Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 270

All polynucleotide polymerases are thought to catalyze synthesis by a two-metal mechanism that requires two conserved aspartic acid residues (illustrated in the figure for a DNA polymerase). The carboxylate groups of these amino acids coordinate two divalent metal ions, shown as Mg²⁺ in the figure. One metal ion promotes deprotonation of the 3′-OH group of the nascent strand, and the other ion stabilizes the transition state at the α-phosphate of the NTP substrate and facilitates the release of pyrophosphate (PP_i).

Two-metal mechanism of DNA polymerase catalysis. Red arrows indicate the net movement of electrons.

Figure 6.4 Protein domain alignments for the four categories of nucleic acid polymerases. (A) Schematic diagrams of polymerases. Numbers at the top are from the poliovirus 3D^pol amino acid sequence. Sequence and structure motifs in each polymerase category are colored. Motif F is found only in RNA-dependent RNA polymerases. The Asp-Asp sequence of motif C is also conserved in RNA-dependent DNA polymerases of retroviruses and in RNA polymerases of double-stranded RNA and segmented (−) strand viruses. The RNA polymerases of nonsegmented (−) strand viruses contain Gly-Asp-Asn instead of Gly-Asp-Asp. Mutational studies have shown that this sequence in the RdRP (L protein) of vesicular stomatitis virus (VSV) is essential for RNA synthesis. The RdRP of birnavirus, an insect virus with a double-stranded RNA genome, has Ala-Asp-Asn instead of Gly-Asp-Asp. (B) Representative structures of each of the four types of nucleic acid polymerases. Ribbon diagrams of the polymerase domain of the large (Klenow) fragment of Escherichia coli DNA polymerase I, a DNA-dependent DNA polymerase; T7 RNA polymerase (T7 RNAP), a DNA-dependent RNA polymerase; human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT), an RNA-dependent DNA polymerase; and polioviral 3D^pol, an RNA-dependent RNA polymerase. The thumb domain is at the right, and the fingers domain is at the left. The conserved structure/sequence motifs A, B, C, D, and E are colored red, green, yellow, purple, and cyan, respectively (PDB files 1QSL, 1S77, 4B3O, and 3OL6).

The crystal structures of the four types of nucleic acid polymerases reveal that the enzymes resemble a right hand consisting of a palm, fingers, and a thumb, with the active site of the enzyme located in the palm (Fig. 6.4B). This shape supports the correct optimal arrangement of substrates and metal ions at the catalytic site and allows the dynamic changes needed during nucleic acid synthesis. The structures of RdRPs differ in detail from those of other polymerases, presumably to accommodate different templates and priming mechanisms. All nucleic acid polymerases have a similar core catalytic domain configuration and evolved from a common ancestor.

High-resolution structures of RdRPs and complexes with RNA in the process of catalysis have been determined for many (+) strand RNA, (–) strand RNA, and double-stranded RNA viruses. The RdRPs of picornaviruses and caliciviruses are the smallest known polymerases. Their structures are at the core of polymerases from larger RNA viruses, which typically contain additional domains that provide other replication-linked functions, such as methyltransferase, RNA capping, a platform for primer-independent initiation, and membrane anchoring.

The fingers and thumb subdomains of RdRPs show extensive interactions that encircle the active site and form a channel in which the template binds (Fig. 6.4). The closed structure creates a nucleoside triphosphate (NTP) entry tunnel on one face of the enzyme and a template-binding site on the other. Residues within motif F, a conserved region unique to RdRPs (Fig. 6.4), form the NTP entry tunnel, while motif G is in a loop that outlines the template entry channel. In contrast, structures of other polynucleotide polymerases resemble an open hand.

Figure 6.5 Structural elements of viral RNA-dependent RNA polymerase. (A) Ribbon representation of poliovirus 3D^pol (PDB file 3OL6). Conserved motifs are colored: motif A, red; motif B, green; motif C, yellow; motif D, cyan; motif E, tan; motif F, blue; motif G, magenta. (B-D) Surface representations of three RdRP enzymes are cut to expose channels that are the entry and exit sites of substrates and reaction products. Motifs A to G are colored as in panel A. (B) Poliovirus RdRP (PDB, 3OLB); (C) reovirus λ3 RdRP (PDB, 1N35). (D) Bacteriophage phi6 RdRP (PDB, 1HI0). Images B–D courtesy of Núria Verdaguer, Molecular Biology Institute of Barcelona.

Three channels can be observed in the structures of RdRPs from (+) strand RNA and some double-stranded RNA viruses, which comprise the entry and exit paths for template and an NTP channel (Fig. 6.5). In the polymerases of reovirus and (–) strand RNA viruses, N- and C-terminal extensions of the core enzyme form cage-like structures. In these enzymes, the buried active sites are connected to the exterior by four channels. The NTP and template entry channels lead to the catalytic site in the palm subdomain. The palm comprises a three-stranded antiparallel β-sheet that is surrounded by three α-helices and contains the four structural motifs in the order A-B-C-D. Motif A contains the consensus sequence DX_4-5C (where X is any amino acid), while motif C includes the amino acid triplet XDD, which is Gly-Asp-Asp in the RdRPs of most (+) strand RNA viruses. The two Asp residues of motif C and the conserved Asp238 of motif A form a cluster that coordinates the triphosphate moiety of the NTP substrate and the metal ions required for catalysis. Motif E, which is present in RNA-dependent but not in DNA-dependent polymerases, lies between the palm and thumb domains (Fig. 6.5). It projects into the active site and helps to position the 3′ end of the RNA primer.

The thumb domains of picornavirus and calicivirus RdRPs are small, and as a consequence, a large central cleft is present on one side of the molecule. This cleft accommodates a protein primer during initiation, and the double-stranded RNA product during elongation. In contrast, the polymerases of flaviviruses have much larger thumb domains with elements that protrude into the template channel and provide priming platforms for de novo initiation (see below).

RdRPs preferentially incorporate NTPs rather than deoxyribonucleoside triphosphates (dNTPs). NTP recognition by poliovirus 3D^pol is regulated by Asp238 of motif A, which forms a hydrogen bond with the ribose 2′-OH (Fig. 6.6). dNTPs are not bound because Asp238 cannot form a hydrogen bond with 2′-deoxyribose. An Asp is present at this position in all RdRPs. A Tyr at this position in RNA-dependent DNA polymerases (reverse transcriptases) is responsible for discriminating against NTPs and selecting dNTPs.

Figure 6.6 Structure of UTP bound to poliovirus 3D^pol. The NTP bridges the fingers (top) and palm (bottom) domains. The base is stacked with Arg163 from the fingers. Hydrogen bonds are shown as dashed lines. The Asp238 of motif A, which is conserved in all RNA-dependent RNA polymerases, hydrogen bonds with the 2′-OH of the ribose moiety; this interaction discriminates NTPs from dNTPs. Asp328 and Asp329, which coordinate Mg²⁺, are also labeled (PDB file 2IM2).