The orientation of the C-terminal domain of the Saccharomyces cerevisiae Rap1 protein is determined by its binding to DNA

Abstract

Rap1 is an essential DNA-binding factor from the yeast Saccharomyces cerevisiae involved in transcription and telomere maintenance. Its binding to DNA targets Rap1 at particular loci, and may optimize its ability to form functional macromolecular assemblies. It is a modular protein, rich in large potentially unfolded regions, and comprising BRCT, Myb and RCT well-structured domains. Here, we present the architectures of Rap1 and a Rap1/DNA complex, built through a step-by-step integration of small angle X-ray scattering, X-ray crystallography and nuclear magnetic resonance data. Our results reveal Rap1 structural adjustment upon DNA binding that involves a specific orientation of the C-terminal (RCT) domain with regard to the DNA binding domain (DBD). Crystal structure of DBD in complex with a long DNA identifies an essential wrapping loop, which constrains the orientation of the RCT and affects Rap1 affinity to DNA. Based on our structural information, we propose a model for Rap1 assembly at telomere.

The Wrapping Loop and Rap1 C-terminal (RCT) Domain of Yeast Rap1 Modulate Access to Different DNA Binding Modes

“…Access to the observed higher stoichiometry and the accompanying change in binding modes is not hampered by the presence of the N-terminal region of Rap1. This is consistent with the reported lack of preferential interactions of this region with the rest of the protein (25).…”

“…1a proposes that the first step for the DBD to transition between binding modes is the transient opening of the C-terminal wrapping loop, shown in the crystal structure to interact with the N-terminal Myb-like domain to form a closed complex on dsDNA (23)(24)(25). A direct prediction of this model is that removal of the C-terminal wrapping loop should favor the transition to the second, lower affinity binding mode.…”

“…The first DBD variant comprises residues 358 -590 (DBD 590 ), which removes the latch that makes direct interaction with the N-terminal Myb-like domain of the DBD (25). The second DBD variant comprises residues 358 -579 (DBD 579 ), which remove the entire C-terminal wrapping loop as defined by the crystal structure of the DBD bound to DNA (23)(24)(25). As a control, we also generated an extended version of the DBD that comprises residues 358 -615 (DBD 615 ) to include part of the toxicity region shown to relieve the growth inhibition phenotype observed by overexpression of Rap1 (26).…”

“…For this, we expressed a region of Rap1 comprising residues 358 -827 (DBD 827 ) (25). In addition to the DBD, this construct contains the latch region, the "tox" region, and the entire RCT domain.…”

“…In addition, formation of the 1:1 complex on DNA is accompanied by a different behavior when monitored by ITC. Matot et al (25) showed that binding of Rap1 to DNA induces a large conformational change of the RCT domain and that the N-terminal region of Rap1 does not interact with the rest of the protein. Therefore, next we asked whether the RCT domain behaves as an independent domain or whether it can interact with any other region of Rap1.…”