Identification of a DNA-binding domain and an active-site residue of pseudorabies virus DNase.
Ontology highlight
ABSTRACT:
SUBMITTER: Ho TY
PROVIDER: S-EPMC1220871 | biostudies-other | 2000 Mar
REPOSITORIES: biostudies-other
Similar Datasets
Project description:The pseudorabies virus (PRV) DNase gene has previously been mapped within the PRV genome. To characterize further the enzymic properties of PRV DNase, this enzyme was expressed in Escherichia coli with the use of a pET expression vector. The protein was purified to homogeneity and assayed for nuclease activity in vitro. Recombinant PRV DNase exhibited an alkaline pH preference and an absolute requirement for Mg2+ ions that could not be replaced by Ca2+ and Na+ ions. Further studies showed that PRV DNase exhibited endonuclease, 5'-exonuclease and 3'-exonuclease activities in both single-stranded and double-stranded DNA. This activity occurred randomly and no significant base preference was demonstrated. The multiple biochemical activities of PRV DNase are similar to the activities of Neurospora crassa endo-exonuclease and E. coli RecBCD, two additional enzymes that are involved in recombination. Taken together, the similarity of action between N. crassa endo-exonuclease, E. coli RecBCD, and PRV DNase suggests that PRV DNase might have a role in the process of recombination that occurs during PRV infection.
| S-EPMC1219107 | biostudies-other
Project description:In silico-docking studies from previous work have suggested that Lys-206 and lys-207 of calreticulin (CR) play a pivotal key role in its well-established transacetylation activity. To experimentally validate this prediction, we introduced three mutations at lysine residues of P-domain of CR: K → A, P mut-1 (K -206, -209), P mut-2 (K -206, -207) and P mut-3 (K -207, -209) and analyzed their immunoreactivity and acetylation potential. The clones of wild-type P-domain (P wt ) and three mutated P-domain (P mut-1, P mut-2 and P mut-3) were expressed in pTrcHis C vector and the recombinant P wt , P mut-1 , P mut-2 and P mut-3 proteins were purified by Ni-NTA affinity chromatography. Screening of the transacylase activity (TAase) by the Glutathione S Transferase (GST) assay revealed that the TAase activity was associated with the P wt and P mut-1 while P mut-2 and P mut-3 did not show any activity. The immune-reactivity to anti-lysine antibody was also retained only by the P mut-1 in which the Lys-207 was intact. Retention of the TAase activity and immunoreactivity of P mut-1 with mutations introduced at Lys-206, Lys-209, while its loss with a mutation at Lys-207 residue indicated that lysine-207 of P-domain constitutes the active site residue controlling TAase activity.Supplementary informationThe online version contains supplementary material available at 10.1007/s13205-021-02659-1.
| S-EPMC7859019 | biostudies-literature
Project description:Phototropins, a class of light-activated protein kinases, are essential for several blue light responses in plants and algae, including phototropism. These proteins contain two internal light, oxygen, and voltage sensitive (LOV) domains, which bind flavin chromophores and undergo a reversible photochemical formation of a cysteinyl-flavin adduct as part of the light sensing process. While the photodynamic properties of such photosensory domains are dictated by interactions between the chromophore and surrounding protein, more distant residues can play a significant role as well. Here we explore the role of the Phe434 residue in the photosensory response of the second LOV domain of Avena sativa phototropin 1 (AsLOV2), a model photochemical system for these LOV domains. Phe434 is more than 6 Å from the FMN chromophore in AsLOV2; nevertheless, an F434Y point mutation is likely to change several structural features of the chromophore binding site, as we demonstrate using molecular dynamics simulations. Transient absorption signals spanning 15 decades in time were compared for wild-type AsLOV2 and the F434Y mutant, showing that the latter has significantly altered photodynamics, including (i) a faster intersystem crossing leading to triplet formation on a nanosecond time scale, (ii) biphasic formation of adduct-state kinetics on the microsecond time scale, and (iii) greatly accelerated ground-state recovery kinetics on a second time scale. We present mechanistic models that link these spectroscopic differences to changes in the configuration of the critical cysteine residue and in the chromophore's accessibility to solvent and oxygen according to MD trajectories and purging experiments. Taken together, these results demonstrate the importance of residues outside the chromophore-binding pocket in modulating LOV domain photodynamics.
| S-EPMC3068209 | biostudies-literature
Project description:Non-conserved amino acids that are far removed from the active site can sometimes have an unexpected effect on enzyme catalysis. We have investigated the effects of alanine replacement of residues distant from the active site of the replicative RB69 DNA polymerase, and identified a substitution in a weakly conserved palm residue (D714A), that renders the enzyme incapable of sustaining phage replication in vivo. D714, located several angstroms away from the active site, does not contact the DNA or the incoming dNTP, and our apoenzyme and ternary crystal structures of the Pol(D714A) mutant demonstrate that D714A does not affect the overall structure of the protein. The structures reveal a conformational change of several amino acid side chains, which cascade out from the site of the substitution towards the catalytic center, substantially perturbing the geometry of the active site. Consistent with these structural observations, the mutant has a significantly reduced k pol for correct incorporation. We propose that the observed structural changes underlie the severe polymerization defect and thus D714 is a remote, non-catalytic residue that is nevertheless critical for maintaining an optimal active site conformation. This represents a striking example of an action-at-a-distance interaction.
| S-EPMC3792054 | biostudies-literature
Project description:Understanding the basis of the DNA-binding specificity of transcription factors (TFs) has been of long-standing interest. Despite extensive efforts to map millions of putative TF binding sequences, identifying the critical determinants for DNA binding specificity remains a major challenge. The coevolution of residues in proteins occurs due to a shared evolutionary history. However, it is unclear how coevolving residues in TFs contribute to DNA binding specificity. Here, we systematically collected publicly available data sets from multiple large-scale high-throughput TF-DNA interaction screening experiments for the major TF families with large numbers of TF members. These families included the Homeobox, HLH, bZIP_1, Ets, HMG_box, ZF-C4, and Zn_clus TFs. We detected TF subclass-determining sites (TSDSs) and showed that the TSDSs were more likely to coevolve with other TSDSs than with non-TSDSs, particularly for the Homeobox, HLH, Ets, bZIP_1, and HMG_box TF families. By in silico modeling, we showed that mutation of the highly coevolving residues could significantly reduce the stability of the TF-DNA complex. The distant residues from the DNA interface also contributed to TF-DNA binding activity. Overall, our study gave evidence that coevolved residues relate to transcriptional regulation and provided insights into the potential application of engineered DNA-binding domains and proteins. IMPORTANCE While unraveling DNA-binding specificity of TFs is the key to understanding the basis and molecular mechanism of gene expression regulation, identifying the critical determinants that contribute to DNA binding specificity remains a major challenge. In this study, we provided evidence showing that coevolving residues in TF domains contributed to DNA binding specificity. We demonstrated that the TSDSs were more likely to coevolve with other TSDSs than with non-TSDSs. Mutation of the coevolving residue pairs (CRPs) could significantly reduce the stability of THE TF-DNA complex, and even the distant residues from the DNA interface contribute to TF-DNA binding activity. Collectively, our study expands our knowledge of the interactions among coevolved residues in TFs, tertiary contacting, and functional importance in refined transcriptional regulation. Understanding the impact of coevolving residues in TFs will help understand the details of transcription of gene regulation and advance the application of engineered DNA-binding domains and protein.
| S-EPMC10100741 | biostudies-literature
Project description:Sequence analysis within the long segment of the pseudorabies virus (PrV) genome identified an open reading frame of 804 bp whose deduced protein product of 268 amino acids exhibited homology to dUTPases of other herpesviruses. The gene was designated UL50 because of its colinearity with the homologous gene of herpes simplex virus type 1. An antiserum raised against a bacterially expressed fragment of PrV UL50 specifically detected a 33-kDa protein in lysates of infected cells, which is in agreement with the predicted molecular mass of the PrV UL50 protein. A UL50-negative PrV mutant (PrV UL50-) was constructed by the insertion of a beta-galactosidase expression cassette into the UL50 coding sequence. A corresponding rescuant (PrV UL50resc) was also isolated. The interruption of the UL50 gene led to the disappearance of the 33-kDa protein, whereas restoration of UL50 gene expression restored detection of the 33-kDa protein. Enzyme activity assays confirmed that UL50 of PrV codes for a dUTPase which copurifies with nuclei of infected cells. PrV UL50- replicated with an only slightly reduced efficiency in epithelial cells in culture compared with that of its parental wild-type virus strain. Our results thus demonstrate that UL50 of PrV encodes a protein of 33 kDa with dUTPase activity which copurifies with nuclei of infected cells and is dispensable for replication in cultured epithelial cells.
| S-EPMC189936 | biostudies-other
Project description:BACKGROUND: Biological evolution conserves protein residues that are important for structure and function. Both protein stability and function often require a certain degree of structural co-operativity between spatially neighboring residues and it has previously been shown that conserved residues occur clustered together in protein tertiary structures, enzyme active sites and protein-DNA interfaces. Residues comprising protein interfaces are often more conserved compared to those occurring elsewhere on the protein surface. We investigate the extent to which conserved residues within protein-protein interfaces are clustered together in three-dimensions. RESULTS: Out of 121 and 392 interfaces in homodimers and heterocomplexes, 96.7 and 86.7%, respectively, have the conserved positions clustered within the overall interface region. The significance of this clustering was established in comparison to what is seen for the subsets of the same size of randomly selected residues from the interface. Conserved residues occurring in larger interfaces could often be sub-divided into two or more distinct sub-clusters. These structural cluster(s) comprising conserved residues indicate functionally important regions within the protein-protein interface that can be targeted for further structural and energetic analysis by experimental scanning mutagenesis. Almost 60% of experimental hot spot residues (with DeltaDeltaG > 2 kcal/mol) were localized to these conserved residue clusters. An analysis of the residue types that are enriched within these conserved subsets compared to the overall interface showed that hydrophobic and aromatic residues are favored, but charged residues (both positive and negative) are less common. The potential use of this method for discriminating binding sites (interfaces) versus random surface patches was explored by comparing the clustering of conserved residues within each of these regions--in about 50% cases the true interface is ranked among the top 10% of all surface patches. CONCLUSIONS: Protein-protein interaction sites are much larger than small molecule biding sites, but still conserved residues are not randomly distributed over the whole interface and are distinctly clustered. The clustered nature of evolutionarily conserved residues within interfaces as compared to those within other surface patches not involved in binding has important implications for the identification of protein-protein binding sites and would have applications in docking studies.
| S-EPMC2894039 | biostudies-literature
Project description:Dengue virus (DENV) protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described.
| S-EPMC4677662 | biostudies-literature
Project description:BackgroundApproximately 5% of Pfam families are enzymatic, but only a small fraction of the sequences within these families (<0.5%) have had the residues responsible for catalysis determined. To increase the active site annotations in the Pfam database, we have developed a strict set of rules, chosen to reduce the rate of false positives, which enable the transfer of experimentally determined active site residue data to other sequences within the same Pfam family.DescriptionWe have created a large database of predicted active site residues. On comparing our active site predictions to those found in UniProtKB, Catalytic Site Atlas, PROSITE and MEROPS we find that we make many novel predictions. On investigating the small subset of predictions made by these databases that are not predicted by us, we found these sequences did not meet our strict criteria for prediction. We assessed the sensitivity and specificity of our methodology and estimate that only 3% of our predicted sequences are false positives.ConclusionWe have predicted 606110 active site residues, of which 94% are not found in UniProtKB, and have increased the active site annotations in Pfam by more than 200 fold. Although implemented for Pfam, the tool we have developed for transferring the data can be applied to any alignment with associated experimental active site data and is available for download. Our active site predictions are re-calculated at each Pfam release to ensure they are comprehensive and up to date. They provide one of the largest available databases of active site annotation.
| S-EPMC2025603 | biostudies-literature
Project description:HapR has been given the status of a high cell density master regulatory protein in Vibrio cholerae. Though many facts are known regarding its structural and functional aspects, much still can be learnt from natural variants of the wild type protein. This work aims at investigating the nature of functional inertness of a HapR natural variant harboring a substitution of a conserved glutamate residue at position 117 which participates in forming a salt bridge by lysine (HapRV2G-E(117)K). Experimental evidence presented here reveals the inability of this variant to interact with various cognate promoters by in vitro gel shift assay. Furthermore, the elution profiles of HapRV2G-E(117)K protein along with the wild type functional HapRV2G in size-exclusion chromatography as well as circular dichroism spectra did not reflect any significant differences in its structure, thereby indicating the intactness of dimer in the variant protein. To gain further insight into the global shape of the proteins, small angle X-ray scattering analysis (SAXS) was performed. Intriguingly, increased radius of gyration of HapRV2G-E(117)K of 27.5 Å in comparison to the wild type protein from SAXS data analyses implied a significant alteration in the global shape of the dimeric HapRV2G-E(117)K protein. Structure reconstruction brought forth that the DNA binding domains were substantially "parted away" in this variant. Taken together, our data illustrates that substitution of the conserved glutamate residue by lysine in the dimerization domain induces separation of the two DNA binding domains from their native-like positioning without altering the dimeric status of HapR variant.
| S-EPMC3796514 | biostudies-literature