Topoisomerase I (TOP1) dynamics: conformational transition from open to closed states

Abstract

Eukaryotic topoisomerases I (TOP1) are ubiquitous enzymes removing DNA torsional stress. However, there is little data concerning the three-dimensional structure of TOP1 in the absence of DNA, nor how the DNA molecule can enter/exit its closed conformation. Here, we solved the structure of thermostable archaeal Caldiarchaeum subterraneum CsTOP1 in an apo-form. The enzyme displays an open conformation resulting from one substantial rotation between the capping (CAP) and the catalytic (CAT) modules. The junction between these two modules is a five-residue loop, the hinge, whose flexibility permits the opening/closing of the enzyme and the entry of DNA. We identified a highly conserved tyrosine near the hinge as mediating the transition from the open to closed conformation upon DNA binding. Directed mutagenesis confirmed the importance of the hinge flexibility, and linked the enzyme dynamics with sensitivity to camptothecin, a TOP1 inhibitor targeting the TOP1 enzyme catalytic site in the closed conformation.

Fig. 1. Domains composition of type IB topoisomerases.

a Schematic view of the domain organization of eukaryotic, archaeal, bacterial, and viral Topo IB. The CAP, CAT, linker, and C-terminal domains are shown as green, pink, yellow, and red boxes, respectively. The N-terminal domains are shown in gray for Eukarya and Archaea Topo IB enzymes and green for Bacteria and Virus for Topo IB enzymes. b Cartoon representation of the Human TOP1 (HsTOP1) crystal structure (PDB 1K4T). Coloring is the same as in a.

Fig. 2. Crystal structure of archaeal CsTOP1.

a Open conformation of CsTOP1 solved by crystallography. b Sequence alignment between CsTOP1 and HsTOP1. The position of the lips are indicated with dotted lines. The residues interacting with DNA are shown with arrows. The coloring scheme is the same as in Fig. 1, apart from the Hinge loop which is shown in orange.

Fig. 3. Comparison of the TOP1 domains in their open and closed conformations.

a Superimposition of the CAP, CAT, Linker, and C-terminal domains of CsTOP1 and HsTOP1. CsTOP1 coloring is the same as in Fig. 1 and HsTOP1 is shown in gray. b Rotation of the CAP domain during DNA binding. The coloring is the same as in Fig. 1. The black arrow represents the rotation of the CAP domain from the open to the closed conformation.

Fig. 4. DNA-binding region of TOP1 in the open and closed conformations.

a Comparison of the relative positions of DNA interacting residues of CsTOP1 and HsTOP1. b Interaction between the lip2 and DNA interacting residues in the open state CsTOP1. CsTOP1 coloring is the same as in Fig. 1. c Position of the linker and DNA in the HsTOP1 crystal structure (PDB 1K4T) (right panel). For the left panel, the putative position of the DNA molecule interacting with CsTop1 is calculated after superposition using Pymol of CsTop1 structure with HsTOP1-DNA structure (PDB 1K4T). The basic residues are colored blue.

Fig. 5. TOP1 hinge loop.

a Alignment of several archaeal and eukaryotic hinge loops, highlighted in orange. The conserved tyrosine is highlighted in green. b Superimposition of the HsTOP1 and CsTop1 CAP domains. The hydrophobic interaction between the conserved leucine from the loop and one conserved hydrophobic pocket in the CAP domain is shown. The coloring scheme is the same as in Fig. 1. c Structural superimposition of the open and closed conformations of CsTOP1. The motion of the hinge allowing the interaction between one conserved tyrosine and DNA is shown. d Relaxation of negatively supercoiled DNA by WT and hinge mutated CsTOP1. The kinetics of relaxation is shown for the three constructs. The experiment was done twice independently.

Fig. 6. TOP1-mediated DNA cleavage assays.

a Sequence of the oligonucleotide used in the assays. The HsTOP1 cleavage site (CPT-dependent) and CsTOP1 cleavage site (CPT-independent) are shown in blue and red, respectively. The star represents the position of ³²P labelling. Ladder numbering represents the size of the DNA in nucleotides. b Cleavage of the oligonucleotide with WT CsTOP1 and HsTOP1 in the presence or absence of CPT. The reversion of TOP1ccs is monitored after the addition of 350 mM NaCl. c Cleavage of the oligonucleotide by WT and the indicated biologically engineered CsTOP1 (chimera) in the presence or absence of CPT. This experiment was done twice.

Fig. 7. Comparison of short Topo IB domains in their open and closed conformations.

Superimposition of Topo IB from Deinococcus radiodurans (PDB 2F4Q) and from Vaccinia (PDB 2H7G). Coloring is the same as in Fig. 1. The black arrow represents the rotation of the CAP domain from the open to the closed conformation.

Fig. 8. Proposed model of DNA entry inside the TOP1 catalytic site.

a In its apo-state, the CAP domain (green) and the CAT, Linker, C-ter domains (pink) can rotate. Lip2 is interacting with the DNA-binding region of the catalytic module. b After the displacement of the Lip2, one DNA molecule can enter the TOP1 catalytic site. c The Capping module rotates to trap DNA in the catalytic site. This rotation relies on the hinge and one conserved tyrosine from the CAP domain. The interaction between the two lips stabilizes the closed conformation.