Laboratory of Protein Structure: Nowotny Laboratory
1. RNases H
Bacterial RNase H2
RNase H2 cleaves the 5’ phosphate of ribonucleotides in RNA-DNA junctions. It is the only known enzyme that can initiate the process of mutation-free removal of single ribonucleotides from genomic DNA. Such ribonucleotides are very often misincorporated by replicative polymerases and lead to genomic instability.
- First crystal structure of substrate complex of RNase H2 obtained using T. maritima protein.
- Unique catalytic mechanism in which the substrate is deformed, so that the phosphate group of the RNA-DNA junction participates in metal ion coordination at the active site. This metal ion positions and activates the nucleophilic water molecule. Hence, the substrate is used to assemble the active site to promote its own cleavage.
The studies of RNases H2 were performed in collaboration with Dr. Robert Crouch (National Institutes of Health, USA).
Rychlik MP, Chon H, Cerritelli SM, Klimek P, Crouch RJ, Nowotny M. Crystal Structures of RNase H2 in complex with nucleic acid reveal the mechanism of RNA-DNA junction recognition and cleavage. Mol. Cell, 2010; 40:658-670
Fig. 1: Crystal structure of T. maritima RNase H2 in complex with the nucleic acid substrate. The catalytic domain is in orange and purple and the helical C-terminal extension in yellow. DNA is shown in blue and single ribonucleotide in red. Metal ions at the active site are shown as green spheres.
Human RNase H2
Human RNase H2 comprises three subunits – the catalytic one is very similar to monomeric bacterial and archaeal enzymes and the sequence of two auxiliary ones do not resemble any know proteins. Mutations in RNase H2 lead to a severe genetic autoimmune disease - Aicardi-Goutieres syndrome.
- Crystal structure of the trimeric human RNase H2.
- All mutations from AGS patients mapped and their effect on the structure and mechanism of the enzyme proposed.
Figiel M, Chon H, Cerritelli SM, Cybulska M, Crouch RJ, Nowotny M. The structural and biochemical characterization of human RNase H2 complex reveals the molecular basis for substrate recognition and Aicardi-Goutieres syndrome defects. J. Biol. Chem., 2011; 286:10540-50
Fig. 2: Crystal structure of human RNase H2. The catalytic subunit is shown in yellow and the auxiliary ones in purple in blue.
RNase H3 is present in some bacterial species. It is closely related to RNases H2 both in sequence and structure but in biochemical properties it is similar to RNases H1 – it prefers to cleave RNA/DNA hybrids in the middle of the RNA sequence. RNase H3 contains a unique N-terminal domain related to TATA-binding protein.
- The first crystal structure of RNase H3 in complex with the RNA/DNA substrate.
- The RNA strand is recognized by contacts between 2’-OH groups of four consecutive ribonucleotides.
- The DNA strand is recognized by deformation to B-form sugar puckers only allowed for DNA.
- The N-domain specifically binds RNA/DNA hybrid by recognizing 2’-OH of RNA and forming stacking interactions with the ribose rings of the DNA.
- The mechanism of N-domain is very similar to the structurally unrelated hybrid-binding domain present in the N-terminus of RNases H1. This is a likely case of convergent evolution of RNA/DNA recognition.
Figiel M, Nowotny M. Crystal structure of RNase H3-substrate complex reveals parallel evolution of RNA/DNA hybrid recognition. Nucleic Acids Res (2014); 42(14):9285-94
Fig. 3: Crystal structure of RNase H3 (catalytic domain in yellow and N-domain in green) interacting with an RNA/DNA hybrid (RNA in red and DNA in blue). The cleaved phosphate is shown as a red sphere and the phosphate group of the deformed DNA residue as a blue sphere.
2. DNA repair
UvrA is a dimeric ATPase which plays a role of DNA damage sensor in nucleotide excision repair (NER) in bacteria. NER is one of the major pathways of DNA repair and its main feature is its ability to recognize a wide spectrum of DNA lesions of various sizes and structures.
- The first crystal structure of UvrA interacting with cDNA obtained using the protein from T. maritima.
- UvrA does not interact with the damage site directly but senses its deformed conformation induced by the presence of the lesion – bending, stretching and unwinding.
- This indirect readout allows UvrA and consequently the whole NER to achieve its broad specificity.
- The mechanism of UvrA is different from its eukaryotic functional equivalents, which probe the DNA lesion directly.
Jaciuk M, Nowak E, Skowronek K, Tanska A, Nowotny M. Structure of UvrA nucleotide excision repair protein in complex with modified DNA. Nat. Struct. Mol. Biol., 2011; 18:191-197
Fig. 4: Crystal structure of UvrA dimer (one monomer in color, the other in gray). One ATPase module is shown in cyan and red and the other in pink and blue. The DNA-binding domain is in green and UvrB-binding domain in yellow. Structural zinc ions are shown as orange spheres.
Eukaryotic nucleotide excision repair (NER) is one of the major DNA repair pathways. It involves the excision of the DNA fragment containing the damage. This is achieved through the action of two nucleases – XPF-ERCC1 complex and XPG (Rad2 in yeast). Rad2/XPG belongs to flap endonuclease metal ion-dependent enzymes along with FLAP1 and EXO1. Its unique feature within this family is the ability to cleave DNA bubbles – substrates with melted single stranded region flanked with double-stranded stretches of DNA.
- The first crystal structure of the catalytic core of Rad2 using a truncated version of the S. cerevisiae enzyme.
- The main substrate specificity determinant of Rad2 is the interaction of the last exposed base pair of the double-stranded region with the so-called hydrophobic wedge of the enzyme. No interactions with the single stranded portion of the substrate are observed.
- The main DNA-binding element is potassium-coordinating helix-two-turn-helix (H2TH) motif. It contains an additional charged helix binding the DNA, which is a unique feature of Rad2.
- The likely explanation for the unique ability of Rad2 to clave DNA bubbles (substrate without a free 5’ DNA end) is the altered structure of the so-called helical arch. In FEN1 and EXO1 this element blocks the exit from the active site preventing cleavage of substrates without a free 5’ end. In Rad2 the helical arch has a different structure forming an exit route from the active site.
Miętus M, Nowak E, Jaciuk M, Kustosz P, Studnicka J, Nowotny M. Crystal structure of the catalytic core of Rad2: insights into the mechanism of substrate binding. Nucleic Acids Res 2014; 42(16):10762-75.
Fig. 5: Crystal structure of Rad2-DNA complex. The complex contains two independent protein molecules – one is shown in color: cyan for H2TH motif, green for hydrophobic wedge and orange for helical arch. The DNA is shown in cyan and blue, potassium ion as a purple sphere and the calcium ion at the active site as a green sphere.
RuvC is a canonical bacterial Holliday junction (HJ) resolvase, which functions as a dimer. HJ are four-way DNA structures formed by the exchange of strands between two helices. They are intermediates in homologous recombination, a process which is used to repair dangerous DNA lesions such as double-strand breaks.
- The first crystal structure of RuvC in complex with a DNA substrate and the first substrate complex structure of a cellular resolvase, solved at 3.8 Å resolution.
- HJ in a novel tetrahedral conformation with two phosphate groups symmetrically located 1 nt from the HJ exchange point interacting with two active sites of RuvC dimer.
- Novel mode of HJ recognition relative to phage enzymes for which complex crystal structures had been available.
Górecka KM, Komorowska W, Nowotny M. Crystal structure of RuvC resolvase in complex with Holliday junction substrate. Nucleic Acids Res., 2013; 41(21):9945-55
Fig. 6: Crystal structure of RuvC in complex with Holliday junction. The two protomers are shown in pink and orange. The DNA is on blue with cleaved phosphates indicated with spheres.
Slx1 is a nuclease which cleaves various DNA structures during DNA repair and recombination. It associates with Slx4 platform protein which coordinates the action of multiple proteins. Slx1 together with Mus81-Eme1 nuclease constitute one of the major Holliday junction resolution pathways in eukaryotes.
- First structural information for Slx1 and Slx4CCD.
- Fungal Slx1 forms a homodimer in which the active site is blocked, explaining why Slx1 alone is inactive.
- Slx4CCD domain binding is mutually exclusive with homodimerization.
- Slx4 binding exposes the active site of Slx1 and activates the nuclease. This mechanism ensures that the promiscuous and potentially dangerous Slx1 nuclease is only active when bound to Slx4 platform which regulates its activity and coordinates it with other proteins.
Gaur V, Wyatt HD, Komorowska W, Szczepanowski RH, de Sanctis D, Gorecka KM, West SC, Nowotny M. Structural and Mechanistic Analysis of the Slx1-Slx4 Endonuclease. Cell Rep. 2015; S2211-1247(15)00165-5
Fig. 7: Crystal structures of Slx1 homodimer and Slx1 in complex with Slx4CCD domain (orange). Slx1 comprises GIY-IYG nuclease domain (yellow) and RING finger zinc-binding domain (green). Upon Slx4 binding the active site of the nuclease domain is exposed and the enzyme is activated.
3. Reverse transcriptases
XMRV reverse transcriptase
Reverse transcriptases use two enzymatic activities – DNA polymerase and RNase H to catalyze the conversion of single-stranded RNA to double-stranded DNA, a process essential for proliferation of retroviruses such as HIV and retrotransposons. Retroviral RTs are divided into two classes – dimeric (i. e. HIV) or monomeric (i. e. gammaretroviral enzyme from mouse Moloney leukemia virus and closely related XMRV).
- The first crystal structure of a monomeric RTs in complex with RNA/DNA hybrid visualizing the polymerase-connection fragment of the enzyme.
- Full-length protein modelled based on small-angle X-ray scattering data.
- SAXS data demonstrated that the RNase H domain is mobile and only occasionally interacts with the substrate to cleave RNA. This is the mechanism to fine tune RNase H activity.
- This mechanism of RNase H fine-tuning is different from dimeric RTs which use substrate deformations for that purpose.
The studies of reverse transcriptases were performed in collaboration with Dr. Stuart Le Grice (National Institutes of Health, USA).
Nowak E, Potrzebowski W, Konarev P, Rausch J, Bona M, Svergun D, Bujnicki JM, Le Grice S, Nowotny M. Structural analysis of monomeric retroviral reverse transcriptase in complex with an RNA/DNA hybrid. Nucleic Acids Res., 2013; 41(6):3874-87
Fig. 8: Model of the full length XMRV RT based on SAXS data, a crystal structure of the polymerase-connection fragment in complex with RNA/DNA hybrid (blue-fingers, red-palm, green-thumb, yellow-connection) and the structure of isolated XMRV RNase H domain (orange, Zhou D, J Struct Biol. 2012). RNA template stand is in purple and DNA primer strand in blue.
Ty3 reverse transcriptase
Retrotransposons are mobile genetic elements that replicate with an RNA intermediate. Reverse activity of element-encoded RT is essential for this process. Retroelements are one of the most potent forces shaping eukaryotic genomes – more than 40% of human genome derives from those elements. Ty3 is a yeast retrolement from long-terminal class termed Ty3/Copia. It is thought that retroviruses evolved from this class of retrotransposons.
- The first crystal structure of a retrotransposon RT.
- Ty3 RT forms an asymmetric homodimer in which one subunit has the polymerase competent configuration and the other has an altered conformation and harbors the RNase H activity.
- RNase H is postulated to undergo a conformational change to reach the position required for RNA/DNA cleavage, which regulates this activity.
- Ty3 and HIV RT architecture differs: HIV enzyme is a constitutive heterodimer with both polymerase and RNase H activities residing in the larger subunit and Ty3 is a substrate-induced homodimer in which the two activities are located in different subunits.
Nowak E, Miller JT, Bona MK, Studnicka J, Szczepanowski RH, Jurkowski J, Le Grice SFJ†, Nowotny M†. Ty3 reverse transcriptase complexed with an RNA-DNA hybrid shows structural and functional asymmetry. Nat. Struct. Mol. Biol., 2014; 21(4):389-96; †corresponding authors
Fig. 9: Crystal structure of Ty3 reverse transcriptase. The subunit with polymerase-competent configuration is shown in darker color (blue-fingers, red-palm, green-thumb, yellow-RNase H) and the subunit with altered conformation in lighter shades of the same colors. RNA template stand is in purple and DNA primer strand in blue. Active site residues for polymerase and RNase H domain are shown as sticks.
Human cap 2’-OH methyltransferase
mRNA contains a cap structure on its 5’ terminus. In higher eukaryotes the first and the second ribonucleotide of the body of the mRNA are methylated on the 2’ oxygen. This is thought to serve as a tag to distinguish self mRNA from the mRNA of invading viruses. To circumvent this mechanism, some viruses such as West Nile and yellow fever viruses encode their own 2’-OH methyltransferases
- The first crystal structure of a cellular 2’-OH cap methyl transferase.
- The mode of cap recognition markedly different from viral counterparts providing hints for antiviral drug development.
The studies of cap 2’-OH methyltransferase were performed in collaboration with Prof. Janusz M. Bujnicki (IIMCB).
Smietanski M, Werner M, Purta E, Kaminska KH, Stepinski J, Darzynkiewicz E, Nowotny M†, Bujnicki JM†. Structural analysis of human 2’-O-ribose methyltransferases involved in mRNA cap structure formation. Nat. Commun., 2014 Jan 9;5:3004; †corresponding authors
Fig. 10: Crystal structure of human cap 2’-OH methyl transferase 1 (hMTR1). A fragment of capped mRNA is shown in red and the S-adenosylmethionine (methyl group donor) in green.