Designed folding pathway of modular coiled-coil-based proteins.
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ABSTRACT:
SUBMITTER: Aupic J
PROVIDER: S-EPMC7878764 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cohesin’s association with and translocation along chromosomal DNAs depend on an ATP hydrolysis cycle driving the association and subsequent release of DNA. This involves DNA being ‘clamped’ by Scc2 and ATP-dependent engagement of cohesin’s Smc1 and Smc3 head domains. Scc2’s replacement by Pds5 abrogates cohesin’s ATPase and has an important role in halting DNA loop extrusion. The ATPase domains of all SMC proteins are separated from their hinge dimerisation domains by 50 nm long coiled coils, which have been observed to zip up along their entire length and fold around an elbow, thereby greatly shortening the distance between hinges and ATPase heads. Whether folding exists in vivo or has any physiological importance is not known. We present here a cryo-EM structure of the apo form of cohesin that reveals the structure of folded and zipped up coils in unprecedented detail and shows that Scc2 can associate with Smc1’s ATPase head even when it is fully disengaged from that of Smc3. Using cysteine-specific cross-linking, we show that cohesin’s coiled coils are frequently folded in vivo, including when cohesin holds sister chromatids together. Moreover, we describe a mutation (SMC1D588Y) within Smc1’s hinge that alters how Scc2 and Pds5 interact with Smc1’s hinge and that enables Scc2 to support loading in the absence of its normal partner Scc4. The mutant phenotype of loading without Scc4 is only explicable if loading depends on an association between Scc2/4 and cohesin’s hinge, which in turn requires coiled coil folding.
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Project description:Collagen triple helices fold slowly and inefficiently, often requiring adjacent globular domains to assist this process. In the Streptococcus pyogenes collagen-like protein Scl2, a V domain predicted to be largely ?-helical, occurs N-terminal to the collagen triple helix (CL). Here, we replace this natural trimerization domain with a de novo designed, hyperstable, parallel, three-stranded, ?-helical coiled coil (CC), either at the N terminus (CC-CL) or the C terminus (CL-CC) of the collagen domain. CD spectra of the constructs are consistent with additivity of independently and fully folded CC and CL domains, and the proteins retain their distinctive thermal stabilities, CL at ?37 °C and CC at >90 °C. Heating the hybrid proteins to 50 °C unfolds CL, leaving CC intact, and upon cooling, the rate of CL refolding is somewhat faster for CL-CC than for CC-CL. A construct with coiled coils on both ends, CC-CL-CC, retains the ?37 °C thermal stability for CL but shows less triple helix at low temperature and less denaturation at 50 °C. Most strikingly however, in CC-CL-CC, the CL refolds slower than in either CC-CL or CL-CC by almost two orders of magnitude. We propose that a single CC promotes folding of the CL domain via nucleation and in-register growth from one end, whereas initiation and growth from both ends in CC-CL-CC results in mismatched registers that frustrate folding. Bioinformatics analysis of natural collagens lends support to this because, where present, there is generally only one coiled-coil domain close to the triple helix, and it is nearly always N-terminal to the collagen repeat.
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Project description:Many meiosis-specific proteins in Schizosaccharomyces pombe contain coiled-coil motifs which play essential roles for meiotic progression. For example, the coiled-coil motifs present in Meu13 and Mcp7 are required for their function as a putative recombinase cofactor complex during meiotic recombination. Mcp6/Hrs1 and Mcp5/Num1 control horsetail chromosome movement by astral microtubule organization and anchoring dynein respectively. Dhc1 and Ssm4 are also required for horsetail chromosome movement. It is clear from these examples that the coiled-coil motif in these proteins plays an important role during the progression of cells through meiosis. However, there are still many unanswered questions on how these proteins operate. In this paper, we briefly review recent studies on the meiotic coiled-coil proteins in Sz. pombe.
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Project description:GemC1, together with Idas and Geminin, an important regulator of DNA-replication licensing and differentiation decisions, constitute a superfamily sharing a homologous central coiled-coil domain. To better understand this family of proteins, the crystal structure of a GemC1 coiled-coil domain variant engineered for better solubility was determined to 2.2 Å resolution. GemC1 shows a less typical coiled coil compared with the Geminin homodimer and the Geminin-Idas heterodimer structures. It is also shown that both in vitro and in cells GemC1 interacts with Geminin through its coiled-coil domain, forming a heterodimer that is more stable that the GemC1 homodimer. Comparative analysis of the thermal stability of all of the possible superfamily complexes, using circular dichroism to follow the unfolding of the entire helix of the coiled coil, or intrinsic tryptophan fluorescence of a unique conserved N-terminal tryptophan, shows that the unfolding of the coiled coil is likely to take place from the C-terminus towards the N-terminus. It is also shown that homodimers show a single-state unfolding, while heterodimers show a two-state unfolding, suggesting that the dimer first falls apart and the helices then unfold according to the stability of each protein. The findings argue that Geminin-family members form homodimers and heterodimers between them, and this ability is likely to be important for modulating their function in cycling and differentiating cells.
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