RNase H mutant D132N (1ZBI) from Bacillus halodurans
Created by: Jason Schwarz
Ribonuclease H (RNase H) mutant D132N (PDB ID: 1ZBI) is an endonuclease that degrades RNA from a RNA/DNA hybrid. RNase H removes RNA primers from Okazaki fragments during DNA replication in cells like those of Bacillus halodurans (Bh). However, RNase H also plays an important role in retroviruses like HIV by destroying the RNA template after the cDNA strand has been created in reverse transcription—a process that allows the virus to replicate (1). RNA degradation occurs through sequence-nonspecific hydrolysis of the P-O3’ bond in the RNA backbone (2). The hydrolysis occurs at the oxygen of the 3’ carbon in one nucleotide and the phosphate of the 5’ carbon of the adjacent nucleotide, regardless of the specific nucleotide base sequence. Thus, hydrolysis yields products with 5’phosphate and 3’ hydroxyl termini (3).
RNase H bound to an RNA/DNA hybrid in Bacillus halodurans is a homo-dimer that consists of two identical polypeptide chains surrounding a nucleotide substrate with one DNA strand and one RNA strand. RNase H catalyzes the RNA hydrolysis by positioning the nucleotides, the metal-ion ligands (Mg 2+ or Mn2+), and the nucleophile all within the active site. The hydrolysis of the phosphodiester bond is driven by nucleophilic attack from a hydroxyl group that forms from a deprotonated water molecule (2). Bh-RNase H has two Mg2+ ions per subunit — four total ions for the homodimer—that facilitate the nucleophilic attack (1).
The primary structure of RNase H plays a big part in the coordination of these metal ligands and the accompanying nucleophile. Both magnesium ions are contained within the active site and held in place through ionic interactions with specific carboxylate residues (1). For example, Asp-71 simultaneously coordinates both the A and B positive metal-ions with its negativel charged oxygens. Likewise, both Glu-188 and Asp-192 help position the A Mg2+ while Glu-109 and Asp-132(Asn) coordinate the B Mg2+ (1). Oxygens along the RNA backbone also help stabilize the metal ligands. One such oxygen is the pro-Sp oxygen of the scissile phosphate—the phosphate that will eventually be hydrolyzed. The two metals serve slightly different purposes, yet both are necessary for the “two-metal-ion catalysis” (1). The A Mg2+ supports the ideal octahedral shell and spurs hydroxide dissociation from the metal-bound water molecule, yielding the nucleophile needed for the attack (2). The B Mg2+ stabilizes the pentacovalent intermediate, which is characterized by a trigonal bipyramidal geometry with three coplanar oxygens around the phosphate; both the nucleophilic oxygen and leaving group oxygen sit perpendicular to the plane on opposite sides (1). The pentacovalent intermediate indicates that the RNA degradation occurs through bimolecular substitution (SN2) when the nucleophile attacks and kicks out the leaving group simultaneously. Therefore, the bimolecular substitution results in an inversion of stereochemistry at the scissile phosphate (1).
The mutant D132N RNase H differs from the wild-type RNase H by having its 132nd residue changed from aspartic acid to asparagine. This alteration from aspartic acid to asparagine skews the coordination of the B site magnesium, which interacts with the 132nd residue. Surprisingly, the mutation causes the metal coordination to deviate significantly from the ideal octahedral shape, completely inactivating the function of the protein (1). Obviously, both metals are critical for the nucleophilic hydrolysis of RNA and must have the correct initial positions. As the hydrolysis occurs, the metal ions move closer together to bring the nucleophile toward the scissile phosphate (2).
The RNA/DNA hybrid substrate has a tertiary structure that matches the shape of the binding site of RNase H. In physiological conditions, the hybrid maximizes its affinity for the protein by adopting a mixed A and B conformation. The tertiary structure of the RNase H allows for two grooves, spaced 8.5 Å apart, to efficiently capture the backbone of the nucleotide complex. The complimentary tertiary structures allow the minor groove of the hybrid to straddle the ridge in between the indentations of the protein. The indentation of RNase H that harbors the RNA strand contains the active site where hydrolysis occurs (1).
The primary structure of RNase H assists in the binding of the nucleotides because many residues form hydrogen bonds to the RNA and DNA strands. For example, Asn-106, Glu-109, Asp-132(Asn), and Gln-134 all form hydrogen bonds with the 2'-OH groups that are directly on the 5' side of the scissile phosphate in the RNA backbone. Simultaneously, Ser-74 and Gly-76 both bond to the 2’-OH on the 3’ side of the scissile phosphate (1). These connections link five adjacent residues (rU7, rG8, rA9, rU10, and rU11) with the protein to amplify the binding between substrate and protein (3). Similar hydrogen bonding extends from residues such as Asn-77, Thr-104, Asn-106, Ser-147, and Thr-148 to the DNA strand of the hybrid (3). The residues create a phosphate-binding pocket that is filled by a DNA phosphate situated two base pairs away from the RNA scissile phosphate (1). All the hydrogen bonds to RNA interact with the 2’ carbon hydroxide groups—the group that is replaced by a single hydrogen in the DNA backbone. The extra hydroxyl group on one strand gives RNA/DNA hybrids a higher binding affinity than DNA duplexes when binding to RNase H. The lack of 2’OH groups on the second DNA strand in a DNA duplex is analogous to “climbing up a ladder while holding on to just one rail” (3).
Bh-RNase H incorporates an ~100 residue core that is seen throughout RNase H molecules among different species such as E.coli and HIV. The core includes a five-stranded beta sheet and three alpha helices. In Bacillus halodurans, the five-stranded beta sheet consists of three antiparallel strands and two parallel strands (1). Secondary structure also plays an indirect role in the binding of the nucleotide substrate. The RNase H binding groove for DNA encompasses the N-terminus of one alpha helix and is situated between the two other helices and two of the beta strands (1).
Hydrophobic regions of RNase H must be kept on the inner face of the molecule because the enzyme interacts constantly with water. Compared with RNase H in other organisms, Bh-RNase H has a shortened C-terminal, which reveals a hydrophobic end to the outer face of the molecule. Consequently, alpha helix D adjusts to reorient that hydrophobic patch to the inner part of the biomolecule, enhancing the protein’s ability to interact with water (1).
Water molecules are essential for the function of RNase H. One water molecule serves as the original nucleophile that is then deprotonated to become a hydroxide group—a more efficient nucleophile than water. The loose proton from water is transferred to the Rp oxygen of the scissile phosphate with the help of three solvating water molecules. After RNA degradation, water molecules shift the proton to the 3’ hydroxy-terminus that was separated from the scissile phosphate (2). Both the metal-ion ligands and carboxylate residues of the active site attract the water molecules that are essential to the activity of RNase H.
The molecular weight of RNase H in Bacillus halodurans is 32,592.2 Da and its isoelectric point occurs at pH6.39 (4). But the optimal pH for RNase activity hovers around pH8 (2). At pH8, the acidic amino acids, aspartic acid and glutamic acid, both hold negative charges on their side chains because their pKa values are 3.9 and 4.1, respectively. The negative charge of the carboxylates is vital for the interactions between these residues and the Mg2+ ions within the active site.
PSI-BLAST compares the primary structure of a given protein with the sequence of a different protein by assigning an E value that is determined by finding gaps in the sequences. Specifically, the gaps represent amino acids that exist in the given protein but not in the comparison protein. Any E value below 0.5 signals a sufficient match between sequences. When PSI-BLAST was run on the FASTA sequence of Bh-RNase H mutant D132N, results showed small E values for many ribonuclease H proteins from other bacterial species. For example, ribonuclease H from Bacillus acalophilus showed an E value of 2e-66, which implied that the sequence of RNase H between the two bacterial organisms was similar (5). Unfortunately, none of the PSI-BLAST results yielded a PDB ID and could not be directly compared with Bh-RNase H in this report.
The Dali server measures similarities in the tertiary structure between the subject protein and an alternate protein. Other servers perform the same test through a least-squares-method, but Dali uses a sum-of-squares technique. The sum-of-squares technique compares intramolecular distances within the proteins to determine similarities in folding (6). The Dali server assigns Z scores for which any score over 2 demonstrates consistencies in folding between the proteins. When Bh-RNase H mutant D132N was tested in the Dali server, reverse transcriptase mutant K103N from human immunodeficiency virus 1 (PDB ID: 1SV5) yielded an acceptable Z score of 10.7 (7).
Reverse transcriptase (RT) in HIV-1 catalyzes the reverse transcription of RNA to DNA, which is an essential process for viral replication. RT is a heterodimer, but its function is mainly derived from the larger of its two subunits. The functional subunit has three domains: N-terminal polymerase, C-terminal RNase H, and a connection domain between the terminals. The main structural difference between RNase H in HIV-1 and RNase H in Bacillus halodurans is that, in the former, it is part of the larger reverse transcriptase protein while it remains a free enzyme in the latter (8). HIV-1 retains the core secondary structure of RNase H with a 5-stranded mixed beta sheet and three alpha helices. In actuality, the core consists of the 5-stranded beta sheet with four alpha helices, but the C helix is absent in many organisms such as HIV-1 and Bacillus halodurans (8). Because RNase H is part of a larger molecule in HIV-1, its activity often depends on that of the polymerase domain in RT. Polymerase-dependent activity results when the polymerase domain is adding nucleotides downstream while the RNase H domain is deleting the RNA template upstream to allow for synthesis of the (+) DNA strand. However, polymerase-independent activity also occurs whereby the RNase H domain deletes leftover RNA strands that are still annealed to the (–) DNA strand (9). In either case, the process of RNA degradation remains the same as in Bacillus halodurans in which two metal-ions catalyze the process. Ion A activates the nucleophile while ion B destabilizes the substrate-protein complex and lowers the activation energy for the transition state (8).
Human immunodeficiency virus-1 is a clear target for drugs because it can lead to AIDS. As of 2010, 25 usable drug complexes existed to treat HIV; 12 of those 25 targeted the reverse transcriptase enzyme in the virus. Of those 12 that focused on RT, all of them tried to inhibit the polymerase activity rather than the RNase H activity. This unbalance arises because the RNase H domain contains fewer binding pockets than the polymerase domain, which makes it harder for drug complexes to bind to RNase H. RNase H must work efficiently for the virus to survive. If RNase H works too quickly, then the RNA template may separate from the primer, halting reverse transcription. If RNase H degrades RNA much slower than the polymerase domain adds nucleotides, then reverse transcription might not be completed (9). Because of its essential role, RNase H has recently been targeted as a way to impede HIV replication. One method to combat the RNase H function revolves around chelating the Mg2+ ligands by a 3-oxygen pharmacophore. Drugs that fixate on the metal-ion ligands are known as active site inhibitors (8).