Mechanism of ribonuclease inhibition by ribonuclease inhibitor protein based on the crystal structure of its complex with ribonuclease A

We describe the mechanism of ribonuclease inhibition by ribonuclease inhibitor, a protein built of leucine-rich repeats, based on the crystal structure of the complex between the inhibitor and ribonuclease A. The structure was determined by molecular replacement and refined to an R(cryst) of 19.4% at 2.5 Angstrom resolution. Ribonuclease A binds to the concave region of the inhibitor protein comprising its parallel beta-sheet and loops. The inhibitor covers the ribonuclease active site and directly contacts several active-site residues. The inhibitor only partially mimics the RNase-nucleotide interaction and does not utilize the pi phosphate-binding pocket of ribonuclease A, where a sulfate ion remains bound. The 2550 Angstrom(2) of accessible surface area buried upon complex formation may be one of the major contributors to the extremely tight association (K-i = 5.9 x 10(-14) M). The interaction is predominantly electrostatic; there is a high chemical complementarity with 18 putative hydrogen bonds and salt links, but the shape complementarity is lower than in most other protein-protein complexes. Ribonuclease inhibitor changes its conformation upon complex formation; the conformational change is unusual in that it is a plastic reorganization of the entire structure without any obvious hinge and reflects the conformational flexibility of the structure of the inhibitor. There is a good agreement between the crystal structure and other biochemical studies of the interaction. The structure suggests that the conformational flexibility of RI and an unusually large contact area that compensates for a lower degree of complementarity may be the principal reasons for the ability of RI to potently inhibit diverse ribonucleases. However, the inhibition is lost with amphibian ribonucleases that have substituted most residues corresponding to inhibitor-binding residues in RNase A, and with bovine seminal ribonuclease that prevents inhibitor binding by forming a dimer. (C) 1996 Academic Press Limited

Centrifugal processing of cell debris and inclusion bodies from recombinant Escherichia coli

The settling characteristics of cell debris and inclusion bodies prior to, and following, fractionation in a disc-stack centrifuge were measured using Cumulative Sedimentation Analysis (CSA) and Centrifugal Disc photosedimentation (CDS). The impact of centrifuge feedrate and repeated homogenisation on both cell debris and inclusion body collection efficiency was investigated. Increasing the normalised centrifuge feedrate (Q/Sigma) from 1.32 x 10(-9) m s(-1) to 3.97 x 10(-9) m s(-1) leads to a 36% increase in inclusion body paste purity. Purity may also be improved by repeated homogenisation. Increasing the number of homogeniser passes results in smaller cell debris size whilst leaves inclusion body size unaltered. At a normalised centrifuge feedrate of 2.65 x 10(-9) m s(-1), increasing the number of homogeniser passes from two (2) to ten (10) improved overall inclusion body paste purity by 58%. Grade-efficiency curves for both the cell debris and inclusion bodies have also been generated in this study. The data are described using an equation developed by Mannweiler (1989) with parameters of k = 0.15-0.26 and n = 2.5-2.6 for inclusion bodies, and k = 0.12-0.14 and n = 2.0-2.2 for cell debris. This is the first accurate experimentally-determined grade efficiency curve for cell debris. Previous studies have simply estimated debris grade efficiency curves using an approximate debris size distribution and grade efficiency curves determined with 'ideal particles' (e.g. spherical PVA particles). The findings of this study may be used to simulate and optimise the centrifugal fractionation of inclusion bodies from cell debris.

Reorganization of terminator DNA upon binding replication terminator protein: Implications for the functional replication fork arrest complex

Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the replication terminator protein (RTP) and DNA terminator sites. While determination of the crystal structure of RTP has facilitated our understanding of how a single RTP dimer interacts with terminator DNA, additional information is required in order to understand the assembly of a functional fork arrest complex, which requires an interaction between two RTP dimers and the terminator site. In this study, we show that the conformation of the major B. subtilis DNA terminator, Terl, becomes considerably distorted upon binding RTP. Binding of the first dimer of RTP to the B site of Terl causes the DNA to become slightly unwound and bent by similar to 40 degrees. Binding of a second dimer of RTP to the A site causes the bend angle to increase to similar to 60 degrees. We have used this new data to construct two plausible models that might explain how the ternary terminator complex can block DNA replication in a polar manner, in the first model, polarity of action is a consequence of the two RTP-DNA half-sites having different conformations. These different conformations result from different RTP-DNA contacts at each half-site (due to the intrinsic asymmetry at the terminator DNA), as well as interactions (direct or indirect) between the RTP dimers on the DNA. In the second model, polar fork arrest activity is a consequence of the different affinities of RTP for the A and B sites of the terminator DNA, modulated significantly by direct or indirect interactions between the RTP dimers.

Identification of the alpha(6) integrin as a candidate receptor for papillomaviruses

Papillomaviruses (PVs) bind in a specific and saturable fashion to a range of epithelial and other cell lines. Treatment of cells with trypsin markedly reduces their ability to bind virus particles, suggesting that binding is mediated via a cell membrane protein. We have investigated the interaction bf human PV type 6b L1 virus-like particles (VLPs) with two epithelial cell lines, CV-1 and HaCaT, which bind VLPs, and a B-cell line (DG75) previously shown not to bind VLPs. Immunoprecipitation of a mixture of PV VLPs with [S-35]methionine-labeled cell extracts and with biotin-labeled cell surface proteins identified four proteins from CV-1 and HaCaT cells of 220, 120, 87, and 35 kDa that reacted with VLPs and were not present in DG75 cells. The alpha(6) beta(4) integrin complex has subunits corresponding to the VLP precipitated proteins, and the tissue distribution of this complex suggested that it was a candidate human PV receptor. Monoclonal antibodies (MAbs) to the alpha(6) or beta(4) integrin subunits precipitated VLPs from a mixture of CV-1 cell proteins and VLPs, whereas MAbs to other integrin subunits did not. An alpha(6) integrin-specific MAb (GoH3) inhibited VLP binding to CV-1 and HaCaT cells, whereas an anti-beta(4) integrin MAb and a range of integrin-specific and other MAbs did not. Furthermore, human laminin, the natural ligand for the alpha(6) beta(4) integrin, was able to block VLP binding. By use of sections of monkey esophagus, the distribution of alpha(6), integrin expression in the basal epithelium was shown to coincide with the distribution of bound VLPs. Taken together, these data suggest that VLPs bind specifically to the alpha(6) integrin subunit and that integrin complexes containing alpha(6) integrin complexed with either beta(1) or beta(4) integrins may act as a receptor for PV binding and entry into epithelial cells.

A peptide-binding motif for I-A(g7), the class II major histocompatibility complex (MHC) molecule of NOD and Biozzi AB/H mice

The class II major histocompatibility complex molecule I-A(g7) is strongly linked to the development of spontaneous insulin-dependent diabetes mellitus (IDDM) in non obese diabetic mice and to the induction of experimental allergic encephalomyelitis in Biozzi AB/H mice. Structurally, it resembles the HLA-DQ molecules associated with human IDDM, in having a non-Asp residue at position 57 in its beta chain. To identify the requirements for peptide binding to I-A(g7) and thereby potentially pathogenic T cell epitopes, we analyzed a known I-A(g7)-restricted T cell epitope, hen egg white lysozyme (HEL) amino acids 9-27. NH2- and COOH-terminal truncations demonstrated that the minimal epitope for activation of the T cell hybridoma 2D12.1 was M12-R21 and the minimum sequence for direct binding to purified I-A(g7) M12-Y20/K13-R21. Alanine (A) scanning revealed two primary anchors for binding at relative positions (p) 6 (L) and 9 (Y) in the HEL epitope. The critical role of both anchors was demonstrated by incorporating L and Y in poly(A) backbones at the same relative positions as in the HEL epitope. Well-tolerated, weakly tolerated, and nontolerated residues were identified by analyzing the binding of peptides containing multiple substitutions at individual positions. Optimally, p6 was a large, hydrophobic residue (L, I, V, M), whereas p9 was aromatic and hydrophobic (Y or F) or positively charged (K, R). Specific residues were not tolerated at these and some other positions. A motif for binding to I-A(g7) deduced from analysis of the model HEL epitope was present in 27/30 (90%) of peptides reported to be I-A(g7)-restricted T cell epitopes or eluted from I-A(g7). Scanning a set of overlapping peptides encompassing human proinsulin revealed the motif in 6/6 good binders (sensitivity = 100%) and 4/13 weak or non-binders (specificity = 70%). This motif should facilitate identification of autoantigenic epitopes relevant to the pathogenesis and immunotherapy of IDDM.

Self-assembly of a tetranuclear macrocyclic copper(II) complex

A dinuclear macrocyclic complex is synthesized via the one-pot reaction of dipotassium nitroacetate, formaldehyde and a linear tetraamine copper(II) complex; the X-ray crystal structure of the product reveals an association of two dinuclear complexes to form a novel tetracopper(II) species.

Haemonchus contortus: The uptake and metabolism of closantel

Closantel is an anthe lmintic which associates with plasma albumin and is useful for the control of sheep parasites, such as Haemonchus contortus, that ingest blood. However, the utility of closantel for parasite control has been threatened by the emergence of resistance. The mechanisms of resistance are unknown. A closantel-resistant and a closantel-susceptible isolate of H. contortus were compared with respect to the distribution and metabolism of closantel. Neither strain appeared to metabolise closantel in vitro or in vivo. Following treatment of infected sheep with radioactively labelled closantel, isotope levels in closantel-resistant adult H. contortus were significantly lower than in susceptible worms. This reduced accumulation of drug could contribute to closantel resistance by mechanisms such as reduced feeding, failure to dissociate the drug-albumin complex in the gut or increased efflux of closantel from resistant worms. (C) 1997 Australian Society for Parasitology.

Spin-orbit mixing and nephelauxetic effects in the electronic spectra of nickel(II)-encapsulating complexes involving nitrogen and sulfur donors

A study of spin-orbit mixing and nephelauxetic effects in the electronic spectra of nickel(II)-encapsulating complexes involving mixed nitrogen and sulfur donors is reported. As the number of sulfur donors is systematically varied through the series [Ni(N6-xSx)](2+) (x = 0-6), the spin-forbidden (3)A(2)g --> E-1(g) and (3)A(2g) --> (1)A(1g) transitions undergo a considerable reduction in energy whereas the spin-allowed transitions are relatively unchanged. The [Ni(diAMN(6)sar)](2+) and [Ni(AMN(5)Ssar)](2+) complexes exhibit an unusual band shape for the (3)A(2g) --> T-3(2g) transition which is shown to arise from spin-orbit mixing of the E spin-orbit levels associated with the E-1(g) and T-3(2g) states. A significant differential nephelauxetic effect also arises from the covalency differences between the t(2g) and e(g) orbitals with the result that no single set of Racah B and C interelectron repulsion parameters adequately fit the observed spectra. Using a differential covalency ligand-field model, the spectral transitions are successfully reproduced with three independent variables corresponding to 10Dq and the covalency parameters f(t) and f(e), associated with the t(2g) and e(g) orbitals, respectively. The small decrease in f(t) from unity is largely attributed to central-field covalency effects whereas the dramatic reduction in f(e) with increasing number of sulfur donors is a direct consequence of the increased metal-ligand covalency associated with the sulfur donors. Covalency differences between the t(2g) and e(g) orbitals also result in larger 10Dq values than those obtained simply from the energy of the (3)A(2g) --> T-3(2g) spin-allowed transition.

Major histocompatibility complex (MHC) class II-positive dendritic cells in the rat iris - In situ development from MHC class II-negative precursors

Purpose. To examine the postnatal development of major histocompatibility complex (MHC) class II-positive dendritic cells (DC) in the iris of the normal rat eye. Methods. Single-and double-color immunomorphologic studies were performed on whole mounts prepared from rat iris taken at selected postnatal ages (2 to 3 days to 78 weeks). Immunopositive cells were enumerated, using a quantitative light microscope, and MHC class II expression on individual cells was assessed by microdensitometric analysis. Results. Major histocompatibility class II-positive DCs in the iris developed in an age-dependent manner and reached adult-equivalent density and structure at approximately 10 weeks of age, considerably later than previously described in other DC populations in the rat. In contrast, the anti-rat DC monoclonal antibody OX62 revealed a population of cells present at adult-equivalent levels as early as 3 weeks after birth. Dual-color immunostaining and microdensitometric analysis demonstrated that during postnatal growth, development of the network of MHC class II-positive DCs was a consequence of the progressive increase in expression of MHC class II antigen by OX62-positive cells. Conclusions. During postnatal growth, the DC population of the iris develops initially as an OX62-positive-MHC class II-negative population, which then develops increasing MHC class II expression in situ and finally resembles classic DC populations in other tissue sites. Maturation of the iris DC population is temporally delayed compared with time to maturation in other tissue sites in the rat.