Residues In Close Proximity Research Articles

Unraveling the Photochemistry of the 5-Azido-2′-deoxyuridine Photoaffinity Label

UV irradiation of 5-azido-2'-deoxyuridine in water provides up to seven products. All likely result from a pivotal azirene, formed by the intramolecular rearrangement of the initially formed nitrene, that undergoes nucleophilic addition at its C5 position. This study strongly suggests that only nucleophilic amino acid residues in close proximity are cross-linkable in photolabeling experiments by using the 5-azidouracil photophore.

Designed Human Serum Hyaluronidase 1 Variant, HYAL1ΔL, Exhibits Activity up to pH 5.9

Hyaluronidases from diverse species and sources have different pH optima. Distinct mechanisms with regard to dynamic structural changes, which control hyaluronidase activity at varying pH, are unknown. Human serum hyaluronidase 1 (HYAL1) is active solely below pH 5.1. Here we report the design of a HYAL1 variant that degrades hyaluronan up to pH 5.9. Besides highly conserved residues in close proximity of the active site of most hyaluronidases, we identified a bulky loop formation located at the end of the substrate binding crevice of HYAL1 to be crucial for substrate hydrolysis. The stretch between cysteine residues 207 and 221, which normally contains 13 amino acids, could be replaced by a tetrapeptide sequence of alternating glycine serine residues, thereby yielding an active enzyme with an extended binding cleft. This variant exhibited hyaluronan degradation at elevated pH. This is indicative for appropriate substrate binding and proper positioning being decisively affected by sites far off from the active center.

Different Roles of C-terminal Cassettes in the Trafficking of Full-length NR1 Subunits to the Cell Surface

N-Methyl-d-aspartate (NMDA) receptors are glutamate-gated ion channels composed of NR1 and NR2 subunits. When expressed alone, the most prevalent NR1 splice variant and all NR2 subunits are retained in the endoplasmic reticulum (ER), whereas other NR1 splice variants reach the cell surface to varying degrees. Because similar trafficking patterns have been seen for single transmembrane domain chimeric proteins with appended C termini of NMDA receptor subunits, these chimeric proteins have been used as a model for studying the mechanisms underlying the ER retention and surface trafficking of NMDA receptors. Using this approach, an RRR motif in the C1 cassette has been identified as a major ER retention signal present in NR1 subunits, and the surface localization of other NR1 splice variants has been explained by the absence of the C1 cassette or by the presence of a PDZ/coatomer protein complex II-binding domain in the C2' cassette. However, when we tested these conclusions using full-length NR1 constructs, a more complex role of the C-terminal cassettes in the trafficking of NR1 subunits emerged. Our experiments showed that two independent ER retention motifs in the C1 cassette, KKK and RRR, are the signals mediating ER retention of the full-length NR1 subunits and that the C2 cassette has an additional inhibitory effect on the forward trafficking of NR1 subunits. On the other hand, C0 and C2' cassettes had an enhancing effect on the trafficking of NR1 subunits to the cell surface. Our observations identify the unique roles of C-terminal cassettes in the trafficking of full-length NR1 subunits.

Identification of Two SET Domain Proteins Required for Methylation of Lysine Residues in Yeast Ribosomal Protein Rpl42ab

We show that the Saccharomyces cerevisiae ribosomal protein Rpl42ab (the identical product of the RPL42A and RPL42B genes) is monomethylated at Lys-40 and Lys-55. The methylation of Lys-40 is dependent upon the Ybr030w gene product; the methylation of Lys-55 is dependent upon the Set7 gene product. Ybr030w and SET7 genes both encode SET domain containing proteins homologous to known protein lysine methyltransferases, suggesting that their products are the specific enzymes responsible for the monomethylation of the two sites in Rpl42ab. We thus designate Ybr030w as Rkm3 and Set7 as Rkm4. Yeast strains with deletions in both the Ybr030w and SET7 genes produce unmethylated Rpl42ab. A slow growth phenotype was seen for the SET7 deletion strain and the double knock-out when grown in low concentrations of the eukaryotic protein synthesis inhibitor, cycloheximide. These results suggest that modification of Rpl42ab at Lys-55 can fine-tune its structure to avoid inhibition. An intact mass fragmentation approach ("top down mass spectrometry") was used to quantitate the extent of methylation of Rpl42ab. In wild-type strains, it was found that 78% was monomethylated at both Lys-40 and Lys-55 and that 22% was a mixture of species with either Lys-40 or Lys-55 monomethylated. The top down approach was also used to reevaluate the methylation sites of Rpl12ab. We found that the yeast Rpl12ab protein is dimethylated at the N-terminal proline residue, trimethylated at Lys-3 by Rkm2, and monomethylated at Arg-66.

Improved prediction of peroxisomal PTS1 proteins from genome sequences based on experimental subcellular targeting analyses as exemplified for protein kinases from Arabidopsis

Due to current experimental limitations in peroxisome proteome research, the identification of low-abundance regulatory proteins such as protein kinases largely relies on computational protein prediction. To test and improve the identification of regulatory proteins by such a prediction-based approach, the Arabidopsis genome was screened for genes that encode protein kinases with predicted type 1 or type 2 peroxisome targeting signals (PTS1 or PTS2). Upon transient expression in onion epidermal cells, the predicted PTS1 domains of four of the seven protein kinases re-directed the reporter protein, enhanced yellow green fluorescent (EYFP), to peroxisomes and were thus verified as functional PTS1 domains. The full-length fusions, however, remained cytosolic, suggesting that PTS1 exposure is induced by specific signals. To investigate why peroxisome targeting of three other kinases was incorrectly predicted and ultimately to improve the prediction algorithms, selected amino acid residues located upstream of PTS1 tripeptides were mutated and the effect on subcellular targeting of the reporter protein was analysed. Acidic residues in close proximity to major PTS1 tripeptides were demonstrated to inhibit protein targeting to plant peroxisomes even in the case of the prototypical PTS1 tripeptide SKL>, whereas basic residues function as essential auxiliary targeting elements in front of weak PTS1 tripeptides such as SHL>. The functional characterization of these inhibitory and essential enhancer-targeting elements allows their consideration in predictive algorithms to improve the prediction accuracy of PTS1 proteins from genome sequences.

Antigenic structure of the hepatitis C virus envelope 2 protein

The antigenic structure of the envelope 2 (e2) protein of the hepatitis C virus (HCV) was characterized by the use of 70 synthetic peptides and 131 human sera from persons with antibodies to HCV. Among 34 overlapping peptides spanning the e2 protein of HCV, two major antigenic regions were located to residues 484-499 and residues 554-569. The sequence of the two major antigenic regions of the e2 protein are partly well conserved within the described types of HCV. Both regions contain two Cys residues in close proximity, and the region at residues 554-569 contains a putative N-glycosylation site, which are factors that previously have been suggested to affect the immune recognition of the e2 protein. Using substitution peptide analogues where each position within residues 484-499 and 554-569 were sequentially substituted by Ala or Gly, the most essential residues for antibody binding were found to be the Pro-498, Ala-499, Ala-566, Pro-567, and Pro-568. All of these, except for the Pro-498 and Ala-566, are conserved among different HCV strains. Also, according to previous studies, position 496 often shows variations, which could be explained by position 496 being contained within the antigenic region at residues 484-499. Interestingly, none of the Cys residues at positions 486, 494, 564 and 569 were found to be essential for antibody binding, indicating that these are not essential in maintaining the e2 antigenicity of the peptides. In a material of 114 confirmed anti-HCV positive sera, derived from patients during the acute or the chronic phase of HCV infection, the prevalence of antibodies to the two major linear antigenic regions of the e2 protein was found to be 55% among HCV RNA-positive sera, and 53% among HCV RNA-negative sera. In conclusion, we have identified and characterized two major linear antigenic regions outside the two hypervariable regions of the e2 protein. Since these regions are accessible to the B cells of the infected host, these two regions are likely to be surface exposed either on the precursor polyprotein or the native e2 protein. Also, we could confirm that antibodies to the e2 protein co-exist with HCV viraemia.

Structural Consideration of the Formation of the Activation Complex between the Staphylokinase-Like Streptococcal Plasminogen Activator PadA and Bovine Plasminogen

The characteristics of a streptococcal plasminogen activator (PA) displaying specificity for ruminant plasminogen (Plg) were defined using molecular approaches. The 16-kDa secreted protein PadA was found to be prevalent in Streptococcus dysgalactiae subspecies dysgalactiae isolated from cases of bovine mastitis and septic arthritis in lambs. PadA was able to activate bovine, ovine and caprine Plg, but not human Plg. Amino acid sequence analysis identified a limited level of homology to other streptococcal PAs, including streptokinase; however, PadA was found to align well with and match in size the staphylococcal PA, staphylokinase. Recombinant PadA was used to investigate interaction with bovine Plg, leading to formation of an activator complex that was capable of recruiting and converting further substrate Plg into plasmin. Individual non-overlapping peptides of PadA or bovine microplasminogen were found to block the interaction between PadA and bovine Plg, preventing the formation of the activation complex. Homology modelling based upon structures of staphylokinase complexed with human microplasminogen supported these findings by placing critical residues in close proximity to the plasmin component of the activation complex.

Structural Interaction Between DsrE-DsrF-DsrH Proteins Involved in the Transport of Electrons in the dsr Operon

Biological redox reactions of inorganic sulfur compounds are important for the proper maintenance of environmental sulfur balance. These reactions are mediated by phylogeneticaly diverse set of microorganisms. The protein complex that is involved in such redox reactions of sulfur compounds is the complex encoded by dsr operon. The ecological and industrial importance of these microorganisms led us to investigate the structural details of the mechanism of the process of electron transport during such redox reactions performed by the dsr operon. Among the gene products of the operon, the proteins DsrE, DsrF, and DsrH are small soluble cytoplasmic proteins acting as α2β2γ2 heterohexamer and are involved in the process of electron transport in these ecologically as well as industrially important microorganisms. Since no structural details of the proteins were available we employed homology modeling to construct the three-dimensional structures of the DsrE, DsrF, and DsrH from Chlorobium tepidum. The putative three dimensional structures of the proteins were predicted from the models. Since DsrE, DsrF, and DsrH proteins act as a hetero-hexameric complex, the modeled proteins were subjected to molecular docking analyses to generate the model of the biochemically active complex. This allowed us to predict the probable binding modes of the proteins as well as the biochemical and the structural basis of the mechanism of the electron transport process by this complex. The hexamerization of the proteins would help to bring the Cys residues in close proximity, which enables the complex to actively take part electron transport process.

Hue-shifted monomeric variants of Clavulariacyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging

BackgroundIn the 15 years that have passed since the cloning of Aequorea victoria green fluorescent protein (avGFP), the expanding set of fluorescent protein (FP) variants has become entrenched as an indispensable toolkit for cell biology research. One of the latest additions to the toolkit is monomeric teal FP (mTFP1), a bright and photostable FP derived from Clavularia cyan FP. To gain insight into the molecular basis for the blue-shifted fluorescence emission we undertook a mutagenesis-based study of residues in the immediate environment of the chromophore. We also employed site-directed and random mutagenesis in combination with library screening to create new hues of mTFP1-derived variants with wavelength-shifted excitation and emission spectra.ResultsOur results demonstrate that the protein-chromophore interactions responsible for blue-shifting the absorbance and emission maxima of mTFP1 operate independently of the chromophore structure. This conclusion is supported by the observation that the Tyr67Trp and Tyr67His mutants of mTFP1 retain a blue-shifted fluorescence emission relative to their avGFP counterparts (that is, Tyr66Trp and Tyr66His). Based on previous work with close homologs, His197 and His163 are likely to be the residues with the greatest contribution towards blue-shifting the fluorescence emission. Indeed we have identified the substitutions His163Met and Thr73Ala that abolish or disrupt the interactions of these residues with the chromophore. The mTFP1-Thr73Ala/His163Met double mutant has an emission peak that is 23 nm red-shifted from that of mTFP1 itself. Directed evolution of this double mutant resulted in the development of mWasabi, a new green fluorescing protein that offers certain advantages over enhanced avGFP (EGFP). To assess the usefulness of mTFP1 and mWasabi in live cell imaging applications, we constructed and imaged more than 20 different fusion proteins.ConclusionBased on the results of our mutagenesis study, we conclude that the two histidine residues in close proximity to the chromophore are approximately equal determinants of the blue-shifted fluorescence emission of mTFP1. With respect to live cell imaging applications, the mTFP1-derived mWasabi should be particularly useful in two-color imaging in conjunction with a Sapphire-type variant or as a fluorescence resonance energy transfer acceptor with a blue FP donor. In all fusions attempted, both mTFP1 and mWasabi give patterns of fluorescent localization indistinguishable from that of well-established avGFP variants.

Crystallographic Studies of Human MitoNEET

MitoNEET was identified as an outer mitochondrial membrane protein that can potentially bind the anti-diabetes drug pioglitazone. The crystal structure of the cytoplasmic mitoNEET (residues 33-108) is determined in this study. The structure presents a novel protein fold and contains a [2Fe-2S] cluster-binding domain. The [2Fe-2S] cluster is coordinated to the protein by Cys-72, Cys-74, Cys-83, and His-87 residues. This coordination is also novel compared with the traditional [2Fe-2S] cluster coordinated by four cysteines or two cysteines and two histidines. The cytoplasmic mitoNEET forms homodimers in solution and in crystal. The dimerization is mainly mediated by hydrophobic interactions as well as hydrogen bonds coordinated by two water molecules binding at the interface. His-87 residue, which plays an important role in the coordination of the [2Fe-2S] cluster, is exposed to the solvent on the dimer surface. It is proposed that mitoNEET dimer may interact with other proteins via the surface residues in close proximity to the [2Fe-2S] cluster.

Epimerization- and Protecting-Group-Free Synthesis of Peptidomimetic Conjugates from Amphoteric Amino Aldehydes

We have developed a novel protecting-group-free strategy for the synthesis of peptidomimetic conjugates. The high degrees of stereocontrol and chemoselectivity achieved in this chemistry hinge upon unusual preferences of the amphoteric amino aldehydes. The resulting conjugates contain reduced amide bonds at defined positions, offering a possibility for peptidomimetic ligation. These findings should allow access to templates for introducing both natural and unnatural amino acid residues in close proximity to the reduced amide bond isosteres.

An Aptly Positioned Azido Group in the Spacer of a Protein Cross‐Linker for Facile Mapping of Lysines in Close Proximity

Cross-links between amino acid residues in close proximity can provide distance constraints for the validation of models of the 3D structure proteins. The mapping of cross-links by the identification of linked peptides in proteolytic digests is facilitated by cleavable cross-linkers that enable isolation of the cleavage products while preserving information about the linkage. We present an amine-specific cross-linker, bis(succinimidyl)-3-azidomethyl glutarate (BAMG), that fulfils these requirements. Two parallel reaction pathways are induced by tris(carboxyethyl)phosphine (TCEP) in cross-linked peptides from BAMG-treated cytochrome c. One pathway leads to cleavage of the cross-linked species, while in the other the azido group of BAMG is reduced to an amino group without cleavage. Cross-linked peptides and peptides modified by partially hydrolysed BAMG yield distinct sets of TCEP-induced reaction products. These can be isolated by reversed-phase diagonal chromatography and identified by mass spectrometry to reveal the identity of the parent compounds. The ease with which cross-link-derived reaction products can be isolated and identified indicates that the mapping of cross-links in complex biological assemblies and mixtures of protein complexes might become feasible in the near future.

The crystal structure of the catalytic domain of the chick retinal neurite inhibitor—receptor protein tyrosine phosphatase CRYP‐2/cPTPRO

The crystal structure of the catalytic domain of the chick retinal neurite inhibitor—receptor protein tyrosine phosphatase CRYP‐2/cPTPRO

High Incidence of Unusual Cysteine Variants in gp120 Envelope Proteins from Early HIV Type 1 Infections from a Phase 3 Vaccine Efficacy Trial

During the course of a large-scale HIV-1 vaccine field trial (VAX004), full-length gp120 sequences were determined for 349 new HIV-1 infections. The data collected represent the largest survey of full-length gp120 sequences from new HIV-1 infections ever assembled. Previous studies have shown that subtype B viruses typically possess 18 cysteine residues that are covalently linked to form 9 conserved disulfide bridges. However, in this study we found that approximately 20% of the trial participants possessed envelope proteins with an unusual number of cysteine residues that could very likely result in unusual protein structures. One class of variants included envelope proteins with two additional cysteine residues in close proximity, potentially yielding additional disulfide-bonded loops. Other classes of variants included envelope proteins where amino acid replacements increased or decreased the number of cysteine residues by one, resulting in molecules with either 19 or 17 cysteines, respectively. Initial functional analysis demonstrated that envelope proteins with 19 cysteine residues bind to CD4 and the CCR5 chemokine coreceptor, and are infectious. These results suggest that the protein structure of gp120 in newly transmitted viruses may be more heterogeneous than previously appreciated and potentially represent a new mechanism of virus variation. The disulfide variation that we report here may have important implications for HIV vaccine and drug development efforts.

Quantification of acetylation at proximal lysine residues using isotopic labeling and tandem mass spectrometry

With the emergence of the histone code as a key determinant in the regulation of gene expression, it has been important to develop tools that can not only identify the types and locations of myriad modifications, but also determine how the levels of these modifications change as a result of various processes in a cell. Mass spectrometry has become a method of choice for the investigation of post-translational modifications in histone proteins. Described in this article is a mass spectrometric method that is useful for direct quantification of levels of acetylation at lysines residues in close proximity to one another, as is the case for the amino terminal tail of histone H4. This method involves fragmentation of peptides into b and y ions that contain one or more sites of modification and isotopic labeling which ensures equivalent ionization and fragmentation.

Selenium-containing enzymes in mammals: Chemical perspectives

The chemical and biochemical route to the synthesis of the 21st amino acid in living systems, selenocysteine, is described. The incorporation of this rare amino acid residue into proteins is described with emphasis on the role of monoselenophosphate as selenium source. The role of selenocysteine moiety in natural mammalian enzymes such as glutathione peroxidase (GPx), iodothyronine deiodinase (ID) and thioredoxin reductase (TrxR) is highlighted and the effect of other amino acid residues located in close proximity to selenocysteine is described. It is evident from various studies that two amino acid residues, tryptophan and glutamine, appear in identical positions in all known members of the GPx family. According to the three-dimensional structure established for bovine GPx, these residues could constitute a catalytic triad in which the selenol group of the selenocysteine is both stabilized and activated by hydrogen bonding with the imino group of the tryptophan (Trp) residue and with the amido group of the glutamine (Gln) residue. The ID enzymes, on the other hand, do not possess any Trp or Gln residues in close proximity to selenium, but contain several histidine residues, which may play important roles in the catalysis. The TrxR enzymes also possess some basic histidines, but the most important amino acid residues are the cysteines which constitute the internal cofactor systems along with the catalytically active selenocysteine. The catalytic activity and substrate specificity of all three selenoenzymes are described. The reactivity of selenocysteine residues in selenoenzymes towards metal-based drugs such as goldthioglucose is also described.

Identification of the Geometric Requirements for Allosteric Communication between the α- and β-Subunits of Tryptophan Synthase

The pyridoxal 5'-phosphate-dependent tryptophan synthase alpha2beta2 complex is a paradigmatic protein for substrate channeling and allosteric regulation. The enzymatic activity is modulated by a ligand-mediated equilibrium between open (inactive) and closed (active) conformations of the alpha- and beta-subunit, predominantly involving the mobile alpha loop 6 and the beta-COMM domain that contains beta helix 6. The alpha ligand-triggered intersubunit communication seems to rely on a single hydrogen bond formed between the carbonyl oxygen of betaSer-178 of beta helix 6 and the NH group of alphaGly-181 of alpha loop 6. We investigated whether and to what extent mutations of alphaGly-181 and betaSer-178 affect allosteric regulation by the replacement of betaSer-178 with Pro or Ala and of alphaGly-181 with either Pro to remove the amidic proton that forms the hydrogen bond or Ala, Val, and Phe to analyze the dependence on steric hindrance of the open-closed conformational transition. The alpha and beta activity assays and the equilibrium distribution of beta-subunit catalytic intermediates indicate that mutations do not significantly influence the intersubunit catalytic activation but completely abolish ligand-induced alpha-to beta-subunit signaling, demonstrating distinct pathways for alpha-beta-site communication. Limited proteolysis experiments indicate that the removal of the interaction between betaSer-178 and alphaGly-181 strongly favors the more trypsin-accessible open conformation of the alpha-active site. When the hydrogen bond cannot be formed, the alpha-subunit is unable to attain the closed conformation, and consequently, the allosteric signal is aborted at the subunit interface.

Substitution of Conserved Residues within the Active Site Alters the Cleavage Religation Equilibrium of DNA Topoisomerase I

Eukaryotic DNA topoisomerase I (Top1p) catalyzes the relaxation of supercoiled DNA and constitutes the cellular target of camptothecin (CPT). Mutation of conserved residues in close proximity to the active site tyrosine (Tyr(727) of yeast Top1p) alters the DNA cleavage religation equilibrium, inducing drug-independent cell lethality. Previous studies indicates that yeast Top1T722Ap and Top1N726Hp cytotoxicity results from elevated levels of covalent enzyme-DNA intermediates. Here we show that Top1T722Ap acts as a CPT mimetic by exhibiting reduced rates of DNA religation, whereas increased Top1N726Hp.DNA complexes result from elevated DNA binding and cleavage. We also report that the combination of the T722A and N726H mutations in a single protein potentiates the cytotoxic action of the enzyme beyond that induced by co-expression of the single mutants. Moreover, the addition of CPT to cells expressing the double top1T722A/N726H mutant did not enhance cell lethality. Thus, independent alterations in DNA cleavage and religation contribute to the lethal phenotype. The formation of distinct cytotoxic lesions was also evidenced by the different responses induced by low levels of these self-poisoning enzymes in isogenic strains defective for the Rad9 DNA damage checkpoint, processive DNA replication, or ubiquitin-mediated proteolysis. Substitution of Asn(726) with Phe or Tyr also produces self-poisoning enzymes, implicating stacking interactions in the increased kinetics of DNA cleavage by Top1N726Hp and Top1N726Fp. In contrast, replacing the amide side chain of Asn(726) with Gln renders Top1N726Qp resistant to CPT, suggesting that the orientation of the amide within the active site is critical for effective CPT binding.

Characterization of Collagenous Peptides Bound to Lysyl Hydroxylase Isoforms

Lysyl hydroxylase (LH, EC 1.14.11.4) is the enzyme catalyzing the formation of hydroxylysyl residues in collagens and other proteins with collagenous domains. Although lower species, such as Caenorhabditis elegans, have only one LH orthologue, LH activity in higher species, such as human, rat, and mouse, is present in three molecules, LH1, LH2, and LH3, encoded by three different genes. In addition, LH2 is present in two alternatively spliced forms (LH2a, LH2b). To understand the functions of the four molecular forms of LH in vertebrates, we analyzed differences in the binding and hydroxylation of various collagenous peptides by the LH isoforms. Nine-amino acid-long synthetic peptides on Pepspot were used for the binding analysis and an activity assay to measure hydroxylation. Our data with 727 collagenous peptides indicated that a positive charge on the peptide and specific amino acid residues in close proximity to the lysyl residues in the collagenous sequences are the key factors promoting peptide binding to the LH isoforms. The data suggest that the LH binding site is not a deep hydrophobic pocket but is open and hydrophilic where acidic amino acids play an important role in the binding. The data do not indicate strict sequence specificity for the LH isoforms, but the data indicated that there was a clear preference for some sequences to be bound and hydroxylated by a certain isoform.

Novel role of tyrosine in catalysis by human AP endonuclease 1

Apurinic/apyrimidinic endonuclease (AP endo, HAP1) recognizes abasic sites in ds DNA and makes a single nick in the backbone 5′ to the abasic site. In this report we examine the roles of three conserved tyrosine residues in close proximity to the active site. We show that Tyr 128 and Tyr 269, which interact upstream and downstream of the abasic site, respectively, are involved in recognition and binding of abasic site-containing double stranded DNA. However, the two residues are not equivalent, as their effects are differentiated by changes in salt concentration. In sharp contrast, Tyr 171 is directly involved in catalysis as well as binding. Y171F, Y171H, and Y171A all show decreased catalytic efficiencies 25,000–50,000-fold from the WT enzyme. Both imidazole and basic pH markedly stimulate the WT enzyme. Imidazole stimulates Tyr 171 mutant enzymes when tyrosine is also present but basic pH eliminates remaining mutant activity. These results underscore the importance of tyrosines in AP endo catalysis. They render the current hypotheses regarding enzyme action unlikely and allow us to consider the possibility that the phenolate of Tyr 171 is the nucleophile that attacks the scissile phosphate.