of dynamic enzymatic behavior, including
homotropic and heterotropic cooperativity, sequential oxidation, and
substrate inhibition.7 Due to their importance in mammalian drug
metabolism and their potential utility as model systems for the study
of complex protein dynamics, we report here the application of a
unique high-resolution 2D NMR technique for the examination of
ligand binding and its contribution to conformational dynamics in
this major enzyme family. CYP119 from Sulfolobus solfataricus
Figure 1. 1H, 13C-HSQC spectra of CYP119-F153MeOF (red) superim-
is a thermophilic P450 that is monomeric and stable at temperatures
1
posed upon the H, 13C-HSQC spectra of CYP119-F162MeOF (blue).
up to 90 °C.8 Ligand-free and ligand-bound crystal structures of
be explained by the existence of two distinct conformers in slow
this protein suggest that dramatic conformational changes occur
chemical exchange, either conformational substates of the protein
upon ligand binding, particularly in the F/G loop region, that result
itself or specific ring-flip rotomers of the 13C-labeled p-methox-
in an overall “closing off” of the active site.8 To better understand
yphenylalanine residue.10 This is further illustrated by coalescence
the structural and thermodynamic contributions of specific residues
of the two resonances with increasing temperature (Figure S7).
in this dynamic region of the protein, we have introduced a 13C-
Ring-flip rotomers of tyrosine and phenylalanine have previously
labeled p-methoxyphenylalanine (13C-MeOF) unnatural amino acid
been shown to be distinguishable on the NMR time scale used in
site-specifically into two unique positions in the F/G loop, F153
these experiments (i.e., ns to ms).10,11 Regardless of the origin of
1
and F162, to monitor the effects of ligand binding using 2D H,
the doublet, the signal occurred in all CYP11913CMeOF mutants
13C-HSQC NMR.9 The advantage of this technique over global
examined. To monitor the dynamic structural changes that occur
upon ligand binding, HSQC experiments were performed with a
model ligand at a variety of concentrations. The ligand 4-phe-
nylimidazole (4-PI) was chosen due to the existence of a crystal
labeling of the protein backbone with 15N is that it allows for site-
selective incorporation of a single, specific amino acid in ViVo,
which results in the production of milligram quantities of protein
and circumvents the need to make spectral residue assignments.
structure and the potential utility of imidazole derivatives as
SDS-PAGE analysis of the CYP119-F162-13C-MeOF mutant
scaffolds for isoform-selective P450 inhibitors.12 Upon addition of
indicated the presence of a single band of the correct molecular
the 4-PI ligand to the F162-13C-MeOF labeled mutant, a single new
weight (Supporting Information, Figure S1). Whole-protein LC-
resonance appeared in the spectrum with a chemical shift of 3.67
MS yielded the predicted mass shift of +31 Da, demonstrating the
incorporation of a single 13C-MeOF residue (Figures S2 and S3).
To assess the integrity of the labeled protein, a variety of ligands
were examined for their ability to bind and induce a spin-state shift
in the heme Soret bands (Figure S4). All ligands examined exhibited
Kd(app) values similar to those of the wild-type enzyme (Table S1).
ppm in the 1H dimension and 57.3 ppm in the 13C dimension
(Figures 2b-d and S5). The intensity of the resonance increased
with increasing concentration of ligand until reaching saturation at
a 1:1 molar stoichiometry. 4-PI is known to form a 1:1 complex
with CYP119 with a Kd(app) of ∼120 nM (Table S1).8 In the absence
of labeled protein, no resonances are detected in this region of the
spectrum, further indicating that this resonance is due to ligand-
bound protein (data not shown). It is interesting to note that, even
at saturation, the resonances representing the ligand-free species
are still present. This phenomenon has been seen previously in a
similar study monitoring the binding of a model compound to the
human fatty acid synthase.9 This may indicate that the “ligand free”
1
Surprisingly, initial examination of the H,13C-HSQC spectra for
both mutants revealed the presence of two symmetrical resonances
1
of equal intensity, separated by ∼0.12 ppm in the H dimension
(Figures 1, S5, and S6).
Since the three protons connected to the 13C-labeled methoxy
moiety are chemically equivalent, these two resonances can only
9
16168 J. AM. CHEM. SOC. 2008, 130, 16168–16169
10.1021/ja8071463 CCC: $40.75
2008 American Chemical Society
Lampe, Jed N.
