UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New Series No. 913; ISSN 0346-6612; ISBN 91-7305-719-3 From the Department of Medical Biochemistry and Biophysics
Umeå University, Umeå, Sweden
Structure and Dynamics of the Hepatitis B Virus Encapsidation Signal Revealed by
NMR Spectroscopy
Sara Flodell 2004
Department of Medical Biochemistry and Biophysics, Umeå University
Umeå, Sweden
Department of Medical Biochemistry and Biophysics Umeå University
SE-901 87 Umeå, Sweden
Copyright © Sara Flodell 2004 ISSN 0346-6612
ISBN 91-7305-719-3
Printed by VMC, KBC, Umeå University, Umeå, Sweden
Table of contents
Abstract 4
List of Papers 6
Abbreviations 7
Introduction 8
Hepatitis B Virus 9
Reverse transcription 11
Epsilon 11
RNA as a Drug Target 14
RNA structure 14
Structure determination of RNA 15
Development of RNA targeted effectors 16
NMR on RNA 17
RNA synthesis and labelling 18
Derivation of structural parameters and constraints 20
Assignments 20
Proton–proton distances 21
Chemical shifts 21
Torsion angles 22
Backbone angles 22
Sugar puckering 22
Glycosidic torsion angles 22
Residual dipolar couplings 23
Motion 25
Results and Conclusions 27
Paper I 27
Paper II 28
Paper III 29
Paper IV 30
Concluding remarks 31
Acknowledgements 32
References 34
Papers
Abstract
Structure and Dynamics of the Hepatitis B Virus Encapsidation Signal Revealed by NMR Spectroscopy
Sara Flodell, Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden
This thesis describes the study of the three-dimensional structure and dynamics of the hepatitis B virus (HBV) encapsidation signal, epsilon, by means of nuclear magnetic resonance (NMR) and mutational data. HBV replicates by reverse transcription of an RNA pregenome into the viral DNA genome, which becomes enclosed in viral particles (encapsidation).
Epsilon is a stem-loop structure within the RNA pregenome and both the primary sequence and secondary structure of epsilon are strongly conserved, in agreement with its essential function of propagating HBV.
Epsilon is therefore a potential target for drug design. Studying the structure of epsilon requires development of new methods in the field of structural biology, as it is such a large RNA. Knowing the structure of epsilon will help to better understand the encapsidation mechanism and priming step of reverse transcription. This will help us in the search for antiviral drugs that block epsilon and prevent the viral reverse transcriptase from binding.
NMR spectroscopy is a method that provides detailed structural and dynamical data in solution under natural conditions. However, the size of the molecules that can be studied with NMR is limited. NMR spectra become more and more difficult to interpret as the size of the molecule increases. To circumvent this problem, large RNA molecules can be divided into smaller parts and only the parts essential for NMR studies are selected. The information obtained from these smaller fragments can then be used to determine the structure of the larger molecule.
Furthermore, a new method of enzymatically synthesizing nucleoside triphosphates with isotopes suitable for NMR has made it possible to specifically label the RNA molecules. Using this method it is possible to derive highly detailed molecular structures of RNA up to a size of 150
nucleotides. The method of selective isotope labelling was applied to different parts of HBV epsilon. Three RNA fragments of 27 (apical loop), 36 (internal bulge) and 61 (whole epsilon) nucleotides (nt) were synthesized in the unlabelled form. The 27-nt and 36-nt RNAs were also synthesized with (13C, 15N, 1', 3', 4', 5', 5"-2H5)-labelled uridines. The 61- nt sequence was (13C, 15N)-guanidine labelled. This labelling allowed unambiguous assignment of otherwise inaccessible parameters. The unlabelled and labelled RNA sequences provided the necessary data for structure derivation of the whole epsilon.
The apical loop of epsilon forms a pseudo-triloop motif. There is only one conformation of the loop that fulfils all the restraints, including experimental chemical shifts. However, the loop adopts several structures that fulfil the experimental distance, torsion angle and residual dipolar coupling restraints. This may reflect true flexibility. Indeed, relaxation studies on the unlabelled and labelled 27-nt sequences show that the residues that show multiple conformations are flexible. This can be an important feature for the recognition and subsequent binding of epsilon to the viral polymerase.
The information gained on the HBV encapsidation signal is useful in our understanding of the initiation of replication of the virus. This can in turn contribute to the search for drugs against HBV.
Keywords: HBV, RNA, isotope labelling, NMR, structure determination
List of Papers
This thesis is based on the papers listed below. They will be referred to in the text by their Roman numerals I - IV.
I Flodell, S., Cromsigt, J., Schleucher, J., Kidd-Ljunggren, K. and Wijmenga, S. (2002) Structure elucidation of the hepatitis B virus encapsidation signal by NMR on selectively labeled RNAs; J.
Biomol. Struct. and Dyn., 19, 627-636.
II Flodell, S., Schleucher, J., Cromsigt, J., Ippel, H., Kidd- Ljunggren, K. and Wijmenga, S. (2002) The apical stem-loop of the hepatitis B virus encapsidation signal folds into a stable tri- loop with two underlying pyrimidine bulges; Nucleic Acids Res.
30, 4803-4811.
III Flodell, S., Petersen, M., Schleucher, J., Zdunek, J., Girard, F., Kidd-Ljunggren, K. and Wijmenga, S. (2004) Structure of the apical loop of the hepatitis B virus encapsidation signal.
Manuscript.
IV Flodell, S., Larsson, G., Kidd-Ljunggren, K., Wijmenga, S. and Schleucher, J. (2004) Mobile nucleotides mediate binding of HBV reverse transcriptase to its RNA target. Manuscript.
Abbreviations
cccDNA circular covalently closed DNA DR1 direct repeat 1
DNA deoxyribonucleic acid HBV hepatitis B virus
HMBC heteronuclear multiple bond correlation HMQC heteronuclear multiple quantum correlation HSQC heteronuclear single quantum correlation mRNA messenger RNA
NMR nuclear magnetic resonance NOE nuclear Overhauser enhancement
NOESY nuclear Overhauser enhancement spectroscopy NTP nucleoside triphosphate
ppm parts per million
RDC residual dipolar coupling rmsd root mean square deviation RNA ribonucleic acid
ROESY rotating frame Overhauser enhancement spectroscopy TOCSY total correlation spectroscopy
TMP tri-methyl phosphate
Introduction
The hepatitis B virus (HBV) is a hepatotropic virus and infection can lead to a chronic carrier state with the risk of developing severe, progressive liver disease and hepatocellular carcinoma. Worldwide, HBV-related morbidity and mortality are considerable, with approximately 350 million chronic HBV carriers. The incidence of HBV infection has not declined in most endemic areas over recent decades and chronic HBV infection remains a global health problem. The treatment available for HBV is not sufficient to clear the infection completely. Therefore new antiviral drugs have to be developed that act via new mechanisms.
HBV replication is triggered by encapsidation of the viral RNA pregenome. The HBV encapsidation signal (epsilon) is a conserved stem- loop structure within the pregenome. Being an essential part of the encapsidation and replication machinery, this RNA structure is a potential drug target. Increasing numbers of RNAs performing regulatory functions in cells are being discovered. Some of these regulatory RNA elements are unique to specific organisms, making it possible to treat infections by targeting these elements. RNA is a relatively new target for structure-based drug design, since the importance of RNA for regulation has not previously been recognized, and RNA sample preparation for structure determination has only been possible for a few years.
Studying the structure of epsilon is important in two ways: firstly, it requires the development of new NMR methods in the field of structural biology, making it possible to investigate larger and more relevant RNA elements; secondly, knowing the structure of epsilon will help us to better understand the encapsidation mechanism and priming step of reverse transcription. This will help in the search for RNA-targeted antiviral drugs that block epsilon and hence prevent viral replication.
Hepatitis B Virus
HBV belongs to the family of Hepadnaviridae, which comprises a number of partially double-stranded, enveloped DNA viruses (Kidd- Ljunggren, 1996; Ganem & Varmus, 1997). Although it is a DNA virus, HBV makes use of reverse transcription during replication, via an RNA intermediate. Several HBV genotypes (A-H) have been described (Okamoto et al., 1988; Burda el al., 2001; Kidd-Ljunggren et al., 2002;
Arauz-Ruiz et al., 2002) which differ from each other in replication rate, clinical features and geographical origin. With a genome of only 3.2 kb, HBV has the smallest genome of all known eukaryotic DNA viruses. The HBV genome is extremely compact. It lacks non-coding regions and has only four extensively overlapping reading frames, encoding DNA polymerase, surface proteins, precore and core proteins, and HBx protein (Varmus & Ganem, 1987; Seeger & Mason, 2000). Therefore, the mutation rate of the HBV genome is not as high as it would have been if the genes had been separated, and the evolution of HBV is therefore somewhat constrained (Okamoto et al., 1987; Bläckberg & Kidd- Ljunggren, 2000; Kidd-Ljunggren et al., 2002). The HBV mutations that have been described are often located in specific genomic regions and some are believed to confer advantages to the virus through immune escape (Locarnini et al., 2003; Pumpens et al., 2002).
Some antiviral substances are available for the treatment of hepatitis B virus infection, e.g. interferon alpha, lamivudine and adefovir (Feld &
Locarnini, 2002; Lai et al., 2003; Marcellin et al., 2003; Karayiannis, 2003; Hadziyannis et al., 2003; Papatheodoridis & Hadziyannis, 2004).
However, these substances are in many cases not sufficient for complete eradication of HBV. Viral resistance to lamivudine has also been reported (Ling & Harrison, 1999; Das et al., 2001, Torresi, 2002). In addition, there is a recombinant vaccine against the surface protein of HBV, providing relatively good protection against HBV infection.
However, there have been reports of vaccine-escape mutations arising in the surface protein gene, lowering the overall efficiency of the vaccine (Chen & Oon, 1999; Burda et al., 2001, Torresi, 2002). This implies that
new antiviral agents must be designed, preferably those that work via new mechanisms, such as blocking the encapsidation signal.
cccDNA
ER
DNA synthesis
Transcription
Encapsidation of pregenomic RNA Minus strand synthesis
Plus strand synthesis Budding
RNAs
Translation Viral proteins
Vesicular export Entry
Recycling
Fig. 1. Hepatitis B virus replication cycle. After infection of a hepatocyte, the incomplete DNA genome is transferred to the nucleus and is repaired to form a circular covalently closed DNA. Both genomic and subgenomic RNAs are transcribed from this DNA, in order to produce the viral proteins. The pregenomic RNA is encapsidated and the DNA synthesis takes place within the capsid. A full minus strand is synthesized, the RNA is degraded, and an incomplete plus-strand is made, before the capsid buds into the endoplasmic reticulum and is secreted via vesicular export.
Reverse transcription
After infection of a hepatocyte (Fig. 1), the virus particle is uncoated and the viral genome is transported to the nucleus where the plus strand of the genome is completed. Host RNA polymerase II transcribes the genome into subgenomic and pregenomic RNAs which are transported to the cytoplasm. The subgenomic RNAs are used for translation of viral proteins, while the pregenomic RNAs act both as mRNA for the synthesis of core protein and viral polymerase, and as a template for reverse transcription. Encapsidation is one of the key events in HBV replication and is triggered by the binding of HBV polymerase to the encapsidation signal, epsilon, at the 5'-end of the RNA pregenome (Fig.
2). Upon capsid assembly, a four-nucleotide DNA primer is synthesized from the bulge within epsilon. The primer–polymerase complex is subsequently translocated to the primer binding site at the 3'-end of the pregenome, where DNA synthesis starts (Wang & Seeger, 1993; Ganem et al. 1994). The polymerase remains covalently bound to the DNA throughout minus strand synthesis (Gerlich & Robinson, 1980; Wang &
Seeger, 1992; Ganem et al., 1994). When the minus strand is complete, the synthesis of the plus strand starts and before the plus strand is fully complete, core particles are selected for export as mature virions. By budding into the endoplasmic reticulum the particles obtain their envelope containing the surface proteins. The viruses are then secreted through the constitutive vesicular transport pathway (Fig. 1) (Ganem et al., 1994).
Epsilon
Epsilon is a 60-nt bulged stem-loop located at the 5'-end of the RNA pregenome (Fig. 2). This stem-loop serves as the signal for capsid assembly. The same sequence is also present at the 3'-end, but this copy does not seem to be essential for viral replication (Rieger & Nassal, 1996). Encapsidation is triggered by the recognition of the upper stem-
loop of epsilon by the viral polymerase. The polymerase binds to epsilon and the RNA–polymerase complex then interacts with core protein, which forms the capsid. Inside the capsid, the DNA primer is synthesized from the UUCA sequence in the six-nucleotide bulge in epsilon.
Encapsidation is crucial for replication of infectious virions. Although the polymerase can perform DNA synthesis outside the capsid (Li & Tyrrell, 1999; Lanford et al., 1995), no viable virions are formed outside the capsid.
5’ RNA pregenome
(A) 3’n 1*
Primer binding site
1
5’
Epsilon
5’ (A) 3’n
(-)-DNA
Primer binding site
Host RNA- polymerase II
transcription
Core protein
Epsilon
Fig. 2. Reverse transcription. The HBV genome is transcribed into pregenomic RNA.
The viral RNA-dependent DNA polymerase and core protein are translated from this RNA. The polymerase binds to epsilon at the 5' -end of the pregenomic RNA, and the capsid forms around the RNA–protein complex. The DNA synthesis takes place within the capsid.
Epsilon has been the subject of several secondary structure prediction studies, which all predict a stem-loop structure (Knaus & Nassal, 1993;
Pollak & Ganem, 1993; Laskus et al., 1994; Kidd & Kidd-Ljunggren, 1996). Enzymatic probing (Knaus & Nassal, 1993) and NMR studies (Paper I) have confirmed this conformation. Mutations which occasionally arise are either very rare and/or structurally silent (Paper II).
However, until recently no structural analysis at molecular detail had been performed. Both the primary sequence and secondary structure of epsilon are highly conserved. With the exception of two nucleotide positions (1850 and 1858) differing in genotype A and some genotype F strains (Kidd-Ljunggren et al., 2002), the sequence of epsilon is generally conserved between HBV genotype strains. This implies that the fold of epsilon is essential for encapsidation and propagation of HBV, and epsilon is therefore a potential target for new RNA-binding drugs.
RNA as a Drug Target
Structural biology is a rapidly growing field, and is of great medical and pharmaceutical interest because of the potential for rational drug design.
Previously, the pharmaceutical industry has focussed on proteins, rather than nucleic acids, as drug targets (Farber, 1999; Klebe, 2000).
Ribonucleic acid (RNA) was considered only to be a carrier of genetic information. However, RNA also takes part in a wide variety of other cellular processes, such as protein synthesis, mRNA splicing and transcriptional regulation, as well as RNA viral replication. In addition, RNA displays great structural diversity in terms of tertiary folding (Chow
& Bogden, 1997; Pearson & Prescott, 1997; Hermann & Westhof, 1998;
Batey et al., 1999; Hermann & Patel, 1999; Hermann & Westhof, 1999a,b; Doudna, 2000; Hermann & Westhof, 2000). Therefore, RNA has a considerable potential for selective structure based targeting of diseases.
RNA structure
The structure of RNA is divided into three main levels: primary, secondary and tertiary. Primary structure refers to the nucleotide sequence. The secondary structure is the two-dimensional representation of the Watson-Crick base pairs and intervening single-stranded regions (Chow & Bogden, 1997; Batey et al., 1999). In folded RNAs more than half of all nucleotides usually participate in Watson-Crick base pairing and the stacking of these base pairs gives rise to A-form helices. Like proteins, RNAs have a large degree of tertiary folding. The tertiary structure is the three-dimensional, global fold of the RNA strand. The canonical Watson-Crick base pairs can be considered as the most basic unit for building three-dimensional frameworks. However, in most cases, it is the single-stranded regions of the RNAs that form unique tertiary interactions. Some RNA structural building blocks that have been defined include non-canonical base pairs, pseudoknots, hairpin loops, bulges, internal loops, mismatches, junctions and triple-strand interactions (Lilley, 1995; Draper, 1996; Chow & Bogden, 1997; Hilbers et al., 1998;
Batey et al., 1999; Hermann & Patel, 1999; Auffinger & Westhof, 1999;
Hermann & Westhof, 1999; Westhof & Fritsch, 2000; Lee et al., 2003;
Leontis & Westhof, 2003).
The HBV epsilon stem-loop contains several of these building blocks (Fig. 3). A six-nucleotide bulge separates the two stems, and the upper stem is capped by a pseudo-triloop. A pseudo-triloop is formed by transloop base pairing of residue number 1 and 5 in a hexaloop, forcing residue number 6 to bulge out from the stem (Haasnoot et al., 2000; Haasnoot et al., 2002; Paper III). The upper stem also contains a single nucleotide bulge. These structural features probably serve as recognition motifs for the viral polymerase at the time of encapsidation and subsequent replication.
Epsilon
Pseudo- triloop Single
nucleotide bulge
Fig. 3. Hepatitis B virus encapsidation signal, epsilon. Epsilon contains many RNA motifs that might be suitable for targeted drug design.
Structure determination of RNA
NMR spectroscopy and X-ray diffraction are the two methods that can provide detailed structural information on biomolecules and form an essential part of the field of structural biology. X-ray diffraction has the advantage of being applicable to large biological systems. However, RNAs can be difficult to crystallize. NMR is the only method that provides detailed structural data in solution under natural conditions. The size of the molecules that can be studied with NMR is limited, since the spectra become more and more difficult to interpret as the size of the molecule increases. The introduction of isotope labels has made it
possible to derive highly detailed molecular structures of RNA up to a size of 150 nucleotides (Varani et al., 1996; Wijmenga & van Buuren, 1998; Cromsigt et al., 2001; Paper I).
Development of RNA-targeting effectors
As in proteins, the specific functions of RNAs are modulated by their distinct three-dimensional structure. Interesting structural insights have been obtained from recently determined structures of RNA, RNA–drug complexes and RNA–protein complexes (Heus, 1997; Jiang et al., 1997;
Zimmerman et al., 1997). Molecular recognition between RNA and small RNA-binding molecules has become a central area of research in recent years (Chow & Bogden, 1997; Pearson & Prescott, 1997; Hermann &
Westhof, 1998; Hermann & Westhof, 1999b; Hermann & Westhof, 2000;
Hermann, 2003; Arzumanov et al., 2003; Davis et al., 2004; Murchie et al., 2004). Advances in RNA synthesis and the development of high- resolution RNA structure analysis, using both X-ray crystallography (Westhof et al., 1985; Cate et al., 1996; Doudna & Cate, 1997) and NMR spectroscopy (Pardi, 1995; Varani et al., 1996; Wijmenga & van Buuren, 1998), together with improved methods for the development of therapeutic agents (Li et al., 1997; Hermann & Westhof, 2000; Xavier et al., 2000; Sucheck et al., 2000; Gallego & Varani, 2001), have facilitated the discovery of RNA-targeting effectors. This has opened the way for the development of new antibiotics and antiviral drugs.
Many of the structural building blocks mentioned above can serve as unique binding sites for other RNA motifs, proteins, metal ions or small molecules, such as aminoglycosides, oxazolidinones and macrolides (Chow & Bogdan, 1997; Ecker & Griffey, 1999; Sucheck & Wong, 2000; Sucheck et al., 2000; Xavier et al., 2000; Gallego & Varani, 2001;
Hermann, 2003). Single non-Watson-Crick base pairs can be complemented by additional RNA motifs, such as bulges or loops, to form recognition surfaces for the substrates (Hermann & Westhof, 1999b). Therefore, these structures could serve as potential targets of therapeutic agents.
NMR on RNA
X-ray diffraction and NMR studies of biomolecules require milligram quantities of material. The easiest and cheapest way to obtain sufficient amounts of RNA is to use in vitro transcription using purified T7 RNA polymerase (Milligan et al., 1987; Milligan & Uhlenbeck, 1989).
However, as the size of the molecule increases, NMR spectra become more and more difficult to interpret, since increasing numbers of peaks tend to overlap. The linewidth also becomes broader with increasing size, causing even more overlap (Fig. 4).
A
B
Fig. 4. Molecular size dependence of the NMR spectra. The H1'/H5 region of 1D spectra of the 27-nt apical stem-loop of epsilon (A) and of the whole 61-nt epsilon (B) are compared. In A the lines are sharper, while in B the lines are broader and more numerous, causing more overlap.
Two ways to circumvent this problem are division of the molecule into smaller parts and/or labelling of the molecule with stable isotopes. With
isotopic labelling it is possible to choose to a greater extent which atoms to study, thus decreasing the complexity of the spectra.
RNA synthesis and labelling
The most common isotopes used for labelling for NMR purposes are 13C and 15N. The use of 13C and 15N labelling of RNA oligomers (Milligan et al., 1987; Milligan & Uhlenbeck, 1989; Batey et al., 1992; Batey et al., 1995; Niconowicz et al., 1992) and heteronuclear NMR techniques (Varani et al., 1996; Wijmenga & van Buuren, 1998) has made it possible to study oligonucleotides up to about 50 nucleotides (Kolk et al., 1998).
The most common way to label nucleotides is to prepare RNA from bacteria grown on 13C glucose and/or 15N ammonium chloride (Batey et al., 1992; Niconowicz et al., 1992; Batey et al., 1995). Unfortunately, the yield of the labelled nucleotides produced in this way is relatively low.
To reduce the number of peaks in a complex proton spectrum, deuterons can be used to replace some protons to reduce the number of peaks.
However, deuteration of the nucleotides reduces the yield of the nucleotides even further, since the bacteria grow poorly in D2O.
Furthermore, these uniformly labelled nucleotides have the disadvantage that RNA labelling can only be varied to a limited degree, i.e. by residue- type specific labelling. By employing different types of labelling in the ribose and bases, a large variety of labelling patterns can be obtained in the RNAs. To achieve this, a new method of synthesizing 13C-/15N-/2H- labelled nucleotides has been developed (Tolbert & Williamson, 1996;
Tolbert & Williamson, 1997; Scott et al., 2000; Cromsigt et al., 2000;
Cromsigt et al., 2002; Paper I). The method is based on converting specifically labelled glucose and bases into nucleotides biochemically by using enzymes from glycolysis, the pentose phosphate pathway and the nucleotide biosynthesis and salvage pathways. The labelled nucleotides are then used in the in vitro transcription of the RNA sequences (Fig. 5).
Phosphoribosyl transferase
Fig. 5. Synthesis of labelled RNA. Labelled nucleotides are made by mixing labelled or unlabelled glucose and base with enzymes from the pentose phosphate and nucleotide salvage pathways. These nucleotides are subsequently used in the RNA transcription from a DNA template by T7 RNA polymerase.
In this way, RNAs can be synthesized which have specific labels at specific positions. In addition, a large variety of different labelling patterns can be constructed, e.g. samples with 13C and/or deuterium labelling of the ribose, with or without 13C-/15N-labelling of the bases. In
combination with isotope editing techniques (Otting et al., 1986; Kay et al., 1990; Paper II; Peterson, et al., 2004) this simplifies crowded spectra and thus facilitates the extraction of crucial structural parameters.
Derivation of structural parameters and constraints Peak assignment
In order to determine the structure of RNA, as much structural information as possible has to be extracted from the NMR spectra. The parameters that can be measured in the spectra are chemical shift, peak intensity, J coupling and linewidth. The information that can subsequently be derived from these parameters includes proton distances and torsion angles. Each atom in a molecule represents one peak in an NMR spectrum (Fig. 4). To be able to derive structural information from a spectrum, one has to determine which peak belongs to which atom.
These “assignments” are the most time-consuming part of structure determination by NMR. The best way to establish the assignments is to use combinations of different spectra (Wijmenga & van Buuren, 1998;
Cromsigt et al., 2001a). The most common techniques for this purpose are total correlation spectroscopy (TOCSY) (Braunschweiler & Ernst, 1983; Shaka et al., 1988), nuclear Overhauser enhancement spectroscopy (NOESY) (Jeener et al., 1979; Hare et al., 1983), and heteronuclear multiple quantum correlation (HMQC) spectroscopy (Bax et al., 1983;
Bendall et al., 1983). These standard techniques can be complemented by the use of recently developed isotope labelling techniques and modified NMR experiments (Cromsigt et al., 2002; Paper I; Paper II). The labelling of RNAs has also made it possible to develop heteronuclear through-bond triple-resonance experiments for RNA to facilitate assignments. However, these through-bond assignment methods complement rather than replace NOE-based assignment (Wijmenga &
van Buuren, 1998; Cromsigt et al., 2001a).
Proton–proton distances
The NOESY technique (Jeener et al., 1979) registers contact between protons in close proximity (<5Å) in space. It is possible to calculate the proton distances from the intensity of the peaks. The intensity of the peak depends on the proton distance (I ~ 1/r6). This gives local information on the structure, which is the main input for structure calculations. The more contacts one can assign, the better defined the structure will be. When determining the structure of nucleic acids, in contrast to proteins, it is most important to find as many contacts as possible, since the proton density is much lower than in proteins. This problem becomes even more important in large systems due to extensive resonance overlap. Isotope labelling of the molecules reduces this overlap to a large extent. Another advantage of isotopic labelling is the possibility of selectively detect protons bound to 12C or 13C nuclei. A modified HMQC-NOESY-HMQC experiment (Kay et al., 1990; Paper II) can be used for this purpose. The experiment allows selection of, or suppression of, 13C-bound protons in different dimensions of the spectrum (Otting et al., 1986; Paper II;
Peterson, et al., 2004).
Chemical shifts
Proton chemical shifts are very sensitive to conformational changes, and in RNA they can be back-calculated from known structures with good accuracy, i.e. with a root mean square deviation (rmsd) of 0.08 ppm (Cromsigt et al., 2001b). Proton shifts can be used for comparison with characteristic shifts observed in helices, to determine whether the structure is helical or not. If no shift deviations larger than about 0.1 ppm are observed, the residue in question has a standard helix conformation.
Furthermore, the shifts can be used directly in the structure calculations for the final local refinements of the structure. The chemical shifts of phosphorus atoms contain information about the torsion angles along the RNA strand (see below).
Torsion angles
Torsion angles are related to the local geometry, and are most often used to define dihedral angles. The torsion angles that can be derived from NMR are the dihedral angles of the phosphate backbone (α-ξ), the conformation of the ribose moieties (sugar puckering) and the angle between the sugar and the base (the glycosidic torsion angle, χ).
Backbone angles: The 31P shifts can be used to derive limits of the backbone torsion angles (Roongata et al., 1990; Wijmenga et al., 1993;
Wijmenga & van Buuren, 1998). When the 31P shift (δ31P) lies in the usual range for helix residues, i.e. between -3.5 ppm and -4.6 ppm (with respect to external tri-methyl phosphate, TMP), the backbone has the regular helix conformation and the backbone torsion angles ε, ξ, α and β, have regular helix values. Downfield shifts are not necessarily but often attributable to changes in the backbone angles next to or penultimate to the phosphorus in question (ε, ξ, α and β). This gives information about the conformation of the backbone in turns bulges and loops.
Sugar puckering: If the H1'–H2' coupling (JH1'–H2') is measurable, the fraction of N puckering, fN, can be calculated according to the equation JH1'–H2' = fNJN + (1-fN)JS. JN and JS, the J coupling values for pure N- and S-pucker sugars, respectively, are taken to be 1.2 Hz and 8.0 Hz (Wijmenga et al., 1993). This gives information on the angles in the sugar moieties and helps to determine the conformation of loops and bulges.
Glycosidic torsion angles: The glycosidic torsion angle, i.e. the angle between the sugar and the base, can be determined in different ways.
Firstly, the H1'–H6/8 and H2'–H6/8 NOE intensities provide broad ranges for the glycosidic torsion angles (Wijmenga et al., 1993). For 150°<χ<340°, the H1'–H6/8 NOE is more intense than the H2'–H6/8 NOE, while for 260°<χ<340°, the H2'–H6/8 NOE is more intense than the H1'–H6/8 NOE. Secondly, the H2' and H3' chemical shifts can provide additional information (Cromsigt et al., 2001b). Thirdly, J coupling between atoms in the ribose and base depend on χ. The χ-angle can be calculated from the intensity of the H1'–C6/8 and H1'–C2/4 cross
peaks in the natural abundance 1H–13C HMBC spectrum. When the H1'–
C6/8 cross peak is more intense than the H1'–C2/4 cross peak, the χ- angle is within anti-range (Flodell et al., unpublished). Other NMR experiments for determination of χ from H1'–C6/8 and H1'–C2/4 have also been proposed for DNA (Schwalbe et al., 1994; Tarantírek et al., 2002; Munzarová & Sklenár, 2003) and for RNA (Duchardt et al., 2003).
Residual dipolar couplings
NOEs can in principle define the relative orientation of structural elements and domains, with respect to each other, if they are close enough in space. However, nucleic acids often adopt extended instead of globular structures. Therefore, these long-range NOEs can not be detected and NOEs fail to correctly determine the global orientation of larger structural elements. In this case residual dipolar couplings (RDCs) can be used (Zhou et al., 1999; de Alba & Tjandra, 2002; McCallum &
Pardi, 2003; Lukavsky et al., 2003). The use of RDCs in structure refinements is a recent method of determining the orientation of dipole vectors with respect to a global coordinate system (alignment tensor).
The dipolar coupling is a through-space interaction between nuclei which is always present when the molecule is located in a magnetic field, but averages to zero when the molecule tumbles isotropically in solution.
When a sample is dissolved in a liquid crystal solution, the molecules become aligned in the magnetic field, and dipolar couplings become visible. However, if the molecules are too highly ordered the resulting spectra are very complex. The use of dilute liquid crystals, e.g. lipid bicelles (Sanders & Schwonek, 1992) or filamentous phages (Hansen et al., 1998; Hansen et al., 2000; Zweckstetter & Bax, 2001), can create sufficient alignment to allow RDCs to be observed while retaining the simplicity of the spectra. The size of the coupling depends on the orientation of an atomic bond relative to the alignment tensor (Fig. 6).
For example, C1'–H1' bonds are approximately oriented along the RNA helix. Therefore, C1'–H1' bonds in the helices H1, H2 and H3 (Fig. 6) will be oriented at different angles relative to the alignment tensor.
Consequently, the RDCs will differ between the helices, making it possible to constrain the inter-helical angles.
Interhelical angle Alignment tensor
Z
X
Y
Interhelical angle
H1 H2
H3
Fig. 6. Global structure determination. The size of the residual dipolar coupling between two atoms depends on the orientation of the bond of the atom pair relative to a coordinate system (alignment tensor). The orientations of different bonds in different residues, e.g. the 1H1'–13C1' bonds, depend on the orientations of the helices H1, H2 and H3 in epsilon. The small arrows indicate the approximate direction of the H1'–C1' bonds in the guanidines (G). These couplings give information about the orientation of individual bases, but more importantly the relative orientation of the helices, i.e. the global fold.
Measurements of RDCs can be made for a variety of atom pairs, including single-bond 15N–1H, 13C–1H and 13C–13C pairs (Tjandra & Bax, 1997). Since the orientation of these atom pairs can be determined, it is possible to globally determine the relative orientation of structural segments and domains. To make use of this information in the structural refinements, the degree of alignment (i.e. the size and orientation of the alignment tensor) has to be estimated. This can be done from the experimental RDC data (Gayathri et al., 1982; Clore et al., 1998a,b;
Meiler et al., 2000; Bryce & Bax, 2004), or from known structures (Losonczy, et al., 1999; Wu et al., 2004).
Motion
Motion in biological molecules influences many of the NMR parameters used for structure determination, such as NOE, RDC, J coupling and chemical shift. These measured parameters will be averaged if there is motion, and this influences structure determination. Firstly, the measured average parameter might not correspond to any physically meaningful structure. Secondly, there may not be a single average structure that agrees with all the data. Thirdly, the data may not be sufficient to define the ensemble of structures.
To investigate the mobility of different residues within a molecule, several relaxation experiments can be performed. The time taken for an atom to relax, and for the NMR signal to disappear, is dependent on the motion of that particular atom. The motion will hence influence the relaxation times. The most important relaxation processes used to analyse motions are: i) the longitudinal relaxation time T1, which describes how fast the magnetization is restored along the applied magnetic field (the z- axis), ii) the transverse relaxation time T2, which corresponds to the dephasing of the magnetization in the xy-plane, and describes how broad the NMR signal is, and iii) cross relaxation rates (NOEs), which describe how fast magnetization is transferred from one nucleus to another (Fischer et al., 1998; Korzhnev et al., 2001). The combination of T1, T2 and NOE allows the determination of how fast a molecule is tumbling in solution and if a particular atom is more mobile than the others. It is possible to measure these relaxation times, and hence obtain an estimate of the flexibility. In general, the faster a molecule, or residue within a molecule, moves the longer T2 relaxation time is observed.
The most common experiments study the relaxation of the heteronuclei 13C and 15N (Kay, 1998; Fischer et al., 1998; Korzhnev et al., 2001). These heteronuclear relaxation experiments are usually applied to isotope-labelled samples. In nucleic acids, the exchangeable
imino N–H can describe only the hydrogen-bound residues. Therefore, the non-exchangeable C–H systems are more powerful reporters. 13C relaxation can be measured in different ways to extract information about the dynamics on time scales ranging from milliseconds to nanoseconds (Yamazaki et al., 1994; Akke & Palmer, 1996; Hall & Tang, 1998;
Mulder et al., 1999; Dayie et al., 2002; Paper IV). In addition, motion can be detected from the analysis of proton NOE intensities, because NOE intensities depend not only on proton distances, but also on motion.
Using a series of off-resonance ROESY experiments, the dependence on motion can be detected (Schleucher & Wijmenga, 2002). The advantage of this method is that no labelling is required.
In conclusion, NMR relaxation data on biomolecules can provide information about the dynamics of the molecules. Since many NMR parameters are influenced by motion, which in turn can influence the structure determination, it is important to investigate the dynamical processes in the molecule.
Results and Conclusions
The research described in this thesis provides detailed structural knowledge about the HBV encapsidation signal, which in turn adds to increased understanding of HBV replication. In addition, it has provided improved NMR tools for structural studies of large RNA molecules. In this section the main results and conclusions of the original research described in Papers I-IV are given.
Paper I
Biologically interesting RNA structures often have a size of a few hundred nucleotides. Currently available NMR methods can only handle up to about 50 nucleotides. The increasing size of molecules results in extensive resonance overlap, hampering the interpretation of the spectra.
NMR studies of large RNA systems (>50 nucleotides) require new approaches, e.g. different labelling schemes and reduction of the system into separate structural building blocks. Recently, a new method of synthesizing 2H-/13C-/15N-labelled nucleotides was developed in our lab.
The method involves the conversion of specifically labelled glucose and bases into nucleotides by using enzymes from the pentose phosphate pathway, and nucleotide and salvage pathways. These NTPs give a large degree of freedom in designing different labelling patterns in in vitro synthesized RNAs for study with NMR. This opens up the way for NMR studies of RNAs that are considerably above the present size limit (up to 150 nucleotides).
Here, this new technique was applied to structural studies of the 61- nucleotide (nt) long encapsidation signal (epsilon) from the hepatitis B virus. This RNA sequence was divided into two smaller fragments, 27 and 36 nucleotides long, representing the apical loop and the internal bulge of epsilon, respectively. The apical loop of epsilon (27-nt) was almost completely assigned from an unlabelled sample via combination of homo- and heteronuclear experiments at natural abundance. Specific
2H-/13C-/15N-uridine labelling of the 27-nt and 36-nt sequences was achieved and resulted in simplification of NOESY and HMQC spectra.
The increased resolution facilitated unambiguous assignments of NOE contacts that would otherwise have been inaccessible, providing additional information that can be used for structure determination of the entire encapsidation signal.
Paper II
The hepatitis B virus encapsidation signal has previously been predicted to form a bulged stem-loop, with a hexaloop at the tip of the upper stem.
To investigate the fold of the apical stem-loop of epsilon, mutational and NMR analyses were performed. Application of new isotope labelling techniques (2H-/13C-/15N-uridine labelling) allowed resolution of many overlapping resonances in 13C–1H HMQC/HSQC, 31P–1H HMBC and NOESY spectra. From these spectra an extensive structural data set could be derived, including torsion angles and proton distances. In addition, using the labelled sample different 13C-edited NOESY spectra could be recorded, establishing the sequential connections between protons bound to 12C or 13C atoms. The NMR data revealed that instead of the predicted hexaloop (residues C11U12G13U14G15C16), the apical stem is capped by a stable UGU triloop closed by a C-G base pair, followed by a bulged C.
The apical stem therefore contains two unpaired pyrimidines (C16 and U23), rather than one, as was predicted. The bulged nucleotides are separated by six nucleotides. C16, the 3'-neighbour of the G of the loop- closing C-G base pair, is completely bulged out while U23 is at least partially intercalated into the stem, as indicated by the H1' and H5 chemical shifts.
Analysis of 205 of our own HBV sequences and 1026 strains from the literature, covering all genotypes, revealed a high degree of conservation of epsilon. In particular, the residues essential for this fold were either totally conserved (e.g. G13, U14, C16 and U23) or showed rare non- disruptive mutations. Taken together, these data strongly indicate that this fold of epsilon is essential for recognition by the reverse transcriptase. Conservation of the structure of the upper stem-loop of free
epsilon in human HBV makes it an interesting target for potential anti- viral drugs.
Paper III
Here the detailed three-dimensional structure of the apical stem-loop of HBV epsilon is presented. The structure is based on NOE, RDC and 1H chemical shift data. The apical stem-loop of 27 nucleotides includes the loop at the tip of epsilon and its underlying stem, which contains a conserved non-paired U residue. NMR data support the formation of a pseudo-triloop at the tip of the stem (C11U12G13U14G15C16), where G15 pairs with C11. This leaves C16 unpaired and bulged out. The residues essential for this fold are either totally conserved or show rare non- disruptive mutations. The pseudo-triloop is well defined apart from the residues U12, G13 and C16. When distance, torsion angle and RDC restraints are used for structure determination, several loop conformations are found that fulfil the restraints. The two first loop residues (U12 and G13) do not converge to one definite position, but alternate between the major and minor groove sides, showing all four possible permutations of minor-major groove conformations. Thus, four major conformational classes (I-IV) were found. This indicates that the apical loop might not be described by a single rigid conformation. In this case, 1H chemical shifts were found to be crucial in defining the loop conformation. When back-calculated chemical shifts for each structure were compared with experimental chemical shifts, one of the conformational classes (class II) fitted better than the others. In class II, U12 stacks onto C11, G13 is positioned in the minor groove, and U14 stacks onto U12. The bulged C16 is located in the major groove. Within class II, three subclasses were found: IIa, IIb and IIc. These subclasses differ in NOE energies and 1H chemical shift rmsd. Subclass IIa is the class that corresponds best to the experimental chemical shifts, with root mean square deviation of 0.23 ppm.
The conserved, non-paired residue U23 is found to be in the major groove, in the minor groove, or partially intercalated in the stem. As
determined from refinements with stem RDCs, the upper and lower stems converge to an average angle of 36° ± 11°. U23 might lower the energetic barrier for unfolding of the apical stem by the viral reverse transcriptase.
It is possible that U23 and the pseudo-triloop provide important anchor points for the initial binding of the reverse transcriptase.
Paper IV
Motion influences many of the NMR parameters used for structure determination, such as NOE, RDC, J coupling and chemical shift.
However, the measured parameters will be averaged if there is motion and will not give true values of proton–proton distances and dipolar couplings, resulting in difficulties when determining the structures.
In this work, the flexibility of the upper stem-loop of the hepatitis B virus encapsidation signal was investigated. The stem of this hairpin structure contains a bulged uridine residue, and is capped by a pseudo- triloop. Previous structure calculations of this stem-loop (Paper III) have shown that it is difficult to make the loop region converge to one single conformation. To establish whether this is due to lack of restraints or true flexibility, several relaxation measurements were performed. Relaxation parameters T1, T1rho and 13C-{1H} NOE were measured for the C5, C6 and C1' carbons of the 13C labelled uridines. These data were subsequently analysed by the modelfree formalism. According to the analysis, the uridines 5, 18 and 19, located in the stems, have rigid bases and riboses. U12 and U14 are located in the loop and U12 shows high mobility (the order parameter, S2, is approximately 0.65 for all carbons), but U14 is nearly as rigid as the nucleotides in the stems. Finally, the non-paired residue U23 also shows significant motions. In agreement with the apparent absence of slower motions, the time constants for internal motions are 200-500 ps.
To get a complete overview over sub-nanosecond internal motions in the apical loop, a series of off-resonance ROESY experiments was recorded. Being sensitive to internal motions on a well-defined time scale of approximately 200 picoseconds, off-resonance ROESY is a good
complement to 13C relaxation experiments. All proton–proton distances that could be analysed in U12, G13, C16 and U23 show that these nucleotides are dynamic. In U12, the S2 derived from 13C relaxation and off-resonance ROESY agree very well. In U14, off-resonance ROESY indicates that the ribose is more flexible than the base. In U23, S2 values from off-resonance ROESY are generally lower than S2 from 13C relaxation. G13 and C16 could only be analysed by off-resonance ROESY, because they were not 13C labelled, and are shown to be the most mobile nucleotides. The upper limits for the S2 of G13 and C16 are 0.5 and 0.6, respectively.
This shows that the reverse transcriptase binds to a flexible target.
Since the UGU triloop and the bulged-out bases in the apical stem-loop are dynamic, the exact conformations of these bases cannot be crucial for the initial binding of the reverse transcriptase. Binding of the reverse transcriptase therefore seems to be an induced fit, either of the mobile bases only, or of larger parts of the apical loop.
Concluding remarks
The main purpose of this work was to determine the structure of the hepatitis B virus encapsidation signal, epsilon. This has required further development of NMR methods for the investigation of large nucleic acids. Although a little too late to be included in this thesis, the structure of the whole 61-nucleotide sequence has now almost been determined.
This was made possible by using new or modified methods, such as selective labelling, isotope-edited NMR methods, residual dipolar coupling and chemical shift calculation. In addition, relaxation measurements have revealed some information about the dynamics of epsilon. Knowing the structure of epsilon will help to better understand the encapsidation mechanism and priming step of reverse transcription.
And, more importantly, it contributes to the relatively new field of research: RNA structure-based drug design.
Acknowledgements
Tack till:
Mina handledare Jürgen Schleucher, Sybren Wijmenga och Karin Kidd-Ljunggren, som gjorde det här projektet möjligt.
Jürgen Schleucher, som förutom bra handledning även har hjälpt till med alla NMR- experiment.
Jenny Cromsigt för all hjälp i labbet under den första tiden.
Frédéric Girard för all hjälp i allmänhet, och för alla franskalektioner.
Michael Petersen och Janusz Zdunek för hjälp med strukturberäkningarna.
Biofysikgruppen, för att ha skapat en trevlig forskningsmiljö, samt diverse sociala aktiviteter. Nuvarande medlemmar: Angela Augusti, Göran Larsson, Jürgen Schleucher, Tatiana Nicol, Tobias Sparrman och Tobias Tengel. Tidigare medlemmar: Bernd van Buuren, Bin Wu, Chinthaka Sanath, Frédéric Girard, Gary Martinez, Hans Ippel, Janusz Zdunek, Jenny Cromsigt, Malin Nording och Sybren Wijmenga.
Mina två sommararbetare år 2000, Malin Eriksson och Jonas Pettersson, för väl utförda arbeten och trevligt sällskap i labbet.
Hela institutionen för Medicinsk kemi och biofysik, för att ha skapat en trevlig arbetsplats. Framför allt Ingrid Råberg, Urban Backman och Janusz Zdunek för all hjälp med pappersexercis, undervisning respektive datorer, samt doktoranderna m.fl.
för alla trevliga diskussioner vid fikaraster och luncher.
Mina vänner på Biofysikalisk kemi, som har gjort att jag inte har känt mig så ensam inom biofysikområdet (även om jag mest har känt mig som den molekylärbiolog jag är).
Framför allt Burkhard Zietz för alla tyskalektioner, och Pär Håkansson för att ha väckt mitt intresse för klättring, men även Gerhard Gröbner, Tomas Byström och många andra för trevliga sammankomster.
Min kompis Johan Broddefalk, som bland annat talat förstånd med mig de gånger jag känt att det kanske vore bättre att sluta doktorera.
Mina vänner på ”fel” sida av vägen, för alla trevliga middagar, biobesök, mm, mm:
Anna Carlsson, Anna-Lena Ström, och Margareta Aili. Även Elsa Gerell, som till skillnad från oss andra insåg att det går att göra annat än att doktorera.
Hela tjocka släkten Flodell för alla trevliga släktträffar. I synnerhet min kusin Åsa Öhagen, som fått stå ut med många gnälliga e-mail när allt har känts fördjävligt.
Och sist men inte minst mina föräldrar, Edina Flodell och Boris Söderberg. Jag kan tänka mig att ni har fått stå ut med åtskilligt från min sida, under hela mitt liv, men ni ställer alltid upp!
Kram /Sara
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