Atomic resolution analysis of the catalytic site of an aspartic proteinase and an unexpected mode of binding by short peptides

The X-ray structures of native endothiapepsin and a complex with a hydroxyethylene transition state analog inhibitor (H261) have been determined at atomic resolution. Unrestrained refinement of the carboxyl groups of the enzyme by using the atomic resolution data indicates that both catalytic aspartates in the native enzyme share a single negative charge equally; that is, in the crystal, one half of the active sites have Asp 32 ionized and the other half have Asp 215 ionized. The electron density map of the native enzyme refined at 0.9 A resolution demonstrates that there is a short peptide (probably Ser-Thr) bound noncovalently in the active site cleft. The N-terminal nitrogen of the dipeptide interacts with the aspartate diad of the enzyme by hydrogen bonds involving the carboxyl of Asp 215 and the catalytic water molecule. This is consistent with classical findings that the aspartic proteinases can be inhibited weakly by short peptides and that these enzymes can catalyze transpeptidation reactions. The dipeptide may originate from autolysis of the N-terminal Ser-Thr sequence of the enzyme during crystallization.     

Detergent structure in crystals of the integral membrane light-harvesting complex LH2 from Rhodopseudomonas acidophila strain 10050

Integral membrane proteins are solubilized by their incorporation into a detergent micelle. The detergent micelle has a critical influence on the formation of a three-dimensional crystal lattice. The bulk detergent phase is not seen in X-ray crystal structures of integral membrane proteins, due to its disordered character. Here, we describe the detergent structure present in crystals of the peripheral light-harvesting complex of the purple bacteria Rhodopseudomonas acidophila strain 10050 at a maximal resolution of 12A as determined by neutron crystallography. The LH2 molecule has a toroidal shape and spans the membrane completely in vivo. A volume of 16% of the unit cell could be ascribed to detergent tails, localized on both the inner and outer hydrophobic surfaces of the molecule. The detergent tail volumes were found to be associated with individual LH2 molecules and had no direct role in the formation of the crystalline lattice.     

None of the integrins known to be present on the mouse egg or to be ADAM receptors are essential for sperm-egg binding and fusion

Antibody inhibition and alpha6beta1 ligand binding experiments indicate that the egg integrin alpha6beta1 functions as a receptor for sperm during gamete fusion; yet, eggs null for the alpha6 integrin exhibit normal fertilization. Alternative integrins may be involved in sperm-egg binding and fusion and could compensate for the absence of alpha6beta1. Various beta1 integrins and alphav integrins are present on mouse eggs. Some of these integrins are also reported to be receptors for ADAMs, which are expressed on sperm. Using alpha3 integrin null eggs, we found that the alpha3beta1 integrin was not essential for sperm-egg binding and fusion. Oocyte-specific, beta1 integrin conditional knockout mice allowed us to obtain mature eggs lacking all beta1 integrins. We found that the beta1 integrin null eggs were fully functional in fertilization both in vivo and in vitro. Furthermore, neither anti-mouse beta3 integrin function-blocking monoclonal antibody (mAb) nor alphav integrin function-blocking mAb inhibited sperm binding to or fusion with beta1 integrin null eggs. Thus, function of beta3 or alphav integrins does not seem to be involved in compensating for the absence of beta1 integrins. These results indicate that none of the integrins known to be present on mouse eggs or to be ADAM receptors are essential for sperm-egg binding/fusion, and thus, egg integrins may not play the role in gamete fusion previously attributed to them.     

Porcine oocyte activation: differing roles of calcium and pH

Intracellular pH has recently been shown to increase during parthenogenetic activation of the porcine oocyte. In the following set of experiments, intracellular pH was monitored during activation and pronuclear development was assessed following activation treatments with calcium, in the absence of calcium, and in oocytes loaded with the calcium chelator BAPTA-AM in calcium-free medium. Intracellular pH increase was not different among groups when treating with 7% ethanol or 50 microM calcium ionophore, or during treatment with thimerosal for 12 or 25 min. Activation with thimerosal (200 microM, 12 min) followed by 8 mM dithiothreitol (DTT, 30 min) resulted in a decreased pronuclear development in calcium-free medium with or without BAPTA-AM loaded oocytes as compared to controls. Activation with 50 microM calcium ionophore resulted in pronuclear development that was different between the calcium-free and BAPTA-AM loaded oocytes in calcium-free medium. Similar incidences of pronuclear formation were observed in all ethanol treatment groups. It was concluded that external calcium as well as large changes in intracellular free calcium are not necessary for the increase in intracellular pH, but normal intracellular calcium signaling is critical for normal levels of pronuclear development. Finally, oocytes were measured for intracellular pH changes for 30 min following subzonal sperm injection. Intracellular pH did not increase, although pronuclear formation was observed 6 hr post SUZI. This suggested that major differences were still present between sperm-induced and parthenogenetic activation of the porcine oocyte.     

Spectral properties of bacterial nitric-oxide reductase: resolution of pH-dependent forms of the active site heme b3

Bacterial nitric-oxide reductase catalyzes the two electron reduction of nitric oxide to nitrous oxide. In the oxidized form the active site non-heme Fe(B) and high spin heme b(3) are mu-oxo bridged. The heme b(3) has a ligand-to-metal charge transfer band centered at 595 nm, which is insensitive to pH over the range of 6.0-8.5. Partial reduction of nitric-oxide reductase yields a three electron-reduced state where only the heme b(3) remains oxidized. This results in a shift of the heme b(3) charge transfer band lambda(max) to longer wavelengths. At pH 6.0 the charge transfer band lambda(max) is 605 nm, whereas at pH 8.5 it is 635 nm. At pH 6.5 and 7.5 the nitric-oxide reductase ferric heme b(3) population is a mixture of both 605- and 635-nm forms. Magnetic circular dichroism spectroscopy suggests that at all pH values examined the proximal ligand to the ferric heme b(3) in the three electron-reduced form is histidine. At pH 8.5 the distal ligand is hydroxide, whereas at pH 6.0, when the enzyme is most active, it is water.     

Two DNA-binding domains of Mga are required for virulence gene activation in the group A streptococcus

Mga is a DNA-binding protein that activates expression of several important virulence genes in the group A streptococcus (GAS), including those encoding M protein (emm), C5a peptidase (scpA) and Mga (mga). To determine the functionality of four potential helix-turn-helix DNA-binding motifs (HTH1-HTH4) identified within the amino-terminus of Mga, alanine substitutions were introduced within each domain in a MBP-Mga fusion allele and purified proteins were assayed for binding to Mga-specific promoter fragments (Pmga, PscpA and Pemm) in vitro. Although HTH-1 and HTH-2 mutations showed wild type DNA-binding activity, an altered HTH-3 domain resulted in reduced binding to the three promoters and an HTH-4 mutant was devoid of detectable binding activity. Plasmid-encoded expression of the HTH-3 and HTH-4 alleles from a constitutive promoter (Pspac) in the mga-deleted GAS strain JRS519 demonstrated that Mga-regulated emm expression correlated directly to the DNA-binding activity observed for each mutant protein in vitro. Single-copy expression of HTH-3 and HTH-4 from their native Pmga resulted in a dramatic reduction in autoregulated mga expression in both mutant strains. Thus, Mga appears to contain two DNA-binding domains (HTH-3 and HTH-4) that are required for direct activation of the Mga virulence regulon in vivo.     

Project Grow-2-Gether: a study of the genetic and environmental influences on child eating and obesity.

"Project Grow-2-Gether" is a child nutrition study of same-sex, 3- to 7-year-old monozygotic and dizygotic twin pairs. The study attempts to bridge two bodies of literature that have rarely interfaced with respect to obesity and ingestive behavior: the first being behavioral genetic approaches to obesity-related traits, and the second being developmental approaches focusing on parent-child relationships. The overarching aim of Project Grow-2-Gether is to disentangle genetic from potential home-environmental influences on child eating behavior and body fat. This paper reviews the rationale for Project Grow-2-Gether, its procedures, and core phenotypic measurement battery. A focus of the study is acquisition of controlled food intake measurements obtained in the laboratory, measurement of specific home environmental variables, and multi-method evaluation of parent-child feeding relations. Future directions may involve longitudinal assessment of child growth and molecular analyses for specific genes that influence child eating behavior.     

Acrosomal status and motility of guinea pig spermatozoa during in vitro penetration of the cumulus oophorus

Previous studies have suggested that both acrosome-intact and acrosome-reacted guinea pig sperm are capable of binding to the zona pellucida of cumulus-free oocytes, but the acrosomal status of guinea pig sperm during penetration of the cumulus has not been reported. We made video recordings of the interaction between capacitated guinea pig sperm and cumulus-invested guinea pig oocytes. The videotapes were analysed to identify sperm with hyperactivated motility and to classify the acrosomal status of sperm during penetration of the cumulus and after binding to the zona pellucida. The resolution of the video recordings was not sufficient to recognise sperm with swollen acrosomes. However, sperm that had completed the acrosome reaction were easily identified. Acrosome-reacted sperm were found adherent to the outer boundary of the cumulus, but were never observed to penetrate the cumulus. The percentage of acrosome-intact, hyperactivated sperm was higher in the cumulus oophorus than in culture medium, suggesting that changes in motility were elicited in response to contact with the cumulus. Fully acrosome-reacted sperm were found adherent to the zona pellucida, and solubilised guinea pig zona pellucida was capable of inducing acrosome reactions in capacitated guinea pig sperm. Acrosome-intact sperm were also observed on the zona, but they were not tightly bound and did not have hyperactivated motility, suggesting that these sperm were not functionally capacitated. Our observations demonstrate that guinea pig sperm penetrate the cumulus matrix in an acrosome-intact state. Although we did not observe sperm undergoing the acrosome reaction, our observations and experimental data suggest that the acrosome reaction of guinea pig sperm is completed on or near the surface of the zona pellucida.     

Relative importance of osmotic-stress and ion-specific effects on ABA-mediated inhibition of leaf expansion growth in Phaseolus vulgaris

The osmotic and ion-specific components of salt-induced inhibition of leaf expansion growth were investigated in beans grown from 12 h to several days in either NaCl-containing solution cultures, an isosmotic concentrated macronutrient solution, or a vermiculite–compost mixture with low Na⁺ but high Cl^– availability. Inhibition of leaf expansion and leaf ABA increase was more intense in the NaCl than in the isosmotic macronutrient treatment. Root Na⁺ was highly correlated to inhibition of leaf expansion and leaf or xylem sap ABA. When Na⁺ was sequestered in soil, salinized plants showed no reduction in leaf expansion or ABA increase, regardless of the presence of high leaf Cl^– concentrations. Stomatal conductance exhibited an exponential relationship with the reciprocal value of xylem sap ABA. Our results indicate that an ion-specific effect caused by Na⁺ in roots may account for an ABA-mediated reponse of both stomatal closure and leaf expansion inhibition.