Laboratory of Protein Structure

Description of Current Research
Our laboratory will focus on structural and biochemical studies of nucleic acid enzymes. Our primary method will be protein crystallography. Our projects can be subdivided into three groups:
1. Structural studies of substrate complexes of members of integrase superfamily.
2. Structural studies of reverse transcriptases.
3. Structural studies of UvrA DNA repair protein.

1. Integrase superfamily
Integrase superfamily (ISF) comprises an important and interesting nucleic acid processing enzyme family, containing transposases, integrases, and various nucleases. They are involved in a wide range of processes such as transposition, replication, and repair of DNA, homologous recombination and the action of siRNAs. One of the best characterized members of ISF is RNase H. It is a nuclease that binds RNA/DNA hybrids in a sequence, non-specifi c manner and degrades the RNA strand. Two types of RNases H have been identified – type 1 (RNase H1) and type 2 (RNase H2). Type 1 enzymes are present in all forms of life from bacteria to animals. They are also an integral part of reverse transcriptases. In HIV reverse transciptase the RNase H activity is essential for viral progression and is, at the same time, one of the least explored and most promising drug targets for the treatment of AIDS. Type 1 RNase H is the only member of the ISF family for which the structures of its complexes with nucleic acids that are bona fi de substrates for the enzymatic reaction are currently available (Nowotny et al. Cell 2005, Nowotny et al. Mol Cell 2007). Th e mechanism of RNA/DNA recognition was revealed and it was shown that the mechanism of catalysis relies on two metal ions. Members of ISF share the same fold of the catalytic core and very similar architecture of the active site, yet they act on a wide range of nucleic acids. In order to see how these different substrates are recognized and reveal the mechanism for their processing, we plan to solve crystal structures of substrate complexes of two ISF enzymes – RNase H2 and RuvC. In biochemical properties, RNase H2 diff ers from type 1 enzyme. For example, it can cleave out single ribonucleotides embedded in DNA and is therefore thought to participate in DNA repair. Recently, it was shown that mutations in human RNase H2 result in Aicardi-Goutičres syndrome (AGS) – an autosomal recessive genetic disorder with symptoms similar to in utero viral infection that severely affects the nervous system. The human enzyme is thus essential. The apo structures of bacterial and archeal are known but there is no structural information about substrate binding and cleavage. To reveal these mechanisms we plan to co-crystallize RNase H2 with RNA/DNA hybrids and solve the structure of this complex. RuvC cleaves Holliday junctions – 4-way DNA structures which are intermediates in homologous recombination. We would like to learn how specifi c binding of Holliday junctions is achieved and to reveal the molecular details of the assembled pre-reaction active site. We would also like to fi nd out how the sequence-specific cleavage is achieved. To this end we plan to solve crystal structures of RuvC in complex with Holliday junctions. Together with known RNase H1 structures, crystallographic studies of RuvC and RNase H2 will allow us to generalize the mode of ISF members’ action and to predict the detailed mechanism for such important enzymes as HIV integrase and Argonaute.

2. Reverse transcriptases
Reverse transcriptases are multifunctional enzymes catalyzing the conversion of single-stranded RNA to dsDNA. Th is process is essential for the life cycle of certain viruses e.g. retroviruses (HIV) or hepadnaviruses (hepatitis B virus). Although the RNase H domain of HIV RT is an important drug target, eff orts to develop its efficient inhibitors failed. One line of our research will be to use novel approaches for the identification of the next generation of inhibitors of HIV RNase H. One of the main problems with known RNase H inhibitors is their lack of specificity. We will exploit the structural differences between human and HIV RNase H to fi nd new inhibitors using the combination of Virtual Screening and protein crystallography. Crystal structures of only two reverse transcriptases have been solved. Only for HIV RT the structures of its complexes with the nucleic acids are available. There is a significant variability of RT architecture between different viruses and several important aspects of the mechanism of RT action remain unclear, e.g. the way in which the polymerase and RNase H activities are coordinated. No structural information is available for hepatitis B virus RT (HBV RT) which is an important drug target. This enzyme cannot be produced in an active form in sufficient quantities to allow structural studies. Therefore, we plan to use bioinformatics to identify its close homologues, crystallize them and next solve their structures. Based on these structures an accurate homology model of HBV RT will be built in collaboration with the bioinformatics group of Dr. Janusz Bujnicki. We will also undertake co-crystallization experiments of these new RTs with their nucleic acid substrates. We hope to identify proteins that will readily form crystals with various nucleic acids corresponding to particular stages of reverse transcription. These snapshots will allow us to reconstruct the detailed mechanism of the reaction.

3. Structural and biochemical studies of UvrA DNA repair protein
DNA molecules – the carriers of genetic information – are susceptible to chemical damage. One of the primary pathways to remove these modifications is nucleotide excision repair (NER), in which a stretch of bases harboring the lesion is cleaved out and the resulting gap is fi lled by a DNA polymerase. The remarkable feature of NER is the fact that it can act on a wide spectrum of unrelated DNA lesions, varying greatly in chemical structure. In bacteria one of its key components is UvrA protein which is thought to be the first to detect the DNA damage. It then recruits other components of NER. Recently, a crystal structure of apoUvrA has been reported but the detailed information about the mechanism of damaged DNA recognition is still lacking. By solving a crystal structure of UvrA with different types of damaged DNA we would like to learn how the remarkably wide specifi city of NER system is achieved. We would like to reveal what features of different lesions are used by UvrA to recognize the damage. The enzyme contains two ATPase domains and ATP hydrolysis is essential for damage recognition. Cocrystallization of UvrA with ATP analogues, ADP and without the nucleotide should reveal the conformational changes during ATP hydrolysis and their consequences for DNA binding. These studies should help explain the central question in the DNA repair – the mechanisms of damage recognition.

Figure 1.
Structure of Bacillus halodurans RNase H1 in complex with RNA/DNA hybrid (RNA in red and DNA in blue). Two magnesium ions involved in catalysis are shown as yellow spheres. The protein is shown as cartoon and surface representations.

Figure 2.
Crystals of the apo form of Bacillus halodurans RNase H1.

Figure 3.
Comparison of the structures of three members of integrase superfamily – RNase H1, RNase H2, and RuvC. Th e common secondary structure element – the central β-sheet is shown in green. The active site residues are shown as an orange ball-and-stick. Two carboxylates that are spatially conserved among the members of integrase superfamily are labeled in purple. PDB codes: B. halodurans RNase H – 1ZBI, A. fulgidus RNase H2 – 1I39, E. coli RuvC – 1HJR.

Selected publications
• Nowotny M, Cerritelli SM, Ghirlando R, Gaidamakov SA, Crouch RJ, Yang W. Specific Recognition of RNA/DNA hybrid and Enhancement of Human RNase H1 Activity by HBD. EMBO J, 2008; in press
• Nowotny M, Gaidamakov SA, Ghirlando R, Cerritelli SM, Crouch RJ, Yang W. Structure of human RNase H1 complexed with an RNA/DNA hybrid: Insight into HIV Reverse Transcription. Mol Cell, 2007; 28:264-276
• Nowotny M, Yang W. Stepwise Analyses of Metal Ions In RNase H Catalysis: From Substrate Destabilization To Product Release. EMBO J, 2006; 25:1924-33
• Yang W, Lee JY, Nowotny M. Making and Breaking Nucleic Acids: Two-Mg2+-ion Catalysis and Substrate Specifi city, (review). Mol Cell, 2006; 22:5-13
• Nowotny M, Gaidamakov SA, Crouch RJ, Yang W. Crystal structures of RNase H bound to an RNA/DNA hybrid: substrate specifi city and metal-dependent catalysis. Cell, 2005; 121:1005-16
• Lee YT, Jacob J, Michowski W, Nowotny M, Kuznicki J, Chazin WJ. Human Sgt1 binds HSP90 through the CHORD-Sgt1 domain and not the tetratricopeptide repeat domain. J Biol Chem, 2004; 279:16511-7
• Nowotny M, Spiechowicz M, Jastrzebska B, Filipek A, Kitagawa K, Kuznicki J. Calcium-regulated interaction of Sgt1 with S100A6 (calcyclin) and other S100 proteins. J Biol Chem, 2003; 278:26923-8
• Filipek A, Jastrzebska B, Nowotny M, Kuznicki J. CacyBP/SIP, a calcyclin and Siah-1-interacting protein, binds EF-hand proteins of the S100 family. J Biol Chem, 2002; 277:28848-52
• Filipek A, Jastrzebska B, Nowotny M, Kwiatkowska K, Hetman M, Surmacz L, Wyroba E, Kuznicki J. Ca2+-dependent translocation of the calcyclin-binding protein in neurons and neuroblastoma NB-2a cells. J Biol Chem, 2002; 277:21103-9
• Nowotny M, Bhattacharya S, Filipek A, Krezel AM, Chazin W, Kuznicki J. Characterization of the interaction of calcyclin (S100A6) and calcyclin-binding protein. J Biol Chem, 2000; 275:31178-82.