Final Report Summary - TOPO (Structural studies of the yeast Topoisomerase III, RMI1 Sgs1 complex.)
Overall architecture. Several TopIII-RMI interfaces coexist due to the crystal packing but according to interface analysis (PISA) and already published data which involved the RMI1 OB domain as well as an insertion loop [4,9,10] inside this OB domain in the binding to TopIIIα, the real biological interface (confirmed recently by our EM experiments, data not shown) between the two proteins is shown in the figure 1.
The human TopIIIα protein retain all the structural features which have been highlighted by the E.coli Top1A [8], IIII [11,12] and the Thermotoga maritima Top1A [13] crystal structures. The crystallized protein is composed of four domains named 1 to 4 (fig1): domain 1 is the toprim (primase fold) domain carrying the acidic cluster involved in the catalytic site, domain 2 forms the so called gate made by two antiparallel topofold domains [14] and generating a ~25 Å diameter hole in the center of the protein, domains 3 and 4 share the same ‘Winged helix’ fold (also named CAP domains for catabolite activator protein) [15]. While domain 4 does not carry any catalytic residues, domain 3 possesses the conserved catalytic Tyrosine responsible for the cutting of ssDNA [12]. It is important to note that the C-terminal region including a putative Zinc finger domain potentially involved in DNA binding and predicted to be highly similar to the Thermotoga maritima Top1A C-terminus is not unambigously buildable in our structure even if some electronic densities are visible. The Nterminal RMI1 is composed of a three-α helices bundle followed by a typical OB domain fold consisting in a β-barrel as already published in [9,10]. The complex has a ‘Door-knocker’ overall shape, RMI1 contacting TopIII at the top of the gate (mainly α9 and the β9- α9 linker) via its OB domain. The so called RMI1 insertion loop (between α3 and β3) creates two other contacting zones to TopIII, the β10, β12 and β12-β13 linker in domain 2, α10 in domain 3, and α8 and 15 as well as α8-β9 linker generating a spread interface and obstructing partially the hole made by TopIII domain 2. This interface involve hydrogen bonds as well as hydrophobic interfacing residues, and one salt bridge for a total interface area of 1319 Å2.