Directed Evolution of Aminoglycoside Phosphotransferase (3′) Type IIIa Variants That Inactivate Amikacin but Impose Significant Fitness Costs

The rules that govern adaptive protein evolution remain incompletely understood. Aminoglycoside aminotransferase (3′) type IIIa (hereafter abbreviated APH(3′)-IIIa) is a good model enzyme because it inactivates kanamycin efficiently; it recognizes other aminoglycoside antibiotics, including amikacin, but not nearly as well. Here we direct the evolution of APH(3′)-IIIa variants with increased activity against amikacin. After four rounds of random mutation and selection in Escherichia coli, the minimum inhibitory concentration of amikacin rose from 18 micrograms/mL (wild-type enzyme) to over 1200 micrograms/mL (clone 4.1). The artificially evolved 4.1 APH(3′)-IIIa variant exhibited 19-fold greater catalytic efficiency (k _cat/K _M) than did the wild-type enzyme in reactions with amikacin. E. coli expressing the evolved 4.1 APH(3′)-IIIa also exhibited a four-fold decrease in fitness (as measured by counting colony forming units in liquid cultures with the same optical density) compared with isogenic cells expressing the wild-type protein under non-selective conditions. We speculate that these fitness costs, in combination with the prevalence of other amikacin-modifying enzymes, hinder the evolution of APH(3′)-IIIa in clinical settings.     

Dynamic RNA Fitness Landscapes of a Group I Ribozyme during Changes to the Experimental Environment

Fitness landscapes of protein and RNA molecules can be studied experimentally using high-throughput techniques to measure the functional effects of numerous combinations of mutations. The rugged topography of these molecular fitness landscapes is important for understanding and predicting natural and experimental evolution. Mutational effects are also dependent upon environmental conditions, but the effects of environmental changes on fitness landscapes remains poorly understood. Here, we investigate the changes to the fitness landscape of a catalytic RNA molecule while changing a single environmental variable that is critical for RNA structure and function. Using high-throughput sequencing of in vitro selections, we mapped a fitness landscape of the Azoarcus group I ribozyme under eight different concentrations of magnesium ions (1–48 mM MgCl₂). The data revealed the magnesium dependence of 16,384 mutational neighbors, and from this, we investigated the magnesium induced changes to the topography of the fitness landscape. The results showed that increasing magnesium concentration improved the relative fitness of sequences at higher mutational distances while also reducing the ruggedness of the mutational trajectories on the landscape. As a result, as magnesium concentration was increased, simulated populations evolved toward higher fitness faster. Curve-fitting of the magnesium dependence of individual ribozymes demonstrated that deep sequencing of in vitro reactions can be used to evaluate the structural stability of thousands of sequences in parallel. Overall, the results highlight how environmental changes that stabilize structures can also alter the ruggedness of fitness landscapes and alter evolutionary processes.     

Revisiting the Nucleotide and Aminoglycoside Substrate Specificity of the Bifunctional Aminoglycoside Acetyltransferase(6′)-Ie/Aminoglycoside Phosphotransferase(2″)-Ia Enzyme

The bifunctional aminoglycoside-modifying enzyme aminoglycoside acetyltransferase(6′)-Ie/aminoglycoside phosphotransferase(2″)-Ia, or AAC(6′)-Ie/APH(2″)-Ia, is the major source of aminoglycoside resistance in Gram-positive bacterial pathogens. In previous studies, using ATP as the cosubstrate, it was reported that the APH(2″)-Ia domain of this enzyme is unique among aminoglycoside phosphotransferases, having the ability to inactivate an unusually broad spectrum of aminoglycosides, including 4,6- and 4,5-disubstituted and atypical. We recently demonstrated that GTP, and not ATP, is the preferred cosubstrate of this enzyme. We now show, using competition assays between ATP and GTP, that GTP is the exclusive phosphate donor at intracellular nucleotide levels. In light of these findings, we reevaluated the substrate profile of the phosphotransferase domain of this clinically important enzyme. Steady-state kinetic characterization using the phosphate donor GTP demonstrates that AAC(6′)-Ie/APH(2″)-Ia phosphorylates 4,6-disubstituted aminoglycosides with high efficiency (k_cat/K_m = 10⁵-10⁷ m⁻¹ s⁻¹). Despite this proficiency, no resistance is conferred to some of these antibiotics by the enzyme in vivo. We now show that phosphorylation of 4,5-disubstituted and atypical aminoglycosides are negligible and thus these antibiotics are not substrates. Instead, these aminoglycosides tend to stimulate an intrinsic GTPase activity of the enzyme. Taken together, our data show that the bifunctional enzyme efficiently phosphorylates only 4,6-disubstituted antibiotics; however, phosphorylation does not necessarily result in bacterial resistance. Hence, the APH(2″)-Ia domain of the bifunctional AAC(6′)-Ie/APH(2″)-Ia enzyme is a bona fide GTP-dependent kinase with a narrow substrate profile, including only 4,6-disubstituted aminoglycosides.     

Mutational fitness landscape and drug resistance

Robust technology has been developed to systematically quantify fitness landscapes that provide valuable opportunities to improve our understanding of drug resistance and define new avenues to develop drugs with reduced resistance susceptibility. We outline the critical importance of drug resistance studies and the potential for fitness landscape approaches to contribute to this effort. We describe the major technical advancements in mutational scanning, which is the primary approach used to quantify protein fitness landscapes. There are many complex steps to consider in planning and executing mutational scanning projects including developing a selection scheme, generating mutant libraries, tracking the frequency of variants using next-generation sequencing, and processing and interpreting the data. Key experimental parameters impacting each of these steps are discussed to aid in planning fitness landscape studies. There is a strong need for improved understanding of drug resistance, and fitness landscapes provide a promising new approach.     

Structure of the Antibiotic Resistance Factor Spectinomycin Phosphotransferase from Legionella pneumophila

Aminoglycoside phosphotransferases (APHs) constitute a diverse group of enzymes that are often the underlying cause of aminoglycoside resistance in the clinical setting. Several APHs have been extensively characterized, including the elucidation of the three-dimensional structure of two APH(3′) isozymes and an APH(2″) enzyme. Although many APHs are plasmid-encoded and are capable of inactivating numerous 2-deoxystreptmaine aminoglycosides with multiple regiospecificity, APH(9)-Ia, isolated from Legionella pneumophila, is an unusual enzyme among the APH family for its chromosomal origin and its specificity for a single non-2-deoxystreptamine aminoglycoside substrate, spectinomycin. We describe here the crystal structures of APH(9)-Ia in its apo form, its binary complex with the nucleotide, AMP, and its ternary complex bound with ADP and spectinomycin. The structures reveal that APH(9)-Ia adopts the bilobal protein kinase-fold, analogous to the APH(3′) and APH(2″) enzymes. However, APH(9)-Ia differs significantly from the other two types of APH enzymes in its substrate binding area and that it undergoes a conformation change upon ligand binding. Moreover, kinetic assay experiments indicate that APH(9)-Ia has stringent substrate specificity as it is unable to phosphorylate substrates of choline kinase or methylthioribose kinase despite high structural resemblance. The crystal structures of APH(9)-Ia demonstrate and expand our understanding of the diversity of the APH family, which in turn will facilitate the development of new antibiotics and inhibitors.     

The Crystal Structures of Substrate and Nucleotide Complexes of Enterococcus faecium Aminoglycoside-2′′-Phosphotransferase-IIa [APH(2′′)-IIa] Provide Insights into Substrate Selectivity in the APH(2′′) Subfamily

Aminoglycoside-2′′-phosphotransferase-IIa [APH(2′′)-IIa] is one of a number of homologous bacterial enzymes responsible for the deactivation of the aminoglycoside family of antibiotics and is thus a major component in bacterial resistance to these compounds. APH(2′′)-IIa produces resistance to several clinically important aminoglycosides (including kanamycin and gentamicin) in both gram-positive and gram-negative bacteria, most notably in Enterococcus species. We have determined the structures of two complexes of APH(2′′)-IIa, the binary gentamicin complex and a ternary complex containing adenosine-5′-(β,γ-methylene)triphosphate (AMPPCP) and streptomycin. This is the first crystal structure of a member of the APH(2′′) family of aminoglycoside phosphotransferases. The structure of the gentamicin-APH(2′′)-IIa complex was solved by multiwavelength anomalous diffraction methods from a single selenomethionine-substituted crystal and was refined to a crystallographic R factor of 0.210 (R_free, 0.271) at a resolution of 2.5 Å. The structure of the AMPPCP-streptomycin complex was solved by molecular replacement using the gentamicin-APH(2′′)-IIa complex as the starting model. The enzyme has a two-domain structure with the substrate binding site located in a cleft in the C-terminal domain. Gentamicin binding is facilitated by a number of conserved acidic residues lining the binding cleft, with the A and B rings of the substrate forming the majority of the interactions. The inhibitor streptomycin, although binding in the same pocket as gentamicin, is orientated such that no potential phosphorylation sites are adjacent to the catalytic aspartate residue. The binding of gentamicin and streptomycin provides structural insights into the substrate selectivity of the APH(2′′) subfamily of aminoglycoside phosphotransferases, specifically, the selectivity between the 4,6-disubstituted and the 4,5-disubstituted aminoglycosides.The emergence of bacteria resistant to several important classes of antibiotics has become a major clinical problem over the last few years. Almost every antibacterial compound in clinical use today has associated examples of resistant bacterial isolates (39), including life-threatening strains of Escherichia coli, Mycobacterium tuberculosis, Pseudomonas aeruginosa, and various enterococci. The latter are among the most common antibiotic-resistance bacteria isolated from patients with nosocomial infections in the United States today. The synergistic use of either ampicillin or vancomycin with an aminoglycoside, such as kanamycin or gentamicin, has long been the optimal therapy for serious enterococcal infections; however, many previously susceptible enterococcal strains have since acquired resistance to the aminoglycosides. The mechanisms of resistance are many and varied, although only three are readily understood: (i) mutation of the ribosomal target, (ii) reduced permeability and/or increased efflux of the drug, and (iii) enzymatic deactivation of the drug. Resistance to the aminoglycosides through enzymatic deactivation, although seemingly straightforward, is in reality a complex problem involving three different classes of enzyme. These enzyme classes are the ATP-dependent phosphotransferases (APH) and adenyltransferases (ANT), and the acetyl coenzyme A-dependent N-acetyltransferases (AAC). This area of research has been extensively reviewed in the past few years (2, 4, 13, 29, 39, 47, 52, 53).Originally isolated from soil bacteria, including various species of Streptomyces and Micromonospora (20), the aminoglycosides are a family of potent, broad-spectrum antibiotics that includes clinically relevant drugs such as gentamicin, neomycin, amikacin, kanamycin, and streptomycin. The structures of these compounds, with the exception of that of streptomycin, are all similar, consisting of a central aminocyclitol ring (the B ring) with two or three substituted aminoglycan rings (A, C, and in some cases, D) attached at either the 4 and 5 positions (the 4,5-disubstituted aminoglycosides, which include neomycin and lividomycin) or the 4 and 6 positions (the 4,6-disubstituted aminoglycosides, such as gentamicin and kanamycin). Streptomycin, a competitive inhibitor of aminoglycoside-2′′-phosphotransferase-IIa [APH(2′′)-IIa] (45), is an atypical aminoglycoside that does not fall into either the 4,5-disubstituted or 4,6-disubstituted classes. It has a modified ribose (ring B) attached to position 4 on a 1,3-diguanidinium-substituted aminocyclitol ring (ring A) with no substituent at the 5 or 6 position. The structures of gentamicin, kanamycin, neomycin, and streptomycin are shown in Fig. ​Fig.1.1. The aminoglycosides are targeted to the 16S rRNA of the bacterial 30S ribosomal subunit, where they selectively bind to the decoding aminoacyl (A) site (31, 51) and stabilize the conformation of the tRNA bound to a cognate mRNA codon. This decreases the dissociation rate of aminoacyl-tRNA and promotes miscoding (28). The structures of a number of the aminoglycosides with either the 30S subunit or oligonucleotides containing minimal A sites are known (51).Open in a separate windowFIG. 1.Structures of gentamicin, kanamycin, streptomycin, and neomycin. Gentamicin and kanamycin are classified as 4,6-disubstituted aminoglycosides, whereas neomycin is an example of a 4,5-disubstituted compound. The three structural variants which comprise gentamicin C are indicated. Amikacin is similar to kanamycin, although the substituent on the N1 amine is a 4-amino-2-hydroxy-1-oxobutyl group. Taken together, the A and B rings of aminoglycosides, such as gentamicin, kanamycin, and neomycin, are commonly known as the neamine moiety.The enzymes which deactivate the aminoglycosides are named according to the reaction they catalyze and the site on the aminoglycoside at which they act. The APH(2′′) enzymes, which give rise to high-level resistance to gentamicin in enterococci, phosphorylate gentamicin and kanamycin at the 2′′-hydroxyl group of the C ring (Fig. ​(Fig.1).1). The APH(3′) enzymes, another major subfamily of the phosphotransferases, phosphorylate kanamycin and neomycin at the 3′-hydroxyl on the A ring but cannot deactivate gentamicin, since it has no corresponding 3′-hydroxyl. The individual members of each family can normally bind only a subset of the available drugs, and this difference in drug specificity is known as the resistance profile, designated with a roman numeral and, in some cases, a letter identifying a specific gene. The first APH(2′′) enzyme discovered for enterococci was the bifunctional AAC(6′)-Ie-APH(2′′)-Ia enzyme, which possesses both 6′-acetylating and 2′′-phosphorylating activities (17, 33). Enterococci with the corresponding gene show resistance to almost all clinically relevant aminoglycosides (38). Four additional APH(2′′) enzymes have since been isolated for Enterococcus spp.; they are designated APH(2′′)-Ib (27), APH(2′′)-Ic (11), APH(2′′)-Id (46), and APH(2′′)-Ie (10) and were initially classified as genetic variants of an APH(2′′)-I-type enzyme. Recently, APH(2′′)-Ib, APH(2′′)-Ic, and APH(2′′)-Id have been reclassified as distinct enzymes with different resistance profiles and, more importantly, different nucleotide specificities, such that they are now named APH(2′′)-IIa, APH(2′′)-IIIa, and APH(2′′)-IVa, respectively (44). APH(2′′)-Ie was not included in the latter study, but based upon the very high sequence similarity with APH(2′′)-IVa (93%) (see Table S1 in the supplemental material), it is possible that it is a genetic variant of APH(2′′)-IVa.Structural details are currently known for only two members of the APH(3′) family, APH(3′)-IIIa (5, 18, 23) and APH(3′)-IIa (37). These enzymes share a two-domain structure similar to the catalytic domains of the eukaryotic Ser/Thr and Tyr protein kinases. Moreover, the phosphotransferases and kinases share several important sequence motifs related to nucleotide binding and phosphoryl transfer, most notably the catalytic loop (HXDXXXXN) and the activation segment (GXIDXG), where X is any amino acid. Not surprisingly, the catalytic mechanisms of the phosphotransferases and the kinases are identical, involving the nucleophilic attack by the target hydroxyl on the γ phosphate of ATP, facilitated by a conserved aspartate residue from the catalytic loop (29, 54). A comparison of the known APH(2′′) and APH(3′) sequences shows that the two families of phosphotransferases share these kinase-like motifs, and there appears to be some partial conservation of acidic residues in the substrate binding region. It has been suggested that their structures may be similar (37). Here, we report the first structure of an APH(2′′) enzyme, APH(2′′)-IIa as the binary complex with the preferred substrate gentamicin and the ternary complex with the nonhydrolyzable ATP analog adenosine-5′-(β,γ-methylene)triphosphate (AMPPCP) and the competitive inhibitor streptomycin.     

Balance between promiscuity and specificity in phage λ host range

As hosts acquire resistance to viruses, viruses must overcome that resistance to re-establish infectivity, or go extinct. Despite the significant hurdles associated with adapting to a resistant host, viruses are evolutionarily successful and maintain stable coevolutionary relationships with their hosts. To investigate the factors underlying how pathogens adapt to their hosts, we performed a deep mutational scan of the region of the λ tail fiber tip protein that mediates contact with the receptor on λ’s host, Escherichia coli. Phages harboring amino acid substitutions were subjected to selection for infectivity on wild type E. coli, revealing a highly restrictive fitness landscape, in which most substitutions completely abrogate function. A subset of positions that are tolerant of mutation in this assay, but diverse over evolutionary time, are associated with host range expansion. Imposing selection for phage infectivity on three λ-resistant hosts, each harboring a different missense mutation in the λ receptor, reveals hundreds of adaptive variants in λ. We distinguish λ variants that confer promiscuity, a general ability to overcome host resistance, from those that drive host-specific infectivity. Both processes may be important in driving adaptation to a novel host.Subject terms: Bacteriophages, Molecular evolution, Viral genetics     

Localization of an Amikacin 3′-Phosphotransferase in Escherichia coli

A plasmid-encoded enzyme reported by us to phosphorylate amikacin in a laboratory strain of Escherichia coli has been localized in the bacterial cell. More than 88% of this amikacin phosphotransferase (APH) activity was retained in spheroplasts formed by ethylenediaminetetraacetate-lysozyme treatment of an APH-containing E. coli transconguant known to form spheroplasts readily. By comparison, the spheroplasts retained 94% of deoxyribonucleic acid polymerase I and 98% of glutamyl-transfer ribonucleic acid synthetase, two internal markers, whereas less than 10% of the activity of a periplasmic marker, acid phosphatase, was present in spheroplasts. Treatment of whole cells of the transconjugant with chemical probes incapable of crossing the plasma membrane obliterated acid phosphatase activity, whereas the internal markers deoxyribonucleic acid polymerase I, glutamyl-transfer ribonucleic acid synthetase, and β-galactosidase were virtually unaffected after treatment for 5 min; more than 60% of the APH activity remained. As a control, similar chemical treatment of sonic extracts, in which enzymes were not protected by bacterial compartmentalization, produced more extensive reduction in the activities of all test enzymes, including APH. Spheroplasts preincubated with adenosine triphosphatase were shown by thin-layer chromatography to phosphorylate amikacin. Spheroplasts of cells grown in the presence of H₃³²PO₄ were shown to utilize internally generated adenosine 5′-triphosphate in the phosphorylation of amikacin. The absence of ³²P-phosphorylated amikacin after incubation of [γ-³²P]adenosine 5′-triphosphate with spheroplasts confirmed that exogenous adenosine 5′-triphosphate was not used in the reaction. These results suggest an internal location for APH. This conclusion has implications for the role of such enzymes in aminoglycoside resistance of gram-negative bacteria.     

Control of 5′,5′-Dinucleoside Triphosphate Catabolism by APH1, a Saccharomyces cerevisiae Analog of Human FHIT

The putative human tumor suppressor gene FHIT (fragile histidine triad) (M. Ohta et al., Cell 84:587–597, 1996) encodes a protein behaving in vitro as a dinucleoside 5′,5′′′-P¹,P³-triphosphate (Ap₃A) hydrolase. In this report, we show that the Saccharomyces cerevisiae APH1 gene product, which resembles human Fhit protein, also hydrolyzes dinucleoside 5′,5′-polyphosphates, with Ap₃A being the preferred substrate. Accordingly, disruption of the APH1 gene produced viable S. cerevisiae cells containing reduced Ap₃A-hydrolyzing activity and a 30-fold-elevated Ap₃N concentration.     

The population genetics of adaptation on correlated fitness landscapes: the block model

Several recent theoretical studies of the genetics of adaptation have focused on the mutational landscape model, which considers evolution on rugged fitness landscapes (i.e., ones having many local optima). Adaptation in this model is characterized by several simple results. Here I ask whether these results also hold on correlated fitness landscapes, which are smoother than those considered in the mutational landscape model. In particular, I study the genetics of adaptation in the block model, a tunably rugged model of fitness landscapes. Considering the scenario in which adaptation begins from a high fitness wild-type DNA sequence, I use extreme value theory and computer simulations to study both single adaptive steps and entire adaptive walks. I show that all previous results characterizing single steps in adaptation in the mutational landscape model hold at least approximately on correlated landscapes in the block model; many entire-walk results, however, do not.     

Oriented Scanning Is the Leading Mechanism Underlying 5′ Splice Site Selection in Mammals

Splice site selection is a key element of pre-mRNA splicing. Although it is known to involve specific recognition of short consensus sequences by the splicing machinery, the mechanisms by which 5′ splice sites are accurately identified remain controversial and incompletely resolved. The human F7 gene contains in its seventh intron (IVS7) a 37-bp VNTR minisatellite whose first element spans the exon7–IVS7 boundary. As a consequence, the IVS7 authentic donor splice site is followed by several cryptic splice sites identical in sequence, referred to as 5′ pseudo-sites, which normally remain silent. This region, therefore, provides a remarkable model to decipher the mechanism underlying 5′ splice site selection in mammals. We previously suggested a model for splice site selection that, in the presence of consecutive splice consensus sequences, would stimulate exclusively the selection of the most upstream 5′ splice site, rather than repressing the 3′ following pseudo-sites. In the present study, we provide experimental support to this hypothesis by using a mutational approach involving a panel of 50 mutant and wild-type F7 constructs expressed in various cell types. We demonstrate that the F7 IVS7 5′ pseudo-sites are functional, but do not compete with the authentic donor splice site. Moreover, we show that the selection of the 5′ splice site follows a scanning-type mechanism, precluding competition with other functional 5′ pseudo-sites available on immediate sequence context downstream of the activated one. In addition, 5′ pseudo-sites with an increased complementarity to U1snRNA up to 91% do not compete with the identified scanning mechanism. Altogether, these findings, which unveil a cell type–independent 5′−3′-oriented scanning process for accurate recognition of the authentic 5′ splice site, reconciliate apparently contradictory observations by establishing a hierarchy of competitiveness among the determinants involved in 5′ splice site selection.     

Alterations to both the Primary and Predicted Secondary Structure of Stem-Loop IIIc of the Hepatitis C Virus 1b 5′ Untranslated Region (5′UTR) Lead to Mutants Severely Defective in Translation Which Cannot Be Complemented in trans by the Wild-Type 5′UTR Sequence

Cap-independent translation of the hepatitis C virus (HCV) genomic RNA is mediated by an internal ribosome entry site (IRES) within the 5′ untranslated region (5′UTR) of the virus RNA. To investigate the effects of alterations to the primary sequence of the 5′UTR on IRES activity, a series of HCV genotype 1b (HCV-1b) variant IRES elements was generated and cloned into a bicistronic reporter construct. Changes from the prototypic HCV-1b 5′UTR sequence were identified at various locations throughout the 5′UTR. The translation efficiencies of these IRES elements were examined by an in vivo transient expression assay in transfected BHK-21 cells and were found to range from 0.4 to 95.8% of the activity of the prototype HCV-1b IRES. Further mutational analysis of the three single-point mutants most severely defective in activity, whose mutations were all located in or near stem-loop IIIc, demonstrated that both the primary sequence and the maintenance of base pairing within this stem structure were critical for HCV IRES function. Complementation studies indicated that defective mutants containing either point mutations or major deletions within the IRES elements could not be complemented in trans by a wild-type IRES.     

Binding of the Bacillus subtilis LexA protein to the SOS operator

The Bacillus subtilis LexA protein represses the SOS response to DNA damage by binding as a dimer to the consensus operator sequence 5′-CGAACN₄GTTCG-3′. To characterize the requirements for LexA binding to SOS operators, we determined the operator bases needed for site-specific binding as well as the LexA amino acids required for operator recognition. Using mobility shift assays to determine equilibrium constants for B.subtilis LexA binding to recA operator mutants, we found that several single base substitutions within the 14 bp recA operator sequence destabilized binding enough to abolish site-specific binding. Our results show that the AT base pairs at the third and fourth positions from the 5′ end of a 7 bp half-site are essential and that the preferred binding site for a LexA dimer is 5′-CGAACATATGTTCG-3′. Binding studies with LexA mutants, in which the solvent accessible amino acid residues in the putative DNA binding domain were mutated, indicate that Arg-49 and His-46 are essential for binding and that Lys-53 and Ala-48 are also involved in operator recognition. Guided by our mutational analyses as well as hydroxyl radical footprinting studies of the dinC and recA operators we docked a computer model of B.subtilis LexA on the preferred operator sequence in silico. Our model suggests that binding by a LexA dimer involves bending of the DNA helix within the internal 4 bp of the operator.     

DNA Sequence Context Effects on the Glycosylase Activity of Human 8-Oxoguanine DNA Glycosylase

Human 8-oxoguanine DNA glycosylase (OGG1) is a key enzyme involved in removing 7,8-dihydro-8-oxoguanine (8-oxoG), a highly mutagenic DNA lesion generated by oxidative stress. The removal of 8-oxoG by OGG1 is affected by the local DNA sequence, and this feature most likely contributes to observed mutational hot spots in genomic DNA. To elucidate the influence of local DNA sequence on 8-oxoG excision activity of OGG1, we conducted steady-state, pre-steady-state, and single turnover kinetic evaluation of OGG1 in alternate DNA sequence contexts. The sequence context effect was studied for a mutational hot spot at a CpG dinucleotide. Altering either the global DNA sequence or the 5′-flanking unmodified base pair failed to influence the excision of 8-oxoG. Methylation of the cytosine 5′ to 8-oxoG also did not affect 8-oxoG excision. In contrast, a 5′-neighboring mismatch strongly decreased the rate of 8-oxoG base removal. Substituting the 5′-C in the CpG dinucleotide with T, A, or tetrahydrofuran (i.e. T:G, A:G, and tetrahydrofuran:G mispairs) resulted in a 10-, 13-, and 4-fold decrease in the rate constant for 8-oxoG excision, respectively. A greater loss in activity was observed when T:C or A:C was positioned 5′ of 8-oxoG (59- and 108-fold, respectively). These results indicate that neighboring structural abnormalities 5′ to 8-oxoG deter its repair thereby enhancing its mutagenic potential.     

Effects of Topology and Sequence in Protein Folding Linked via Conformational Fluctuations

Experiments have compared the folding of proteins with different amino acid sequences but the same basic structure, or fold. Results indicate that folding is robust to sequence variations for proteins with some nonlocal folds, such as all-β, whereas the folding of more local, all-α proteins typically exhibits a stronger sequence dependence. Here, we use a coarse-grained model to systematically study how variations in sequence perturb the folding energy landscapes of three model sequences with 3α, 4β + α, and β-barrel folds, respectively. These three proteins exhibit folding features in line with experiments, including expected rank order in the cooperativity of the folding transition and stability-dependent shifts in the location of the free-energy barrier to folding. Using a generalized-ensemble simulation approach, we determine the thermodynamics of around 2000 sequence variants representing all possible hydrophobic or polar single- and double-point mutations. From an analysis of the subset of stability-neutral mutations, we find that folding is perturbed in a topology-dependent manner, with the β-barrel protein being the most robust. Our analysis shows, in particular, that the magnitude of mutational perturbations of the transition state is controlled in part by the size or “width” of the underlying conformational ensemble. This result suggests that the mutational robustness of the folding of the β-barrel protein is underpinned by its conformationally restricted transition state ensemble, revealing a link between sequence and topological effects in protein folding.     

A tRNA splicing operon: Archease endows RtcB with dual GTP/ATP cofactor specificity and accelerates RNA ligation

Archease is a 16-kDa protein that is conserved in all three domains of life. In diverse bacteria and archaea, the genes encoding Archease and the tRNA ligase RtcB are localized into an operon. Here we provide a rationale for this operon organization by showing that Archease and RtcB from Pyrococcus horikoshii function in tandem, with Archease altering the catalytic properties of the RNA ligase. RtcB catalyzes the GTP and Mn(II)-dependent joining of either 2′,3′-cyclic phosphate or 3′-phosphate termini to 5′-hydroxyl termini. We find that catalytic concentrations of Archease are sufficient to activate RtcB, and that Archease accelerates both the RNA 3′-P guanylylation and ligation steps. In addition, we show that Archease can alter the NTP specificity of RtcB such that ATP, dGTP or ITP is used efficiently. Moreover, RtcB variants that have inactivating substitutions in the guanine-binding pocket can be rescued by the addition of Archease. We also present a 1.4 Å-resolution crystal structure of P. horikoshii Archease that reveals a metal-binding site consisting of conserved carboxylates located at the protein tip. Substitution of the Archease metal-binding residues drastically reduced Archease-dependent activation of RtcB. Thus, evolution has sought to co-express archease and rtcB by creating a tRNA splicing operon.     

Adaptation in Tunably Rugged Fitness Landscapes: The Rough Mount Fuji Model

Much of the current theory of adaptation is based on Gillespie’s mutational landscape model (MLM), which assumes that the fitness values of genotypes linked by single mutational steps are independent random variables. On the other hand, a growing body of empirical evidence shows that real fitness landscapes, while possessing a considerable amount of ruggedness, are smoother than predicted by the MLM. In the present article we propose and analyze a simple fitness landscape model with tunable ruggedness based on the rough Mount Fuji (RMF) model originally introduced by Aita et al. in the context of protein evolution. We provide a comprehensive collection of results pertaining to the topographical structure of RMF landscapes, including explicit formulas for the expected number of local fitness maxima, the location of the global peak, and the fitness correlation function. The statistics of single and multiple adaptive steps on the RMF landscape are explored mainly through simulations, and the results are compared to the known behavior in the MLM model. Finally, we show that the RMF model can explain the large number of second-step mutations observed on a highly fit first-step background in a recent evolution experiment with a microvirid bacteriophage.     

Evolutionary accessibility of mutational pathways

Functional effects of different mutations are known to combine to the total effect in highly nontrivial ways. For the trait under evolutionary selection ('fitness'), measured values over all possible combinations of a set of mutations yield a fitness landscape that determines which mutational states can be reached from a given initial genotype. Understanding the accessibility properties of fitness landscapes is conceptually important in answering questions about the predictability and repeatability of evolutionary adaptation. Here we theoretically investigate accessibility of the globally optimal state on a wide variety of model landscapes, including landscapes with tunable ruggedness as well as neutral 'holey' landscapes. We define a mutational pathway to be accessible if it contains the minimal number of mutations required to reach the target genotype, and if fitness increases in each mutational step. Under this definition accessibility is high, in the sense that at least one accessible pathway exists with a substantial probability that approaches unity as the dimensionality of the fitness landscape (set by the number of mutational loci) becomes large. At the same time the number of alternative accessible pathways grows without bounds. We test the model predictions against an empirical 8-locus fitness landscape obtained for the filamentous fungus Aspergillus niger. By analyzing subgraphs of the full landscape containing different subsets of mutations, we are able to probe the mutational distance scale in the empirical data. The predicted effect of high accessibility is supported by the empirical data and is very robust, which we argue reflects the generic topology of sequence spaces. Together with the restrictive assumptions that lie in our definition of accessibility, this implies that the globally optimal configuration should be accessible to genome wide evolution, but the repeatability of evolutionary trajectories is limited owing to the presence of a large number of alternative mutational pathways.