Tables for
Volume F
Crystallography of biological macromolecules
Edited by M. G. Rossmann and E. Arnold

International Tables for Crystallography (2006). Vol. F. ch. 23.4, pp. 638-639   | 1 | 2 |

Section Protein–DNA recognition

C. Mattosa* and D. Ringeb

aDepartment of Molecular and Structural Biochemistry, North Carolina State University, 128 Polk Hall, Raleigh, NC 02795, USA, and  bRosenstiel Basic Medical Sciences Research Center, Brandeis University, 415 South St, Waltham, MA 02254, USA
Correspondence e-mail: Protein–DNA recognition

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The trp repressor binds specifically to the target DNA sequence ACTAGT, resulting in the transcriptional control of L-tryptophan levels in bacteria. The crystal structure of the trp repressor/operator complex was solved to 1.9 Å resolution (Otwinowski et al., 1988[link]). Although the structure revealed hydrogen-bonding interactions between the protein and the backbone phosphate groups, no direct hydrogen bonds or non-polar contacts between the protein and DNA bases were observed. Specificity was therefore attributed to the effect of the sequence on the geometry of the phosphate backbone and to water-mediated polar contacts between protein atoms and specific DNA bases. To confirm this hypothesis, the 1.95 Å resolution crystal structure of the free decamer CCACTAGTGG was obtained, containing the recognition six-base-pair sequence (Shakked et al., 1994[link]). A comparative analysis of the free and complexed DNA showed that, when bound to the trp repressor, the six-base-pair region is bent by about 15° so as to compress the major groove, with concomitant expansion of the minor groove relative to the uncomplexed DNA (Shakked et al., 1994[link]). However, both free and complexed DNA are underwound, with 10.6 base pairs per turn, rather than the usual 10.0 base pairs per turn. This feature is presumably a result of the particular DNA sequence and is thought to decrease the energy barrier for the binding interaction with the trp repressor protein (Shakked et al., 1994[link]). Another specificity component suggested by the authors is conferred by the hydration of the consensus bases. Ten water molecules are observed to interact in the major groove at similar positions in both the free and complexed DNA. Three of these mediate in four hydrogen-bonding interactions to the protein in the complex. Interestingly, the DNA bases to which these three water molecules are bound are among the most conserved and mutationally sensitive bases of the operator. In effect, these three water molecules can be regarded as extensions of the DNA bases and part of the specific recognition elements of the target DNA sequence (Shakked et al., 1994[link]).

The idea of water molecules as mediators of interactions conferring specificity in protein–DNA associations is further supported by the co-crystal structure of the HNF-3/fork head DNA-recognition motif in complex with DNA, solved to 2.5 Å resolution (Clark et al., 1993[link]). Although the lower resolution of this protein–DNA complex may limit the unambiguous determination of water molecules to those that are tightly bound, a series of water molecules are observed in the major groove, bridging specific DNA bases to amino-acid side chains in one of the α-helices of the protein. In this case, direct hydrogen bonding between DNA bases and protein side chains also exists.

The involvement of water in specific protein–DNA recognition was further confirmed in a study of the accuracy of specific DNA cleavage by the restriction endonuclease EcoRI under different osmotic pressures (Robinson & Siglar, 1993[link]). Changes in osmotic pressure, resulting from changes in osmolite concentrations, have direct effects on the number of water molecules associated with macromolecules (Rand, 1992[link]). The EcoRI experiments show that water activity affects site-specific DNA recognition, with an increase in osmotic pressure leading to a decrease in accuracy of protein–DNA recognition, as observed by DNA cleavage at sites containing an incorrect base pair (Robinson & Siglar, 1993[link]). The results of this study strongly imply a role for one or more water molecules in recognition of specific sequences of DNA. The authors suggest that water mediation may constitute a general motif for sequence-specific DNA recognition by DNA-binding proteins (Robinson & Siglar, 1993[link]).

The role of water molecules as mediators of sequence-specific DNA recognition may be a general motif, but not a necessary one. The solution NMR structure of the complex of erythroid transcription factor GATA-1 with the 16-base-pair DNA fragment GTTGCAGATAAACATT, containing the recognition sequence, shows that the specific interactions between GATA-1 and the major groove of the DNA are dominated by van der Waals interactions hydrophobic in nature (Omichinski et al., 1993[link]). Furthermore, NMR experiments designed to identify the location of water molecules in the complex detected clusters of water molecules bridging the protein to the DNA phosphate backbone, but showed that water was excluded from the hydrophobic interface between the protein and the DNA bases (Clore et al., 1994[link]). Although many of the existing crystal structures of protein–DNA complexes support the general view that water molecules are often integral components of the specific recognition between the protein and the target DNA, this solution structure provides an important example of exclusion of water molecules from the specificity determinants. In the GATA-1–DNA complex, however, water molecules do mediate non-specific binding of the protein to the DNA backbone. It appears, not surprisingly, that water molecules play a variety of roles in the mediation of protein–DNA interactions and that these roles are specific to each particular case.


First citation Clark, K. L., Halay, E. D., Lai, E. & Burley, S. K. (1993). Co-crystal structure of the HNF-3/fork head DNA-recognition motif resembles histone H5. Nature (London), 364, 412–420.Google Scholar
First citation Clore, G. M., Bax, A., Omichinski, J. G. & Gronenborn, A. M. (1994). Localization of bound water in the solution structure of a complex of the erythroid transcription factor GATA-1 with DNA. Curr. Biol. 2, 89–94.Google Scholar
First citation Omichinski, J. G., Clore, G. M., Schaad, O., Felsenfeld, G., Trainor, C., Appella, E., Stahl, S. J. & Gronenborn, A. (1993). NMR structure of a specific DNA complex of Zn-containing DNA binding domain of GATA-1. Science, 261, 438–446.Google Scholar
First citation Otwinowski, Z., Schevitz, R. W., Zhang, R.-G., Lawson, C. L., Joachimiak, A., Marmorstein, R. Q., Luisi, B. F. & Sigler, P. B. (1988). Crystal structure of trp repressor/operator complex at atomic resolution. Nature (London), 335, 321–329.Google Scholar
First citation Rand, R. P. (1992). Raising water to new heights. Science, 256, 618.Google Scholar
First citation Robinson, C. R. & Siglar, S. G. (1993). Molecular recognition mediated by bound water. A mechanism for star activity of the restriction endonuclease EcoRI. J. Mol. Biol. 234, 302–306.Google Scholar
First citation Shakked, Z., Guzikevich-Guerstein, G., Frolow, F., Rabinovich, D., Joachimiak, A. & Sigler, P. B. (1994). Determinants of repressor/operator recognition from the structure of the trp operator binding site. Nature (London), 368, 469–473.Google Scholar

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