Abstract
High-resolution structural analysis of flexible proteins is frequently challenging and requires the synergistic application of different experimental techniques. For these proteins, small-angle X-ray scattering (SAXS) allows for a quantitative assessment and modeling of potentially flexible and heterogeneous structural states. Here, we report SAXS characterization of the condensin HEAT-repeat subunit Ycg1Cnd3 in solution, complementing currently available high-resolution crystallographic models. We show that the free Ycg1 subunit is flexible in solution but becomes considerably more rigid when bound to its kleisin-binding partner protein Brn1Cnd2 The analysis of SAXS and dynamic and static multiangle light scattering data furthermore reveals that Ycg1 tends to oligomerize with increasing concentrations in the absence of Brn1. Based on these data, we present a model of the free Ycg1 protein constructed by normal mode analysis, as well as tentative models of Ycg1 dimers and tetramers. These models enable visualization of the conformational transitions that Ycg1 has to undergo to adopt a closed rigid shape and thereby create a DNA-binding surface in the condensin complex.
Highlights
High-resolution structural analysis of flexible proteins is frequently challenging and requires the synergistic application of different experimental techniques
The normalized Kratky plots for Ycg1 and Ycg1–Brn1515–634 (Fig. 2d) suggest that unbound Ycg1 exhibits more flexibility than the Ycg1–Brn1515–634 complex. The latter plot is bell-shaped, which is characteristic of globular proteins, whereas the unbound protein reveals elevated scattering at higher angles, pointing to a higher anisometry/flexibility
It has been suggested that this recruitment triggers the subsequent ATP-dependent entrapment of DNA strands within the lumen of the large ring structure created by the SMC and kleisin subunits of condensin
Summary
HEAT-repeat proteins have been shown to exhibit significant flexibility, both in response to binding events and as a result of external forces [9, 10] It remains unclear whether the inability of Ycg to bind DNA by itself is merely due to the missing positive charges that are contributed by Brn residues and/or its safety-belt entrapment or whether binding to Brn induces structural transitions in the HEAT-repeat solenoid to create a DNA-binding site. The latter option would be analogous to the large conformational changes that other HEAT-repeat proteins undergo upon ligand binding [11, 12]. Utilizing the high-resolution structure of Sc Ycg1–Brn as a starting point, we constructed models of monomeric Ct Ycg and the Ct Ycg1–Brn1515–634 complex in solution by a normal mode analysis against the SAXS data
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