Ammonia Channeling in Plasmodium falciparum GMP Synthetase: Investigation by NMR Spectroscopy and Biochemical Assays

GMP synthetase, a class I amidotransferase, catalyzes the last step of the purine biosynthetic pathway, where ammonia from glutamine is incorporated into xanthosine 5'-monophospate to yield guanosine 5'-monnophosphate as the main product. Combined biochemical, structural, and computational studies of glutamine amidotransferases have revealed the existence of physically separate active sites connected by molecular tunnels that efficiently transfer ammonia from the glutaminase site to the synthetase site. Here, we have investigated aspects of ammonia channeling in P. falciparum GMP synthetase using biochemical assays in conjunction with N-15-edited proton NMR spectroscopy. Our results suggest that (1) ammonia released from glutamine is not equilibrated with the external medium (2) saturating concentrations of glutamine do not obliterate the incorporation of external ammonia into GMP, and (3) ammonia in the external medium can access the thioester intermediate when the ATPPase domain is bound to substrates. Further, mutation of Cys-102 to alanine confirmed its identity as the catalytic residue in the glutaminase domain, and ammonia-dependent assays on the mutant indicated glutamine to be a partial uncompetitive inhibitor of the enzyme.

DNA recognition by the EcoP15I and EcoPI modification methyltransferases

The DNA-binding properties of the EcoP15I DNA methyltransferase (M . EcoP15I; MTase) were studied using electrophoretic mobility shift assays. We show by molecular size-exclusion chromatography and dimethyl suberimidate crosslinking that M . EcoP15I is a dimer in solution. While M . EcoP15I binds approx. threefold more tightly to its recognition sequence, 5'-CAGCAG-3', than to non-specific sequences in the presence of AdoMet or its analogs, the discrimination between specific and non-specific sequences significantly increases in presence of ATP. These results suggest for the first time a role for ATP in DNA recognition by type-III restriction-modification enzymes. Furthermore, we show that although c2 EcoPI mutant MTases are defective in AdoMet binding, they are still able to bind DNA in a sequence-specific manner.

Probing the role of the fully conserved Cys126 in triosephosphate isomerase by site-specific mutagenesis - distal effects on dimer stability

Cys126 is a completely conserved residue in triosephosphate isomerase that is proximal to the active site but has been ascribed no specific role in catalysis. A previous study of the C126S and C126A mutants of yeast TIM reported substantial catalytic activity for the mutant enzymes, leading to the suggestion that this residue is implicated in folding and stability [Gonzalez-Mondragon E et al. (2004) Biochemistry43, 3255–3263]. We re-examined the role of Cys126 with the Plasmodium falciparum enzyme as a model. Five mutants, C126S, C126A, C126V, C126M, and C126T, were characterized. Crystal structures of the 3-phosphoglycolate-bound C126S mutant and the unliganded forms of the C126S and C126A mutants were determined at a resolution of 1.7–2.1 Å. Kinetic studies revealed an approximately five-fold drop in kcat for the C126S and C126A mutants, whereas an approximately 10-fold drop was observed for the other three mutants. At ambient temperature, the wild-type enzyme and all five mutants showed no concentration dependence of activity. At higher temperatures (> 40 °C), the mutants showed a significant concentration dependence, with a dramatic loss in activity below 15 μm. The mutants also had diminished thermal stability at low concentration, as monitored by far-UV CD. These results suggest that Cys126 contributes to the stability of the dimer interface through a network of interactions involving His95, Glu97, and Arg98, which form direct contacts across the dimer interface.

Mutations in beta ` subunit of Escherichia coli RNA polymerase perturb the activator polymerase functional interaction required for promoter clearance

P>Transcription activator C employs a unique mechanism to activate mom gene of bacteriophage Mu. The activation process involves, facilitating the recruitment of RNA polymerase (RNAP) by altering the topology of the promoter and enhancing the promoter clearance by reducing the abortive transcription. To understand the basis of this multi-step activation mechanism, we investigated the nature of the physical interaction between C and RNAP during the process. A variety of assays revealed that only DNA-bound C contacts the beta' subunit of RNAP. Consistent to these results, we have also isolated RNAP mutants having mutations in the beta' subunit which were compromised in C-mediated activation. Mutant RNAPs show reduced productive transcription and increased abortive initiation specifically at the C-dependent mom promoter. Positive control (pc) mutants of C, defective in interaction with RNAP, retained the property of recruiting RNAP to the promoter but were unable to enhance promoter clearance. These results strongly suggest that the recruitment of RNAP to the mom promoter does not require physical interaction with C, whereas a contact between the beta' subunit and the activator, and the subsequent allosteric changes in the active site of the enzyme are essential for the enhancement of promoter clearance.

Mutations in the Res subunit of the EcoPI restriction enzyme that affect ATP-dependent reactions

The Res subunits of the type III restriction-modification enzymes share a statistically significant amino acid sequence similarity with several RNA and DNA helicases of the so-called DEAD family. It was postulated that in type III restriction enzymes a DNA helicase activity may be required for local unwinding at the cleavage site. The members of this family share seven conserved motifs, all of which are found in the Res subunit of the type III restriction enzymes. To determine the contribution, if any, of these motifs in DNA cleavage by EcoPI, a type III restriction enzyme, we have made changes in motifs I and II. While mutations in motif I (GTGKT) clearly affected ATP hydrolysis and resulted in loss of DNA cleavage activity, mutation in motif II (DEPH) significantly decreased ATP hydrolysis but had no effect on DNA cleavage. The double mutant R.EcoPIK90R-H229K showed no significant ATPase or DNA restriction activity though ATP binding was not affected. These results imply that there are at least two ATPase reaction centres in EcoPI restriction enzyme. Motif I appears to be involved in coupling DNA restriction to ATP hydrolysis. Our results indicate that EcoPI restriction enzyme does not have a strand separation activity. We suggest that these motifs play a role in the ATP-dependent translocation that has been proposed to occur in the type III restriction enzymes. (C) 1997 Academic Press Limited.

The role of lysine-256 in the structure and function of sheep liver recombinant serine hydroxymethyltransferase

The active site lysine residue, K256, involved in Schiffs base linkage with pyridoxal-5'-phosphate (PEP) in sheep liver recombinant serine hydroxymethyltransferase (rSHMT) was changed to glutamine or arginine by site-directed mutagenesis. The purified K256Q and K256R SHMTs had less than 0.1% of catalytic activity with serine and H(4)folate as substrates compared to rSHMT. The mutant enzymes also failed to exhibit the characteristic visible absorbance spectrum (lambda(max) 425 nm) and did not produce the quinonoid intermediate (lambda(max) 495 nm) upon the addition of glycine and H(4)folate. The mutant enzymes were unable to catalyze aldol cleavage of beta-phenylserine and transamination of D-alanine. These results suggested that the mutation of the lysine had resulted in the inability of the enzyme to bind to the cofactor. Therefore, the K256Q SHMT was isolated as a dimer and the K256R SHMT as a mixture of dimers and tetramers which were converted to dimers slowly. On the other hand, rSHMT was stable as a tetramer for several months, further confirming the role of PLP in maintenance of oligomeric structure. The mutant enzymes also failed to exhibit the increased thermal stability upon the addition of serine, normally observed with rSHMT. The enhanced thermal stability has been attributed to a change in conformation of the enzyme from open to closed form leading to reaction specificity. The mutant enzymes were unable to undergo this conformational change probably because of the absence of bound cofactor.

A gradient PCR-based screen for use in site-directed mutagenesis

Site-directed mutagenesis is widely used to study protein and nucleic acid structure and function. Despite recent advancements in the efficiency of procedures for site-directed mutagenesis, the fraction of site-directed mutants by most procedures rarely exceeds 50% on a routine basis and is never 100%. Hence it is typically necessary to sequence two or three clones each time a site-directed mutant is constructed. We describe a simple and robust gradient-PCR-based screen for distinguishing site-directed mutants from the starting, unmutated plasmid. The procedure can use either purified plasmid DNA or colony PCR, starting from a single colony. The screen utilizes the primer used for mutagenesis and a common outside primer that can be used for all other mutants constructed with the same template. Over 30 site-specific mutants in a variety of templates were successfully screened and all of the mutations detected were subsequently confirmed by DNA sequencing. A single base pair mismatch could be detected in an oligonucleotide of 36 bases. Detection efficiency was relatively independent of starting template concentration and the nature of the outside primer used. (C) 2003 Elsevier Science (USA). All rights reserved.

Autonomous and non-autonomous traits mediate social cooperation in Dictyostelium discoideum

In the trishanku (triA(-)) mutant of the social amoeba Dictyostelium discoideum, aggregates are smaller than usual and the spore mass is located mid-way up the stalk, not at the apex. We have monitored aggregate territory size, spore allocation and fruiting body morphology in chimaeric groups of (quasi-wild-type) Ax2 and triA(-) cells. Developmental canalisation breaks down in chimaeras and leads to an increase in phenotypic variation. A minority of triA(-) cells causes largely Ax2 aggregation streams to break up; the effect is not due to the counting factor. Most chimaeric fruiting bodies resemble those of Ax2 or triA(-). Others are double-deckers with a single stalk and two spore masses, one each at the terminus and midway along the stalk. The relative number of spores belonging to the two genotypes depends both on the mixing ratio and on the fruiting body morphology. In double-deckers formed from 1:1 chimaeras, the upper spore mass has more Ax2 spores, and the lower spore mass more triA(-) spores, than expected. Thus, the traits under study depend partly on the cells' own genotype and partly on the phenotypes, and so genotypes, of other cells: they are both autonomous and non-autonomous. These findings strengthen the parallels between multicellular development and behaviour in social groups. Besides that, they reinforce the point that a trait can be associated with a genotype only in a specified context.

Conformational basis for substrate recognition and regulation of catalytic activity in Staphylococcus aureus nucleoside di-phosphate kinase

Nucleoside diphosphate kinases (NDK) are characterized by high catalytic turnover rates and diverse substrate specificity. These features make this enzyme an effective activator of a pro-drug an application that has been actively pursued for a variety of therapeutic strategies. The catalytic mechanism of this enzyme is governed by a conserved histidine that coordinates a magnesium ion at the active site. Despite substantial structural and biochemical information on NDK, the mechanistic feature of the phospho-transfer that leads to auto-phosphorylation remains unclear. While the role of the histidine residue is well documented, the other active site residues, in particular the conserved serine remains poorly characterized. Studies on some homologues suggest no role for the serine residue at the active site, while others suggest a crucial role for this serine in the regulation and quaternary association of this enzyme in some species. Here we report the biochemical features of the Staphylococcus aureus NDK and the mutant enzymes. We also describe the crystal structures of the apo-NDK, as a transition state mimic with vanadate and in complex with different nucleotide substrates. These structures formed the basis for molecular dynamics simulations to understand the broad substrate specificity of this enzyme and the role of active site residues in the phospho-transfer mechanism and oligomerization. Put together, these data suggest that concerted changes in the conformation of specific residues facilitate the stabilization of nucleotide complexes thereby enabling the steps involved in the ping-pong reaction mechanism without large changes to the overall structure of this enzyme. (C) 2011 Elsevier B.V. All rights reserved.

Design and Characterization of Stabilized Derivatives of Human CD4D12 and CD4D1

CD4 is present on the surface of T-lymphocytes and is the primary cellular receptor for HIV-1. CD4 consists of a cytoplasmic tail, one transmembrane region, and four extracellular domains, D1-D4. A construct consisting of the first two domains of CD4 (CD4D12) is folded and binds gp120 with similar affinity as soluble 4-domain CD4 (sCD4). However, the first domain alone (CD4D1) was previously shown to be largely unfolded and had 3-fold weaker affinity for gp120 when compared to sCD4 [Sharma, D.; et al. (2005) Biochemistry 44, 16192-16202]. We now report the design and characterization of three single-site mutants of CD4D12 (G6A, L51I, and V86L) and one multisite mutant of CD4D1 (G6A/L511/L5K/F98T). G6A, L51I, and V86L are cavity-filling mutations while L5K and F98T are surface mutations which were introduced to minimize the aggregation of CD4D1 upon removal of the second domain. Two mutations, G6A and V86L in CD4D12 increased the stability and yield of the protein relative to the wild-type protein. The mutant CD4D1 (CD4D1a) with the 4 mutations was folded and more stable compared to the original CD4D1, but both bound gp120 with comparable affinity. In in vitro neutralization assays, both CD4D1a and G6A-CD4D12 were able to neutralize diverse HIV-1 viruses with similar IC(50)s as 4-domain CD4. These stabilized derivatives of human CD4 can be useful starting points for the design of other more complex viral entry inhibitors.

Revisiting the Mechanism of the Triosephosphate Isomerase Reaction: The Role of the Fully Conserved Glutamic Acid 97 Residue

An analysis of 503 available triosephosphate isomerase sequences revealed nine fully conserved residues. Of these, four residues-K12, H95, E97 and E165-are capable of proton transfer and are all arrayed around the dihydroxyacetone phosphate substrate in the three-dimensional structure. Specific roles have been assigned to the residues K12, H95 and E165, but the nature of the involvement of E97 has not been established. Kinetic and structural characterization is reported for the E97Q and E97D mutants of Plasmodium falciparum triosephosphate isomerase (Pf TIM). A 4000-fold reduction in k(cat) is observed for E97Q, whereas the E97D mutant shows a 100-fold reduction. The control mutant, E165A, which lacks the key catalytic base, shows an approximately 9000-fold drop in activity. The integrity of the overall fold and stability of the dimeric structure have been demonstrated by biophysical studies. Crystal structures of E97Q and E97D mutants have been determined at 2.0 angstrom resolution. In the case of the isosteric replacement of glutamic acid by glutamine in the E97Q mutant a large conformational change for the critical K12 side chain is observed, corresponding to a trans-to-gauche transition about the C gamma-C delta (chi(3)) bond. In the E97D mutant, the K12 side chain maintains the wild-type orientation, but the hydrogen bond between K12 and D97 is lost. The results are interpreted as a direct role for E97 in the catalytic proton transfer cycle. The proposed mechanism eliminates the need to invoke the formation of the energetically unfavourable imidazolate anion at H95, a key feature of the classical mechanism.

Cryptic AUG is important for 48S ribosomal assembly during internal initiation of translation of coxsackievirus B3 RNA

We have investigated the possible role of a conserved cis-acting element, the cryptic AUG, present in the 5' UTR of coxsackievirus B3 (CVB3) RNA. CVB3 5' UTR contains multiple AUG codons upstream of the initiator AUG, which are not used for the initiation of translation. The 48S ribosomal assembly takes place upstream of the cryptic AUG. We show here that mutation in the cryptic AUG results in reduced efficiency of translation mediated by the CVB3 IRES; mutation also reduces the interaction of mutant IRES with a well characterized IRES trans-acting factor, the human La protein. Furthermore, partial silencing of the La gene showed a decrease in IRES activity in the case of both the wild-type and mutant. We have demonstrated here that the interaction of the 48S ribosomal complex with mutant RNA was weaker compared with wild-type RNA by ribosome assembly analysis. We have also investigated by chemical and enzymic modifications the possible alteration in secondary structure in the mutant RNA. Results suggest that the secondary structure of mutant RNA was only marginally altered. Additionally, we have demonstrated by generating compensatory and non-specific mutations the specific function of the cryptic AUG in internal initiation. Results suggest that the effect of the cryptic AUG is specific and translation could not be rescued. However, a possibility of tertiary interaction of the cryptic AUG with other cis-acting elements cannot be ruled out. Taken together, it appears that the integrity of the cryptic AUG is important for efficient translation initiation by the CVB3 IRES RNA.

Novel Pentameric Structure of the Diarrhea-Inducing Region of the Rotavirus Enterotoxigenic Protein NSP4

A novel pentameric structure which differs from the previously reported tetrameric form of the diarrhea-inducing region of the rotavirus enterotoxin NSP4 is reported here. A significant feature of this pentameric form is the absence of the calcium ion located in the core region of the tetrameric structures. The lysis of cells, the crystallization of the region spanning residues 95 to 146 of NSP4 (NSP4(95-146)) of strain ST3 (ST3: NSP4(95-146)) at acidic pH, and comparative studies of the recombinant purified peptide under different conditions by size-exclusion chromatography (SEC) and of the crystal structures suggested pH-, Ca(2+)-, and protein concentration-dependent oligomeric transitions in the peptide. Since the NSP4(95-146) mutant lacks the N-terminal amphipathic domain (AD) and most of the C-terminal flexible region (FR), to demonstrate that the pentameric transition is not a consequence of the lack of the N- and C-terminal regions, glutaraldehyde cross-linking of the Delta N72 and Delta N94 mutant proteins, which contain or lack the AD, respectively, but possess the complete C-terminal FR, was carried out. The results indicate the presence of pentamers in preparations of these longer mutants. Detailed SEC analyses of Delta N94 prepared under different conditions, however, revealed protein concentration-dependent but metal ion-and pH-independent pentamer accumulation at high concentrations which dissociated into tetramers and lower oligomers at low protein concentrations. While calcium appeared to stabilize the tetramer, magnesium in particular stabilized the dimer. Delta N72 existed primarily in the multimeric form under all conditions. These findings of a calcium-free NSP4 pentamer and its concentration-dependent and largely calcium-independent oligomeric transitions open up a new dimension in an understanding of the structural basis of its multitude of functions.

UDP-glucose 4, 6-dehydratase Activity Plays an Important Role in Maintaining Cell Wall Integrity and Virulence of Candida albicans

Candida albicans, a human fungal pathogen, undergoes morphogenetic changes that are associated with virulence. We report here that GAL102 in C. albicans encodes a homolog of dTDP-glucose 4,6-dehydratase, an enzyme that affects cell wall properties as well as virulence of many pathogenic bacteria. We found that GAL102 deletion leads to greater sensitivity to antifungal drugs and cell wall destabilizing agents like Calcofluor white and Congo red. The mutant also formed biofilms consisting mainly of hyphal cells that show less turgor. The NMR analysis of cell wall mannans of gal102 deletion strain revealed that a major constituent of mannan is missing and the phosphomannan component known to affect virulence is greatly reduced. We also observed that there was a substantial reduction in the expression of genes involved in biofilm formation but increase in the expression of genes encoding glycosylphosphatidylinositol-anchored proteins in the mutant. These, along with altered mannosylation of cell wall proteins together might be responsible for multiple phenotypes displayed by the mutant. Finally, the mutant was unable to grow in the presence of resident peritoneal macrophages and elicited a weak pro-inflammatory cytokine response in vitro. Similarly, this mutant elicited a poor serum pro-inflammatory cytokine response as judged by IFN gamma and TNF alpha levels and showed reduced virulence in a mouse model of systemic candidiasis. Importantly, an Ala substitution for a conserved Lys residue in the active site motif YXXXK, that abrogates the enzyme activity also showed reduced virulence and increased filamentation similar to the gal102 deletion strain. Since inactivating the enzyme encoded by GAL102 makes the cells sensitive to antifungal drugs and reduces its virulence, it can serve as a potential drug target in combination therapies for C. albicans and related pathogens.

Protein Model Discrimination Using Mutational Sensitivity Derived from Deep Sequencing

A major bottleneck in protein structure prediction is the selection of correct models from a pool of decoys. Relative activities of similar to 1,200 individual single-site mutants in a saturation library of the bacterial toxin CcdB were estimated by determining their relative populations using deep sequencing. This phenotypic information was used to define an empirical score for each residue (Rank Score), which correlated with the residue depth, and identify active-site residues. Using these correlations, similar to 98% of correct models of CcdB (RMSD <= 4 angstrom) were identified from a large set of decoys. The model-discrimination methodology was further validated on eleven different monomeric proteins using simulated RankScore values. The methodology is also a rapid, accurate way to obtain relative activities of each mutant in a large pool and derive sequence-structure-function relationships without protein isolation or characterization. It can be applied to any system in which mutational effects can be monitored by a phenotypic readout.