Distant Mutations Research Articles

Outside‐binding site mutations modify the active site's shapes in neuraminidase from influenza A H1N1

The recent occurrence of 2009 influenza A (H1N1) pandemic as well as others has raised concern of a far more dangerous outcome should this virus becomes resistant to current drug therapies. The number of clinical cases that are resistant to oseltamivir (Tamiflu®) is larger than the limited number of neuraminidase (NA) mutations (H275Y, N295S, and I223R) that have been identified at the active site and that are associated to oseltamivir resistance. In this study, we have performed a comparative analysis between a set of NAs that have the most representative mutations located outside the active site. The recently crystallized NA-oseltamivir complex (PDB ID: 3NSS) was used as a wild-type structure. After selecting the target NA sequences, their three-dimensional (3D) structure was built using 3NSS as a template by homology modeling. The 3D NA models were refined by molecular dynamics (MD) simulations. The refined models were used to perform a docking study, using oseltamivir as a ligand. Furthermore, the docking results were refined by free-energy analysis using the MM-PBSA method. The analysis of the MD simulation results showed that the NA models reached convergence during the first 10 ns. Visual inspection and structural measures showed that the mutated NA active sites show structural variations. The docking and MM-PBSA results from the complexes showed different binding modes and free energy values. These results suggest that distant mutations located outside the active site of NA affect its structure and could be considered to be a new source of resistance to oseltamivir, which agrees with reports in the clinical literature.

Combining different design strategies for rational affinity maturation of the MICA‐NKG2D interface

We redesigned residues on the surface of MICA, a protein that binds the homodimeric immunoreceptor NKG2D, to increase binding affinity with a series of rational, incremental changes. A fixed-backbone RosettaDesign protocol scored a set of initial mutations, which we tested by surface plasmon resonance for thermodynamics and kinetics of NKG2D binding, both singly and in combination. We combined the best four mutations at the surface with three affinity-enhancing mutations below the binding interface found with a previous design strategy. After curating design scores with three cross-validated tests, we found a linear relationship between free energy of binding and design score, and to a lesser extent, enthalpy and design score. Multiple mutants bound with substantial subadditivity, but in at least one case full additivity was observed when combining distant mutations. Altogether, combining the best mutations from the two strategies into a septuple mutant enhanced affinity by 50-fold, to 50 nM, demonstrating a simple, effective protocol for affinity enhancement.

Nucleotide Dynamics at the A-Site Cleft in the Peptidyltransferase Center of H. marismortui 50S Ribosomal Subunits.

Resistance mutations to antibiotics targeting rRNA can be far from the drug-binding site. Crystallography studies revealed that the antibiotic resistance mutation G2482A (G2447A in E. coli ) in Haloarcula marismortui 50S ribosomes does not directly contact the drug or introduce changes to the ribosomal structure except for losing a potassium ion coordinated to a base triple at the drug-binding site. Using molecular dynamics simulations, we tested hypotheses regarding the effects of the G2482A mutation and ion coordination on the conformational dynamics of the 50S ribosome. Simulations show that the mutation enhances conformational fluctuation at the antibiotic binding site, weakens the hydrogen-bonding network, and increases flexibility at the 50S peptidyl transferase center (PTC). Our data supports the view that distant mutations can perturb the dynamic network in the ribosomal PTC, thereby raising the entropic cost of antibiotic binding. These results underscore the importance of considering conformational dynamics in rational drug design.

Spliceman—a computational web server that predicts sequence variations in pre-mRNA splicing

It was previously demonstrated that splicing elements are positional dependent. We exploited this relationship between location and function by comparing positional distributions between all possible 4096 hexamers around a database of human splice sites. The distance measure used in this study found point mutations that produced higher distances disrupted splicing, whereas point mutations with smaller distances generally had no effect on splicing. Reasoning the idea that functional splicing elements have signature positional distributions around constitutively spliced exons, we introduce Spliceman-an online tool that predicts how likely distant mutations around annotated splice sites were to disrupt splicing. Spliceman takes a set of DNA sequences with point mutations and returns a ranked list to predict the effects of point mutations on pre-mRNA splicing. The current implementation included the analyses of 11 genomes: human, chimp, rhesus, mouse, rat, dog, cat, chicken, guinea pig, frog and zebrafish. Freely available on the web at http://fairbrother.biomed.brown.edu/spliceman/ fairbrother@brown.edu.

Distant and New Mutations in CTX-M-1 β-Lactamase Affect Cefotaxime Hydrolysis

The CTX-M β-lactamases are an increasingly prevalent group of extended-spectrum β-lactamases (ESBL). Point mutations in CTX-M β-lactamases are considered critical for enhanced hydrolysis of cefotaxime. In order to clarify the structural determinants of the activity against cefotaxime in CTX-M β-lactamases, screening for random mutations was carried out to search for decreased activity against cefotaxime, with the CTX-M-1 gene as a model. Thirteen single mutants with a considerable reduction in cefotaxime MICs were selected for biochemical and stability studies. The 13 mutated genes of the CTX-M-1 β-lactamase were expressed, and the proteins were purified for kinetic studies against cephalothin and cefotaxime (as the main antibiotics). Some of the positions, such as Val103Asp, Asn104Asp, Asn106Lys, and Pro107Ser, are located in the (103)VNYN(106) loop, which had been described as important in cefotaxime hydrolysis, although this has not been experimentally confirmed. There are four mutations located close to catalytic residues-Thr71Ile, Met135Ile, Arg164His, and Asn244Asp-that may affect the positioning of these residues. We show here that some distant mutations, such as Ala219Val, are critical for cefotaxime hydrolysis and highlight the role of this loop at the top of the active site. Other distant substitutions, such as Val80Ala, Arg191, Ala247Ser, and Val260Leu, are in hydrophobic cores and may affect the dynamics and flexibility of the enzyme. We describe here, in conclusion, new residues involved in cefotaxime hydrolysis in CTX-M β-lactamases, five of which are in positions distant from the catalytic center.

The Cat and Mouse of HIV-1 Antibody Escape

The Cat and Mouse of HIV-1 Antibody Escape

Saturation-mutagenesis in two positions distant from active site of a Klebsiella pneumoniae glycerol dehydratase identifies some highly active mutants

Synthesis of 1,3-propanediol (1,3-PD) from glycerol through the biotransformation process requires two steps, catalyzed by glycerol dehydratase (GDHt) and 1,3-PD oxidoreductase. GDHt is the rate-limiting enzyme in this process. All recombinant microorganisms for production of 1,3-PD so far utilized the natural genes that may not have been optimized. Two positions, which are 19.3 Å and 29.6 Å away from the active site in GDHt from Klebsiella pneumoniae, were subjected to saturation-mutagenesis and 38 mutants were characterized. The catalytic activity of a mutant in β-subunit (β-Q42F, 29.6 Å from the active site) was 8.3-fold higher than the wild type, and the enzyme efficiency of other two mutants β-Q42L and β-Q42S for substrate glycerol was 336-fold and 80-fold higher than that for 1,2-propanediol. This investigation supplied further evidence that distant mutations could be a good source of diversity and therefore, made a contribution to the toolbox of industrial enzyme improvement.

Probing the Flexibility of the Bacterial Reaction Center: The Wild-Type Protein Is More Rigid Than Two Site-Specific Mutants

Experimental and theoretical studies have stressed the importance of flexibility for protein function. However, more local studies of protein dynamics, using temperature factors from crystallographic data or elastic models of protein mechanics, suggest that active sites are among the most rigid parts of proteins. We have used quasielastic neutron scattering to study the native reaction center protein from the purple bacterium Rhodobacter sphaeroides, over a temperature range of 4-260 K, in parallel with two nonfunctional mutants both carrying the mutations L212Glu/L213Asp --> Ala/Ala (one mutant carrying, in addition, the M249Ala --> Tyr mutation). The so-called dynamical transition temperature, Td, remains the same for the three proteins around 230 K. Below Td the mean square displacement, u2, and the dynamical structure factor, S(Q,omega), as measured respectively by backscattering and time-of-flight techniques are identical. However, we report that above Td, where anharmonicity and diffusive motions take place, the native protein is more rigid than the two nonfunctional mutants. The higher flexibility of both mutant proteins is demonstrated by either their higher u2 values or the notable quasielastic broadening of S(Q,omega) that reveals the diffusive nature of the motions involved. Remarkably, we demonstrate here that in proteins, point genetic mutations may notably affect the overall protein dynamics, and this effect can be quantified by neutron scattering. Our results suggest a new direction of investigation for further understanding of the relationship between fast dynamics and activity in proteins. Brownian dynamics simulations we have carried out are consistent with the neutron experiments, suggesting that a rigid core within the native protein is specifically softened by distant point mutations. L212Glu, which is systematically conserved in all photosynthetic bacteria, seems to be one of the key residues that exerts a distant control over the rigidity of the core of the protein.

The Catalytic Effect of Dihydrofolate Reductase and Its Mutants Is Determined by Reorganization Energies

The effect of distant mutations on the catalytic reaction of dihydrofolate reductase (DHFR) is reexamined by empirical valence bond simulations. The simulations reproduce for the first time the changes in the observed rate constants (without the use of adjustable parameters for this purpose) and show that the changes in activation barriers are strongly correlated with the corresponding changes in the reorganization energy. The preorganization of the polar groups of enzymes is the key catalytic factor, and anticatalytic mutations destroy this preorganization. Some anticatalytic mutations in DHFR also increase the distance between the donor and acceptor, but this effect is not directly related to catalysis since the native enzyme and the uncatalyzed reaction in water have similar average donor-acceptor distances. Insight into the effect of a mutation is provided by constructing the relevant free energy surfaces in terms of the generalized solute-solvent coordinates. It is shown how the mutations change the reaction coordinate and the activation barrier, and it is clarified that the corresponding changes do not reflect dynamical effects. It is also pointed out that all reactions in a condensed phase involve correlated motions (both in enzymes and in solution) and that the change of such motions upon mutations is a result of the change in the shape of the multidimensional reaction path on the solute-solvent surface, rather than the reason for the change in rate constant. Thus, as far as catalysis is concerned, the change in the activation barrier is due to the change in the electrostatic preorganization energy.

Improving enzyme properties: when are closer mutations better?

Study of mutations that improve enzyme properties reveals that in many, but not all, cases closer mutations are more effective than distant ones. For enantioselectivity, substrate selectivity and new catalytic activity (catalytic promiscuity) closer mutations improved enzymes more effectively than distant ones. However, both close and distant mutations can improve activity, thermal stability and also probably stability toward organic solvents. Typical random mutagenesis methods, such as error-prone PCR, create greater numbers of distant mutations than close mutations because enzymes contain more amino acids distant from the active site than close to the active site. This suggests that instead of mutating the entire enzyme, focusing mutations near the substrate-binding site might dramatically increase the success rate in many directed evolution experiments.

Focusing Mutations into the P. fluorescens Esterase Binding Site Increases Enantioselectivity More Effectively than Distant Mutations

Rational design of enzymes with improved properties, such as enantioselectivity, usually focuses mutations within the substrate binding site. On the other hand, directed evolution of enzymes usually targets the entire protein and discovers beneficial mutations far from the substrate binding site. In this paper, we propose an explanation for this discrepancy and show that a combined approach--random mutagenesis within the substrate binding site--is better. To increase the enantioselectivity (E) of a Pseudomonas fluorescens esterase (PFE) toward methyl 3-bromo-2-methylpropionate, we focused mutagenesis into the substrate binding site at Trp28, Val121, Phe198, and Val225. Five of the catalytically active mutants (13%) showed better enantioselectivity than wild-type PFE. The increases in enantioselectivity were higher (up to 5-fold, reaching E = 61) than with mutants identified by random mutagenesis of the entire enzyme.

Mechanism for multiple ligand recognition by the human transferrin receptor.

Transferrin receptor 1 (TfR) plays a critical role in cellular iron import for most higher organisms. Cell surface TfR binds to circulating iron-loaded transferrin (Fe-Tf) and transports it to acidic endosomes, where low pH promotes iron to dissociate from transferrin (Tf) in a TfR-assisted process. The iron-free form of Tf (apo-Tf) remains bound to TfR and is recycled to the cell surface, where the complex dissociates upon exposure to the slightly basic pH of the blood. Fe-Tf competes for binding to TfR with HFE, the protein mutated in the iron-overload disease hereditary hemochromatosis. We used a quantitative surface plasmon resonance assay to determine the binding affinities of an extensive set of site-directed TfR mutants to HFE and Fe-Tf at pH 7.4 and to apo-Tf at pH 6.3. These results confirm the previous finding that Fe-Tf and HFE compete for the receptor by binding to an overlapping site on the TfR helical domain. Spatially distant mutations in the TfR protease-like domain affect binding of Fe-Tf, but not iron-loaded Tf C-lobe, apo-Tf, or HFE, and mutations at the edge of the TfR helical domain affect binding of apo-Tf, but not Fe-Tf or HFE. The binding data presented here reveal the binding footprints on TfR for Fe-Tf and apo-Tf. These data support a model in which the Tf C-lobe contacts the TfR helical domain and the Tf N-lobe contacts the base of the TfR protease-like domain. The differential effects of some TfR mutations on binding to Fe-Tf and apo-Tf suggest differences in the contact points between TfR and the two forms of Tf that could be caused by pH-dependent conformational changes in Tf, TfR, or both. From these data, we propose a structure-based model for the mechanism of TfR-assisted iron release from Fe-Tf.

Mutations in Distant Residues Moderately Increase the Enantioselectivity of Pseudomonas fluorescens Esterase towards Methyl 3Bromo‐2‐methylpropanoate and Ethyl 3Phenylbutyrate

Directed evolution combined with saturation mutagenesis identified six different point mutations that each moderately increases the enantioselectivity of an esterase from Pseudomonas fluorescens (PFE) towards either of two chiral synthons. Directed evolution identified a Thr230Ile mutation that increased the enantioselectivity from 12 to 19 towards methyl (S)-3-bromo-2-methylpropanoate. Saturation mutagenesis at Thr230 identified another mutant, Thr230Pro, with higher-than-wild-type enantioselectivity (E=17). Previous directed evolution identified mutants Asp158Asn and Leu181Gln that increased the enantioselectivity from 3.5 to 5.8 and 6.6, respectively, towards ethyl (R)-3-phenylbutyrate. In this work, saturation mutagenesis identified other mutations that further increase the enantioselectivity to 12 (Asp158Leu) and 10 (Leu181Ser). A homology model of PFE indicates that all mutations lie outside the active site, 12-14 A from the substrate and suggests how the distant mutations might indirectly change the substrate-binding site. Since proteins contain many more residues far from the active site than close to the active site, random mutagenesis is strongly biased in favor of distant mutations. Directed evolution rarely screens all mutations, so it usually finds the distant mutations because they are more common, but probably not the most effective.

Concordant modulation of neutralization resistance and high infectivity of the primary human immunodeficiency virus type 1 MN strain and definition of a potential gp41 binding site in gp120.

Efforts to develop a vaccine against human immunodeficiency virus type 1 (HIV-1) are complicated by resistance of virus to neutralization. The neutralization resistance phenotype of HIV-1 has been linked to high infectivity. We studied the mechanisms determining this phenotype using clones of the T-cell-line-adapted (TCLA) MN strain (MN-TCLA) and the neutralization-resistant, primary MN strain (MN-P). Mutations in the amino- and carboxy-terminal halves of gp120 and the carboxy terminus of gp41 contributed to the neutralization resistance, high-infectivity phenotype but depended upon sequences in the leucine zipper (LZ) domain of gp41. Among 23 clones constructed to map the contributing mutations, there was a very strong correlation between infectivity and neutralization resistance (R(2) = 0.81; P < 0.0001). Mutations that distinguished the gp120s of MN-P and MN-TCLA clones were clustered in or near the CD4 and coreceptor binding sites and in regions distant from those binding sites. To test the hypothesis that some of these distant mutations may interact with gp41, we determined which of them contributed to high infectivity and whether those mutations modulated gp120-gp41 association in the context of MN-P LZ sequences. In one clone, six mutations in the amino terminus of gp120, at least four of which clustered closely on the inner domain, modulated infectivity. This clone had a gp120-gp41 association phenotype like MN-P: in comparison to MN-TCLA, spontaneous dissociation was low, and dissociation induced by soluble CD4 binding was high. These results identify a region of the gp120 inner domain that may be a binding site for gp41. Our studies clarify mechanisms of primary virus neutralization resistance.

The structure of a mutant photosynthetic reaction center shows unexpected changes in main chain orientations and quinone position.

We report on the unexpected structural changes caused by substitution of acidic amino acids in the Q(B) binding pocket of the bacterial photosynthetic reaction center by alanines. The mutations targeted key residues L212Glu and L213Asp of this transmembrane protein-cofactor complex. The amino acid substitutions in the L212Ala-L213Ala mutant reaction center ("AA") were known to affect the delivery of protons after the light-induced generation of Q(B)(-), which renders the AA strain incapable of photosynthetic growth. The AA structure not only revealed side chain rearrangements but also showed movement of the main chain segments that are contiguous with the mutation sites. The alanine substitutions caused an expansion of the cavity rather than its collapse. In addition, Q(B) is found mainly in the binding site that is proximal to the iron-ligand complex (closest to Q(A)) as opposed to its distal binding site (furthest from Q(A)) in the structure of the wild-type reaction center. The observed rearrangements in the structure of the AA reaction center establish a new balance between charged residues of an interactive network near Q(B). This structurally and electrostatically altered complex forms the basis for future understanding of the structural basis for proton transfer in active reaction centers which retain the alanine substitutions but carry a distant compensatory mutation.

Fragile T-stem in Disease-associated Human Mitochondrial tRNA Sensitizes Structure to Local and Distant Mutations

Mutations in human mitochondrial isoleucine tRNA (hs mt tRNA(Ile)) are associated with cardiomyopathy and opthalmoplegia. A recent study showed that opthalmoplegia-related mutations gave rise to severe decreases in aminoacylation efficiencies and that the defective mutant tRNAs were effective inhibitors of aminoacylation of the wild-type substrate. The results suggested that the effectiveness of the mutations was due in large part to an inherently fragile mitochondrial tRNA structure. Here, we investigate mutant tRNAs associated with cardiomyopathy, and a series of rationally designed second-site substitutions introduced into both opthalmoplegia- and cardiomyopathy-related mutant tRNAs. A source of structural fragility was uncovered. An inherently unstable T-stem appears susceptible to misalignments. This susceptibility sensitizes both domains of the L-shaped tRNA structure to base substitutions that are deleterious. Thus, the fragile T-stem makes the structure of this human mitochondrial tRNA particularly vulnerable to local and distant mutations.

Structural analysis of affinity matured antibodies and laboratory-evolved enzymes

This chapter reveals that directed evolution and antibody affinity maturation offer efficient routes to redesigning proteins for new functional characteristics. Adaptive mutations and well-defined selection pressures allow structural analysis of the evolved products to provide insights into the molecular basis of protein structure and function. The chapter describes the structural studies that highlight the importance of mutations away from the active site. Importantly, both close and distant productive mutations in both affinity-matured and directed evolution generated enzymes are found to ‘‘tweak’’ the desired enzyme function, by interacting with key areas of the protein scaffolding. X-ray structural characterization of both the ‘‘before’’ and ‘‘after’’ evolved protein structures provided the first opportunity to point out the location of critical mutations and key areas of the protein scaffold that have been found to modulate and attenuate overall enzyme function.

Intragenic suppressors that restore the splicing and homing activities of the protein encoded by the second intron of the Saccharomyces capensis cyt b gene

The second (bi2) intron of the mitochondrial cyt b gene from Saccharomyces capensis encodes a bifunctional protein which acts both as a maturase, promoting intron splicing, and as a homing-endonuclease, I-ScaI, promoting intron mobility. In this work we isolated and characterized revertants from a respiratory-deficient mutant in which both functions of the protein have been lost. Intragenic revertants resulted mainly from monosubstitutions in the mutated codon and in one case from a distant second site mutation. All novel variants of the S. capensis bi2 intron-encoded protein are competent for the maturase activity but only two of them can partially complement the homing function.

Proton uptake by bacterial reaction centers: the protein complex responds in a similar manner to the reduction of either quinone acceptor.

In bacterial photosynthetic reaction centers, the protonation events associated with the different reduction states of the two quinone molecules constitute intrinsic probes of both the electrostatic interactions and the different kinetic events occurring within the protein in response to the light-generated introduction of a charge. The kinetics and stoichiometries of proton uptake on formation of the primary semiquinone Q(A)(-) and the secondary acceptor Q(B)(-) after the first and second flashes have been measured, at pH 7.5, in reaction centers from genetically modified strains and from the wild type. The modified strains are mutated at the L212Glu and/or at the L213Asp sites near Q(B); some of them carry additional mutations distant from the quinone sites (M231Arg --> Leu, M43Asn --> Asp, M5Asn --> Asp) that compensate for the loss of L213Asp. Our data show that the mutations perturb the response of the protein system to the formation of a semiquinone, how distant compensatory mutations can restore the normal response, and the activity of a tyrosine residue (M247Ala --> Tyr) in increasing and accelerating proton uptake. The data demonstrate a direct correlation between the kinetic events of proton uptake that are observed with the formation of either Q(A)(-) or Q(B)(-), suggesting that the same residues respond to the generation of either semiquinone species. Therefore, the efficiency of transferring the first proton to Q(B) is evident from examination of the pattern of H(+)/Q(A)(-) proton uptake. This delocalized response of the protein complex to the introduction of a charge is coordinated by an interactive network that links the Q(-) species, polarizable residues, and numerous water molecules that are located in this region of the reaction center structure. This could be a general property of transmembrane redox proteins that couple electron transfer to proton uptake/release reactions.

Protein-chromophore interactions: spectral shifts report the consequences of mutations in the bacterial photosynthetic reaction center

Features of the low temperature absorption spectra of reaction centers from a large family of Rhodobacter capsulatus strains carrying site-specific mutations at the M208Tyr and L181Phe positions are presented. Through systematic analysis of the observed electronic transitions (with accompanying vibronic bands), the primary effects of many of the mutations have been identified and explained in terms of the reaction center structure. Some of the observed shifts are comparable to those that have been seen in other systems as a result of formation of hydrogen bonds or hexacoordination of the central Mg atom in bacteriochlorophyll species. Shifts of the Q y and Q x bacteriochlorophyll bands are also seen as a result of distant mutations near the quinones that change the charge distribution of the reaction center. We include kinetic data which show that the spectral and kinetic characteristics of different cofactors can be modulated depending on the particular amino acid that is substituted at the M208 site.