A single amino acid substitution alters ClpS2 binding specificity

ClpS2 is a small protein under development as a probe for selectively recognizing N-terminal amino acids of N-degron peptide fragments. To understand the structural basis of ClpS2 specificity for an N-terminal amino acid, all atom molecular dynamics (MD) simulations were conducted using the sequence of a bench-stable mutant of ClpS2, called PROSS. We predicted that a single amino acid leucine to asparagine substitution would switch the specificity of PROSS ClpS2 to an N-terminal tyrosine over the preferred phenylalanine. Experimental validation of the mutant using a fluorescent yeast-display assay showed an increase in tyrosine binding over phenylalanine, in support of the proposed hypothesis.     

A single amino acid residue can determine the sensitivity of SERCAs to artemisinins

Artemisinins are the most important class of antimalarial drugs. They specifically inhibit PfATP6, a SERCA-type ATPase of Plasmodium falciparum. Here we show that a single amino acid in transmembrane segment 3 of SERCAs can determine susceptibility to artemisinin. An L263E replacement of a malarial by a mammalian residue abolishes inhibition by artemisinins. Introducing residues found in other Plasmodium spp. also modulates artemisinin sensitivity, suggesting that artemisinins interact with the thapsigargin-binding cleft of susceptible SERCAs.     

Six amino acids determine the sequence-specific DNA binding and replication specificity of the initiator proteins of the pT181 family.

The replication of pT181 and related plasmids of Staphylococcus aureus proceeds by a rolling circle mechanisms. The initiator proteins encoded by the plasmids of the pT181 family have sequence-specific DNA binding and topoisomerase activities. These proteins nick one strand of the DNA at the origin of replication. The free 3'-hydroxyl end at the nick is then used as a primer for the replication of the leading strand of the DNA. Although these initiator proteins are highly homologous, they show specificity in DNA binding and replication for their cognate DNAs. In this study, we have generated hybrid initiator proteins and studied their various biochemical activities in vitro. Our results show that 6 amino acids are sufficient to determine the DNA binding and replication specificities of such initiator proteins.     

Nucleotides flanking a conserved TAAT core dictate the DNA binding specificity of three murine homeodomain proteins.

Murine homeobox genes play a fundamental role in directing embryogenesis by controlling gene expression during development. The homeobox encodes a DNA binding domain (the homeodomain) which presumably mediates interactions of homeodomain proteins with specific DNA sites in the control regions of target genes. However, the bases for these selective DNA-protein interactions are not well defined. In this report, we have characterized the DNA binding specificities of three murine homeodomain proteins, Hox 7.1, Hox 1.5, and En-1. We have identified optimal DNA binding sites for each of these proteins by using a random oligonucleotide selection strategy. Comparison of the sequences of the selected binding sites predicted a common consensus site that contained the motif (C/G)TAATTG. The TAAT core was essential for DNA binding activity, and the nucleotides flanking this core directed binding specificity. Whereas variations in the nucleotides flanking the 5' side of the TAAT core produced modest alterations in binding activity for all three proteins, perturbations of the nucleotides directly 3' of the core distinguished the binding specificity of Hox 1.5 from those of Hox 7.1 and En-1. These differences in binding activity reflected differences in the dissociation rates rather than the equilibrium constants of the protein-DNA complexes. Differences in DNA binding specificities observed in vitro may contribute to selective interactions of homeodomain proteins with potential binding sites in the control regions of target genes.     

A single amino acid substitution in 1918 influenza virus hemagglutinin changes receptor binding specificity

The receptor binding specificity of influenza viruses may be important for host restriction of human and avian viruses. Here, we show that the hemagglutinin (HA) of the virus that caused the 1918 influenza pandemic has strain-specific differences in its receptor binding specificity. The A/South Carolina/1/18 HA preferentially binds the alpha2,6 sialic acid (human) cellular receptor, whereas the A/New York/1/18 HA, which differs by only one amino acid, binds both the alpha2,6 and the alpha2,3 sialic acid (avian) cellular receptors. Compared to the conserved consensus sequence in the receptor binding site of avian HAs, only a single amino acid at position 190 was changed in the A/New York/1/18 HA. Mutation of this single amino acid back to the avian consensus resulted in a preference for the avian receptor.     

Single amino acids determine specificity of binding of protein kinase A regulatory subunits by protein kinase A anchoring proteins.

Cyclic AMP-dependent protein kinase is tethered to protein kinase A anchoring proteins (AKAPs) through regulatory subunits (R) by RIalpha-specific, RIIalpha-specific, or RIalpha/RIIalpha dual-specific binding. Ala- and Val-scanning mutagenesis determined that hydrophobic amino acids at three homologous positions are required for binding of RIalpha to FSC1/AKAP82 domain B and RIIalpha to AKAP Ht31. A mutation at the middle position reversed the binding specificity of both AKAPs, and mutations at this same position of the dual-specific domain A of FSC1/AKAP82 converted it into either an RIalpha or RIIalpha binding domain. This suggests that hydrophobic amino acids at three conserved positions within the primary sequence and an amphipathic helix of AKAPs are required for cyclic AMP-dependent protein kinase binding, with the size of the aliphatic side chain at the middle position determining RIalpha or RIIalpha binding specificity.     

Differential DNA binding properties of three human homeodomain proteins.

The products of three human homeobox containing (HOX) genes, 2C, 3C and 4B, were produced in insect cells using the Baculovirus expression system and purified to near homogeneity. In this system we observed that the DNA binding forms of the three proteins are not glycosylated. HOX 3C and 4B are phosphorylated in insect cells, while HOX 2C is not. The three HOX proteins bind to a DNA sequence known to be a target site for Antennapedia protein with a very similar affinity (Kd = 1-2 x 10(-9) M). We then measured their binding properties to four human sequences present in the HOX 3D, 4C, 1C and 4B promoters. Two of these sequences have been reported to be binding sites for HOX proteins. HOX 2C, 3C and 4B behaved quite differently, showing low affinity for promoters of genes located upstream from their own gene in the HOX clusters and a higher affinity for regulatory sequences of their own gene and downstream HOX genes.     

Influence of amino acid numbers between two ligand cysteines of zinc finger proteins on affinity and specificity of DNA binding

Hypaphorine, an indolic alkaloid from an ectomycorrhizal fungus is a putative antagonist of indole-3-acetic acid (IAA) known to inhibit the effect of IAA in growing roots of Eucalyptus seedling. Previously we have used horseradish peroxidase-C (HRP) as a sensitive reporter of IAA-binding to the IAA-binding domain, and reported that hypaphorine specifically inhibits the HRP-catalyzed superoxide generation coupled to oxidation of IAA [Kawano et al., Biochem. Biophys. Res. Commun. 288]. Since binding of IAA to the auxin-binding domain is the key step required for IAA oxidation by HRP, it was assumed that the inhibitory effect of hypaphorine is due to its competitive binding to the auxin-binding domain in HRP. Here, we obtained further evidence in support of our assumption that hypaphorine specifically inhibits binding of IAA to HRP. In this study, HRP arrested at the temporal inactive form known as Compound III was used as a sensitive indicator for binding of IAA to HRP. Addition of IAA to the preformed Compound III resulted in rapid decreases in absorption maxima at 415, 545, and 578 nm characteristic to Compound III, and in turn a rapid increase in absorption maximum at 670 nm representing the formation of P-670, the irreversibly inactivated form of hemoproteins, was induced. In contrast, the IAA-dependent irreversible inactivation of HRP was inhibited in the presence of hypaphorine. In addition, the mode of interaction between IAA and hypaphorine was determined to be competitive inhibition, further confirming that hypaphorine is an IAA antagonist which specifically compete with IAA in binding to the IAA-binding site in plant peroxidases.     

The specificity of TIMP-2 for matrix metalloproteinases can be modified by single amino acid mutations.

Residues 1-127 of human TIMP-2 (N-TIMP-2), comprising three of the disulfide-bonded loops of the TIMP-2 molecule, is a discrete protein domain that folds independently of the C-terminal domain. This domain has been shown to be necessary and sufficient for metalloproteinase inhibition and contains the major sites of interaction with the catalytic N-terminal domain of active matrix metalloproteinases (MMPs). Residues identified as being involved in the interaction with MMPs by NMR chemical shift perturbation studies and TIMP/MMP crystal structures have been altered by site-directed mutagenesis. We show, by measurement of association rates and apparent inhibition constants, that the specificity of these N-TIMP-2 mutants for a range of MMPs can be altered by single site mutations in either the TIMP "ridge" (Cys1-Cys3 and Ser68-Cys72) or the flexible AB loop (Ser31-Ile41). This work demonstrates that it is possible to engineer TIMPs with altered specificity and suggests that this form of protein engineering may be useful in the treatment of diseases such as arthritis and cancer where the selective inhibition of key MMPs is desirable.     

Conservation of binding site specificity of three yeast DNA binding proteins.

Sequence specific binding of protein extracts from 13 different yeast species to three oligonucleotide probes and two points mutants derived from Saccharomyces cerevisiae DNA binding proteins were tested using mobility shift assays. The probes were high affinity binding sites for GRF1/RAP1/ABF1 and CP1/CPF1. Most yeasts in the genus Saccharomyces showed specific binding to all three probes and also displayed similar sequence requirements when challenged by molar excesses of mutant probes. The affinities for the probes varied amongst the other yeasts tested, but in general, CPF1 binding activity was the most widespread, while the other two were more limited.     

A single amino acid substitution in the capsid of foot-and-mouth disease virus can increase acid resistance

Foot-and-mouth disease virus (FMDV) particles lose infectivity due to their disassembly at pH values slightly below neutrality. This acid-dependent disassembly process is required for viral RNA release inside endosomes. To study the molecular determinants of viral resistance to acid-induced disassembly, six FMDV variants with increased resistance to acid inactivation were isolated. Infection by these mutants was more sensitive to drugs that raise the endosomal pH (NH(4)Cl and concanamycin A) than was infection by the parental C-S8c1 virus, confirming that the increase in acid resistance is related to a lower pH requirement for productive uncoating. Amino acid replacement N17D at the N terminus of VP1 capsid protein was found in all six mutants. This single substitution was shown to be responsible for increased acid resistance when introduced into an infectious FMDV clone. The increased resistance of this mutant against acid-induced inactivation was shown to be due to its increased resistance against capsid dissociation into pentameric subunits. Interestingly, the N17D mutation was located close to but not at the interpentamer interfaces. The mutants described here extend the panel of FMDV variants exhibiting different pH sensitivities and illustrate the adaptive flexibility of viral quasispecies to pH variations.     

A single amino acid change in the plant alternative oxidase alters the specificity of organic acid activation.

The alternative oxidase is a quinol oxidase of the respiratory chain of plants and some fungi and protists. Its activity is regulated by redox-sensitive disulphide bond formation between neighbouring subunits and direct interaction with certain alpha-ketoacids. To investigate these regulatory mechanisms, we undertook site-directed mutagenesis of soybean and Arabidopsis alternative oxidase cDNAs, and expressed them in tobacco plants and Escherichia coli, respectively. The homologous C99 and C127 residues of GmAOX3 and AtAOX1a, respectively, were changed to serine. In the plant system, this substitution prevented oxidative inactivation of alternative oxidase and rendered the protein insensitive to pyruvate activation, in agreement with the recent results from other laboratories [Rhoads et al. (1998) J. Biol. Chem. 273, 30750-30756; Vanlerberghe et al. (1998) Plant Cell 10, 1551-1560]. However, the mutated protein is instead activated specifically by succinate. Measurements of AtAOX1a activity in bacterial membranes lacking succinate dehydrogenase confirmed that the stimulation of the mutant protein's activity by succinate did not involve its metabolism. Examples of alternative oxidase proteins with the C to S substitution occur in nature and these oxidases are expected to be activated under most conditions in vivo, with implications for the efficiency of respiration in the tissues which express them.     

Study of the specificity of amino acid binding protein fromSaccharomyces cerevisiae membranes

Membranes obtained from exponentially growing yeast were extracted with buffered solution of 1% Tween 80 plus 0.1m MgCl₂. The soluble proteins, saparated from detergent and fine membrane particles on Saphadex G-100 colunn, were fond, to have binding affinity for Val, Lau, Ileu, Asp, Lys, His, Arg, Phe and Met. The occurrence of individual binding activities in different preparations was irregular. This and the fast inactivation by aging showed the extrame sensitivity of the molecules of binding proteins towards deleterious agents. Saturation of the binding proteins with aspartic acid and phenylalanine was of an identical biphasic shape indicating the change ofK _diss and suggesting the existence of several binding sites for each amino acid. In our opinion the above listed observations imply low specificity of amino acid binding proteins in yeast.     

A single residue in leucyl-tRNA synthetase affecting amino acid specificity and tRNA aminoacylation

Human mitochondrial leucyl-tRNA synthetase (hs mt LeuRS) achieves high aminoacylation fidelity without a functional editing active site, representing a rare example of a class I aminoacyl-tRNA synthetase (aaRS) that does not proofread its products. Previous studies demonstrated that the enzyme achieves high selectivity by using a more specific synthetic active site that is not prone to errors under physiological conditions. Interestingly, the synthetic active site of hs mt LeuRS displays a high degree of homology with prokaryotic, lower eukaryotic, and other mitochondrial LeuRSs that are less specific. However, there is one residue that differs between hs mt and Escherichia coli LeuRSs located on a flexible closing loop near the signature KMSKS motif. Here we describe studies indicating that this particular residue (K600 in hs mt LeuRS and L570 in E. coli LeuRS) strongly impacts aminoacylation in two ways: it affects both amino acid discrimination and transfer RNA (tRNA) binding. While this residue may not be in direct contact with the amino acid or tRNA substrate, substitutions of this position in both enzymes lead to altered catalytic efficiency and perturbations to the discrimination of leucine and isoleucine. In addition, tRNA recognition and aminoacylation is affected. These findings indicate that the conformation of the synthetic active site, modulated by this residue, may be coupled to specificity and provide new insights into the origins of selectivity without editing.     

A single amino acid limits the substrate specificity of Thermus thermophilus uridine-cytidine kinase to cytidine

The salvage pathways of nucleotide biosynthesis are more diverse and are less well understood as compared with de novo pathways. Uridine-cytidine kinase (UCK) is the rate-limiting enzyme in the pyrimidine-nucleotide salvage pathway. In this study, we have characterized a UCK homologue of Thermus thermophilus HB8 (ttCK) biochemically and structurally. Unlike other UCKs, ttCK had substrate specificity toward only cytidine and showed no inhibition by UTP, suggesting uridine does not bind to ttCK as substrate. Structural analysis revealed that the histidine residue located near the functional group at position 4 of cytidine or uridine in most UCKs is substituted with tyrosine, Tyr93, in ttCK. Replacement of Tyr93 by histidine or glutamine endowed ttCK with phosphorylation activity toward uridine. These results suggested that a single amino acid residue, Tyr93, gives cytidine-limited specificity to ttCK. However, replacement of Tyr93 by Phe or Leu did not change the substrate specificity of ttCK. Therefore, we conclude that a residue at this position is essential for the recognition of uridine by UCK. In addition, thymidine phosphorylase from T. thermophilus HB8 was equally active with thymidine and uridine, which indicates that this protein is the sole enzyme metabolizing uridine in T. Thermophilus HB8. On the basis of these results, we discuss the pyrimidine-salvage pathway in T. thermophilus HB8.     

A few amino acid substitutions can convert deoxyribonucleoside kinase specificity from pyrimidines to purines

In mammals, the four native deoxyribonucleosides are phosphorylated to the corresponding monophosphates by four deoxyribonucleoside kinases, which have specialized substrate specificities. These four enzymes are likely to originate from a common progenitor kinase. Insects appear to have only one multisubstrate deoxyribonucleoside kinase (dNK, EC 2.7.1.145), which prefers pyrimidine nucleosides, but can also phosphorylate purine substrates. When the structures of the human deoxyguanosine kinase (dGK, EC 2.7.1.113) and the dNK from Drosophila melanogaster were compared, a limited number of amino acid residues were identified and proposed to be responsible for the substrate specificity. Three of these key residues in Drosophila dNK were then mutagenized and the mutant enzymes were characterized regarding their ability to phosphorylate native deoxyribonucleosides and nucleoside analogs. The mutations converted the dNK substrate specificity from predominantly pyrimidine specific into purine specific. A similar scenario could have been followed during the evolution of kinases. Upon gene duplication of the progenitor kinase, only a limited number of single amino acid changes has taken place in each copy and resulted in substrate-specialized enzymes.