The 0.78 A structure of a serine protease: Bacillus lentus subtilisin

Ultrahigh-resolution X-ray diffraction data from cryo-cooled, B. lentus subtilisin crystals has been collected to a resolution of 0.78 A. The refined model coordinates have a rms deviation of 0.22 A relative to the same structure determined at room temperature and 2.0 A resolution. Several regions of main-chain and side-chain disorder have been identified for 21 out of 269 residues in one polypeptide chain. Hydrogen atoms appear as significant peaks in the Fo - Fc difference electron density map, and carbon, nitrogen, and oxygen atoms can be differentiated. The estimated standard deviation (ESD) for all main-chain non-hydrogen bond lengths is 0.009 A and 0.5 degrees for bond angles based on an unrestrained full-matrix least-squares refinement. Hydrogen bonds are resolved in the serine protease catalytic triad (Ser-His-Asp). Electron density is observed for an unusual, short hydrogen bond between aspartic acid and histidine in the catalytic triad. The hydrogen atom, identified by NMR in numerous serine proteases, appears to be shared by the heteroatoms in the bond. This represents the first reported correlation between detailed chemical features identified by NMR and those in a cryo-cooled crystallographic structure determination at ultrahigh resolution. The short hydrogen bond, designated "catalytic hydrogen bond", occurs as part of an elaborate hydrogen bond network, involving Asp of the catalytic triad. While unusual, these features appear to have conserved analogues in other serine protease families although specific details differ from family to family.     

Primary structure of a putative serine protease specific for IGF-binding proteins

From a subtracted cDNA library we have isolated a cDNA clone coding for a novel transformation-sensitive protein which is expressed by human fibroblasts, but not by their matched SV40 transformed counterparts. This protein has a molecular mass of 51 kDa and is highly related to the HtrA family of serine proteases from bacteria. At the N-terminal end, it contains an IGF-binding domain which may modulate the activity of the associated serine protease. Our data are consistent with the assumption that the novel protein represents one of the proteases that regulate the availability of IGFs by cleaving IGF-binding proteins.     

Cold-active serine alkaline protease from the psychrotrophic bacterium Shewanella strain ac10: gene cloning and enzyme purification and characterization

The gene encoding serine alkaline protease (SapSh) of the psychrotrophic bacterium Shewanella strain Ac10 was cloned in Escherichia coli. The amino acid sequence deduced from the 2,442-bp nucleotide sequence revealed that the protein was 814 amino acids long and had an estimated molecular weight of 85,113. SapSh exhibited sequence similarities with members of the subtilisin family of proteases, and there was a high level of conservation in the regions around a putative catalytic triad consisting of Asp-30, His-65, and Ser-369. The amino acid sequence contained the following regions which were assigned on the basis of homology to previously described sequences: a signal peptide (26 residues), a propeptide (117 residues), and an extension up to the C terminus (about 250 residues). Another feature of SapSh is the fact that the space between His-65 and Ser-369 is approximately 150 residues longer than the corresponding spaces in other proteases belonging to the subtilisin family. SapSh was purified to homogeneity from the culture supernatant of E. coli recombinant cells by affinity chromatography with a bacitracin-Sepharose column. The recombinant SapSh (rSapSh) was found to have a molecular weight of about 44,000 and to be highly active in the alkaline region (optimum pH, around 9.0) when azocasein and synthetic peptides were used as substrates. rSapSh was characterized by its high levels of activity at low temperatures; it was five times more active than subtilisin Carlsberg at temperatures ranging from 5 to 15 degreesC. The activation energy for hydrolysis of azocasein by rSapSh was much lower than the activation energy for hydrolysis of azocasein by the subtilisin. However, rSapSh was far less stable than the subtilisin.     

The crystal structure of bikunin from the inter-alpha-inhibitor complex: a serine protease inhibitor with two Kunitz domains

Bikunin is a serine protease inhibitor found in the blood serum and urine of humans and other animals. Its sequence shows internal repetition, suggesting that it contains two domains that resemble bovine pancreatic trypsin inhibitor (BPTI). A fragment of bikunin has been crystallised, its structure solved and subsequently refined against 2.5 A data. The two BPTI-like domains pack closely together and are related by an approximate 60 degrees rotation combined with a translation. These domains are very similar to each other and other proteins with this fold. The largest variations occur in the loops responsible for protease recognition. The loops of the first domain are unobstructed by the remaining protein. However, the loops of the second domain are close to the first domain and it is possible that protease binding may be affected or, in some cases, abolished by the presence of the first domain. Thus, cleavage of the two domains could alter the substrate specificity of domain II. Bikunin has a hydrophobic patch close to the N terminus of domain I, which is the most likely site for cell-surface receptor binding. In addition, there is a basic patch at one end of domain II that may be responsible for the inhibition of calcium oxalate crystallization in urine.     

An objective comparison of 3-D image interpolation methods

To aid in the display, manipulation, and analysis of biomedical image data, they usually need to he converted to data of isotropic discretization through the process of interpolation. Traditional techniques consist of direct interpolation of the grey values. When user interaction is called for in image segmentation, as a consequence of these interpolation methods, the user needs to segment a much greater (typically 4-10x) amount of data. To mitigate this problem, a method called shape-based interpolation of binary data was developed 121. Besides significantly reducing user time, this method has been shown to provide more accurate results than grey-level interpolation. We proposed an approach for the interpolation of grey data of arbitrary dimensionality that generalized the shape-based method from binary to grey data. This method has characteristics similar to those of the binary shape-based method. In particular, we showed preliminary evidence that it produced more accurate results than conventional grey-level interpolation methods. In this paper, concentrating on the three-dimensional (3-D) interpolation problem, we compare statistically the accuracy of eight different methods: nearest-neighbor, linear grey-level, grey-level cubic spline, grey-level modified cubic spline, Goshtasby et al., and three methods from the grey-level shape-based class. A population of patient magnetic resonance and computed tomography images, corresponding to different parts of the human anatomy, coming from different three-dimensional imaging applications, are utilized for comparison. Each slice in these data sets is estimated by each interpolation method and compared to the original slice at the same location using three measures: mean-squared difference, number of sites of disagreement, and largest difference. The methods are statistically compared pairwise based on these measures. The shape-based methods statistically significantly outperformed all other methods in all measures in all applications considered here with a statistical relevance ranging from 10% to 32% (mean = 15%) for mean-squared difference.     

The future of protein secondary structure prediction accuracy

In order to investigate the path of medical education in Iran, indicators of medical education were searched from 1970 to 1994. There have been rises in the number of educational institutions from 10 to 46; student admissions in programmes of medical sciences from 1387 to 18,141; medical student admissions from 632 to 3630; teaching staff from 1573 to 7979; and teaching-bed to student ratio from 1.05 to 2.08. The numbers of students in clinical specialty and MS degrees have increased, and various programmes in clinical sub-specialty and PhD degrees have been initiated. The quality of medical education has improved with increasing field and ambulatory care training, with more emphasis on teaching preventive medicine and a significant rise in the research activities. Most qualitative and quantitative progress has been achieved following the establishment of a joint Ministry of Health and Medical Education in 1985. The results of this review demonstrate the success of Iran in upgrading medical education by the unification of health services and medical education in one ministry.     

A comparison of 2-D and 3-D FSE imaging in MR of the cervical spine

Angiotensin affects the growth of many cell types and there have been suggestions that it could influence cells which respond to erythropoietin. During first trimester, human fetal haematopoietic cells are exquisitely responsive to erythropoietin and we have assessed the potential for angiotensin to also influence the growth of these cell types in a standard culture system. The data obtained from these studies indicate that angiotensin does not influence fetal haematopoiesis over a range of concentrations.     

A molecular model of the serine protease domain of activated protein C: application to the study of missense mutations causing protein C deficiency

A molecular model of the serine protease domain of protein C was constructed by standard comparative methods. Individual missense mutations were inserted into the model and plausible explanations for their interference with protein C structure/function were derived through consideration of location, steric effects and protein stability. A hydrophilic cluster of many Arg and Lys residues, found adjacent to the active site cleft, is proposed to be involved in thrombomodulin and/or protein S interactions. Analysis of comparative binding studies also suggested the presence of an extended substrate binding pocket in the model.     

Update on protein structure prediction: results of the 1995 IRBM workshop

Computational tools for protein structure prediction are of great interest to molecular, structural and theoretical biologists due to a rapidly increasing number of protein sequences with no known structure. In October 1995, a workshop was held at IRBM to predict as much as possible about a number of proteins of biological interest using ab initio prediction of fold recognition methods. 112 protein sequences were collected via an open invitation for target submissions. 17 were selected for prediction during the workshop and for 11 of these a prediction of some reliability could be made. We believe that this was a worthwhile experiment showing that the use of a range of independent prediction methods and thorough use of existing databases can lead to credible and useful ab initio structure predictions.     

Band 3 protein: physiology, function and structure

Band 3 protein is a typical polytropic membrane protein and mediates the exchange of the cellular HCO3- with CI- in plasma, which has been known as the "Chloride Shift". Owing to the "Chloride Shift", red blood cells can discriminate the metabolically active cells from inactive cells and deliver oxygen particularly to metabolically active tissues that produce carbon dioxide. Thus, band 3 protein is a sensor for metabolically active tissues and no excess oxygen is supplied to tissues as far as oxygen is delivered by red blood cells. In this chapter, we review the physiological role of the anion exchange mediated by band 3 protein and our work concerning the structure and function relationship in band 3 protein, that, is, affinity labeling of the active center for the anion exchange with pyridoxal phosphate, conformational change during the anion exchange process, examination of fidelity of hydropathy prediction on band 3 protein, and phosphoenolpyruvate transport mediated by band 3 protein and its clinical application.     

Band 3 protein: structure, flexibility and function

The oxidation of antibody carbohydrate residues is a common approach used for site-specific antibody immobilization or modification. In this study a flow injection analysis system (FIA) was developed for monitoring antibody oxidation. Antibodies were oxidized with periodate and the resulting aldehyde groups were labeled with Lucifer yellow CH (LyCH). The labeled antibodies were then injected onto an FIA system where the amount of LyCH label was determined by absorbance measurements at 428 nm and the amount of antibody was determined using an on-line bicinchoninic acid protein assay. The analysis time was 2 min per 20 microliters sample injection. The limits of detection for rabbit immunoglobulin G (IgG) and LyCH were 1 x 10(-8) and 4 x 10(-7) M, respectively. The dynamic ranges for IgG and LyCH extended to 2 x 10(-5) and 7 x 10(-3) M. The within-run precision was +/- 5% or less for both analytes. Studies with known LyCH/antibody mixtures indicated that the FIA system had greater accuracy than manual methods at high LyCH levels. One specific application studied for this system was its use in monitoring the time course of periodate-antibody oxidation.     

cDNA cloning and expression of a novel serine protease, TLSP

A cDNA for a putative novel serine protease, TLSP, was cloned from human hippocampus cDNA with polymerase chain reaction based strategies. The putative amino acid sequence of TLSP is similar to the trypsin-type serine proteases. TLSP mRNA is expressed in keratinocytes. Overexpressed TLSP protein in neuro2a cells was detected in culture medium.     

Solution structure of nodularin. An inhibitor of serine/threonine-specific protein phosphatases

The three-dimensional solution structure of nodularin was studied by NMR and molecular dynamics simulations. The conformation in water was determined from the distance and dihedral data by distance geometry and refined by iterative relaxation matrix analysis. The cyclic backbone adopts a well defined conformation but the remote parts of the side chains of arginine as well as the amino acid derivative Adda have a large spatial dispersion. For the unusual amino acids the partial charges were calculated and nodularin was subjected to molecular dynamic simulations in water. A good agreement was found between experimental and computational data with hydrogen bonds, solvent accessibility, molecular motion, and conformational exchange. The three-dimensional structure resembles very closely that of microcystin-LR in the chemically equivalent segment. Therefore, it is expected that the binding of both microcystins and nodularins to serine/threonine-specific protein phosphatases is similar on an atomic level.     

Nonpeptide cyclic cyanoguanidines as HIV-1 protease inhibitors: synthesis, structure-activity relationships, and X-ray crystal structure studies

Comparison of the high-resolution X-ray structures of the native HIV-1 protease and its complexes with the inhibitors suggested that the enzyme flaps are flexible. The movement at the tip of the flaps could be as large as 7 A. On the basis of this observation, cyclic cyanoguanidines have been designed, synthesized, and evaluated as HIV-1 protease (PR) inhibitors. Cyclic cyanoguanidines were found to be very potent inhibitors of HIV-1 protease. The choice of cyclic cyanoguanidines over cyclic guanidines was based on the reduced basicity of the former. X-ray structure studies of the HIV PR complex with cyclic cyanoguanidine demonstrated that in analogy to cyclic urea, cyclic cyanoguanidines also displace the unique structural water molecule. The structure-activity relationship of the cyclic cyanoguanidines is compared with that of the corresponding cyclic urea analogues. The differences in binding constants of the two series of compounds have been rationalized using high-resolution X-ray structure information.     

Structure of the human cytomegalovirus protease catalytic domain reveals a novel serine protease fold and catalytic triad

Proteolytic processing of capsid assembly protein precursors by herpesvirus proteases is essential for virion maturation. A 2.5 A crystal structure of the human cytomegalovirus protease catalytic domain has been determined by X-ray diffraction. The structure defines a new class of serine protease with respect to global-fold topology and has a catalytic triad consisting of Ser-132, His-63, and His-157 in contrast with the Ser-His-Asp triads found in other serine proteases. However, catalytic machinery for activating the serine nucleophile and stabilizing a tetrahedral transition state is oriented similarly to that for members of the trypsin-like and subtilisin-like serine protease families. Formation of the active dimer is mediated primarily by burying a helix of one protomer into a deep cleft in the protein surface of the other.     

The perception of 3-D structure from contradictory optical patterns

Two experiments investigated observers' perception of 3-D structure when optical sources of information were contradictory. When motion and stereoscopic disparities specified different surfaces, the perceptual outcome dependent strongly on the direction of curvature present within each modality. Previous research has shown that the perception of surface slant and curvature is anisotropic for both motion and stereo and that it depends on the direction in which it takes place. In the present experiments, the modality with the "effective" direction of curvature tended to dominate or suppress the perception of surfaces in the other modality with less effective curvatures. The results have implications for models which attempt to combine 3-D data from different optical sources.     

Effect of exceptional valine replacement for highly conserved alanine-55 on the catalytic site structure of chymotrypsin-like serine protease

The catalytic triad consisting of His57, Asp102 and Ser195, which is completely conserved within the chymotrypsin-like serine protease family, plays a central role in catalysis. Highly conserved Ala55 also likely plays an important role in catalysis due to its location just behind the catalytic triad. The only exception to the conserved Ala55 in mammalian serine proteases is Val55 in bovine protein C. Interestingly, it has been demonstrated that the replacement of Ala55 with Thr results in the reduced activity of plasmin in patients with venous thrombosis and with retinochoroidal vascular disorders, which indicates the importance of Ala55 in catalysis. In the present study, we constructed a bovine protein C model which shows that Val55 causes no serious rearrangement of the catalytic site structure. We also constructed an A55T variant model of trypsin for comparison. The A55T substitution alters His57 into an inactive conformation, forming an unusual hydrogen bond between Thr55 O gamma 1 and His57 N epsilon 2. The present study shows that the Ala/Val55 residue contributes heavily to the active conformation of His57 and enables His57 to accept a proton from Ser195 O gamma effectively.     

Crystal structure of the unliganded alkaline protease from Pseudomonas aeruginosa IFO3080 and its conformational changes on ligand binding

Since volatile anesthetics, barbiturates, and local anesthetics have been reported to inhibit endothelium-dependent relaxation, we hypothesized that any drug with anesthetic action would suppress this relaxation. In the present study, using rat thoracic aortae, we attempted to determine whether nonbarbiturate intravenous anesthetics, including midazolam, propofol, and ketamine, suppress endothelium-dependent relaxation, and to clarify the mechanism(s) involved. Acetylcholine-induced, endothelium-dependent relaxation was significantly attenuated by propofol and ketamine, but was unaffected by midazolam. Sodium nitroprusside (SNP)-induced relaxation was attenuated by propofol, but not by midazolam or ketamine. The acetylcholine-stimulated 3',5'-cyclic guanosine monophosphate (cGMP) level was reduced by pretreatment with propofol and ketamine but not by midazolam, and that stimulated by SNP was reduced by propofol but not by ketamine or midazolam. We conclude that propofol and ketamine suppress endothelium-dependent relaxation, whereas midazolam has no influence. Moreover, the suppressive effect of ketamine on endothelium-dependent relaxation is mediated by suppression of nitrous oxide (NO) formation, whereas that of propofol may be mediated at least partly by suppression of NO function.     

Addiction protein Phd of plasmid prophage P1 is a substrate of the ClpXP serine protease of Escherichia coli

Plasmid-encoded addiction genes augment the apparent stability of various low copy number bacterial plasmids by selectively killing plasmid-free (cured) segregants or their progeny. The addiction module of plasmid prophage P1 consists of a pair of genes called phd and doc. Phd serves to prevent host death when the prophage is retained and, should retention mechanisms fail, Doc causes death on curing. Doc acts as a cell toxin to which Phd is an antidote. In this study we show that host mutants with defects in either subunit of the ClpXP protease survive the loss of a plasmid that contains a P1 addiction module. The small antidote protein Phd is fully stable in these two mutant hosts, whereas it is labile in a wild-type host. We conclude that the role of ClpXP in the addiction mechanism of P1 is to degrade the Phd protein. This conclusion situates P1 among plasmids that elicit severe withdrawal symptoms and are able to do so because they encode both a cell toxin and an actively degraded macromolecule that blocks the synthesis or function of the toxin.