TOP3β – TDRD3 Complex (PDB ID: 5GVE) from Homo sapiens

Created by: Daniel Song

            Human topoisomerase β – tudor domain-containing protein 3 (PDB ID: 5GVE) is a type IA topoisomerase protein from Homo sapiens.  Type IA topoisomerases relax supercoiled DNA and resolve recombinant DNA intermediates in meiosis and cross overs (1).  Topoisomerase 3β, TOP3β, is complexed with its auxiliary factor, tudor domain-containing protein 3, TDRD3 (1, 2).  TOP3β-TDRD3 has a molecular weight of 96,661.96 daltons and an isoelectric point of 8.47, both of which were calculated with Expasy (3).  TOP3β contains 25 alpha helices and 22 beta strands, whereas TDRD3 contains 4 alpha helices and 10 beta strands (2).  These two subunits interact to form the TOP3β -TDRD3 complex that is able to bind with both DNA and RNA (1).

            TOP3β was crystallized by the sitting drop vapor diffusion method (1, 2).  The structural data was obtained by X-ray diffraction and solved using the molecular replacement method (2).  TOP3β is composed of a topoisomerase domain (TOPO), five zinc finger regions, and an RGG domain.  The TOPO domain is a toroidal structure, formed by four subdomains (1).  Subdomains I, III, and IV form a Mg2+ bound catalytic pocket, which is found in other type IA topoisomerases.  This catalytic pocket relaxes supercoiled DNA through an enzyme-bridged strand-passage mechanism (4).  Upon cleavage of DNA, the 5’ end of the cleaved strand is bound to the catalytic pocket by a phosphotyrosine covalent bond in the TOPO domain (1, 4).  The unbound strand is then passed through TOP3β before the break is resealed (4).  The catalytic site is exposed through the pivot motion of subdomains II and IV, which separates subdomains I and III (1).  This motion is accomplished due to the tight stacking interactions of TOP3β Phe-235 and Phe-265, and TDRD3 Pro-82 (1).

            Although not essential for viability, TOP3β is involved with proper neural development and promoting transcription (1).  TOP3β binds to DNA at its active site in the TOPO domain.  The catalytic pocket consists of functionally essential residues that assist in DNA binding such as Asp-119, Glu-9, Asp-117, Lys-10, Glu-326, Tyr-336, His-387, and Arg-338 (1).  Specifically, Tyr-336 is essential for DNA topoisomerase activity by holding the cleaved DNA in place at the catalytic site through a phosphotyrosine bond (5).  The Mg2+ bound in the catalytic pocket also contributes to stability of the TOPO domain active site (1).  In addition, although not fully understood, the four zinc finger regions present may also be essential for catalytic function and structural stability (1).  TOP3β also shows greater RNA binding affinity at its C-terminal RGG domain, which is unique to TOP3β.  It is believed that this region is an RNA binding motif and is essential for RNA topoisomerase activity (1, 5).

TDRD3 is the auxiliary factor of TOP3β and coexists with TOP3β in the cell.  TDRD3 is composed of a DUF1767 domain, a beta-barrel OB-fold domain, a UBA domain, and a Tudor domain (1, 2).  The N-terminal of the DUF1767 domain is composed of four alpha helices and is linked to the C-terminal of the OB-fold domain, which is unfolded in solution (1).  The OB domain also contains an insertion loop from Val-79 to Pro-92, which binds with TOP3β (1).  This insertion loop plays a role in distinguishing TOP3β from TOP3α.  Replacing the insertion loop of RMI1, the auxiliary factor of TOP3Aα, with that of TDRD3 resulted in a 30% increase in TOP3β binding (1).  Other specific core residues are also essential for TOP3β binding.  Mutating TDRD3 Arg-96, Val-109, and Phe-139 resulted in a 35% decrease in TOP3β binding (1).  Mutation of these core, hydrophobic residues and the replacement of the TDRD3 insertion loop with that of RMI1 resulted in a complete elimination of TOP3β binding by TDRD3 (1).

The TOP3β-TDRD3 interaction is further maintained by essential residues.  Hydrogen bonds are formed between TDRD3 Val-79 and TOP3β Phe-265, and TDRD3 Arg-96 and TOP3β Asp-266 (1).  Core and insertion loop mediated hydrophobic interactions help maintain the complex structure and contribute to TDRD3 specificity towards TOP3β.  Arg-96, Val-109, Phe-111 and Phe-139 of TDRD3 are essential for core mediated interactions.  In the N-terminal region of TDRD3, Val-l79, Ala-80, Ala-81, and Pro-82 form a hydrophobic surface to interact with Val-264, Phe-265, and Ile-269 of TOP3Β.  Finally, in the C-terminal region of TDRD3, Ala-90, Ala-91, Pro-92 and Met-94 of TDRD3 form a hydrophobic surface to interact with Val-262, Val-264, Ile-269 and Phe-273 of TOP3β (1).

In humans, TOP3β interacts with the fragile X mental retardation protein (FMRP) to function as an RNA topoisomerase (1, 5).  Ile-304 of TOP3β is essential for FMRP interaction (5).  FMRP binds to over 840 mRNA’s, and one missense mutation in FMRP results in the onset of fragile X syndrome (1, 5).  Through its interaction with FMRP, TOP3β regulates mRNA expression (1).  This can be accomplished by resolving topological problems with RNA such as the decatenation of circularized mRNA (5).  In addition, TOP3β is necessary for proper synaptogenesis, since the knockout of TOP3β resulted in reduced synapse density in Drosophila (5).  Although both FMRP and TOP3β contain binding sites on several mRNAs that contain genes related to autism and schizophrenia, it is believed that the two interact antagonistically to prevent the expression of these neurological disorders (5).

TOP3β -TDRD3 is also essential for epigenetic regulation.  Previous studies show that mice lacking TOP3β contain increased levels of autoantibodies, as a result of chromosomal problems that arise from a failure to resolve recombinant pairs of chromosomes (6).  The apoptotic cell death that results from this can result in development of autoimmunity (6).  Mice that lacked TOP3β also were shown to have a mean lifespan of 15 months, compared to the two-year life span of wild type mice (7).  It is known that type IA DNA topoisomerases form complexes with RecQ helicases (7).  Therefore, this shortened life span suggests that lack of TOP3β may result in development of progeroid diseases from RecQ helicase mutation (7).  Finally, TOP3β-TDRD3 is essential for promoting transcription at methylated arginine sites.  The complex is recruited to the c-MYC locus to promote transcription and resolve R loops.  These R loops result from negative supercoiled DNA, formed by newly transcribed RNA that anneals back to the template DNA (1, 7).

TDRD3 is also essential for TOP3β function.  TDRD3 functions as a scaffolding, recruiting TOP3β and DNA or RNA to bind and interact with one another. (1).  The C-terminal of the Tudor domain interacts with methyl-histone marks in DNA (8).  The mutation of the Tudor domain of TDRD3 resulted in a reduction of TOP3β recruitment to chromatin, and TDRD3 deficient mice were shown to have decreased TOP3β and genomic stability (8).  This demonstrates that TDRD3 links and stabilizes TOP3β to bind DNA and RNA (8).  This is further shown by increased translocation between the c-MYC and Igh loci in TDRD3 deficient mice, which is driven by R loop accumulation as a result of decreased TOP3β activity.  (8).  Finally, TDRD3 is also shown to promote TOP3β and FMRP interaction by binding its C-terminal Tudor region to FMRP and its N-terminal Tudor region to TOP3β (5).

TOP3β-TDRD3 shows remarkable similarity to Escherichia coli DNA Topoisomerase III (PDB ID: 1D6M) (2).  The Z-score calculated using the Dali server indicates significant similarity in tertiary structures if it is greater than 2.  The Z-score of 28.3 for Escherichia coli DNA topoisomerase III indicates that the tertiary structure is very similar to that of TOP3β (9).  In addition, the primary structures are very similar as determined from the Position-Specific Iterated Basic Local Assignment Search Tool (PSI-BLAST) (10).  An E value is assigned using gaps in the protein primary structure to indicate levels of similarity.  The subject protein, Escherichia coli DNA topoisomerase III, was assigned an E value of 0.0.  This E value is less than a significant E value of 0.05, indicating that the protein sequence aligns closely with that of TOP3β-TDRD3 (10).  Based on these tests, topoisomerase III is a protein of great similarity to TOP3β, and can reveal significant information about the mechanism of action and structure of the protein.

Escherichia coli topoisomerase III, like TOP3β-TDRD3, is a type IA topoisomerase and thus functions to catenate and decatenate DNA and relax negatively supercoiled DNA (11).  This is essential for proper DNA replication in Escherichia coli since DNA unwinding results in supercoiled DNA that prevents proper DNA replication initiation (12).  This function is similar to that of TOP3β-TDRD3, which resolves R loop accumulation (1, 7).  Because Escherichia coli topoisomerase III is also a type IA topoisomerase, key structural features are similar to those of TOP3β.  For instance, the core toroidal shape formed by four subdomain structures is found in topoisomerase III, and other members of the type IA topoisomerase family (4).  The active site also contains a tyrosine residue at position 328, which binds to one end of a cleaved DNA strand like in TOP3β (4).  The Lys-8, Glu-7, Asp-103, Asp-105 and Arg-330 residues also are similar to those found in TOP3Β, which bind to DNA in the active site (4, 11).  Finally, the mechanism of activity for topoisomerase III can be applied for other type IA topoisomerases (11).

There are also structural and functional differences between TOP3β-TDRD3, and Escherichia coli topoisomerase III.  TOP3β contains an RGG region that binds to RNA and contributes to RNA topoisomerase activity, which is not found in E.Coli topoisomerase III (5, 11).  This RGG region is unique to TOP3β and shows the functional uniqueness of TOP3β as an RNA topoisomerase (5).  Also, while topoisomerase III functions independently, TOP3β interacts with its auxiliary factor, TDRD3 (1, 11).  TDRD3 not only affects the function of TOP3β by recruiting DNA and RNA to the TOP3β TOPO domain, but also has its own DNA and RNA binding capabilities (1, 5, 8).  As a result, TOP3β can potentially function in more ways than topoisomerase III.  Finally, topoisomerase III is an effective decatenating enzyme and is suggested to be involved in the unlinking of nascent daughter chromosomes, as opposed to TOP3β-TDRD3, which works to relax supercoiled DNA (4, 11).

TOP3β-TDRD3 is a type IA topoisomerase found in Homo sapiens that coexists with its auxiliary factor TDRD3.  This complex is capable of binding to both DNA and RNA at the TOPO and RGG domains of TOP3β respectively.  TDRD3 regulates this binding and function by acting as a scaffolding that recruits DNA and RNA to TOP3β.  A similar protein, Escherichia coli topoisomerase III, provides valuable insight into the structure and functional mechanism of TOP3β.  As a whole, the TOP3β-TDRD3 complex is essential for both proper neural development and epigenetic regulation and has been linked to preventing disorders such as schizophrenia and autism.