U1A Spliceosomal
Protein/Hepatitis Delta Virus Genomic Ribozyme Complex (PDB ID: 1DRZ) from Homo sapiens and Hepatitis Delta Virus
Created by: Kira
Guth
The U1A Spliceosomal Protein/Hepatitis Delta Virus Genomic Ribozyme Complex is composed
of two subunits, a self-catalytic ribozyme found in Hepatitis delta virus (HDV) and a spliceosomal protein found in Homo sapiens that was used to
crystallize the ribozyme through co-crystallization. The ribozyme is used by
the virus to cleave the initial linear multimer products of replication into
unit-length RNA circles which undergo further replication (Figure 1). This
ribozyme is the only self-catalytic piece of RNA identified as a requirement for
human pathogen viability (1-3). The Chang Bioscience Calculator was used to
obtain a molecular weight of 23163.4 Da for the ribozyme which is composed of
72 nucleotides (2, 4). The complex is assigned the PDB ID 1DRZ by the Protein
Data Bank (2).
The
HDV genomic ribozyme functions through the catalysis of a transesterification
reaction (Figure 2) which yields one product with a 5’-hydroxyl terminus and a
second with a 2’,3’-cyclic phosphate terminus. The HDV ribozyme is the only
ribozyme identified in a human pathogen although similar self-cleaving RNA molecules
are found in plant viroids and some eukaryotic genomes. These similar ribozymes
perform the same self-cleavage reaction although at a slower rate. In contrast
to the RNA motifs common in viroid ribozymes, notably the hairpin and
hammerhead ribozymes, the HDV ribozyme exhibits extreme hardiness and stability
along with a unique double pseudoknot structure (1, 5).
The
pseudoknots observed in the HDV ribozyme are composed of characteristic
base-paired regions denoted P1 through P4 in a 5’ to 3’ direction with a two
base-pair helix P1.1. Paired regions 3 and 4 are capped by loops L3 and L4
respectively while single stranded regions J1/2, J1.1/4 and J2/4 connect the
indicated helices. P1 was identified as the substrate bearing helix with a G-1 – U-37 wobble pair at the bottom of the base stack serving as the active site
(1). Protonated C-75 acts as the general acid in the self-cleavage reaction and
a hydrated magnesium ion as the general base. Repulsion between the hydrated
metal ion and the protonated nucleotide may help drive the reaction (6).
A
complex “trefoil” turn incorporated into single-stranded J2/4 serves to
position the nucleotides of the active site. G-74, C-75 and G-76 form three
leaflets of a clover with the base of G-76 fully extruded into the solvent and
C-75 projected into the core of the structure (1). C-75 participates in the
self-cleavage reaction as a general acid. Protonation of this base is coupled
with the N7 of G-1(6). Raman spectra show that the pKa of C-75 is
shifted from a typical cytosine residue value of around 4 to a pKa
of 6.15-6.40 depending on magnesium concentration. This shift in pKa
supports the conclusion that C-75 acts as a general acid rather than a general
base (7). This increase in pKa value may be caused by the presence
of three polar groups, the N4 of C-75, the pro-Rp phosphate oxygen
atom of C-22 and the O2’ group of U-20, and only two hydrogen atoms arranged in
a tight triangle (1).
A strand-crossover extending from P3 to P1 overhangs the active site, protecting
it from hydroxyl radicals. This strand exits the P3 helix at C-30 and
experiences a sharp curve around the phosphate of G-31 which then enters the P1
base stack. A set of four hydrogen bonds between A-78, the last nucleotide of
the J2/4 region, and the penultimate base pair of the P3 helix, C-18 and G-29,
stabilizes the crossover. The N6 of A-78 is not involved in this hydrogen
bonding and instead points towards the active site where it is available for
coordination and participation in catalysis (1).
Helical
region P3 and loop L3 together form a niche flanking the active site composed
of the minor groove face of the helix and unpaired bases U-20, C-24, and G-25.
Bases U-20 and G-25 are too far apart for any hydrogen bonds to form between
their Watson-Crick faces. Mutagenesis studies suggest this additional space
allows for correct positioning of the G-74, C-75, G-76 cloverleaf (14).
Hydrogen bonds between U-20, C-75, C-22, and C-24 stabilize the niche. Residue U-23 extrudes from the base stack creating a pocket for the base of a
nucleotide without the need for specific interactions (1).
Three magnesium ions (Mg2+) were found to be the only ligands bound to the ribozyme (2).
Kinetic experiments and Raman crystallographic studies indicate that a hydrated
magnesium ion acts as the general base in the self-cleavage reaction (6) Data
suggest that the identity of the catalytic metal ion is nonspecific as ribozyme
activity was not inhibited by the replacement of the divalent cation with two thallium
(Tl+) ions although both hydrated magnesium ions and a mimic, cobalt
hexamine, outcompeted monovalent cations (8).
In
order to identify proteins comparable to the HDV ribozyme, the n-BLAST server
was used for bioinformatics searches. This server functions by accepting a nucleotide
query and seeking results with similar sequences from a database. These results
are then ranked based on sequence homology and gaps. An E value is assigned to
each comparable nucleotide with strong homology lowering that value and gaps
increasing it. Results with an E value of less than 0.05 are considered to have
a significantly similar sequence to the query. Only six results were produced
by the n-BLAST server, all of which displayed 99% or 100% homology with the HDV
ribozyme and were found in Hepatitis
Delta Virus, implying that they were either different crystallizations of
the protein or were the HDV ribozyme with different ligands (9). The Dali
server is another tool for conducting bioinformatics searches. Unlike n-BLAST,
the Dali server uses the amino acid backbone to identify proteins with a
similar tertiary structure to the query protein. Results are assigned a Z-score
to quantify similarity. Z-scores of 2 or above indicate that the resultant
protein shares a similar folding pattern with the query. As the functional
portion of the HDV ribozyme is composed exclusively of RNA, a nucleotide, it
lacks the necessary amino acid backbone. Therefore, the Dali server was not a
useful tool to identify similar proteins (10).
Articles
published in the literature along with the n-BLAST results obtained suggest
that the HDV ribozyme has a unique structure with no known homologs (1, 9). However,
at least four additional, structurally distinct, RNA motifs have been
identified in self-cleaving ribozymes: the hairpin, hammerhead, Varkud
satellite (VS) and glmS motifs. Of
these four ribozymes, only the hairpin and hammerhead were discovered before the
characterization of the structure of the HDV ribozyme.
The RNA hammerhead ribozyme (PDB ID: 300D) was first found in plant viroids but has since been
reported in eukaryotic genomes (2, 5, 11). As is observed in the HDV ribozyme, the
hammerhead is composed entirely of RNA with divalent manganese ion (Mn2+) ligands (12). These cations ionize a 2’-hydroxyl at the active site to generate
the nucleophile used in the self-catalysis. The residue C-17 acts as the actual
cleavage site. The self-cleavage reaction is catalyzed by binding of a
manganese ion to the Pro-R phosphate oxygen of A-1.1. Binding of this metal ion
induces the conformational change required for attack of the C-17 2’-hydroxyl
moiety and also provides the hydroxide ion for the base-catalyzed step of the
reaction (12).
Although the
mechanism of reaction is similar, the hammerhead ribozyme is structurally very
different from the HDV variant (12). The hammerhead ribozyme is composed of two subunits, a 16-nucelotide enzymatic strand and a 25-nucelotide substrate
strand. Displaying a much simpler structure than the HDV ribozyme, the
hammerhead ribozyme is characterized by three helical segments, stems I, II and
II, connected by a loop that flanks a highly conserved catalytic pocket. The
uridine turn of that pocket is stabilized by aromatic interactions between C-17 and U-4 as well as hydrogen bonding between C-17 and C-3. The conformational change induced by the binding of a magnesium ion breaks this hydrogen bond by
shifting C-17 an additional 0.45 Å from C-3 but compensates by promoting the
formation of a hydrogen bond between C-17 and U-16. Mutagenesis studies
replacing U-16 with deoxythymine reduced the activity of the protein,
indicating that this bond stabilizes the intermediate conformation of the ribozyme,
supporting its function (12). Overall, the hammerhead ribozyme exhibits a much smaller and simpler structure than the HDV ribozyme with fewer non-canonical base pairs, fewer turns, and less complex sets of hydrogen bonds.
The hairpin ribozyme (PDB ID: 1M5K) is also a common ribozyme motif (2). It catalyzes
the same self-cleavage transesterification reaction as the hammerhead and HDV
ribozymes but without the involvement of metal ions (13). A calcium ion (Ca2+) was found bound to the major groove of a B-form helical segment but it does not
participate in catalysis. The hairpin ribozyme is largest of the three with a 21-nucleotide substrate RNA strand and a 92-nucelotide ribozyme. Structurally,
it is very different from the other molecules with four helical stems radiating
from a four-way junction and an artificial U1A spliceosome binding site which,
as with the HDV ribozyme, was added to allow for co-crystallization. These
helices stack in pairs which cross each other at a 60° angle and are connected
by a pair of anti-parallel crossovers (13).
The active site itself, composed entirely of RNA, arises at the junction of two pairs of
helices (13). The scissile bond is between G-13, a nucleotide in the syn
conformation with a C2’-endo ribose pucker (twists of the furanose ring), and A-12,
another nucleotide with a C2’-endo ribose pucker and an artificial 2’-methoxy modification
to prevent cleavage. The unusual conformation of this nucleotide pair produces
an in-line arrangement of the nucleophile (2’-OH of A-12, artificially replaced
with a methoxy moiety) and the leaving group (5’-oxo of G-13), both components
of the transesterification reaction (13). As with the hammerhead ribozyme, the
hairpin ribozyme catalyzes the same reaction as the HDV ribozyme but is overall
less complex in structure despite its larger size. It does not use a metal ion
in catalysis and features fewer non-canonical base pairs with fewer complex
turns (13).
The
study of self-cleaving ribozymes is valuable both to improving the
characterization of modern organisms and to developing an understanding of very
early life. Ribozymes, as catalytic RNA molecules, have played an invaluable
role in the construction of the RNA world hypothesis (11). As the only ribozyme
identified in a human pathogen, the HDV ribozyme occupies a uniquely important place in both the creation of potential therapies and the elucidation of clues about
the first life on Earth.