The role of His-134, -147, and -150 residues in subunit assembly, cofactor binding, and catalysis of sheep liver cytosolic serine hydroxymethyltransferase

In an attempt to unravel the role of conserved histidine residues in the structure-function of sheep liver cytosolic serine hydroxymethyltransferase (SHMT), three site-specific mutants (H134N, H147N, and H150N) were constructed and expressed, H134N and H147N SHMTs had K-m values for L-serine, L-allo-threonine and beta-phenylserine similar to that of wild type enzyme, although the k(cat) values were markedly decreased, H134N SHMT was obtained in a dimeric form with only 6% of bound pyridoxal 5'-phosphate (PLP) compared with the wild type enzyme, Increasing concentrations of PLP (up to 500 mu M) enhanced the enzyme activity without changing its oligomeric structure, indicating that His-134 may be involved in dimer-dimer interactions, H147N SHMT was obtained in a tetrameric form but with very little PLP (3%) bound to it, suggesting that this residue was probably involved in cofactor binding, Unlike the wild type enzyme, the cofactor could be easily removed by dialysis from H147N SHMT, and the apoenzyme thus formed was present predominantly in the dimeric form, indicating that PLP binding is at the dimer-dimer interface, H150N SHMT was obtained in a tetrameric form with bound PLP, However, the mutant had very little enzyme activity (<2%). The k(cat)/K-m values for L-serine, L-allo-threonine and beta-phenylserine were 80-, 56-, and SS-fold less compared with wild type enzyme, Unlike the wild type enzyme, it failed to form the characteristic quinonoid intermediate and was unable to carry out the exchange of 2-S proton from glycine in the presence of H-4-folate. However, it could form an external aldimine with serine and glycine, The wild type and the mutant enzyme had similar K-d values for serine and glycine, These results suggest that His-150 may be the base that abstracts the alpha-proton of the substrate, leading to formation of the quinonoid intermediate in the reaction catalyzed by SHMT.

Role of Arg-401 of cytosolic serine hydroxymethyltransferase in subunit assembly and interaction with the substrate carboxy group.

In an attempt to identify the arginine residue involved in binding of the carboxylate group of serine to mammalian serine hydroxymethyltransferase, a highly conserved Arg-401 was mutated to Ala by site-directed mutagenesis. The mutant enzyme had a characteristic visible absorbance at 425 nm indicative of the presence of bound pyridoxal 5'-phosphate as an internal aldimine with a lysine residue. However, it had only 0.003% of the catalytic activity of the wild-type enzyme. It was also unable to perform reactions with glycine, beta-phenylserine or d-alanine, suggesting that the binding of these substrates to the mutant enzyme was affected. This was also evident from the interaction of amino-oxyacetic acid, which was very slow (8.4x10(-4) s-1 at 50 microM) for the R401A mutant enzyme compared with the wild-type enzyme (44.6 s-1 at 50 microM). In contrast, methoxyamine (which lacks the carboxy group) reacted with the mutant enzyme (1.72 s-1 at 250 microM) more rapidly than the wild-type enzyme (0.2 s-1 at 250 microM). Further, both wild-type and the mutant enzymes were capable of forming unique quinonoid intermediates absorbing at 440 and 464 nm on interaction with thiosemicarbazide, which also does not have a carboxy group. These results implicate Arg-401 in the binding of the substrate carboxy group. In addition, gel-filtration profiles of the apoenzyme and the reconstituted holoenzyme of R401A and the wild-type enzyme showed that the mutant enzyme remained in a tetrameric form even when the cofactor had been removed. However, the wild-type enzyme underwent partial dissociation to a dimer, suggesting that the oligomeric structure was rendered more stable by the mutation of Arg-401. The increased stability of the mutant enzyme was also reflected in the higher apparent melting temperature (Tm) (61 degrees C) than that of the wild-type enzyme (56 degrees C). The addition of serine or serinamide did not change the apparent Tm of R401A mutant enzyme. These results suggest that the mutant enzyme might be in a permanently 'open' form and the increased apparent Tm could be due to enhanced subunit interactions.

Ligand dependent intra and inter subunit communication in human tryptophanyl tRNA synthetase as deduced from the dynamics of structure networks

Homodimeric protein tryptophanyl tRNA synthetase (TrpRS) has a Rossmann fold domain and belongs to the 1c subclass of aminoacyl tRNA synthetases. This enzyme performs the function of acylating the cognate tRNA. This process involves a number of molecules (2 protein subunits, 2 tRNAs and 2 activated Trps) and thus it is difficult to follow the complex steps in this process. Structures of human TrpRS complexed with certain ligands are available. Based on structural and biochemical data, mechanism of activation of Trp has been speculated. However, no structure has yet been solved in the presence of both the tRNA(Trp) and the activated Trp (TrpAMP). In this study, we have modeled the structure of human TrpRS bound to the activated ligand and the cognate tRNA. In addition, we have performed molecular dynamics (MD) simulations on these models as well as other complexes to capture the dynamical process of ligand induced conformational changes. We have analyzed both the local and global changes in the protein conformation from the protein structure network (PSN) of MD snapshots, by a method which was recently developed in our laboratory in the context of the functionally monomeric protein, methionyl tRNA synthetase. From these investigations, we obtain important information such as the ligand induced correlation between different residues of this protein, asymmetric binding of the ligands to the two subunits of the protein as seen in the crystal structure analysis, and the path of communication between the anticodon region and the aminoacylation site. Here we are able to elucidate the role of dimer interface at a level of detail, which has not been captured so far.

Kinetic characterization of rat brain type IIA sodium channel alpha-subunit stably expressed in a somatic cell line

1. The rat brain type IIA Na+ channel alpha-subunit was stably expressed in Chinese hamster ovary (CHO) cells. Current through the expressed Na+ channels was studied using the whole-cell configuration of the patch clamp technique. The transient Na+ current was sensitive to TTX and showed a bell-shaped peak current vs. membrane potential relation. 2. Na+ current inactivation was better described by the sum of two exponentials in the potential range -30 to +40 mV, with. a dominating fast component and a small slower component. 3. The steady-state inactivation, h(infinity), was related to potential by a Boltzmann distribution, underlying thr ee states of the inactivation gate. 4. Recovery of the channels from inactivation at different potentials in the range -70 to -120 mV were characterized by al? initial delay which decreased with hyperpolarization. The time course was well fitted by the sum of two exponentials. In this case the slower exponential was the major component, and both time constants decreased with hyperpolarization. 5. For a working description of the Na+ channel inactivation in this preparation, with a minimal deviation from the Hodgkin-Huxley model, a three-state scheme of the form O reversible arrow I-1 reversible arrow I-2 was proposed, replacing the original two-state scheme of the Hodgkin-Huxley model, and the rate constants are reported. 6. The instantaneous current-voltage relationship showed marked deviation from linearity and was satisfactorily fitted by the constant-field equation. 7. The time course of activation was described by an m(x) model. However, the best-fitted value of x varied with the membrane potential and had a mean value of 2. 8. Effective gating charge was determined to be 4.7e from the slope of the activation plot, plotted on a logarithmic scale. 9. The rate constants of activation, alpha(m) and beta(m), were determined. Their functional dependence on the membrane potential was investigated.

Identification and characterization of DdRPB4, a subunit of Dictyostelium discoideum RNA polymerase II

Rpb4, the fourth largest subunit of the eukaryotic RNA polymerase II (RNAPII), is required for growth at extreme temperatures and for an appropriate response to nutrient starvation in yeast. Sequence homologs of Rpb4 are found in most sequenced genomes from yeast to humans. To elucidate the role of this subunit in nutrient starvation, we chose Dictyostelium discoideum, a soil amoeba, which responds to nutrient deprivation by undergoing a complex developmental program. Here we report the identification of homolog of Saccharomyces cerevisiae RPB4 in D. discoideum. Localization and complementation studies suggest that Rpb4 is functionally conserved. DdRPB4 transcript and protein levels are developmentally regulated. Although DdRPB4 could not be deleted, overexpression revealed that the Rpb4 protein is essential for cell survival and is regulated stringently at the post-transcriptional level in D. discoideum. Thus maintaining a critical level of Rpb4 is important for this organism.

Supramolecular Organization in Tetra Aqua (mu-8-Hydroxyquinoline-5-sulfonate) Barium (II) and Ag center dot center dot center dot I Interactions in a Pseudopolymorphic Form of (7-Iodo-8-hydroxyquinoline-5-sulfonate) Silver (I) Monohydrate

The complexes, Ba (HQS) (H2O)(4) (HQS = 8-hydroxyquinoline-5-sulfonic acid) (1) and Ag (HIQS) (H2O) (Ferron = 7-iodo-8-hydroxyquinoline-5-sulfonic acid) (2) have been synthesized and characterized by X-ray diffraction analysis and spectroscopic studies. In compound 1, Ba2+ ion has a nine-coordinate monocapped antiprismatic geometry. In compound 2, Ag+ has distorted tetrahedral coordination and Ag center dot center dot center dot I interactions generate the supramolecular architectures. The complexes have been characterized by FT-IR and UV-Visible measurements. In both the structures, the inversion-related organic ligands are stacked over one another leading to three-dimensional networks.

Unstructured N Terminus of the RNA Polymerase II Subunit Rpb4 Contributes to the Interaction of Rpb4·Rpb7 Subcomplex with the Core RNA Polymerase II of Saccharomyces cerevisiae

Two subunits of eukaryotic RNA polymerase II, Rpb7 and Rpb4, form a subcomplex that has counterparts in RNA polymerases I and III. Although a medium resolution structure has been solved for the 12-subunit RNA polymerase II, the relative contributions of the contact regions between the subcomplex and the core polymerase and the consequences of disrupting them have not been studied in detail. We have identified mutations in the N-terminal ribonucleoprotein-like domain of Saccharomyces cerevisiae Rpb7 that affect its role in certain stress responses, such as growth at high temperature and sporulation. These mutations increase the dependence of Rpb7 on Rpb4 for interaction with the rest of the polymerase. Complementation analysis and RNA polymerase pulldown assays reveal that the Rpb4 center dot Rbp7 subcomplex associates with the rest of the core RNA polymerase II through two crucial interaction points: one at the N-terminal ribonucleoprotein-like domain of Rpb7 and the other at the partially ordered N-terminal region of Rpb4. These findings are in agreement with the crystal structure of the 12-subunit polymerase. We show here that the weak interaction predicted for the N-terminal region of Rpb4 with Rpb2 in the crystal structure actually plays a significant role in interaction of the subcomplex with the core in vivo. Our mutant analysis also suggests that Rpb7 plays an essential role in the cell through its ability to interact with the rest of the polymerase.

Glycosphingolipids in microdomain formation and their spatial organization

Plasma membranes regulate the influx and efflux of molecules across themselves and are also responsible for primary signal transduction between cells or within the same cell. Presence of lateral heterogeneity and the ability of reorganization are essential requirements for effective functioning of biomembranes. Lipid rafts are small, heterogeneous, dynamic domains enriched in glycosphingolipids, sphingomyelin and cholesterol, and profoundly influence membrane organization. Glycosphingolipids are inclined towards formation of liquid-ordered phases in membranes, both with and without cholesterol; they are therefore prime players in domain formation. Here, we discuss the role of glycosphingolipids in microdomain formation and their spatial organization within these rafts.

Structure-function relationships of icosahedral plant

X-ray diffraction studies on single crystals of a few viruses have led to the elucidation of their three dimensional structure at near atomic resolution. Both the tertiary structure of the coat protein subunit and the quaternary organization of the icosahedral capsid in these viruses are remarkably similar. These studies have led to a critical re-examination of the structural principles in the architecture of isometric viruses and suggestions of alternative mechanisms of assembly. Apart from their role in the assembly of the virus particle, the coat proteins of certian viruses have been shown to inhibit the replication of the cognate RNA leading to cross-protection. The coat protein amino acid sequence and the genomic sequence of several spherical plant RNA viruses have been determined in the last decade. Experimental data on the mechanisms of uncoating, gene expression and replication of several classes of viruses have also become available. The function of the non-structural proteins of some viruses have been determined. This rapid progress has provided a wealth of information on several key steps in the life cycle of RNA viruses. The function of the viral coat protein, capsid architecture, assembly and disassembly and replication of isometric RNA plant viruses are discussed in the light of this accumulated knowledge.

Relative ability of ovine follicle stimulating hormone and its beta-subunit to generate antibodies having bioneutralization potential in nonhuman primates

The relative ability of ovine follicle stimulating hormone and its beta-subunit, two potential candidates for male contraceptive vaccine, to generate antibodies in monkeys capable of bioneutralizing follicle stimulating hormone was assessed using in vitro model systems. Antiserum against native ovine follicle stimulating hormone was found to be highly specific to the intact form with no cross-reactivity with either of the two subunits while the antiserum against beta-subunit of follicle stimulating hormone could bind to the beta-subunit in its free form as well as when it is combined with alpha-subunit to form the intact hormone. Both antisera could block the binding of the hormone to the receptor if the hormone was preincubated with the antibody. However, the follicle stimulating hormone beta-antisera could only inhibit the binding of the hormone partially (33 percent inhibition) if the antibody and receptor were mixed prior to the addition of the hormone, while antisera to the native follicle stimulating hormone could block the binding completely (100 percent inhibition) in the same experiment. Similarly antisera to the native follicle stimulating hormone was significantly effective in blocking (100 percent) response to follicle stimulating hormone but not the beta-subunit antisera (0 percent) as checked using an in vitro granulosa cell system. Thus the probability of obtaining antibodies of greater bioneutralization potential is much higher if intact hormone is used as an antigen rather than its beta-subunit as a vaccine.

Ability of human chorionic gonadotropin beta-subunit to inhibit the steroidogenic response to lutropin

Ability of the beta-subunit of human chorionic gonadotropin to inhibit the response to lutropin (luteinizing hormone, LH) was tested in the immature rat ovarian system and pregnant-mare-serum-gonadotropin-primed rat ovarian system with progesterone production being used as the response. Human chorionic gonadotropin beta-subunit was found to inhibit human and ovine lutropin-stimulated progesterone production. At a constant dose of lutropin, inhibition was dependent on the concentration of beta-subunit. When concentration of the beta-subunit was kept constant at 5.0 microgram/ml and the concentration of lutropin was varied, the inhibition was maximum at the saturating concentration of the native hormone. The alpha-subunit of the human chorionic gonadotropin did not inhibit the response to lutropin. The lutropin/beta-subunit ratio required to produce an inhibition of response was much lower than that required to bring about an observable inhibition of binding.

DNA Clasping by Mycobacterial HU: The C-Terminal Region of HupB Mediates Increased Specificity of DNA Binding

Background: HU a small, basic, histone like protein is a major component of the bacterial nucleoid. E. coli has two subunits of HU coded by hupA and hupB genes whereas Mycobacterium tuberculosis (Mtb) has only one subunit of HU coded by ORF Rv2986c (hupB gene). One noticeable feature regarding Mtb HupB, based on sequence alignment of HU orthologs from different bacteria, was that HupB(Mtb) bears at its C-terminal end, a highly basic extension and this prompted an examination of its role in Mtb HupB function. Methodology/Principal Findings: With this objective two clones of Mtb HupB were generated; one expressing full length HupB protein (HupB(Mtb)) and another which expresses only the N terminal region (first 95 amino acid) of hupB (HupB(MtbN)). Gel retardation assays revealed that HupBMtbN is almost like E. coli HU (heat stable nucleoid protein) in terms of its DNA binding, with a binding constant (K-d) for linear dsDNA greater than 1000 nM, a value comparable to that obtained for the HU alpha alpha and HU alpha beta forms. However CTR (C-terminal Region) of HupB(Mtb) imparts greater specificity in DNA binding. HupB(Mtb) protein binds more strongly to supercoiled plasmid DNA than to linear DNA, also this binding is very stable as it provides DNase I protection even up to 5 minutes. Similar results were obtained when the abilities of both proteins to mediate protection against DNA strand cleavage by hydroxyl radicals generated by the Fenton's reaction, were compared. It was also observed that both the proteins have DNA binding preference for A: T rich DNA which may occur at the regulatory regions of ORFs and the oriC region of Mtb. Conclusions/Significance: These data thus point that HupB(Mtb) may participate in chromosome organization in-vivo, it may also play a passive, possibly an architectural role.

Passive immunization with an antibody to the beta-subunit of ovine luteinizing hormone as a method of early abortion--a feasibility study in monkeys (Macaca radiata)

Antiserum to the beta-subunit of ovine luteinizing hormone (oLH-beta) raised in monkeys (Macaca radiata) has been tested by a variety of criteria both in vivo and in vitro to establish its ability to neutralize oLH, hLH, and human chorionic gonadotropin (hCG). Passive administration of this antiserum caused inhibition of ovulation and termination of pregnancy in recipient monkeys as indicated by premature vaginal bleeding and a significant reduction in serum progesterone and estrogen levels. The results suggest that antiserum raised in monkeys against oLH-beta can neutralize monkey LH as well as monkey CG.