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Exploring a New Approach for Discovery of Conformational
Heterogeneity in HomeodomainDNA Complexes
Mark Rance*
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, Ohio 45267, United States
T he molecular basis of proteinDNA interactions con-
tinues to be a very important subject of investigation in
the eld of structural biology. In particular, one aspect of such
interactions that requires further exploration is the general
question regarding the contributions of conformational
heterogeneity and molecular dynamics to molecular recognition
and binding anity. Despite the large number of structural and
thermodynamic studies that have been reported for a variety of
proteinDNA systems, critical and substantial gaps exist in our
understanding of the roles played by molecular dynamics and
exibility in proteinDNA interactions. A general problem in
the eld of molecular recognition is that structural studies
reveal relatively little about the entropic component of the free
energy of complex formation. Thus, it is very important to
complement available structural information by undertaking
studies designed to elucidate details concerning side-chain
dynamics in the proteinDNA interface. As mentioned by
Fraenkel and Pabo in their work on the Antennapedia (Antp)
homeodomainDNA complex, it will be interesting to
compare other protein/DNA complexes as we try to integrate
X-ray and NMR data to understand the respective roles of
exibility and of discrete, favorable contacts in macromolecular
recognition.1 Billeter et al. hypothesized that Antp achieves
specicity through a uctuating network of short-lived contacts
that allows it to recognize the DNA without the entropic cost
that would result if side chains were immobilized upon DNA
binding.2 To date, detailed structural studies of homeodomains
and their complexes with DNA have predominantly been
performed via X-ray crystallography and nuclear magnetic
resonance (NMR) spectroscopy. However, in a recent paper
from Romesbergs group,3 they indicated that infrared (IR)
spectroscopy can be a valuable, complementary tool for
discovering conformational heterogeneity in proteinDNA
complexes. A principal advantage of IR spectroscopy is the
subpicosecond time resolution it oers, which means that it can
potentially capture any conformational heterogeneity that
occurs on biologically relevant time scales. A key to the
approach of Romesbergs group was the incorporation of
nonperturbing, site-specic spectroscopic probes, namely,
carbondeuterium (CD) bonds, which eliminates the
problems that would otherwise exist because of overlapping
absorptions. It had been established previously that isotopic
substitution of D for H shifts the IR stretching absorptions into
a spectral region that is free of other signals (see ref 4 for the
origins of this approach).
In their work, Adhikary et al. demonstrated the potential
value of IR measurements in studies utilizing the Bicoid
homeodomain.3 The Drosophila protein Bicoid is responsible
for embryonic anterior structure development and recognizes
DNA sequences present in enhancer elements of a wide variety
2017 American Chemical Society
of Bicoid-responsive genes. The homeodomain of Bicoid has
been the most extensively studied member of the so-called
Lys50 class of homeodomains, those homeodomains having a
lysine at the key position 50 (see ref 5 and references cited
therein). Homeodomains are often classied according to the
identity of residue 50 because of the key role of this residue in
DNA binding specicity. Much attention has been focused on
the consequences of lysine being located at position 50, largely
due to the fact that the most dramatic examples of altered DNA
specicity occur when a lysine is either introduced or replaced
at position 50. The tightest and most specic binding occurs
when lysine is present at position 50.
For the FTIR (Fourier transform infrared) studies, isotopic
labeling of the Bicoid homeodomain was accomplished via the
use of deuterated amino acids combined with chemical peptide
synthesis and native chemical ligation. The authors chose to
perform their IR measurements on samples labeled individually
at the Leu16, Leu21, Leu31, Val45, and Lys50 positions. The
canonical homeodomain structure consists of three helices and
an N-terminal tail, with helix 3 binding in the major groove of a
DNA binding partner and the N-terminal tail making contacts
with the minor groove. Residues Leu16 and Leu21 are located
in helix 1. Leu31 is located in helix 2. Val45 and Lys50 are
located in helix 3. FTIR data were recorded for the free amino
acids and for the Bicoid homeodomain in the free state and in a
complex with a TAATCC DNA binding site. Data analysis
focused on the IR signals from the symmetric and asymmetric
stretch absorptions of the methyl CD3 groups (Leu and Val) or
the CD2 methylene groups (Lys).
The data for the (d7)Leu residues in the homeodomain gave
rise to relatively simple looking FTIR spectra and indicated
little change occurred upon DNA binding, in good agreement
with the usual observation that the homeodomain is generally a
well-folded entity even in the absence of DNA. On the other
hand, the FTIR spectra for the (d8)Val45 and (d8)Lys50
samples were considerably more complex in appearance, in
both bound and unbound states. The complexity of the Lys50
spectra is consistent with prior evidence of multiple
conformations of the Lys50 side chain in the Bicoid
homeodomainDNA complex,5 where NMR and molecular
dynamics simulation data showed that the exibility of the
Lys50 side chain allows it to make uctuating contacts with
multiple bases in the DNA binding site (TAATCC/ATTAGG).
Given the surface-exposed position of Lys50 in the unbound
homeodomain, it is not surprising that multiple conformations
would also exist in this free state. The situation for Val45 is less
obvious. For the Leu and Val residues, both methyl groups in
Received: August 8, 2017
Published: September 13, 2017
5033 DOI: 10.1021/acs.biochem.7b00760
Biochemistry 2017, 56, 50335034
Biochemistry Viewpoint

each residue type were deuterated. The Val45 side chain forms Bicoid homeodomain bound to the consensus TAATCC DNA-
part of the hydrophobic core of the Bicoid homeodomain, with binding site. J. Mol. Biol. 356, 11371151.
one methyl group being in close contact with the side chains of
Leu40 and Phe8, and the other methyl group in close contact
with Leu31, Ser35, and the highly conserved residue Leu16. In
this case, it is somewhat surprising to observe signicant
conformational heterogeneity for Val45, at least in terms of
signicant, multiple rotamer populations. The authors do not
provide a structural interpretation of their data and acknowl-
edge that further work is necessary to support a conclusion that
conformational heterogeneity contributes in a signicant way to
the complexity of the FTIR signals for Val45 and Lys50.
Perhaps an alternate possibility to explain the Val45 spectral
complexity might be some uctuation of other residues in the
vicinity of the Val45 methyl groups, such as Leu31, Ser35, and
Leu40. One wonders whether data interpretation for Val45
would have been simplied via the use of stereospecically
labeled Val, to eliminate the question of whether slightly
dierent chemical environments of the two methyl groups
contributed to the complexity of the FTIR spectrum.
In summary, this work demonstrates the value of FTIR
spectroscopy in structural studies of proteinDNA complexes,
to complement more commonly used biophysical tools.
Conformational heterogeneity can often manifest itself as
missing electron density in X-ray crystallographic studies, and
in NMR approaches, such heterogeneity can lead to exchange-
broadened resonances or motionally averaged results that
require more extensive studies to unravel. In contrast, the
inherently high temporal and spatial resolution of FTIR
measurements provides the potential for discovery of
conformational heterogeneity on the fastest time scales, thus
allowing for important insights into the molecular mechanisms
of proteinDNA recognition.

AUTHOR INFORMATION
Corresponding Author
*E-mail: mark.rance@uc.edu.
ORCID
Mark Rance: 0000-0003-0664-6024
Funding
M.R. acknowledges support from the National Institute of
General Medical Sciences of the National Institutes of Health
(R01 GM063855).
Notes
The author declares no competing nancial interest.

REFERENCES
(1) Fraenkel, E., and Pabo, C. O. (1998) Comparison of X-ray and
NMR structures for the Antennapedia homeodomain-DNA complex.
Nat. Struct. Mol. Biol. 5, 692697.
(2) Billeter, M., Guntert, P., Luginbuhl, P., and Wuthrich, K. (1996)
Hydration and DNA recognition by homeodomains. Cell 85, 1057
1065.
(3) Adhikary, R., Tan, Y. X., Liu, J., Zimmermann, J., Holcomb, M.,
Yvellez, C., Dawson, P. E., and Romesberg, F. E. (2017) Conforma-
tional heterogeneity and DNA recognition by the morphogen Bicoid.
Biochemistry 56, 27872793.
(4) Adhikary, R., Zimmermann, J., and Romesberg, F. E. (2017)
Transparent window vibrational probes for the characterization of
proteins with high structural and temporal resolution. Chem. Rev. 117,
19271969.
(5) Baird-Titus, J. M., Clark-Baldwin, K., Dave, V., Caperelli, C. A.,
Ma, J., and Rance, M. (2006) The solution structure of the native K50

5034 DOI: 10.1021/acs.biochem.7b00760

Biochemistry 2017, 56, 50335034