A role of the Nse4 kleisin and Nse1/Nse3 KITE subunits in the ATPase cycle of SMC5/6

Abstract

The SMC (Structural Maintenance of Chromosomes) complexes are composed of SMC dimers, kleisin and kleisin-interacting (HAWK or KITE) subunits. Mutual interactions of these subunits constitute the basal architecture of the SMC complexes. In addition, binding of ATP molecules to the SMC subunits and their hydrolysis drive dynamics of these complexes. Here, we developed new systems to follow the interactions between SMC5/6 subunits and the relative stability of the complex. First, we show that the N-terminal domain of the Nse4 kleisin molecule binds to the SMC6 neck and bridges it to the SMC5 head. Second, binding of the Nse1 and Nse3 KITE proteins to the Nse4 linker increased stability of the ATP-free SMC5/6 complex. In contrast, binding of ATP to SMC5/6 containing KITE subunits significantly decreased its stability. Elongation of the Nse4 linker partially suppressed instability of the ATP-bound complex, suggesting that the binding of the KITE proteins to the Nse4 linker constrains its limited size. Our data suggest that the KITE proteins may shape the Nse4 linker to fit the ATP-free complex optimally and to facilitate opening of the complex upon ATP binding. This mechanism suggests an important role of the KITE subunits in the dynamics of the SMC5/6 complexes.

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

“…Multiple inter‐domain cross‐links of the Nse1/3/4 module to the Smc5 and Smc6 proteins, mostly to the heads, were lost upon ATP and plasmid DNA addition. We postulate that Nse1/3/4 relocates by binding to DNA using the proposed DNA‐binding residues in Nse3 (Zabrady et al , 2016 ; Vondrova et al , 2020 ), possibly assisting to evict Nse5/6 from the heads and if so underscoring the central role of Nse5/6 in regulating the enzymatic activities of the Smc5/6 complex.…”

“…Another invariably conserved subunit, called “kleisin”, asymmetrically bridges the two SMC proteins at their head domains to create a tripartite ring structure capable of entrapping DNA in its lumen (Haering et al , 2004 ; Palecek et al , 2006 ; Burmann et al , 2013 ; Gligoris et al , 2014 ; Wilhelm et al , 2015 ). Kleisin also serves as an attachment point for additional proteins from the KITE (Kleisin‐Interacting Tandem winged‐helix Element) or HAWK (HEAT‐protein Associated With Kleisin) families (Palecek & Gruber, 2015 ; Wells et al , 2017 ) for which functions related to DNA substrate interactions and ATPase regulation are emerging (Zabrady et al , 2016 ; Kschonsak et al , 2017 ; Li et al , 2018 ; Vondrova et al , 2020 ).…”

Nse5/6 inhibits the Smc5/6 ATPase to facilitate DNA substrate selection

“…Indeed, DNA binding sites on top of the engaged heads were described for several related complexes ( Another invariably conserved subunit, called 'kleisin', asymmetrically bridges the two SMC proteins at their head domains to create a tripartite ring structure capable of entrapping DNA in its lumen (Burmann et al, 2013;Gligoris et al, 2014;Haering et al, 2004;Palecek et al, 2006;Wilhelm et al, 2015). Kleisin also serves as an attachment point for additional proteins from the KITE (Kleisin-Interacting Tandem winged-helix Element) or HAWK (HEAT-protein Associated With Kleisin) families (Palecek and Gruber, 2015;Wells et al, 2017) for which functions related to DNA substrate interactions and ATPase regulation are emerging (Kschonsak et al, 2017;Vondrova et al, 2020;Zabrady et al, 2016).…”

Role of Nse1 Subunit of SMC5/6 Complex as a Ubiquitin Ligase

“…The Smc5-Smc6 heterodimer forms the SMC5/6 complex along with six Nse (non-Smc element) subunits [ 6 ]. The Nse4 kleisin molecule binds SMC6 neck and SMC5 cap via its N- and C-terminal domains, respectively, analogously to the other SMC complexes [ 7 , 8 ]. Similar to prokaryotic SMC complexes, the Nse1-Nse3 kite subunits bind the Nse4 middle region via their winged-helix B (WHB) domains and regulate the dynamics of the SMC5/6 ATPase cycle [ 8 , 9 , 10 , 11 , 12 , 13 ].…”

“…The Nse4 kleisin molecule binds SMC6 neck and SMC5 cap via its N- and C-terminal domains, respectively, analogously to the other SMC complexes [ 7 , 8 ]. Similar to prokaryotic SMC complexes, the Nse1-Nse3 kite subunits bind the Nse4 middle region via their winged-helix B (WHB) domains and regulate the dynamics of the SMC5/6 ATPase cycle [ 8 , 9 , 10 , 11 , 12 , 13 ]. Uniquely for SMC complexes, Nse1 and Nse2 subunits possess enzymatic activities (reviewed in [ 4 , 14 ]).…”