Probing the mechanism of recognition of ssDNA by the Cdc13-DBD

Abstract

The Saccharomyces cerevisiae protein Cdc13 tightly and specifically binds the conserved G-rich single-stranded overhang at telomeres and plays an essential role in telomere end-protection and length regulation. The 200 residue DNA-binding domain of Cdc13 (Cdc13-DBD) binds an 11mer single-stranded representative of the yeast telomeric sequence [Tel11, d(GTGTGGGTGTG)] with a 3 pM affinity and specificity for three bases (underlined) at the 5' end. The structure of the Cdc13-DBD bound to Tel11 revealed a large, predominantly aromatic protein interface with several unusual features. The DNA adopts an irregular, extended structure, and the binding interface includes a long ( approximately 30 amino acids) structured loop between strands beta2-beta3 (L(2-3)) of an OB-fold. To investigate the mechanism of ssDNA binding, we studied the free and bound states of Cdc13-DBD using NMR spectroscopy. Chemical shift changes indicate that the basic topology of the domain, including L(2-3), is essentially intact in the free state. Changes in slow and intermediate time scale dynamics, however, occur in L(2-3), while conformational changes distant from the DNA interface suggest an induced fit mechanism for binding in the 'hot spot' for binding affinity and specificity. These data point to an overall binding mechanism well adapted to the heterogeneous nature of yeast telomeres.

Figure 1.

¹H-¹⁵N HSQC spectrum of free Cdc13-DBD with assignments labeled. Boxed region labelled in inset.

Figure 2.

Graphs showing the difference in Cα chemical shift from random coil values for the free state (a) and the bound state (b). Positive values indicate helical propensity and negative values show regions in β-strands. The difference of free to bound differences to random coil values shows virtually no change in chemical shift index value (c).

Figure 3.

Graph of combined chemical shift difference from free to bound showing the improved analysis of using actual chemical shift values (black) over minimal analysis (red) (a). Combined chemical shift difference values were determined using the following equation Δδ = [(ΔH)² + (0.17*(ΔN))²]^1/2 (11). Chemical shift differences mapped onto the structure of Cdc13-DBD/Tel11 [PDB accession number 1S40 (12)] (b and c). The differences are shown in a color ramp from red to yellow with the largest changes in red (>0.3 ppm), residues with very small changes are shown in white, and residues with no data are in grey [figure was prepared using PyMol (46)].

Figure 4.

Exponential decay curves of selected hydrogen exchange data showing that some residues have similar rates in free and bound whether slow (T53) or fast (E184) (a). Residue V128 is an example of a residue with very different rates between free and bound. Mapping the difference in the free and bound rates shows that residues in red near the 5′-end of the DNA and in α1 have faster hydrogen exchange rates in the free state (b).

Figure 5.

Residues with average {¹H}¹⁵N NOE values lower than 0.7 are mapped onto the Cdc13-DBD/Tel11 structure (a). Residues with low NOE values in both free and bound are shown in purple, residues with only low values in free are in red, only in bound are in cyan and residues with no data are in grey. R_ex values are mapped on the Cdc13-DBD/Tel11 structure (b). More residues have slow and intermediate dynamics in the free state (residues in red have measurable R_ex values and in salmon are the exchange broadened unassigned residues in L_2–3) versus the bound (cyan). Residues exhibiting relaxation dispersion in both the free and bound states are shown in purple.