Palm Subdomains Research Articles

Solution structural dynamics of HIV-1 reverse transcriptase heterodimer.

Crystal structures and simulations suggest that conformational changes are critical for the function of HIV-1 reverse transcriptase. The enzyme is an asymmetric heterodimer of two subunits, p66 and p51. The two subunits have the same N-terminal sequence, with the p51 subunit lacking the C-terminal RNase H domain. We used hydrogen exchange mass spectrometry to probe the structural dynamics of RT. H/D exchange revealed that the fingers and palm subdomains of both subunits form the stable core of the heterodimer. In the crystal structure, the tertiary fold of the p51 subunit is more compact than that of the polymerase domain of the p66 subunit, yet both subunits show similar flexibility. The p66 subunit contains the polymerase and RNase H catalytic sites. H/D exchange indicated that the RNase H domain of p66 is very flexible. The beta-sheet beta12-beta13-beta14 lies at the base of the thumb subdomain of p66 and contains highly conserved residues involved in template/primer binding and NNRTI binding. Using the unique ability of hydrogen exchange mass spectrometry to resolve slowly interconverting species, we found that beta-sheet beta12-beta13-beta14 undergoes slow cooperative unfolding with a t(1/2) of <20 s. The H/D exchange results are discussed in relation to existing structural, simulation, and sequence information.

Disease Mutations in the Human Mitochondrial DNA Polymerase Thumb Subdomain Impart Severe Defects in Mitochondrial DNA Replication

Forty-five different point mutations in POLG, the gene encoding the catalytic subunit of the human mitochondrial DNA polymerase (pol gamma), cause the early onset mitochondrial DNA depletion disorder, Alpers syndrome. Sequence analysis of the C-terminal polymerase region of pol gamma revealed a cluster of four Alpers mutations at highly conserved residues in the thumb subdomain (G848S, c.2542g-->a; T851A, c.2551a-->g; R852C, c.2554c-->t; R853Q, c.2558g-->a) and two Alpers mutations at less conserved positions in the adjacent palm subdomain (Q879H, c.2637g-->t and T885S, c.2653a-->t). Biochemical characterization of purified, recombinant forms of pol gamma revealed that Alpers mutations in the thumb subdomain reduced polymerase activity more than 99% relative to the wild-type enzyme, whereas the palm subdomain mutations retained 50-70% wild-type polymerase activity. All six mutant enzymes retained physical and functional interaction with the pol gamma accessory subunit (p55), and none of the six mutants exhibited defects in misinsertion fidelity in vitro. However, differential DNA binding by these mutants suggests a possible orientation of the DNA with respect to the polymerase during catalysis. To our knowledge this study represents the first structure-function analysis of the thumb subdomain in pol gamma and examines the consequences of mitochondrial disease mutations in this region.

HIV-1 reverse transcriptase thumb subdomain polymorphisms associated with virological failure to nucleoside drug combinations

The aim of this study was to identify mutations within the fingers, palm and thumb subdomains of the HIV-1 reverse transcriptase (RT) associated with therapy failure to various combinations of two nucleoside analogues. RT nucleotide sequences of 1893 HIV-1 isolates from 1680 persons with different treatment histories (including naive and treated patients) were analysed. chi(2) contingency tests were performed to detect mutations within positions 1-333 of HIV-1 RT, associated with therapy failure, and correlated mutations were identified using statistical methods. Thymidine analogue resistance mutations were strongly associated with therapy failure to all nucleoside analogue combinations analysed. Previously identified accessory mutations at positions 35, 39, 43, 90, 98, 101, 122, 178, 196, 203, 208, 221, 223 and 228 were associated with therapy failure with at least one of the drug combinations studied. Interestingly, several mutations affecting RT thumb subdomain polymorphisms were strongly associated (P < 10(-3)) with therapy failure to abacavir/stavudine, stavudine/didanosine and abacavir/stavudine/didanosine. Mutations A272P, K277R and V293I were more prevalent in patients failing treatment with abacavir/stavudine than in naive individuals, while mutation frequencies of T286A, V292I and to a lesser extent E291D and E297K decreased in the treated population. These effects were also detected for A272P, T286A and V292I, when analysing didanosine/stavudine therapy failure, although statistical significance was higher for T286A. RT thumb subdomain polymorphisms are strongly associated with therapy failure to nucleoside analogues. Based on their structural location, we suggest a role for those residues in controlling the balance between RNase H degradation and nucleotide excision during DNA polymerization.

Slow Binding Inhibition and Mechanism of Resistance of Non-nucleoside Polymerase Inhibitors of Hepatitis C Virus

The binding affinity of four palm and thumb site representative non-nucleoside inhibitors (NNIs) of HCV polymerase NS5B to wild-type and resistant NS5B polymerase proteins was determined, and the influence of RNA binding on NNI binding affinity was investigated. NNIs with high binding affinity potently inhibited HCV RNA polymerase activity and replicon replication. Among the compounds tested, HCV-796 showed slow binding kinetics to NS5B. The binding affinity of HCV-796 to NS5B increased 27-fold over a 3-h incubation period with an equilibrium Kd of 71 +/- 2 nm. Slow binding kinetics of HCV-796 was driven by slow dissociation from NS5B with a k(off) of 4.9 +/- 0.5 x 10(-4) s(-1). NS5B bound a long, 378-nucleotide HCV RNA oligonucleotide with high affinity (Kd = 6.9 +/- 0.3 nm), whereas the binding affinity was significantly lower for a short, 21-nucleotide RNA (Kd = 155.1 +/- 16.2 nm). The formation of the NS5B-HCV RNA complex did not affect the slow binding kinetics profile and only slightly reduced NS5B binding affinity of HCV-796. The magnitude of reduction of NNI binding affinity for the NS5B proteins with various resistance mutations in the palm and thumb binding sites correlated well with resistance -fold shifts in NS5B polymerase activity and replicon assays. Co-crystal structures of NS5B-Con1 and NS5B-BK with HCV-796 revealed a deep hydrophobic binding pocket at the palm region of NS5B. HCV-796 interaction with the induced binding pocket on NS5B is consistent with slow binding kinetics and loss of binding affinity with mutations at amino acid position 316.

A New Generation of Peptide-based Inhibitors Targeting HIV-1 Reverse Transcriptase Conformational Flexibility

The biologically active form of human immunodeficiency virus (HIV) type 1 reverse transcriptase (RT) is a heterodimer. The formation of RT is a two-step mechanism, including a rapid protein-protein interaction "the dimerization step," followed by conformational changes "the maturation step," yielding the biologically active form of the enzyme. We have previously proposed that the heterodimeric organization of RT constitutes an interesting target for the design of new inhibitors. Here, we propose a new class of RT inhibitors that targets protein-protein interactions and conformational changes involved in the maturation of heterodimeric reverse transcriptase. Based on a screen of peptides derived from the thumb domain of this enzyme, we have identified a short peptide P(AW) that inhibits the maturation step and blocks viral replication at subnanomolar concentrations. P(AW) only binds dimeric RT and stabilizes it in an inactive/non-processive conformation. From a mechanistic point of view, P(AW) prevents proper binding of primer/template by affecting the structural dynamics of the thumb/fingers of p66 subunit. Taken together, these results demonstrate that HIV-1 RT maturation constitutes an attractive target for AIDS chemotherapeutics.

Involvement of the TPR2 subdomain movement in the activities of 29 DNA polymerase

The polymerization domain of ϕ29 DNA polymerase acquires a toroidal shape by means of an arch-like structure formed by the specific insertion TPR2 (Terminal Protein Region 2) and the thumb subdomain. TPR2 is connected to the fingers and palm subdomains through flexible regions, suggesting that it can undergo conformational changes. To examine whether such changes take place, we have constructed a ϕ29 DNA polymerase mutant able to form a disulfide bond between the apexes of TPR2 and thumb to limit the mobility of TPR2. Biochemical analysis of the mutant led us to conclude that TPR2 moves away from the thumb to allow the DNA polymerase to replicate circular ssDNA. Despite the fact that no TPR2 motion is needed to allow the polymerase to use the terminal protein (TP) as primer during the initiation of ϕ29 TP–DNA replication, the disulfide bond prevents the DNA polymerase from entering the elongation phase, suggesting that TPR2 movements are necessary to allow the TP priming domain to move out from the polymerase during transition from initiation to elongation. Furthermore, the TPR2-thumb bond does not affect the equilibrium between the polymerization and exonuclease activities, leading us to propose a primer-terminus transference model between both active sites.

X-ray crystal structure of the polymerase domain of the bacteriophage N4 virion RNA polymerase

Coliphage N4 virion RNA polymerase (vRNAP), which is injected into the host upon infection, transcribes the phage early genes from promoters that have a 5-bp stem-3 nt loop hairpin structure. Here, we describe the 2.0-A resolution x-ray crystal structure of N4 mini-vRNAP, a member of the T7-like, single-unit RNAP family and the minimal component having all RNAP functions of the full-length vRNAP. The structure resembles a "fisted right hand" with Fingers, Palm and Thumb subdomains connected to an N-terminal domain. We established that the specificity loop extending from the Fingers along with W129 of the N-terminal domain play critical roles in hairpin-promoter recognition. A comparison with the structure of the T7 RNAP initiation complex reveals that the pathway of the DNA to the active site is blocked in the apo-form vRNAP, indicating that vRNAP must undergo a large-scale conformational change upon promoter DNA binding and explaining the highly restricted promoter specificity of vRNAP that is essential for phage early transcription.

Activation mechanism of a noncanonical RNA-dependent RNA polymerase.

Two lineages of viral RNA-dependent RNA polymerases (RDRPs) differing in the organization (canonical vs. noncanonical) of the palm subdomain have been identified. Phylogenetic analyses indicate that both lineages diverged at a very early stage of the evolution of the enzyme [Gorbalenya AE, Pringle FM, Zeddam JL, Luke BT, Cameron CE, Kalmakoff J, Hanzlik TN, Gordon KH, Ward VK (2002) J Mol Biol 324:47-62]. Here, we report the x-ray structure of a noncanonical birnaviral RDRP, named VP1, in its free form, bound to Mg(2+) ions, and bound to a peptide representing the polymerase-binding motif of the regulatory viral protein VP3. The structure of VP1 reveals that the noncanonical connectivity of the palm subdomain maintains the geometry of the catalytic residues found in canonical polymerases but results in a partial blocking of the active site cavity. The VP1-VP3 peptide complex shows a mode of polymerase activation in which VP3 binding promotes a conformational change that removes the steric blockade of the VP1 active site, facilitating the accommodation of the template and incoming nucleotides for catalysis. The striking structural similarities between birnavirus (dsRNA) and the positive-stranded RNA picornavirus and calicivirus RDRPs provide evidence supporting the existence of functional and evolutionary relationships between these two virus groups.

The bacteriophage ϕ29 DNA polymerase

The bacteriophage ϕ29 DNA polymerase

Structures of phi29 DNA polymerase complexed with substrate: the mechanism of translocation in B-family polymerases

Replicative DNA polymerases (DNAPs) move along template DNA in a processive manner. The structural basis of the mechanism of translocation has been better studied in the A-family of polymerases than in the B-family of replicative polymerases. To address this issue, we have determined the X-ray crystal structures of phi29 DNAP, a member of the protein-primed subgroup of the B-family of polymerases, complexed with primer-template DNA in the presence or absence of the incoming nucleoside triphosphate, the pre- and post-translocated states, respectively. Comparison of these structures reveals a mechanism of translocation that appears to be facilitated by the coordinated movement of two conserved tyrosine residues into the insertion site. This differs from the mechanism employed by the A-family polymerases, in which a conserved tyrosine moves into the templating and insertion sites during the translocation step. Polymerases from the two families also interact with downstream single-stranded template DNA in very different ways.

Ability of Polymerase η and T7 DNA Polymerase to Bypass Bulge Structures

DNA misalignment occurs in homopolymer tracts during replication and can lead to frameshift mutations. Polymerase (pol) recognition of primer-templates containing bulge structures and the transmission of a bulge through a polymerase binding site or replication complex are important components of frameshift mutagenesis. In this report, we describe the interaction of the catalytic core of pol eta with primer-templates containing bulge structures by single round primer extension. We found that pol eta could stabilize a frayed primer terminus, which enhances its ability to extend primer-templates containing bulges. Based on methylphosphonate-DNA mapping, pol eta interacts with the single strand template but not appreciably with the template strand of the DNA stem greater than two nucleotides from the primer terminus. These latter characteristics, combined with the ability to stabilize a frayed primer terminus, may explain why primer-templates containing template bulges are extended so efficiently by pol eta. Although pol eta could accommodate large bulges and continue synthesis without obstruction, bulge structures in the template, but not in the primer, caused termination of the T7 DNA replication complex. Terminations occurred when the template bulge neared the helix-loop-helix domain of the polymerase thumb. Terminations were not observed, however, when bulge structures approached the site of interaction of the DNA with the extended thumb and thioredoxin. At low temperature, however, terminations did occur at this site.

Investigating the “Steric Gate” of Human Immunodeficiency Virus Type 1 (HIV-1) Reverse Transcriptase by Targeted Insertion of Unnatural Amino Acids

To investigate how structural changes in the amino acid side chain affect nucleotide substrate selection in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), a variety of non-natural tyrosine analogues were substituted for Tyr115 of p66 RT. RT variants containing meta-Tyr, nor-Tyr, aminomethyl-Phe, and 1- and 2-naphthyl-Tyr were produced in an Escherichia coli coupled transcription/translation system. Mutant p66 subunits were reconstituted with wild-type (WT) p51 RT and purified by affinity chromatography. Each modified enzyme retained DNA polymerase activity following this procedure. Aminomethyl-Phe115 RT incorporated dCTP more efficiently than the WT and was resistant to the chain terminator (-)-beta-2',3'-dideoxy-3'-thiacytidine triphosphate (3TCTP) when examined in a steady-state fidelity assay. However, 2-naphthyl-Tyr115 RT inefficiently incorporated dCTP at low concentrations and was kinetically slower with all dCTP analogues tested. Models of RT containing these side chains suggest that the aminomethyl-Phe115 substitution provides new hydrogen bonds through the minor groove to the incoming dNTP and the template residue of the terminal base pair. These hydrogen bonds likely contribute to the increased efficiency of dCTP incorporation. In contrast, models of HIV-1 RT containing 2-naphthyl-Tyr115 reveal significant steric clashes with Pro157 of the p66 palm subdomain, necessitating rearrangement of the active site.

Analysis of Amino Acids in the β7–β8 Loop of Human Immunodeficiency Virus Type 1 Reverse Transcriptase for their Role in Virus Replication

The HIV-1 p51/p66 reverse transcriptase (RT) heterodimer interface comprises, in part, intermolecular interaction of the loop region between β-strands 7 and 8 (β7–β8 loop) in the p51 fingers subdomain with the p66 palm subdomain. In this study, for the first time in the context of infectious HIV-1 particles, we analyzed the contribution of amino acid residues (S134, I135, N136, N137, T139 and P140) in the β7–β8 loop for RT heterodimerization, enzymatic activity, and virus infectivity. Mutating asparagine 136 to alanine (N136A) reduced viral infectivity and enzyme activity dramatically. The N136A mutation appeared to destabilize the RT heterodimer and render both the p66 and p51 subunits susceptible to aberrant cleavage by the viral protease. Subunit-specific mutagenesis demonstrated that the presence of the N136A mutation in the p51 subunit alone was sufficient to cause degradation of RT within the virus particle. Alanine mutation at other residues of the β7–β8 loop did not affect either RT stability or virus infectivity significantly. None of the β7–β8 loop alanine mutations affected the sensitivity of virus to inhibition by NNRTIs. In the context of infectious virions, our results indicate a critical role of the p51 N136 residue within the β7–β8 loop for RT heterodimer stability and function. These findings suggest the interface comprising N136 in p51 and interacting residues in p66 as a possible target for rational drug design.

A second, non-canonical RNA-dependent RNA polymerase in SARS Coronavirus

In (+) RNA coronaviruses, replication and transcription of the giant ∼30 kb genome to produce genome- and subgenome-size RNAs of both polarities are mediated by a cognate membrane-bound enzymatic complex. Its RNA-dependent RNA polymerase (RdRp) activity appears to be supplied by non-structural protein 12 (nsp12) that includes an RdRp domain conserved in all RNA viruses. Using SARS coronavirus, we now show that coronaviruses uniquely encode a second RdRp residing in nsp8. This protein strongly prefers the internal 5′-(G/U)CC-3′ trinucleotides on RNA templates to initiate the synthesis of complementary oligonucleotides of <6 residues in a reaction whose fidelity is relatively low. Distant structural homology between the C-terminal domain of nsp8 and the catalytic palm subdomain of RdRps of RNA viruses suggests a common origin of the two coronavirus RdRps, which however may have evolved different sets of catalytic residues. A parallel between the nsp8 RdRp and cellular DNA-dependent RNA primases is drawn to propose that the nsp8 RdRp produces primers utilized by the primer-dependent nsp12 RdRp.

Phosphorylation of Viral RNA-dependent RNA Polymerase and Its Role in Replication of a Plus-strand RNA Virus

Central to the process of plus-strand RNA virus genome amplification is the viral RNA-dependent RNA polymerase (RdRp). Understanding its regulation is of great importance given its essential function in viral replication and the common architecture and catalytic mechanism of polymerases. Here we show that Turnip yellow mosaic virus (TYMV) RdRp is phosphorylated, when expressed both individually and in the context of viral infection. Using a comprehensive biochemical approach, including metabolic labeling and mass spectrometry analyses, phosphorylation sites were mapped within an N-terminal PEST sequence and within the highly conserved palm subdomain of RNA polymerases. Systematic mutational analysis of the corresponding residues in a reverse genetic system demonstrated their importance for TYMV infectivity. Upon mutation of the phosphorylation sites, distinct steps of the viral cycle appeared affected, but in contrast to other plus-strand RNA viruses, the interaction between viral replication proteins was unaltered. Our results also highlighted the role of another TYMV-encoded replication protein as an antagonistic protein that may prevent the inhibitory effect of RdRp phosphorylation on viral infectivity. Based on these data, we propose that phosphorylation-dependent regulatory mechanisms are essential for viral RdRp function and virus replication.

Structural Determinants of Slippage-mediated Mutations by Human Immunodeficiency Virus Type 1 Reverse Transcriptase

Single-base deletions at nucleotide runs or -1 frameshifting by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) result from template slippage during polymerization. In crystal structures of HIV-1 RT complexed with DNA-DNA template-primer, the palm subdomain in the template cleft contacts the template backbone near the proposed site of slippage via the Glu(89) side chain. We investigated the role of Glu(89) in frameshifting by perturbing this interaction. Substitutions with Asp, Gly, Ala, Val, Ser, Thr, Asn, or Lys were created in recombinant HIV RT, and frameshift frequencies of the resulting mutant RTs were measured. All substitutions led to reduced -1 frameshifting by HIV-1 RT (2-40-fold). Interestingly, the suppression of -1 frameshifting frequently coincided with an enhancement of +1 frameshifting (3-47-fold) suggesting that Glu(89) can influence the slippage of both strands. Glu(89) substitutions also led to reduced rates of dNTP misincorporation that paralleled reductions in -1 frameshifting, suggesting a common structural mechanism for both classes of RT error. Our results reveal a major influence of Glu(89) on slippage-mediated errors and dNTP incorporation fidelity. The crystal structure of HIV-1 RT reveals a salt bridge between Glu(89) and Lys(154), which may facilitate -1 frameshifting; this concept is supported by the observed reduction in -1 frameshifting for K154A and K154R mutants.

Bioinformatic prediction of polymerase elements in the rotavirus VP1 protein

Rotaviruses are the major cause of acute gastroenteritis in infants world-wide. The genome consists of eleven double stranded RNA segments. The major segment encodes the structural protein VP1, the viral RNA-dependent RNA polymerase (RdRp), which is a minor component of the viral inner core. This study is a detailed bioinformatic assessment of the VP1 sequence. Using various methods we have identified canonical motifs within the VP1 sequence which correspond to motifs previously identified within RdRps of other positive strand, double-strand RNA viruses. The study also predicts an overall structural conservation in the middle region that may correspond to the palm subdomain and part of the fingers and thumb subdomains, which comprise the polymerase core of the protein. Based on this analysis, we suggest that the rotavirus replicase has the minimal elements to function as an RNA-dependent RNA polymerase. VP1, besides having common RdRp features, also contains large unique regions that might be responsible for characteristic features observed in the Reoviridae family.

Interaction of the Ty3 Reverse Transcriptase Thumb Subdomain with Template-Primer

Amino acid sequence alignment was used to identify the putative thumb subdomain of reverse transcriptase (RT) from the Saccharomyces cerevisiae long terminal repeat-containing retrotransposon Ty3. The counterpart to helix alphaH of human immunodeficiency virus type 1 (HIV-1) RT, which mediates important interactions with a duplex nucleic acid approximately 3-6 bp behind the DNA polymerase catalytic center, was identified between amino acids 290 and 298 of the Ty3 enzyme. The consequences of substituting Ty3 RT Gln290, Phe292, Gly294, Asn297, and Tyr298 (the counterparts of HIV-1 RT Gln258, Leu260, Gly262, Asn265, and Trp266, respectively) for both DNA polymerase and RNase H activities were examined. DNA-dependent DNA synthesis was evaluated on unmodified substrates and on duplexes containing targeted insertion of locked nucleic acid analogs and abasic lesions in either the template or primer. Based on this combined strategy, our data suggest an interaction of Ty3 RT Tyr298 with primer nucleotide -3, Gly294 with primer nucleotide -4, and Asn297 with template nucleotide -6. Substitution of Ala for Gln290 was well tolerated, despite the high degree of conservation at this position. Mutations in the thumb subdomain of Ty3 also affected RNase H activity, suggesting a closer spatial relationship between its N- and C-terminal catalytic centers compared with HIV-1 RT.

Substitution of proline 306 in the reverse transcriptase domain of hepatitis B virus regulates replication

The proline residue at position 306 in hepatitis B virus (HBV) reverse transcriptase (rtP306) has been suggested to constrain the conformation of the alpha-helices in the thumb subdomain that interacts with the viral DNA template-primer. To study the impact of residue rt306 in HBV replication further, 11 site-directed mutants were constructed that substituted rtP306 with different amino acids. The replicative competencies of these mutants were assayed by HepG2 cell transfection and real-time PCR. When rtP306 was substituted with glycine or threonine, the replication competency of these mutants was drastically reduced to 1.96 and 4.51% of the wild-type HBV level, respectively. When rtP306 was substituted with glutamic acid, the replicative competency of the mutant increased up to 9.4-fold compared with wild-type virus. The results also showed that changes in the replicative competency of these constructed mutants were not associated with functional changes of HBV enhancer I. These results indicate the importance of amino acid(s) at the interface between the thumb and palm subdomains in modulating the replicative competency of HBV isolates. This regulatory residue(s) could serve as a new target for the development of anti-HBV drugs.

Mutations in the Spacer Region of Drosophila Mitochondrial DNA Polymerase Affect DNA Binding, Processivity, and the Balance between Pol and Exo Function

The catalytic subunit (alpha) of mitochondrial DNA polymerase (pol gamma) shares conserved DNA polymerase and 3'-5' exonuclease active site motifs with Escherichia coli DNA polymerase I and bacteriophage T7 DNA polymerase. A major difference between the prokaryotic and mitochondrial proteins is the size and sequence of the region between the exonuclease and DNA polymerase domains, referred to as the spacer in pol gamma-alpha. Four gamma-specific conserved sequence elements are located within the spacer region of the catalytic subunit in eukaryotic species from yeast to humans. To elucidate the functional roles of the spacer region, we pursued deletion and site-directed mutagenesis of Drosophila pol gamma. Mutant proteins were expressed from baculovirus constructs in insect cells, purified to near homogeneity, and analyzed biochemically. We find that mutations in three of the four conserved sequence elements within the spacer alter enzyme activity, processivity, and/or DNA binding affinity. In addition, several mutations affect differentially DNA polymerase and exonuclease activity and/or functional interactions with mitochondrial single-stranded DNA-binding protein. Based on these results and crystallographic evidence showing that the template-primer binds in a cleft between the exonuclease and DNA polymerase domains in family A DNA polymerases, we propose that conserved sequences within the spacer of pol gamma may position the substrate with respect to the enzyme catalytic domains.