Hox proteins are transcriptional regulators that bind consensus DNA sequences. The DNA-binding specificity of many of these Hox proteins is modulated by the heterodimerization with partners, such as the Pbx proteins. This cooperative heterodimerization is accomplished through a conserved hexapeptide motif found N-terminal to the Hox DNA-binding homeodomain. Several human leukemias have been associated with a chromosomal translocation involving either the Hox gene (i.e., NUP98/HOXA9) or the gene encoding Pbx1 (E2A/PBX1). The transforming ability of these fusion oncoproteins relies at least partially on the ability to interact with one another through this hexapeptide motif. Herein we describe NMR structural calculations of the hexapeptide of HoxB1 (N␣-acetyl-Thr-Phe-Asp-Trp-Met-Lys-amide) that has been shown to mediate binding between HoxB1 and Pbx1 and a hexapeptide consensus sequence (N␣-acetyl-Leu-Phe-Pro-TrpMet-Arg-amide). The consensus peptide exists in two conformations caused by cis-trans isomerization of the Phe-Pro peptide bond. The structures of the HoxB1 peptide and the trans form of the consensus peptide reveal a turn very similar to that found as part of the HoxB1/Pbx1/DNA complex in the X-ray crystal structure. This observation implies that this region is at least partially 'preformed' and thus ready to interact with Pbx1 and stabilize binding of Pbx1 and HoxB1 to DNA. The structural results presented here provide a starting point for synthesizing potential nonpeptide or cyclical peptide antagonists that mimic the interaction of these transcriptional cofactors resulting in a potential chemotherapeutic for certain types of leukemias.Keywords: Hox; Pbx; DNA; transcription; inhibitor; NMR; peptide The Homeobox genes are a family of developmental regulatory genes that encode nuclear homeoproteins that act as transcription factors (Gehring et al. 1994). These proteins contain a highly conserved common 60-63-residue DNAbinding homeodomain (HD) that is capable of binding DNA as a monomer (Laughon 1991). Although the homeobox genes were initially described as crucial to the correct anteroposterior patterning of the embryo in vertebrates, Drosophila, and Caenorhabditis elegans (for review, see McGinnis and Krumlauf 1992), the homeobox has subsequently been identified in a wide range of ∼100 mammalian proteins (Stein et al. 1996).The class-I homeobox genes are defined by homology with the Drosophila Antennapeida (Antp) homeodomain (Akam 1987). The 40 mammalian class-I genes (Hox genes) have attracted the most attention because of their clustered organization on four chromosomes (Scott et al. 1989). The Hox proteins are very specific regulators of transcription, and yet, as monomers in vitro, they exhibit similar DNAReprint requests to: Dr. Brian D. Sykes, Protein Engineering, Network Centers of Excellence, 713 Heritage Medical Research Building, University of Alberta, Edmonton, Alberta T6G 2H7, Canada; e-mail: brian.sykes@ualberta.ca; fax: 780-492-1473.Abbreviations: NMR, nuclear magnetic resonance; D...

The potential for using paramagnetic lanthanide ions to partially align troponin C in solution as a tool for the structure determination of bound troponin I peptides has been investigated. A prerequisite for these studies is an understanding of the order of lanthanide ion occupancy in the metal binding sites of the protein. , and Yb 3+ to both apo-and holo-forms of human cardiac troponin C (cTnC) and of Ce 3+ to holochicken skeletal troponin C (sTnC). The disappearance of cross-peak resonances in the HSQC spectrum was used to determine the order of occupation of the binding sites in both cTnC and sTnC by each lanthanide. For the lanthanides tested, the binding order follows that of the net charge of the binding site residues from most to least negative; the N-domain calcium binding sites are the first to be filled followed by the C-domain sites. Given this binding order for lanthanide ions, it was demonstrated that it is possible to create a cTnC species with one lanthanide in the N-domain site and two Ca 2+ ions in the C-domain binding sites. By using the species cTnC·Yb