Invariant Acidic Residues Research Articles

Investigation of Ionization Pattern of the Adjacent Acidic Residues in the DXDXE Motif of GH-18 Chitinases Using Theoretical pKa Calculations.

GH-18 chitinases are chitinolytic enzymes, primarily responsible for the recycling of insoluble chitin biomaterials. These enzymes contain three invariant acidic active-site residues within a DXDXE motif, which play a synergistic role in the catalytic cycle of chitin degradation. We employed a pKa calculation approach to approximate the protonation states of residues D1, D2, and E in the DXDXE motif of 75 GH-18 chitinases. Theoretical pH-activity profiles of these enzymes were subsequently constructed and compared with the experimentally determined pH-activity profiles. Theoretical pKa data indicate that in the majority of chitinases the D1 side-chain is in the "up" and the E side-chain in the "down" position, while the position of the D2 side-chain is versatile and depends on the state of the enzyme. The pKa values in 75 GH-18 chitinases were predicted to be <0 for D1, 8-13 for D2, and 6-9 for E, indicating that the D1-D2 pair holds exactly one net negative charge. On the other hand, the catalytic acid E is protonated over the active pH-range, agreeing with the pH-activity curves reported previously for most chitinases. The results obtained from this study help to elucidate the mechanistic details of the concerted participation of D1, D2, and E in the catalytic cycle of chitin hydrolysis by GH-18 chitinases.

Functional role of conserved residues in the characteristic secretion NTPase motifs of the Pseudomonas aeruginosa type IV pilus motor proteins PilB, PilT and PilU

Type IV pili are retractable protein fibres used by many bacterial pathogens for adherence, twitching motility, biofilm development and host colonization. In Pseudomonas aeruginosa, PilB and PilT are bipolar proteins belonging to the secretion NTPase superfamily, and power pilus extension and retraction, respectively, while the unipolar PilT paralogue PilU supports pilus retraction in an unknown manner. Assay of purified 6xHis-tagged PilB, PilT and PilU from P. aeruginosa showed that all three proteins have ATPase activities in vitro. Conserved residues in the Walker A (WA), Walker B (WB), Asp Box and His Box motifs characteristic of secretion NTPases were mutated, and complementation of twitching motility was tested. Mutation of conserved WA or WB residues in any of the three ATPases abrogated twitching motility, and for the WA mutant of PilT caused loss of polar localization. The requirement for three invariant acidic residues in the Asp Box motif, and for two invariant His residues in the His Box motif varied, with PilB being the least tolerant of changes. In all three proteins, the third acidic residue in the Asp Box and the second His of the His Box were crucial for function; mutation of these residues caused loss of PilT ATPase activity in vitro. Modelling of the effects of these mutations on the crystal structures of Aquifex aeolicus PilT and Vibrio cholerae EpsE (a PilB homologue) showed that the critical Asp Box and His Box residues contribute to a catalytic pocket that surrounds the ligand. These results provide experimental evidence differentiating widely conserved Asp and His Box residues that are essential for function from those whose roles are modulated by specific local environments.

Crystal Structure of the Translocation ATPase SecA from Thermus thermophilus Reveals a Parallel, Head-to-Head Dimer

The mechanism of pre-protein export through the bacterial cytoplasmic membrane, in which the SecA ATPase plays a crucial role as an “energy supplier”, is poorly understood. In particular, biochemical and structural studies provide contradictory data as to the oligomeric state of SecA when it is integrated into the active trans-membrane translocase. Here, we report the 2.8 Å resolution crystal structure of the Thermus thermophilus SecA protein (TtSecA). Whereas the structure of the TtSecA monomer closely resembles that from other bacteria, the oligomeric state of TtSecA is strikingly distinct. In contrast to the antiparallel (head-to-tail) dimerization reported previously for the other bacterial systems, TtSecA forms parallel (head-to-head) dimers that are reminiscent of open scissors. The dimer interface is abundant in bulky Arg and Lys side-chains from both subunits, which stack on one another to form an unusual “basic zipper” that is highly conserved, as revealed by homology modeling and sequence analysis. The basic zipper is sealed on both ends by two pairs of the salt bridges formed between the basic side-chains from the zipper and two invariant acidic residues. The organization of the dimers, in which the two pre-protein binding domains are located proximal to each other at the tip of the “scissors”, might allow a concerted mode of substrate recognition while the opening/closing of the scissors might facilitate translocation.

Regulation through the RNA Polymerase Secondary Channel: STRUCTURAL AND FUNCTIONAL VARIABILITY OF THE COILED-COIL TRANSCRIPTION FACTORS

Gre factors enhance the intrinsic endonucleolytic activity of RNA polymerase to rescue arrested transcription complexes and are thought to confer the high fidelity and processivity of RNA synthesis. The Gre factors insert the extended alpha-helical coiled-coil domains into the RNA polymerase secondary channel to position two invariant acidic residues at the coiled-coil tip near the active site to stabilize the catalytic metal ion. Gfh1, a GreA homolog from Thermus thermophilus, inhibits rather than activates RNA cleavage. Here we report the structure of the T. thermophilus Gfh1 at 2.4 A resolution revealing a two-domain architecture closely resembling that of GreA. However, the interdomain orientation is strikingly distinct (approximately 162 degrees rotation) between the two proteins. In contrast to GreA, which has two acidic residues on a well fixed self-stabilized alpha-turn, the tip of the Gfh1 coiled-coil is flexible and contains four acidic residues. This difference is likely the key to the Gre functional diversity, while Gfh1 inhibits exo- and endonucleolytic cleavage, RNA synthesis, and pyrophosphorolysis, GreA enhances only the endonucleolytic cleavage. We propose that Gfh1 acidic residues stabilize the RNA polymerase active center in a catalytically inactive configuration through Mg2+-mediated interactions. The excess of the acidic residues and inherent flexibility of the coiled-coil tip might allow Gfh1 to adjust its activity to structurally distinct substrates, thereby inhibiting diverse catalytic reactions of RNA polymerase.

Kinetic and Mechanistic Studies of a Cell Cycle Protein Phosphatase Cdc14

The Cdc14 family of protein phosphatases is conserved within eukaryotes and antagonizes the action of cyclin-dependent kinases, thereby promoting mitotic exit and cytokinesis. We performed a detailed kinetic and mechanistic study of the Cdc14 phosphatases with both small molecule aryl phosphates and a physiological protein substrate hCdh1. We found that Cdc14 displays a strong preference for two-ringed aryl phosphates over smaller one-ringed or larger, multi-ringed substrates, a finding that may have important implications for inhibitor design. Results from both leaving group and pH dependence of the Cdc14-catalyzed reaction are consistent with a general acid-independent mechanism for substrates with leaving group pKa < 7 and a general acid-dependent mechanism for substrates with leaving group pKa > 7. The use of both low and high leaving group pKa substrates, in combination with steady-state and pre-steady-state kinetic techniques enabled the isolation and analysis of both the phosphoenzyme (E-P) formation and hydrolysis step. We established the requirement of general acid catalysis for E-P formation in reactions with high leaving group pKa substrates, and the presence of general base catalysis in E-P hydrolysis. Mutational study of invariant acidic residues in Cdc14 identified Asp253 as the general acid during E-P formation and the general base in E-P hydrolysis. We also identified several residues including Asp50, Asp129, Glu168, Glu171, and Asp177 in the Cdc14 active site cleft that are required for efficient dephosphorylation of hCdh1.

Architecture of the RNA Polymerase II-TFIIS Complex and Implications for mRNA Cleavage

The transcription elongation factor TFIIS induces mRNA cleavage by enhancing the intrinsic nuclease activity of RNA polymerase (Pol) II. We have diffused TFIIS into Pol II crystals and derived a model of the Pol II-TFIIS complex from X-ray diffraction data to 3.8 Å resolution. TFIIS extends from the polymerase surface via a pore to the internal active site, spanning a distance of 100 Å. Two essential and invariant acidic residues in a TFIIS loop complement the Pol II active site and could position a metal ion and a water molecule for hydrolytic RNA cleavage. TFIIS also induces extensive structural changes in Pol II that would realign nucleic acids in the active center. Our results support the idea that Pol II contains a single tunable active site for RNA polymerization and cleavage, in contrast to DNA polymerases with two separate active sites for DNA polymerization and cleavage.

Mechanism of Action of RNase T : I. IDENTIFICATION OF RESIDUES REQUIRED FOR CATALYSIS, SUBSTRATE BINDING, AND DIMERIZATION

Escherichia coli RNase T, an RNA-processing enzyme and a member of the DEDD exonuclease superfamily, was examined using sequence analysis and site-directed mutagenesis. Like other DEDD exonucleases, RNase T was found to contain three conserved Exo motifs that included four invariant acidic residues. Mutagenesis of these motifs revealed that they are essential for RNase T activity, indicating that they probably form the RNase T catalytic center in a manner similar to that found in other DEDD exonucleases. We also identified by sequence analysis three short, but highly conserved, sequence segments rich in positively charged residues. Site-directed mutagenesis of these regions indicated that they are involved in substrate binding. Additional analysis revealed that residues within the C-terminal region of RNase T are essential for RNase T dimerization and, consequently, for RNase T activity. These data define the domains necessary for RNase T action, and together with information in the accompanying article, have led to the formulation of a detailed model for the structure and mechanism of action of RNase T.

Structure and Mechanism of E. coli Rnase.

INTRODUCTION. RNase T is one of eight distinct 3’ to 5’ exoribonucleases identified in Escherichia coli[1]. It belongs to the DEDD exonuclease superfamily[2] characterized by common motifs consisting of four invariant acidic residues, which in DNA polymerases were shown to form the exonuclease active site[3]. RNase T plays an important role in stable RNA metabolism, including tRNA end turnover and 3’ maturation of many stable RNAs[1]. RNase T proteins are small polypeptides of about 220 amino acids. They are closely related to the proofreading domains/subunits of bacterial DNA polymerase III, and interestingly, E. coli RNase T also displays strong DNA exonuclease activity[4,5]. E. coli RNase T must form a homodimer in order to function[1].

Crystallographic Studies of Bacterial Exoribonucleases

INTRODUCTION. Ribonucleases (RNases) play a central role in all cellular RNA processes. These processes include mRNA degradation, and maturation and turnover of stable RNAs, which are vital for the proper functioning of all cells. E. coli has served as a model system for understanding the role of ribonucleases in RNA metabolism, and eight distinct exoribonucleases have been identified in this bacterium. Of these, three (RNase T, RNase D, and oligoribonuclease) are members of a larger exonuclease superfamily (named the DEDD exonuclease family, after the four invariant acidic residues in these proteins) that includes the proof-reading domains of DNA polymerases[1].

3-Hydroxy-3-methylglutaryl-CoA Synthase: Participation of Invariant Acidic Residues in Formation of the Acetyl-S-Enzyme Reaction Intermediate‡

Inactivation of HMG-CoA synthase by a carboxyl-directed reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), in a concentration-dependent and substrate-protectable manner suggested that the active site contains reactive acidic amino acids. This observation prompted functional evaluation of 11 invariant acidic amino acids by site-directed mutagenesis. Characterization of the isolated synthase variants' ability to catalyze overall and partial reactions identified three mutant synthases (D99A, D159A, and D203A) that exhibit significant diminution of k(cat) for the overall reaction (10(2)-, 10(3)-, and 10(4)-fold decreases, respectively). D99A, D159A, and D203A form the acetyl-S-enzyme intermediate very slowly (0.0025, 0.0026, 0.0015 U/mg, respectively, measured at pH 7. 0 and 22 degrees C) as compared to the wild-type synthase (1.59 U/mg), where intermediate formation approaches rate-limiting status. Differences in substrate saturation do not account for impaired activities or rates of intermediate formation. The structural integrity of the purified mutants' active sites is demonstrated by their abilities to bind a spin-labeled acyl-CoA analogue (R.CoA) with affinities and stoichiometries comparable to values measured for wild-type synthase. The impact of three distinct amino acids on reaction intermediate formation supports a mechanism of acetyl-S-enzyme formation that probably requires formation and directed collapse of a tetrahedral adduct. (18)O-induced shift of the (13)C NMR signal of (13)C acetyl-S-enzyme demonstrates that an analogous tetrahedral species is produced upon solvent exchange with the acetyl-S-enzyme. Partial discrimination between the functions of D99, D159, and D203 becomes possible based on the observation that D159A and D203A synthases exhibit retarded kinetics of solvent (18)O exchange while D99A fails to support (18)O exchange.

Identification of three aspartic acid residues essential for catalysis by the RusA holliday junction resolvase

RusA is a Holliday junction resolvase encoded by the cryptic prophage DLP12 of Escherichia coli K-12 that can be activated to promote homologous recombination and DNA repair in resolution-deficient mutants lacking the RuvABC proteins. Database searches with the 120 amino acid residue RusA sequence identified 11 homologues from diverse species, including one from the extreme thermophile Aquifex aeolicus, which suggests that RusA may be of ancient bacterial ancestry. A multiple alignment of these sequences revealed seven conserved or invariant acidic residues in the C-terminal half of the E. coli protein. By making site-directed mutations at these positions and analysing the ability of the mutant proteins to promote DNA repair in vivo and to resolve junctions in vitro, we identified three aspartic acid residues (D70, D72 and D91) that are essential for catalysis and that provide the first insight into the active-site mechanism of junction resolution by RusA. Substitution of any one of these three residues with asparagine reduces resolution activity >80-fold. The mutant proteins retain the ability to bind junction DNA regardless of the DNA sequence or of the mobility of the crossover. They interfere with the function of the RuvABC proteins in vivo, when expressed from a multicopy plasmid, an effect that is reproducible in vitro and that reflects the fact that the RusA proteins have a higher affinity for junction DNA in the presence of Mg2+ than do the RuvA and RuvC proteins. The D70N protein has a greater affinity for junctions in Mg2+ than does the wild-type, which indicates that the negatively charged carboxyl group of the aspartate residue plays a critical role at the active site of RusA. Electrostatic repulsions between D70, D72 and D91 may help to form a classical Mg2+-binding pocket.

Calcium binding to the class I alpha-1,2-mannosidase from Saccharomyces cerevisiae occurs outside the EF hand motif.

Class I alpha-1,2-mannosidases are a family of Ca2+-dependent enzymes that have been conserved through eukaryotic evolution. These enzymes contain a conserved putative EF hand Ca2+-binding motif and nine invariant acidic residues. The catalytic domain of the alpha-1, 2-mannosidase from Saccharomyces cerevisiae was expressed in Pichia pastoris and was shown by atomic absorption and equilibrium dialysis to bind one Ca2+ ion with high affinity (KD = 4 x 10(-)7 M). Ca2+ protected the enzyme from thermal denaturation. Mutation of the 1st and 12th residues of the putative EF hand Ca2+ binding loop (D121N, D121A, E132Q, E132V, and D121A/E132V) had no effect on Ca2+ binding, demonstrating that the EF hand motif is not the site of Ca2+ binding. In contrast, three invariant acidic residue mutants (D275N, E279Q, and E438Q) lost the ability to bind 45Ca2+ following nondenaturing polyacrylamide gel electrophoresis whereas D86N, E132Q, E503Q, and E526Q mutants exhibited binding of 45Ca2+ similar to the wild-type enzyme. The wild-type enzyme had a Km and kcat of 0.5 mM and 12 s-1, respectively. The Km of E526Q was greatly increased to 4 mM with a small reduction in kcat to 5 s-1 whereas the kcat values of D86N and E132Q(V) were greatly reduced (0.005-0.007 s-1) with a decrease in Km (0.07-0.3 mM). The E503Q mutant is completely inactive. Asp275, Glu279, and Glu438 are therefore required for Ca2+ binding whereas Asp86, Glu132, and Glu503 are required for catalysis.

Deoxy- and dideoxynucleotide discrimination and identification of critical 5' nuclease domain residues of the DNA polymerase I from Mycobacterium tuberculosis.

The DNA polymerase I (PolI) from Mycobacterium tuberculosis (Mtb) was overproduced in Escherichia coli as an enzymatically active, recombinant protein with or without an N-terminal His-tag. The proteins catalysed both the DNA polymerisation of homo- and heteropolymer template-primers and the 5'-3' exonucleolytic hydrolysis of gapped and nicked substrates but lacked an associated proofreading activity. In accordance with recent predictions [Tabor, S. and Richardson, C.C. (1995) Proc. Natl. Acad. Sci. USA, 92, 6339-6343], both recombinant forms of the M. tuberculosis enzyme were unable to discriminate against dideoxynucleotide 5'-triphosphates and were thus efficiently inhibited by these chain-terminating nucleotide analogues during DNA synthesis. This unusual property might be potentially exploitable in terms of novel anti-mycobacterial drug design. A mutational analysis of 5' nuclease domain residues allowed the roles of nine invariant acidic residues to be evaluated. Acidic side chain neutralisation resulted in a > or = 20-fold reduction in activity, with the most profound reduction (> or = 10(4)-fold) being caused by neutralisation of the Asp125, Asp148 and Asp150 residues.

タンパク質結晶学と構造生物学 新しいタンパク質立体構造 ニワトリ卵黄膜由来の蛋白質ＶＭＯ‐Ｉの結晶構造解析

The crystal structure of vitelline membrane outer layer protein I (VMO-I), which was isolated from the outer layer of the vitelline membrane of hen's eggs, has been determined by X-ray analysis. VMO-I is composed of three homologous structures, each containing a β-sheet forming Greek key motif, which are in accordance with the three repeats in the sequence. These three homologous structures are related by a pseudo three-fold symmetry. This new folding motif, recently designated as the β-prism, has also been observed in the second domain of δ-endotoxin. Thus, VMO-I and δ-endotoxin constitute a new family with a novel folding motif. The VMO-I molecule has a groove-like cavity. This region contains invariant acidic residues in the three repeats. VMO-I is assumed to be an oligosaccharide binding protein like lysozyme, although details of its catalytic function remain to be solved. A docking model of an oligosaccharide to VMO-I has been constructed. This model shows that the cavity has a sufficient space to bind roughly pentamer saccharides. The structural features strongly suggest that the negatively-charged cavity in the top region of the protein may be involved in an unknown catalytic reaction.

Identification of two acidic residues involved in the catalysis of xylanase A from Streptomyces lividans

On the basis of similarities between known xylanase sequences of the F family, three invariant acidic residues of xylanase A from Streptomyces lividans were investigated. Site-directed-mutagenesis experiments were carried out in Escherichia coli after engineering the xylanase A gene to allow its expression. Replacement of Glu-128 or Glu-236 by their isosteric form (Gln) completely abolished enzyme activity with xylan and p-nitrophenyl beta-D-cellobioside, indicating that the two substrates are hydrolysed at the same site. These two amino acids probably represent the catalytic residues. Immunological studies, which showed that the two mutants retained the same epitopes, indicate that the lack of activity is the result of the mutation rather than misfolding of the protein. Mutation D124E did not affect the kinetic parameters with xylan as substrate, but D124N reduced the Km 16-fold and the Vmax. 14-fold when compared with the wild-type enzyme. The mutations had a more pronounced effect with p-nitrophenyl beta-D-cellobioside as the substrate. Mutation D124E increased the Km and decreased the Vmax. 5-fold each, while D124N reduced the Km 4.5-fold and the Vmax. 75-fold. The mutations had no effect on the cleavage mode of xylopentaose.

Domains of the integrase protein of human immunodeficiency virus type 1 responsible for polynucleotidyl transfer and zinc binding.

The integrase protein of human immunodeficiency virus type 1 carries out a set of polynucleotidyl transfer reactions that result in the covalent attachment of the retroviral cDNA to host DNA. We have analyzed the activities of a set of deletion derivatives of the integrase protein. The analysis reveals that a central domain of only 137 amino acids is sufficient in vitro to catalyze a subset of the reactions carried out by the complete protein. This polypeptide contains an amino acid sequence motif, Asp-Xaa39-58-Asp-Xaa35-Glu (DX39-58DX35E, where X and the subscript indicate the intervening amino acids between the invariant acidic residues), that is found in the integrases of retroviruses and retrotransposons and also the transposase proteins of some bacterial transposable elements. We also find that the integrase protein can bind Zn2+, and the histidine and cysteine residues of another conserved motif (HX3-7HX23-32CX2C) are required for efficient Zn2+ binding. The activities displayed by deletion mutants suggest to us possible functions for the various parts of integrase.

Structural details of ribonuclease H from Escherichia coli as refined to an atomic resolution

The crystal structure of RNase H from Escherichia coli has been determined by the multiple isomorphous replacement method, and refined by the stereochemically restrained leastsquares procedure to a crystallographic R-factor of 0.196 at 1.48 Å resolution. In the final structure, the root-mean-square (r.m.s.) deviation for bond lengths is 0.017 Å, and for angle distances 0.036 Å. The structure is composed of a five-stranded β-sheet and five α-helices, and reveals the details of hydrogen bonding, electrostatic and hydrophobic interactions between intra- and intermolecular residues. The refined structure allows an explanation of the particular interactions between the basic protrusion, consisting of helix αIII and the following loop, and the remaining major domain. The β-sheet, αII, αIII and αIV form a central hydrophobic cleft that contains all six tryptophan residues, and presumably serves to fix the orientation of the basic protrusion. Two parallel adjacent helices, αI and αIV, are associated with a few triads of hydrophobic interactions, including many leucine residues, that are similar to the repeated leucine motif. The well-defined electron density map allows detailed discussion of amino acid residues likely to be involved in binding a DNA/RNA hybrid, and construction of a putative model of the enzyme complexed with a DNA/RNA hybrid oligomer. In this model, a protein region, from the Mg 2+-binding site to the basic protrusion, covers roughly two turns of a DNA/RNA hybrid double helix. A segment (11–23) containing six glycine residues forms a long loop between the βA and βB strands. This loop, which protrudes into the solvent region, lies on the interface between the enzyme and a DNA/RNA hybrid in the model of the complex. The mean temperature factors of main-chain atoms show remarkably high values in helix αIII that constitutes the basic protrusion, suggesting some correlation between its flexibility and the nucleic acid binding function. The Mg 2+-binding site, surrounded by four invariant acidic residues, can now be described more precisely in conjunction with the catalytic activity. The arrangement of molecules within the crystal appears to be dominated by the cancelling out of a remarkably biased charge distribution on the molecular surface, which is derived in particular from the separation between the acidic Mg 2+-binding site and the basic protrusion.

The gene encoding cytochrome c oxidase subunit II from Rhodobacter sphaeroides; comparison of the deduced amino acid sequence with sequences of corresponding peptides from other species

The gene ( coxII) encoding subunit II of Rhodobacter sphaeroides cytochrome c oxidase (cytochrome aa 3) has been isolated by screening a genomic DNA library in phage λ with a probe derived from coxII of Paracoccus denitrificans. A 2-kb fragment containing coxII DNA was subcloned into the phage M13mp18 and the sequence determined. The 2-kb insert contains the entire coding region for coxII gene, including the ATG start codon and a TGA stop codon. The deduced amino acid (aa) sequence of subunit II of R. sphaeroides shows regions of substantial homology to the corresponding subunit of the bovine mitochondrial oxidase (63 % overall) and P. denitrificans oxidase (68 % overall). The postulated redox-active copper ion (Cu A binding site involving two Cys and two His residues (as well as an alternative Met residue) is conserved among these species, along with four invariant acidic aa residues (two Asp and two Glu) that may be involved in interactions with cytochrome c, and a region of aromatic residues (Tyr-Gln-Trp-Tyr-Trp-Gly-Tyr-Glu-Tyr) which is postulated to play a role in electron transfer. Hydropathy profile analysis suggests that while the bovine COXII secondary structure contains two transmembrane helices, the R. sphaeroides subunit II has a third such helix that may function as part of a signal sequence, as suggested for P. denitrificans.