Three-dimensional topology of the SMC2/SMC4 subcomplex from chicken condensin I revealed by cross-linking and molecular modelling

Abstract

SMC proteins are essential components of three protein complexes that are important for chromosome structure and function. The cohesin complex holds replicated sister chromatids together, whereas the condensin complex has an essential role in mitotic chromosome architecture. Both are involved in interphase genome organization. SMC-containing complexes are large (more than 650 kDa for condensin) and contain long anti-parallel coiled-coils. They are thus difficult subjects for conventional crystallographic and electron cryomicroscopic studies. Here, we have used amino acid-selective cross-linking and mass spectrometry combined with structure prediction to develop a full-length molecular draft three-dimensional structure of the SMC2/SMC4 dimeric backbone of chicken condensin. We assembled homology-based molecular models of the globular heads and hinges with the lengthy coiled-coils modelled in fragments, using numerous high-confidence cross-links and accounting for potential irregularities. Our experiments reveal that isolated condensin complexes can exist with their coiled-coil segments closely apposed to one another along their lengths and define the relative spatial alignment of the two anti-parallel coils. The centres of the coiled-coils can also approach one another closely in situ in mitotic chromosomes. In addition to revealing structural information, our cross-linking data suggest that both H2A and H4 may have roles in condensin interactions with chromatin.

Cross-linking reveals laminin coiled-coil architecture

“…This technique is particularly suitable for elongated protein assemblies such as coiled coils (30), which have high surface-to-volume ratios. The laminin coiled coil is rich in amino acids with cross-linkable functional groups (Fig.…”

Development of Large-scale Cross-linking Mass Spectrometry

“…This approach allows focusing the analysis on proteins of interest and significantly reduces the complexity of the sample (38). SDS-PAGE is also used to optimize the cross-linking protocol.…”

“…The software visualizes the spectra matches so they can be manually validated and their confidence assigned. Xi, works well when used with lysine reactive cross-linkers, and has been shown to produce meaningful data for datasets of the size of the chicken chromosome (38). Kojak is an open-source software that works with variety of cross-linker chemistries, and it was shown to correctly identify cross-links for 26S ribosome (39).…”

“…In this study the same strategy to identify cross-linked peptides as in (35) Ideal search engine should be able to search any type of cross-linking data, provide a score reflecting the confidence of the matches, annotate spectra matches, extract the high confidence spectra and provide FDR. Cross-links can be directly used for building models of protein structure or PPI network (38). …”

The torments of the cohesin ring

“…16,[24][25][26] Thus, it is likely that all protein complexes containing Smc-like subunits are able to transition between the "arms together" and "arms apart" forms and this transition is important for their function. The long-range allosteric regulation involving coiled coils was studied in the cohesin-related Rad50/Mre11 complex which tethers the ends of broken DNA molecules to facilitate DNA repair.…”