Ace is a collagen-binding MSCRAMM from Enterococcus faecalis.

A putative collagen-binding MSCRAMM, Ace, of Enterococcus faecalis was identified by searching bacterial genome data bases for proteins containing domains homologous to the ligand-binding region of Cna, the collagen-binding MSCRAMM from Staphylococcus aureus. Ace was predicted to have a molecular mass of 71 kDa and contains features characteristic of cell surface proteins on Gram-positive bacteria, including a LPXTG motif for cross-linking to the cell wall. The N-terminal region of Ace contained a region (residues 174-319) in which 56% of the residues are identical or similar when compared with the minimal ligand-binding region of Cna (Cna 151-318); the remainder of the Ace A domain has 46% similarity with the corresponding region of the Cna A domain.

Trench-shaped binding sites promote multiple classes of interactions between collagen and the adherence receptors, alpha(1)beta(1) integrin and Staphylococcus aureus cna MSCRAMM.

Most mammalian cells and some pathogenic bacteria are capable of adhering to collagenous substrates in processes mediated by specific cell surface adherence molecules. Crystal structures of collagen-binding regions of the human integrin alpha(2)beta(1) and a Staphylococcus aureus adhesin reveal a "trench" on the surface of both of these proteins. This trench can accommodate a collagen triple-helical structure and presumably represents the ligand-binding site (Emsley, J.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of uracil phosphoribosyltransferase of Toxoplasma gondii.

Recombinant uracil phosphoribosyltransferase (UPRT) enzyme of Toxoplasma gondii was expressed in Escherichia coli and purified from the cell-free extract by a combination of chromatographic steps. The recombinant protein was enzymatically active when tested in an in vitro UPRT assay. The purified protein was crystallized using the hanging-drop vapor-diffusion technique with ammonium phosphate as precipitant.

Crystallization of ClfA and ClfB fragments: the fibrinogen-binding surface proteins of Staphylococcus aureus.

Recombinant constructs encoding the fibrinogen-binding domains of ClfA and ClfB from Staphylococcus aureus have been crystallized. ClfA was crystallized in the orthorhombic space group P212121 with unit-cell parameters a = 39.58, b = 81.

Crystallization and preliminary X-ray analysis of B-domain fragments of a Staphylococcus aureus collagen-binding protein.

Recombinant proteins of monomeric and dimeric B-domain repeats of a Staphylococcus aureus FDA 574 collagen-binding adhesin have been crystallized. The single repeat unit (B1) was crystallized in a body-centered orthorhombic lattice with a = 96.9, b = 101.

Structural basis of profactor D activation: from a highly flexible zymogen to a novel self-inhibited serine protease, complement factor D.

The crystal structure of profactor D, determined at 2.1 A resolution with an Rfree and an R-factor of 25.1 and 20.

Domain structure of the Staphylococcus aureus collagen adhesin.

Sequence analysis of surface proteins from Gram-positive bacteria indicates a composite organization consisting of unique and repeated segments. Thus, these proteins may contain discrete domains that could fold independently. In this paper, we have used a panel of biophysical methods, including gel permeation chromatography, analytical ultracentrifugation, circular dichroism, and fluorescence spectroscopy, to analyze the structural organization of the Staphylococcus aureus collagen adhesin, CNA.

Crystallization and preliminary crystallographic investigation of porcine quinolinate phosphoribosyltransferase.

Quinolinate phosphoribosyltransferase (QPRT), purified from hog liver, has been crystallized using PEG 8000 as the precipitant. The crystals form long hexagonal rods in the space group P6322 with cell dimensions a = b = 121.7, c = 94.

Structures of native and complexed complement factor D: implications of the atypical His57 conformation and self-inhibitory loop in the regulation of specific serine protease activity.

Factor D is a serine protease essential for the activation of the alternative pathway of complement. The structures of native factor D and a complex formed with isatoic anhydride inhibitor were determined at resolution of 2.3 and 1.

Construction of a whole body blood flow model for use in positron emission tomography imaging with [15O]water.

A whole body blood flow model (WBBFM) was developed and tested using STELLA II, an icon-driven mathematical simulation software package. The WBBFM uses parallel chambers to represent gray and white areas of the brain, body organs such as lungs, heart (right and left halves), injection site, and blood sampling sites. Input values to the WBBFM include organ blood flows, organ volumes, tissue:blood partition coefficients, injected activity, and data acquisition times for a positron emission tomography (PET) camera.

Current concepts of thyroid function and laboratory evaluation.

The role of triodothyronine (T(3) in regulating thyroid function is paramount in that at cellular level it interacts with receptors in the nucleus and thereby modulates gene expression. The multiple steps in the processing of lodide by the thyroid gland is under the influence of the thyroid stimulating hormone (TSH). In terms of laboratory evaluation of thyroid function the primary test is TSH.

Structure of the collagen-binding domain from a Staphylococcus aureus adhesin.

The crystal structure of the recombinant 19,000 M(r) binding domain from the Staphylococcus aureus collagen adhesin has been determined at 2 A resolution. The domain fold is a jelly-roll, composed of two antiparallel beta-sheets and two short alpha-helices. Triple-helical collagen model probes were used in a systematic docking search to identify the collagen-binding site.

Preliminary crystallographic study on a low molecular weight form of bacterial plasminogen activator staphylokinase.

Staphylokinase, a 17 kDa protein, produced by certain strains of Staphylococcus aureus functions as a fibrin-specific plasminogen activator. During its interaction with plasminogen, staphylokinase is converted into a low molecular weight form by loss of ten amino-terminal residues. This low molecular weight form of recombinant staphylokinase has been crystallized using the hanging-drop vapor-diffusion technique with polyethylene glycol 4000 as precipitant.

Purification, crystallization and preliminary X-ray diffraction studies of recombinant calcium-binding domain of the small subunit of porcine calpain.

The calcium-binding domain of the small subunit of porcine calpain (domain VI) has been expressed in Escherichia coli, purified, and crystallized in the presence of Ca(2+). Two crystal forms have been obtained by the vapor-diffusion method using PEG 6000 as the precipitant. Crystal form I, belonging to trigonal space group P3(1)21 (or P3(2)21) with cell dimensions a = b = 79.

Crystal structure of calcium bound domain VI of calpain at 1.9 A resolution and its role in enzyme assembly, regulation, and inhibitor binding.

The three dimensional structure of calcium-bound domain VI of porcine calpain has been determined to 1.9 A resolution. The crystal structure reveals five EF-hands, one more than previously suggested.

Structure of diisopropyl fluorophosphate-inhibited factor D.

Factor D (D) is a serine protease, crucial for the activation of the alternative complement pathway. Only a limited number of general serine protease inhibitors are known to inhibit D, most of which covalently bind to the serine hydroxyl of the catalytic triad. The structure of the first enzyme:inhibitor covalent adduct of D with diisopropyl fluorophosphate (DIP:D) to a resolution of 2.

Refined structure of purine nucleoside phosphorylase at 2.75 A resolution.

Human purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of ribonucleosides and 2'-deoxyribonucleosides to the free base and (2'-deoxy)ribose-l-phosphate. The crystal structure previously determined at 3.2 A resolution by multiple isomorphous replacement methods [Ealick, Rule, Carter, Greenhough, Babu, Cook, Habash, Helliwell, Stoeckler, Parks, Chen & Bugg (1990).

Site-directed mutagenesis of the phosphocholine-binding site of human C-reactive protein: role of Thr76 and Trp67.

We have reported previously that residues Lys57, Arg58, and Trp67 of human C-reactive protein (CRP) contribute to the structure of the phosphocholine (PCh)-binding site. In this study, based on the three-dimensional structures of human CRP and serum amyloid P, we constructed an additional mutant, T76Y, to probe the structural determinants of the PCh-binding site of CRP. Binding properties of four mutant CRPs, K57Q/R58G, W67K, K57Q/R58G/W67K, and T76Y were compared.

Complement factor D, a novel serine protease.

Factor D is unique among serine proteases in that it requires neither enzymatic cleavage for expression of proteolytic activity nor inactivation by a serpin for its control. Regulation of factor D activity is instead attained by a novel mechanism that depends on reversible conformational changes for expression and control of catalytic activity. These conformational changes are believed to be induced by the single natural substrate, C3bB, and to result in realignment of the catalytic triad, the specificity pocket, and the nonspecific substrate binding site, all of which have atypical conformations.

Dosimetry of [15O]water: a physiologic approach.

Earlier dosimetry estimates for [15O]water assumed its instantaneous equilibrium with total body water. This assumption leads to an underestimation of the absorbed doses to organs with high blood flows, since the biodistribution of this short-lived radiopharmaceutical is dependent upon blood flow to organs. We have developed a physiologically based whole body blood flow model (WBBFM) using a commercially available icon-driven mathematical simulation software package and applied it to the reevaluation of [15O]water dosimetry in humans.

Crystal structure of a complement factor D mutant expressing enhanced catalytic activity.

Complement factor D is a serine protease regulated by a novel mechanism that depends on conformational changes rather than cleavage of a zymogen for expression of proteolytic activity. The conformational changes are presumed to be induced by the single natural substrate, C3bB, and to result in reversible reorientation of the catalytic center and of the substrate binding site of factor D, both of which have atypical conformations. Here we report that replacement of Ser94, Thr214, and Ser215 of factor D (chymotrypsinogen numbering has been used for comparison purposes) with the corresponding residues of trypsin, Tyr, Ser, and Trp, is sufficient to induce substantially higher catalytic activity associated with a typical serine protease alignment of the catalytic triad residues His57, Asp102, and Ser195.

Catalytic role of a surface loop of the complement serine protease factor D.

We have investigated the structural determinants of the unique functional properties of complement factor D by constructing and testing a series of trypsin-like mutants of the enzyme. Mutational replacement of the primary substrate-binding pocket of factor D with that of trypsin resulted in a mutant (M1) with greatly reduced proteolytic activity and slightly reduced reactivity toward small thioester substrates. Combining the M1 mutations with substitution of Tyr for Ser94, previously shown to enhance substantially both the proteolytic and esterolytic activities of factor D, produced a mutant (M2) with reactivities similar to M1.

Mutational analysis of the substrate binding site of human complement factor D.

Complement factor D is a serine protease with a single natural substrate, C3b-complexed factor B, and very low catalytic activity against synthetic esters. The recently solved X-ray crystal structure of factor D has demonstrated certain key differences from other serine protease in the conformation of residues of the catalytic triad and the substrate-binding regions. To investigate possible contributions of unique amino acid substitutions to these distinct structural and functional features of factor D, we constructed a series of mutants by substituting trypsin substrate-binding residues for the corresponding factor D residues.