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Exploring a New Approach for Discovery of Conformational Heterogeneity in Homeodomain−DNA Complexes Mark Rance* Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, Ohio 45267, United States
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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 classified according to the identity of residue 50 because of the key role of this residue in DNA binding specificity. 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 specificity occur when a lysine is either introduced or replaced at position 50. The tightest and most specific 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 homeodomain−DNA complex,5 where NMR and molecular dynamics simulation data showed that the flexibility of the Lys50 side chain allows it to make fluctuating 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
he molecular basis of protein−DNA interactions continues to be a very important subject of investigation in the field 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 affinity. Despite the large number of structural and thermodynamic studies that have been reported for a variety of protein−DNA systems, critical and substantial gaps exist in our understanding of the roles played by molecular dynamics and flexibility in protein−DNA interactions. A general problem in the field 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 protein−DNA interface. As mentioned by Fraenkel and Pabo in their work on the Antennapedia (Antp) homeodomain−DNA 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 flexibility and of discrete, favorable contacts in macromolecular recognition”.1 Billeter et al. hypothesized that Antp achieves specificity through a fluctuating 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 Romesberg’s group,3 they indicated that infrared (IR) spectroscopy can be a valuable, complementary tool for discovering conformational heterogeneity in protein−DNA complexes. A principal advantage of IR spectroscopy is the subpicosecond time resolution it offers, which means that it can potentially capture any conformational heterogeneity that occurs on biologically relevant time scales. A key to the approach of Romesberg’s group was the incorporation of nonperturbing, site-specific spectroscopic probes, namely, carbon−deuterium (C−D) 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 © XXXX American Chemical Society
Received: August 8, 2017
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DOI: 10.1021/acs.biochem.7b00760 Biochemistry XXXX, XXX, XXX−XXX
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Biochemistry
Bicoid homeodomain bound to the consensus TAATCC DNAbinding site. J. Mol. Biol. 356, 1137−1151.
each residue type were deuterated. The Val45 side chain forms part of the hydrophobic core of the Bicoid homeodomain, with 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 significant conformational heterogeneity for Val45, at least in terms of significant, multiple rotamer populations. The authors do not provide a structural interpretation of their data and acknowledge that further work is necessary to support a conclusion that conformational heterogeneity contributes in a significant 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 fluctuation 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 simplified via the use of stereospecifically labeled Val, to eliminate the question of whether slightly different 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 protein−DNA 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 exchangebroadened 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 protein−DNA recognition.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. 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 financial interest.
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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, 692−697. (2) Billeter, M., Guntert, P., Luginbuhl, P., and Wüthrich, 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) Conformational heterogeneity and DNA recognition by the morphogen Bicoid. Biochemistry 56, 2787−2793. (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, 1927−1969. (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 B
DOI: 10.1021/acs.biochem.7b00760 Biochemistry XXXX, XXX, XXX−XXX
