Abstract
In Sterkiella nova, alpha and beta telomere proteins bind cooperatively with single-stranded DNA to form a ternary alpha.beta.DNA complex. Association of telomere protein subunits is DNA-dependent, and alpha-beta association enhances DNA affinity. To further understand the molecular basis for binding cooperativity, we characterized several possible stepwise assembly pathways using isothermal titration calorimetry. In one path, alpha and DNA first form a stable alpha.DNA complex followed by the addition of beta in a second step. Binding energy accumulates with nearly equal free energy of association for each of these steps. Heat capacity is nonetheless dramatically different, with DeltaCp = -305 +/- 3 cal mol(-1) K(-1) for alpha binding with DNA and DeltaCp = -2010 +/- 20 cal mol(-1) K(-1) for the addition of beta to complete the alpha.beta.DNA complex. By examining alternate routes including titration of single-stranded DNA with a preformed alpha.beta complex, a significant portion of binding energy and heat capacity could be assigned to structural reorganization involving protein-protein interactions and repositioning of the DNA. Structural reorganization probably affords a mechanism to regulate high affinity binding of telomere single-stranded DNA with important implications for telomere biology. Regulation of telomere complex dissociation is thought to involve post-translational modifications in the lysine-rich C-terminal portion of beta. We observed no difference in binding energetics or crystal structure when comparing complexes prepared with full-length beta or a C-terminally truncated form, supporting interesting parallels between the intrinsically disordered regions of histones and this portion of beta.
Highlights
Protein enzyme telomerase [8]
As elaborated under “Discussion,” these characteristics make the C-terminal tail of  intriguingly comparable with intrinsically disordered protein (IDP) regions such as those found in histones [35]
The ITC method allowed examination of all binding reactions leading to ␣1⁄71⁄7DNA telomere complex formation without labels, tags, or postequilibrium handling steps. We relate these thermodynamic measurements to structural information provided by x-ray crystallography (18 –20), build a model for structural transitions that attend each binding reaction, and discuss broader implications of this work for telomere biology
Summary
Preparation of Protein and Single-stranded DNA—␣ (495 aa), the N-terminal domain of ␣ (aa 1–326, ␣-N),  (385 aa), and the protease-resistant core of  (aa 1–260, 28-kDa) proteins were expressed in Escherichia coli and purified to homogeneity through ammonium sulfate fractionation, ion exchange, and size exclusion chromatography as described previously [17, 18]. Titrations of ␣1⁄7 into solutions of singlestranded DNA were treated with a more complete binding model so as to take into account heat evolved or absorbed as a consequence of multiple potential reactions, including dissociation of the ␣1⁄7 complex upon dilution into the sample cell and protein-protein association coupled with DNA binding. For this analysis, numerical methods encoded in a C language program Rfree values were computed with the same set of structure factors reserved for cross-validation of the previously determined structure, Protein Data Bank code 1JB7
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