Integrative analysis reveals unique structural and functional features of the Smc5/6 complex

Abstract

Structural maintenance of chromosomes (SMC) complexes are critical chromatin modulators. In eukaryotes, the cohesin and condensin SMC complexes organize chromatin, while the Smc5/6 complex directly regulates DNA replication and repair. The molecular basis for the distinct functions of Smc5/6 is poorly understood. Here, we report an integrative structural study of the budding yeast Smc5/6 holo-complex using electron microscopy, cross-linking mass spectrometry, and computational modeling. We show that the Smc5/6 complex possesses several unique features, while sharing some architectural characteristics with other SMC complexes. In contrast to arm-folded structures of cohesin and condensin, Smc5 and Smc6 arm regions do not fold back on themselves. Instead, these long filamentous regions interact with subunits uniquely acquired by the Smc5/6 complex, namely the Nse2 SUMO ligase and the Nse5/Nse6 subcomplex, with the latter also serving as a linchpin connecting distal parts of the complex. Our 3.0-Å resolution cryoelectron microscopy structure of the Nse5/Nse6 core further reveals a clasped-hand topology and a dimeric interface important for cell growth. Finally, we provide evidence that Nse5/Nse6 uses its SUMO-binding motifs to contribute to Nse2-mediated sumoylation. Collectively, our integrative study identifies distinct structural features of the Smc5/6 complex and functional cooperation among its coevolved unique subunits.

Nse5/6 inhibits the Smc5/6 ATPase and modulates DNA substrate binding

“…To rule out that this lack of effect was caused by a lack of interaction under our experimental conditions, we measured the affinity for dsDNA and ssDNA substrates by fluorescence anisotropy (Fig 4C ). In agreement with independent reports (Hallett et al , 2021 ; Yu et al , 2021 ), the Nse5/6 dimer did not detectably bind DNA in the tested concentration range, whereas the Smc5/6 hexamer interacted strongly (K d around 150 nM) with both types of substrates. The reconstituted octamer also had no preference for either substrate and displayed a slightly higher affinity (lowered K d of around 100 nM), potentially because the Nse5/6 complex stabilizes a Smc5/6 conformation that is favourable for DNA binding.…”

“…In our XL‐MS data, we detected multiple inter‐domain cross‐links between Nse5/6 and the Smc5/6 hexamer (Fig 3C ), which are in line with the putative interface determined by pulldown assays and also with independent XL‐MS experiments published while this work was in progress (Gutierrez‐Escribano et al , 2020 ; Yu et al , 2021 ). Of note, cross‐links between Nse5 and Nse6 were surprisingly rare and only observed under the ATPase buffer conditions (see below).…”

“…We speculate, however, that Nse5 and Nse6 have evolved from larger ancestral proteins—being reminiscent of Slf1 and Slf2—by the loss of HEAT repeats. While this work was under review an independent study reported the cryo‐EM structure of the budding yeast Nse5/6 complex (Yu et al , 2021 ), which is in excellent agreement with the crystal structure presented here (rmsd 0.829 Å; see Fig EV1F ).…”

“…Structural information on the Nse5/6 complex is currently limited to the disordered N‐terminus of Nse6 that interacts with Rtt107 (Wan et al , 2019 ) (Fig 2A ). Compounding this, there are conflicting reports in the literature on the presence of α‐helical HEAT repeats in Nse6 and its putative relatedness to human Slf2, a subunit of the Smc5/6 associated Slf1/Slf2 complex (Pebernard et al , 2006 ; Raschle et al , 2015 ; Adamus et al , 2020 ; Yu et al , 2021 ). Limited proteolysis of purified Nse5/6 (Fig 2B ) led to the design of two N‐terminally truncated Nse6 fragments (154‐C and 177‐C) which were easily co‐purified with Nse5 indicating a stable interaction with Nse5.…”

“…Due to the large interaction surface and the fact that neither Nse6 nor Nse5 could be produced in isolation, we did not attempt to disrupt this interface by mutagenesis. Such mutations, however, have been reported based on a related structure obtained by cryo‐electron microscopy recently (Yu et al , 2021 ). To evaluate our crystal structure using a different approach, we mutated H368 in Nse6 as well as G56 in Nse5 to cysteines.…”