Search

In certain maize genotypes (nulls), beta-glucosidase specifically interacts with a chimeric lectin called beta-glucosidase aggregating factor (BGAF), resulting in high molecular weight complexes. Previously, we showed that three regions (S1-T29, E50-N127, and F466-A512) on the maize beta-glucosidase isozyme Glu1 are involved in interaction and aggregation with BGAF. Recently, we found that the peptide span I72-T82 within E50-N127 is essential and sufficient for BGAF binding, whereas the S1-T29 and F466-A512 regions are required for formation of large complexes. To define the contribution of individual amino acids in the above three regions to BGAF binding, we constructed mutant beta-glucosidases based on sequence differences between maize beta-glucosidase and sorghum beta-glucosidase (dhurrinase 2, Dhr2), which does not bind BGAF. Binding was evaluated by gel-shift assay and affinity by frontal affinity chromatography (FAC). In the gel-shift assay, Glu1 mutants K81E and T82Y failed to bind BGAF, and their FAC profiles were essentially similar to that of Dhr2, indicating that these two amino acids within the I72-T82 region are important for BGAF binding. Substitution of N481 with E (as in Dhr2) lowered affinity for BGAF, whereas none of the mutations in the S1-T29 region showed any effect on BGAF binding. To further confirm the importance of K81 and T82 for BGAF binding, we produced a number of Dhr2 mutants, and the results showed that all four amino acids (I72, N75, K81, and T82) that differ between Glu1 and Dhr2 in the peptide span I72-T82 are required to impart BGAF-binding ability to Dhr2.

RTBP1 is a rice telomeric protein that binds to the duplex array of TTTAGGG repeats at chromosome ends. The DNA binding domain of RTBP1 contains a Myb-type DNA binding motif and a highly conserved C-terminal Myb extension that is unique to plant telomeric proteins. Using an electrophoretic mobility shift assay, we identified the C-terminal 110-amino acid region (RTBP1(506-615)) as the minimal telomeric DNA binding domain, suggesting that the Myb extension is required for binding plant telomeric DNA. Like other telomeric proteins such as human TRF1 and yeast Rap1, RTBP1 induced a DNA bending in the telomeric repeat sequence, suggesting that RTBP1 may play a role in establishing and/or maintaining an active telomere configuration in vivo. To elucidate the DNA binding mode of RTBP1, we determined the three-dimensional structure of RTBP1(506-615) in solution by NMR spectroscopy. The overall structure of RTBP1(506-615) is composed of four alpha-helices and stabilized by three hydrophobic patches. The second and third helices in RTBP1 form a helix-turn-helix motif that interacts directly with DNA. The fourth helix located in the Myb extension is essential for binding to telomeric DNA via stabilization of the overall structure of the RTBP1 DNA binding domain. When DNA bound to RTBP1(506-615), large chemical shift perturbations were induced in the N-terminal arm, helix 3, and the loop between helices 3 and 4. These results suggest that helix 3 functions as a sequence-specific recognition helix while the N-terminal arm stabilizes the DNA binding.