Analysis of a conformation-specific epitope of the alpha subunit of human chorionic gonadotropin: Study using monoclonal antibody probes

Monoclonal antibodies (MAbs) have been used extensively for identification of sequence-specific epitopes using either the ELISA or/and IRMA methods, However, attempts to use MAbs for identification of conformation-specific epitopes have been very few as they are considered very labile. We have investigated the stability of conformation-specific epitopes of human chorionic gonadotropin (hCG) using a quantitative solid-phase radioimmnunoassay (SPRIA) technique. Several epitopes are stable to mild modification (chemical and proteolytic) conditions, and epitopes show differential stability for these modifications. Based on these observations, a monoclonal antibody (MAb 16) for an a-subunit-specific epitope of hCG has been used to monitor changes at the epitopic site (identified as epitope 16) on modification of hCG, using SPRIA with immobilized MAb 16. Modifications of amino groups, hydroxyl group of tyrosine as well as carboxyl group of Asp/Glu all bring about sufficient changes in the epitope integrity. Peptide bond hydrolysis at lysine residues damages the epitope, but not at arginine residues, Hydrolysis at tyrosine does not affect the epitope, though modification of the side-chain of tyrosine inactivates the epitope. Destruction of the epitope occurs on reduction of the disulphide bonds. Partial retention of the epitope activity is seen on modification of carboxyl or the epsilon-amino groups of lysine. Based on these results four to six amino acids have been identified to be at the epitopic site, and the data suggest that two peptide segments are brought together by the disulphide bond Cys10-Cys60 to form the epitope.

Functional analysis of conserved motifs in EcoP15I DNA methyltransferase

EcoP15I DNA methyltransferase recognizes the sequence 5'-CAGCAG-3' and transfers a methyl group to N-6 of the second adenine residue in the recognition sequence. All N-6 adenine methyltransferases contain two highly conserved sequences, FxGxG (motif I), postulated to form part of the S-adenosyl-L-methionine binding site and (D/N/S)PP(Y/F) (motif IV) involved in catalysis. We have altered the second glycine residue in motif I to arginine and serine, and substituted tyrosine in motif IV with tryptophan in EcoP15I DNA methyltransferase, using site-directed mutagenesis. The mutant enzymes were overexpressed, purified and characterized by biochemical methods. The mutations in motif I completely abolished AdoMet binding but left target DNA recognition unaltered. Although the mutation in motif IV resulted in loss of enzyme activity, we observed enhanced crosslinking of S-adenosyl-L-methionine and DNA. This implies that DNA and AdoMet binding sites are close to motif IV. Taken together, these results reinforce the importance of motif I in AdoMet binding and motif IV in catalysis. Additionally, limited proteolysis and UV crosslinking experiments with EcoP15I DNA methyltransferase imply that DNA binds in a cleft formed by two domains in the protein. Methylation protection analysis provides evidence for the fact that EcoP15I DNA MTase makes contacts in the major groove of its substrate DNA. Interestingly, hypermethylation of the guanine residue next to the target adenine residue indicates that the protein probably flips out the target adenine residue. (C) 1996 Academic Press Limited

Role of Water in the Enzymatic Catalysis: Study of ATP plus AMP -> 2ADP Conversion by Adenylate Kinase

The catalytic conversion ATP + AMP -> 2ADP by the enzyme adenylate kinase (ADK) involves the binding of one ATP. molecule to the LID domain and one AMP molecule to the NMP domain. The latter is followed by a. phosphate transfer and then the release of two ADP molecules. We have computed a novel two-dimensional configurational free energy surface (2DCFES), with one reaction coordinate each for the LID and the NMP domain motions, while considering explicit water interactions. Our computed 2DCFES clearly reveals the existence of a stable half-open half-closed (HOHC) intermediate stale of the enzyme. Cycling of the enzyme through the HOHC state reduces the conformational free energy barrier for. the reaction by about 20 kJ/mol. We find that the stability of the HOHC state (missed in all earlier studies with implicit solvent model) is largely because of the increase of specific interactions of the polar amino acid side chains with water, particularly with the arginine and the histidine residues. Free energy surface of the LID domain is rather rugged, which can conveniently slow down LID's conformational motion, thus facilitating a new substrate capture after the product release in the catalytic cycle.

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.

Location and conformation of pantothenate and its derivatives in Mycobacterium tuberculosis pantothenate kinase: insights into enzyme action

Previous studies of complexes of Mycobacterium tuberculosis PanK (MtPanK) with nucleotide diphosphates and non-hydrolysable analogues of nucleoside triphosphates in the presence or the absence of pantothenate established that the enzyme has dual specificity for ATP and GTP, revealed the unusual movement of ligands during enzyme action and provided information on the effect of pantothenate on the location and conformation of the nucleotides at the beginning and the end of enzyme action. The X-ray analyses of the binary complexes of MtPanK with pantothenate, pantothenol and N-nonylpantothenamide reported here demonstrate that in the absence of nucleotide these ligands occupy, with a somewhat open conformation, a location similar to that occupied by phosphopantothenate in the `end' complexes, which differs distinctly from the location of pantothenate in the closed conformation in the ternary `initiation' complexes. The conformation and the location of the nucleotide were also different in the initiation and end complexes. An invariant arginine appears to play a critical role in the movement of ligands that takes place during enzyme action. The work presented here completes the description of the locations and conformations of nucleoside diphosphates and triphosphates and pantothenate in different binary and ternary complexes, and suggests a structural rationale for the movement of ligands during enzyme action. The present investigation also suggests that N-alkylpantothenamides could be phosphorylated by the enzyme in the same manner as pantothenate.

Lysozyme: A model protein for amyloid research

Ever since lysozyme was discovered by Fleming in 1922, this protein has emerged as a model for investigations on protein structure and function. Over the years, several high-resolution structures have yielded a wealth of structural data on this protein. Extensive studies on folding of lysozyme have shown how different regions of this protein dynamically interact with one another. Data is also available from numerous biotechnological studies wherein lysozyme has been employed as a model protein for recovering active recombinant protein from inclusion bodies using small molecules like L-arginine. A variety of conditions have been developed in vitro to induce fibrillation in hen lysozyme. They include (a) acidic pH at elevated temperature, (b) concentrated solutions of ethanol, (c) moderate concentrations of guanidinium hydrochloride at moderate temperature, and (d) alkaline pH at room temperature. This review aims to bring together similarities and differences in aggregation mechanisms, morphology of aggregates, and related issues that arise using the different conditions mentioned above to improve our understanding. The alkaline pH condition (pH 12.2), discovered and studied extensively in our lab, shall receive special attention. More than a decade ago, it was revealed that mutations in human lysozyme can cause accumulation of large quantities of amyloid in liver, kidney, and other regions of gastrointestinal tract. Understanding the mechanism of lysozyme aggregation will probably have therapeutic implications for the treatment of systemic nonneuropathic amyloidosis. Numerous studies have begun to focus attention on inhibition of lysozyme aggregation using antibody or small molecules. The enzymatic activity of lysozyme presents a convenient handle to quantify the native population of lysozyme in a sample where aggregation has been inhibited. The rich information available on lysozyme coupled with the multiple conditions that have been successful in inducing/inhibiting its aggregation in vitro makes lysozyme an ideal model protein to investigate amyloidogenesis.

15-deoxyspergualin hinders physical interaction between basic residues of transit peptide in PfENR and Hsp70-1

The apicoplast of Plasmodium harbors several metabolic pathways. The enzymes required to perform these reactions are all nuclearly encoded and apicoplast targeted (NEAT) proteins. Plasmodium falciparum Enoyl-ACP Reductase (PfENR) is one such NEAT protein. The NEAT proteins have a transit peptide which is required for crossing the membranes of apicoplast. We studied the importance of basic residues like Arginine and Lysine within the transit peptide. Previous studies have suggested that all basic residues are essential for apicoplast trafficking. In this study, we demonstrate that only some of these residues are essential (K44, R48, K51, and R52), whereas others are dispensable (R40, K42, and K49). On mutating these specific residues, PfENR is not imported into the apicoplast and is mislocalized to the cytoplasm. We also demonstrate that these residues are also crucial for interaction with Hsp70-1, implying that interactions of Lysine 44, Arginine 48, Lysine 51, and Arginine 52 of the transit peptide with PfHsp70-1 are required for apicoplast trafficking. 15-Deoxyspergualin, which has earlier been proposed to interact with EEVD motif of PfHsp70-1 hinders the physical interaction between these cationic residues of PfENR and Hsp70-1. Hence, we propose that in the transport competent state of NEAT proteins some specific positively charged amino acids in the transit peptide interact with PfHsp70-1, and this interaction is essential for apicoplast targeting.

Functional characterization of UvrD helicases from Haemophilus influenzae and Helicobacter pylori

Haemophilus influenzae and Helicobacter pylori are major bacterial pathogens that face high levels of genotoxic stress within their host. UvrD, a ubiquitous bacterial helicase that plays important roles in multiple DNA metabolic pathways, is essential for genome stability and might, therefore, be crucial in bacterial physiology and pathogenesis. In this study, the functional characterization of UvrD helicase from Haemophilus influenzae and Helicobacter pylori is reported. UvrD from Haemophilus influenzae (HiUvrD) and Helicobacter pylori (HpUvrD) exhibit strong single-stranded DNA-specific ATPase and 3'5' helicase activities. Mutation of highly conserved arginine (R288) in HiUvrD and glutamate (E206) in HpUvrD abrogated their activities. Both the proteins were able to bind and unwind a variety of DNA structures including duplexes with strand discontinuities and branches, three- and four-way junctions that underpin their role in DNA replication, repair and recombination. HiUvrD required a minimum of 12 nucleotides, whereas HpUvrD preferred 20 or more nucleotides of 3'-single-stranded DNA tail for efficient unwinding of duplex DNA. Interestingly, HpUvrD was able to hydrolyze and utilize GTP for its helicase activity although not as effectively as ATP, which has not been reported to date for UvrD characterized from other organisms. HiUvrD and HpUvrD were found to exist predominantly as monomers in solution together with multimeric forms. Noticeably, deletion of distal C-terminal 48 amino acid residues disrupted the oligomerization of HiUvrD, whereas deletion of 63 amino acids from C-terminus of HpUvrD had no effect on its oligomerization. This study presents the characteristic features and comparative analysis of Haemophilus influenzae and Helicobacter pylori UvrD, and constitutes the basis for understanding the role of UvrD in the biology and virulence of these pathogens.

IDH1 Mutations in Diffusely Infiltrating Astrocytomas Grade Specificity, Association With Protein Expression, and Clinical Relevance

IDH1 mutations are frequent genetic alterations in low-grade diffuse gliomas and secondary glioblastoma (GBM). To validate mutation frequency, IDH1 gene at codon 132 was sequenced in 74 diffusely infiltrating astrocytomas: diffuse astrocytoma (DA; World Health Organization WHO] grade II), anaplastic astrocytoma (AA; WHO grade III), and GBM (WHO grade IV). All cases were immunostained with IDH1-R132H monoclonal antibody. Mutational status was correlated with mutant protein expression, patient age, duration of symptoms, and prognosis of patients with GBM. We detected 31 (41.9%) heterozygous IDH1 mutations resulting in arginine-to-histidine substitution (R132H;CGT-CAT). All 12 DAs (100%), 13 of 14 AAs (92.9%), and 6 of 48 GBMs (12.5%) (5/6 83.3%] secondary, and 1/42 2.4%] primary) harbored IDH1 mutations. The correlation between mutational status and protein expression was significant (P < .001). IDH1 mutation status, though not associated with prognosis of patients with GBM, showed significant association with younger age and longer duration of symptoms in the whole cohort (P < .001). Our study validates IDH1 mutant protein expression across various grades of astrocytoma, and demonstrates a high incidence of IDH1 mutations in DA, AA, and secondary GBM.

Theoretical and in vitro studies of a C-terminal peptide from PGKC of Leishmania mexicana mexicana

Trypanosomatids cause deadly diseases in humans. Of the various biochemical pathways in trypanosomatids, glycolysis, has received special attention because of being sequestered in peroxisome like organelles critical for the survival of the parasites. This study focuses on phosphoglycerate kinase (PGK) from Leishmania spp. which, exists in two isoforms, the cytoplasmic PGKB and glycosomal PGKC differing in their biochemical properties. Computational analysis predicted the likelihood of a transmembrane helix only in the glycosomal isoform PGKC, of approximate length 20 residues in the 62-residue extension, ending at, arginine residues R471 and R472. From experimental studies using circular dichroism and NMR with deuterated sodium dodecyl sulfate, we find that the transmembrane helix spans residues 448 +/- 2 to 476 in Leishmania mexicana PGKC. The significance of this observation is discussed in the context of glycosomal transport and substrate tunneling. (C) 2012 Elsevier B.V. All rights reserved.

Biochemical characterization of C4 protein of cotton leaf curl Kokhran virus-Dabawali

Background: Cotton leaf curl Kokhran Virus-Dabawali (CLCuKV-Dab) is a monopartite begomovirus encoding two proteins V1 and V2 in the virion sense and four proteins Cl, C2, C3 and C4 in the complementary sense. The C4 protein of monopartite begomoviruses has been implicated to play a role in symptom determination and virus movement. The present work aims at the biochemical characterization of this protein. Methods: The C4 protein of CLCuKV-Dab was purified in fusion with GST and tested for the ability to hydrolyze ATP and other phosphate containing compounds. ATPase activity was assayed by using radiolabeled gamma-32P]-ATP and separating the product of reaction by thin layer chromatography. The hydrolysis of other compounds was monitored by the formation of a blue colored phosphomolybdate complex which was estimated by measuring the absorbance at 655 nm. Results: The purified GST-C4 protein exhibited metal ion dependent ATPase and inorganic pyrophosphatase activities. Deletion of a sequence resembling the catalytic motif present in phosphotyrosine phosphatases resulted in 70% reduction in both the activities. Mutational analysis suggested arginine 13 to be catalytically important for the ATPase and cysteine 8 for the pyrophosphatase activity of GST-C4. Interaction of V2 with GST-C4 resulted in an increase in both the enzymatic activities of GST-C4. Conclusions: The residues important for the enzymatic activities of GST-C4 are present in a motif different from the classical Walker motifs and the non-classical ATP binding motifs reported so far. General significance: The C4 protein of CLCuKV-Dab, a putative natively unfolded protein, exhibits enzymatic activities.

A novel adeno-associated virus serotype (AAV)-8 capsid mutant improves human coagulation factor IX expression in vivo

Recombinant AAV-8 vectors have shown significant promise for hepatic gene therapy of hemophilia B. However, the theme of AAV vector dose dependent immunotoxicity seen with AAV2 vectors earlier seem to re-emerge with AAV8 vectors as well. It is therefore important to develop novel AAV8 vectors that provide enhanced gene expression at significantly less vector doses. We hypothesized that AAV8 during its intracellular trafficking, are targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal degradation machinery and modification of specific serine/threonine kinase or ubiquitination targets on AAV8 capsid (Fig.1A) may improve its transduction efficiency. To test this, point mutations at specific serine (S)/threonine (T) > alanine (A) or lysine (K)>arginine (R) residues were generated on AAV8 capsid. scAAV8-EGFP vectors containing the wild-type (WT) and each one of the 5 S/T/K-mutant(S276A, S501A, S671A, T251A and K137R) capsids were evaluated for their liver transduction efficiency at a dose of 5 X 1010 vgs/ animal in C57BL/6 mice in vivo. The best performing mutant was found to be the K137R vector in terms of either the gene expression (46-fold) or the vector copy numbers in the hepatocytes (22-fold) compared to WT-AAV8 (Fig.1B). The K137R-AAV8 vector that showed significantly decreased ubiquitination of the viral capsid had reduced activation of markers of innate immune response [IL-6, IL-12, tumor necrosis factor α, Kupffer cells and TLR-9]. In addition, animals injected with the K137R mutant also demonstrated decreased (2-fold) levels of cross-neutralizing antibodies when compared to animals that received the WT-AAV8 vector. To study further the utility of the novel AAV8-K137R mutant in a therapeutic setting, we delivered human coagulation factor IX (h.FIX) under the control of liver specific promoters (LP1 or hAAT) at two different doses (2.5x10^10 and 1x10^11 vgs per mouse) in 8-12 weeks old male C57BL/6 mice. As can be seen in Fig.1C/D, the circulating levels of h.FIX were higher in all the K137R-AAV8 treated groups as compared to the WT-AAV8 treated groups either at 2 weeks (62% vs 37% for hAAT constructs and 47% vs 21% for LP1 constructs) or 4 weeks (78% vs 56% for hAAT constructs and 64% vs 30% for LP1 constructs) post hepatic gene transfer. These studies demonstrate the feasibility of the use of this novel vector for potential gene therapy of hemophilia B.

Cloning, expression, purification, crystallization and preliminary X-ray studies of argininosuccinate lyase (Rv1659) from Mycobacterium tuberculosis

The last enzyme in the arginine-biosynthesis pathway, argininosuccinate lyase, from Mycobacterium tuberculosis has been cloned, expressed, purified and crystallized, and preliminary X-ray studies have been carried out on the crystals. The His-tagged tetrameric enzyme with a subunit molecular weight of 50.9 kDa crystallized with two tetramers in the asymmetric unit of the orthorhombic unit cell, space group P2(1)2(1)2(1). Molecular-replacement calculations and self-rotation calculations confirmed the space group and the tetrameric nature of the molecule.

The N-terminal region containing the zinc finger domain of tobacco streak virus coat protein is essential for the formation of virus-like particles

Tobacco streak virus (TSV), a member of the genus Ilarvirus (family Bromoviridae), has a tripartite genome and forms quasi-isometric virions. All three viral capsids, encapsidating RNA 1, RNA 2 or RNA 3 and subgenomic RNA 4, are constituted of a single species of coat protein (CP). Formation of virus-like particles (VLPs) could be observed when the TSV CP gene was cloned and the recombinant CP (rCP) was expressed in E. coli. TSV VLPs were found to be stabilized by Zn2+ ions and could be disassembled in the presence of 500 mM CaCl2. Mutational analysis corroborated previous studies that showed that an N-terminal arginine-rich motif was crucial for RNA binding; however, the results presented here demonstrate that the presence of RNA is not a prerequisite for assembly of TSV VLPs. Instead, the N-terminal region containing the zinc finger domain preceding the arginine-rich motif is essential for assembly of these VLPs.

Structural and kinetic studies on adenylosuccinate lyase from Mycobacterium smegmatis and Mycobacterium tuberculosis provide new insights on the catalytic residues of the enzyme

Adenylosuccinate lyase (ASL), an enzyme involved in purine biosynthesis, has been recognized as a drug target against microbial infections. In the present study, ASL from Mycobacteriumsmegmatis (MsASL) and Mycobacteriumtuberculosis (MtbASL) were cloned, purified and crystallized. The X-ray crystal structure of MsASL was determined at a resolution of 2.16 angstrom. It is the first report of an apo-ASL structure with a partially ordered active site C3 loop. Diffracting crystals of MtbASL could not be obtained and a model for its structure was derived using MsASL as a template. These structures suggest that His149 and either Lys285 or Ser279 of MsASL are the residues most likely to function as the catalytic acid and base, respectively. Most of the active site residues were found to be conserved, with the exception of Ser148 and Gly319 of MsASL. Ser148 is structurally equivalent to a threonine in most other ASLs. Gly319 is replaced by an arginine residue in most ASLs. The two enzymes were catalytically much less active compared to ASLs from other organisms. Arg319Gly substitution and reduced flexibility of the C3 loop might account for the low catalytic activity of mycobacterial ASLs. The low activity is consistent with the slow growth rate of Mycobacteria and their high GC containing genomes, as well as their dependence on other salvage pathways for the supply of purine nucleotides. Structured digital abstract andby()