Abstract
Cdt1 is a mixed folded protein critical for DNA replication licensing and it also has a “moonlighting” role at the kinetochore via direct binding to microtubules and the Ndc80 complex. However, it is unknown how the structure and conformations of Cdt1 could allow it to participate in these multiple, unique sets of protein complexes. While robust methods exist to study entirely folded or unfolded proteins, structure–function studies of combined, mixed folded/disordered proteins remain challenging. In this work, we employ orthogonal biophysical and computational techniques to provide structural characterization of mitosis-competent human Cdt1. Thermal stability analyses shows that both folded winged helix domains1 are unstable. CD and NMR show that the N-terminal and linker regions are intrinsically disordered. DLS shows that Cdt1 is monomeric and polydisperse, while SEC-MALS confirms that it is monomeric at high concentrations, but without any apparent inter-molecular self-association. SEC-SAXS enabled computational modeling of the protein structures. Using the program SASSIE, we performed rigid body Monte Carlo simulations to generate a conformational ensemble of structures. We observe that neither fully extended nor extremely compact Cdt1 conformations are consistent with SAXS. The best-fit models have the N-terminal and linker disordered regions extended into the solution and the two folded domains close to each other in apparent “folded over” conformations. We hypothesize the best-fit Cdt1 conformations could be consistent with a function as a scaffold protein that may be sterically blocked without binding partners. Our study also provides a template for combining experimental and computational techniques to study mixed-folded proteins.
| Original language | English (US) |
|---|---|
| Journal | Cytoskeleton |
| DOIs | |
| State | Accepted/In press - 2024 |
Funding
We thank Dr. Cara Gottardi for her support and mentorship. We thank Anita Varma for laboratory operations. We thank Dr. Sergii Pshenychnyi of the Recombinant Protein Production Core for supplying TEV protease. We thank Dr. Ronald Soriano, James Casey, and the Northwestern Keck Biophysics Facility for assistance with data collection. This work used resources from the Northwestern University Keck Biophysics Facility, supported by NCI\u2010CCSG\u2010P30\u2010CA060553 and was awarded to the Robert H. Lurie Comprehensive Cancer Center. This study made use of the National Magnetic Resonance Facility at Madison; an NIH Biomedical Technology Research Resource Center supported by NIH R24GM141526. Helium recovery equipment, computers, and infrastructure for data archive were funded by the University of Wisconsin\u2010Madison, NIH P41GM136463, R24GM141526, and by the United States National Science Foundation Mid\u2010Scale Research Infrastructure\u20101 Program Grant No. 1946970. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE\u2010AC02\u201006CH11357. The use of Pilatus 3 1M detector was provided by grant 1S10OD018090\u201001 from NIGMS. This work was supported by NIGMS grant R01GM135391 to D.V. Helium recovery equipment, computers, and infrastructure for data archive were funded by the University of Wisconsin\u2010Madison, NIH P41GM136463, R24GM141526, and by the United States National Science Foundation Mid\u2010Scale Research Infrastructure\u20101 program under Grant No. 1946970. The use of Pilatus 3 1M detector was provided by grant 1S10OD018090\u201001 from NIGMS. This work was supported by NIGMS grant R01GM135391 to Dileep Varma. Funding:
Keywords
- Monte Carlo
- conformational dynamics
- intrinsically disordered protein
- kinetochore
- rigid body modeling
- small angle x-ray scattering
ASJC Scopus subject areas
- Structural Biology
- Cell Biology