The protease domain of procollagen C-proteinase (BMP1) lacks substrate selectivity, which is conferred by non-proteolytic domains

Procollagen C-proteinase (PCP) removes the C-terminal pro-peptides of procollagens and also processes other matrix proteins. The major splice form of the PCP is termed BMP1 (bone morphogenetic protein 1). Active BMP1 is composed of an astacin-like protease domain, three CUB (complement, sea urchin Uegf, BMP1) domains and one EGF-like domain. Here we compare the recombinant human full-length BMP1 with its isolated proteolytic domain to further unravel the functional influence of the CUB and EGF domains. We show that the protease domain alone cleaves truncated procollagen VII within the short telopeptide region into fragments of similar size as the full-length enzyme does. However, unlike full-length BMP1, the protease domain does not stop at this point, but degrades its substrate completely. Moreover, the protease domain cleaves other matrix proteins such as fibronectin, collagen I and collagen IV, which are left intact by the full-length enzyme. In addition, we show for the first time that thrombospondin-1 is differently cleaved by both BMP1 and its catalytic domain. In summary, our data support the concept that the C-terminal domains of BMP1 are important for substrate recognition and for controlling and restricting its proteolytic activity via exosite binding.     

NMR structure of the netrin-like domain (NTR) of human type I procollagen C-proteinase enhancer defines structural consensus of NTR domains and assesses potential proteinase inhibitory activity and ligand binding

Procollagen C-proteinase enhancer (PCOLCE) proteins are extracellular matrix proteins that enhance the activities of procollagen C-proteinases by binding to the C-propeptide of procollagen I. PCOLCE proteins are built of three structural modules, consisting of two CUB domains followed by a C-terminal netrin-like (NTR) domain. While the enhancement of proteinase activity can be ascribed solely to the CUB domains, sequence homology of the NTR domain with tissue inhibitors of metalloproteinases suggest proteinase inhibitory activity for the NTR domain. Here we present the three-dimensional structure of the NTR domain of human PCOLCE1 as the first example of a structural domain with the canonical features of an NTR module. The structure rules out a binding mode to metalloproteinases similar to that of tissue inhibitors of metalloproteinases but suggests possible inhibitory function toward specific serine proteinases. Sequence conservation between 13 PCOLCE proteins from different organisms suggests a conserved binding surface for other protein partners.     

Protein conformation of potato (Solanum tuberosum) lectin determined by circular dichroism.

The structure of potato (Solanum tuberosum) lectin, which is a hydroxyproline-rich glycoprotein, has been investigated by circular dichroism. The spectra of the native lectin, and of the oxidized, reduced and carboxymethylated and deglycosylated derivatives were examined, as was a hydroxyproline-rich glycopeptide and its deglycosylated derivative. It is concluded that the lectin contains about 35% polyproline II conformation, 34% type II beta-turn and 31% irregular conformation. No indications were found for the presence of alpha-helix or beta-sheet conformations. The polyproline II conformation is heat-stable, but is markedly destabilized by deglycosylation. The type II beta-turn is destabilized by cleavage of disulphide bonds.     

The NTR module: domains of netrins, secreted frizzled related proteins, and type I procollagen C-proteinase enhancer protein are homologous with tissue inhibitors of metalloproteases.

Using homology search, structure prediction, and structural characterization methods we show that the C-terminal domains of (1) netrins, (2) complement proteins C3, C4, C5, (3) secreted frizzled-related proteins, and (4) type I procollagen C-proteinase enhancer proteins (PCOLCEs) are homologous with the N-terminal domains of (5) tissue inhibitors of metalloproteinases (TIMPs). The proteins harboring this netrin module (NTR module) fulfill diverse biological roles ranging from axon guidance, regulation of Wnt signaling, to the control of the activity of metalloproteases. With the exception of TIMPs, it is not known at present what role the NTR modules play in these processes. In view of the fact that the NTR modules of TIMPs are involved in the inhibition of matrixin-type metalloproteases and that the NTR module of PCOLCEs is involved in the control of the activity of the astacin-type metalloprotease BMP1, it seems possible that interaction with metzincins could be a shared property of NTR modules and could be critical for the biological roles of the host proteins.     

Probing the binding of scutellarin to human serum albumin by circular dichroism, fluorescence spectroscopy, FTIR, and molecular modeling method

The binding of scutellarin with human serum albumin (HSA) was investigated at four temperatures, 296, 303, 310, and 318 K, by fluorescence, circular dichroism (CD), Fourier transform infrared spectroscopy (FT-IR), and molecular modeling study at pH 7.40. The binding parameters were determined by Scatchard's procedure, which are approximately consistent with the results of Stern-Volmer equation. The thermodynamic parameters were calculated according to the dependence of enthalpy change on the temperature as follows: DeltaH degrees is a small negative value (-8.55 kJ/mol), whereas DeltaS degrees is a positive value (65.15 J/mol K). Quenching of the fluorescence HSA in the presence of scutellarin was observed. Data obtained by fluorescence spectroscopy and CD experiment, FT-IR experiment, and molecular modeling method suggested that scutellarin can strongly bind to the HSA and the primary binding site of scutellarin is located in site I of HSA. It is considered that scutellarin binds to site I (subdomain II) mainly by a hydrophobic interaction and there are hydrogen bond interactions between the scutellarin and the residues Arg222 and Arg257.     

Structure,stability, and function of hDim1 investigated by NMR,circular dichroism,and mutational analysis

The 142 amino acid Dim1p protein is a component of the U4/U6.U5 tri-snRNP complex required for pre-mRNA splicing and interacts with multiple splicing-associated proteins. To gain further insight into the structural basis of its function, we determined the solution structure of the reduced form of the dominant negative human hDim1 (hDim1(1)(-)(128)) using multidimensional NMR spectroscopy. This dominant negative hDim1 assumes a thioredoxin-like fold, confirming previous NMR and crystallographic results. However, in contrast to a recent crystal structure, the NMR solution structure for the reduced form of hDim1(1)(-)(128) presented here, along with thermodynamic data, indicates that the presence of a disulfide bond between Cys38 and Cys79 is structurally and functionally unimportant. Comparison of the truncated hDim1(1)(-)(128) with the full-length protein, using NMR and circular dichroism spectroscopy, indicates that the 14 C-terminal residues can undergo a local unfolding transition and assume alternative conformations, which appear to play a functional role. Other residues essential for hDim1 function are identified by using mutational and genetic approaches. The residues thus identified are not identical with those previously shown to govern Dim1 interaction with defined protein partners.     

Characterization of the cadmium(II) binding site in Cd,Zn-metallothionein by magnetic circular dichroism spectroscopy

Absorption and magnetic circular dichroism spectra of rat liver Cd, Zn-metallothionein, and the cadmium complexes of propanethiolate and 1,2 propanedithiolate are reported. Observation of the same derivative-like MCD signal in the 250 nm region of each of these species provides experimental evidence for the assignment of the 250 nm shoulder in the Cd, Zn-metallothionein absorption spectrum as a sulfur to cadmium charge transfer band.     

Solution conformations of cyclosporins and magnesium-cyclosporin complexes determined by vibrational circular dichroism

Vibrational circular dichroism (VCD) spectroscopy was used to investigate the solution conformations of cyclosporins A, C, D, G, and H in CDCl(3), in the amide I and NH/OH-stretching regions, and their corresponding magnesium complexes in CD(3)CN, in the amide I region. VCD spectra are sensitive to the chiral arrangement of Cdbond;O and NH bonds in this cyclic undecapeptide. Calculations of molecular geometries, as well as IR and VCD intensities of model cyclosporin fragments that include the intramolecular hydrogen bonds of the crystal conformations of cyclosporins A and H (CsA and CsH), were carried out at the density functional theory (DFT; BPW91 functional/6-31G* basis set) level. The good agreement between IR and VCD spectra from experiment and DFT calculations provides evidence that the crystal conformation of CsA is dominant in CDCl(3) solution; CsH, however, assumes both an intramolecularly hydrogen-bonded crystal conformation and more open forms in solution. Comparisons of the experimental and calculated VCD spectra in the NH/OH-stretching region of the noncomplexed cyclosporins indicate that conformers with both free and hydrogen-bonded NH and OH groups are present in solution. Differences between the IR and VCD spectra for the metal-free and magnesium-complexed cyclosporins are indicative of strong interactions between cyclosporins and magnesium ions.     

The secondary structure of bacteriorhodopsin determined by Raman and circular dichroism spectroscopy

The secondary structure of bacterio-opsin (BO), the retinal free protein-component of bacteriorhodopsin (BR), has been determined by Raman spectroscopy. Additional circular dichroism (CD) measurements have revealed only negligible conformational differences between BO in apomembranes and BR in purple membranes. Therefore, the secondary structure of BR was derived from the Raman data of BO. The protein conformation was determined to consist of 72-82% helices, 2-11% beta-strands, and 11-17% beta-turns. Only about half of the helical structures correspond to alpha 1-helices, the other half possess non-alpha 1-helical structures. According to the analysis of the Raman data, the derived secondary structure of BR was obtained with high reliability for all structure classes which can be distinguished by this method within the given uncertainty range. This is a remarkable difference from recently published secondary structural data derived from CD measurements where the helix content was reported to be between 50 and 80%. The inherent experimental and methodological uncertainties of the CD-technique leading to such a range of variation are critically discussed in comparison to the method of Raman spectroscopy. The combined application of Raman and CD spectroscopy, as performed here, is demonstrated to be a substantial improvement in the secondary structure determination of retinal-containing membrane proteins. On the basis of our results, some of the recently proposed structural models of BR with a beta-strand content of more than 11% can be ruled out.     

Stabilization of RNA strands in protein synthesis by type I procollagen C-proteinase enhancer protein, a potential RNA-binding protein, in hepatic stellate cells.

Type I procollagen C-proteinase enhancer (PCPE) exists in hepatic stellate cells (HSCs) which can produce collagen. The deduced amino acid sequence of PCPE contains motifs specific for RNA-binding proteins. The effect of PCPE on the syntheses of collagen and noncollagenous protein was studied using an HSC clone derived from cirrhotic rat liver. When the cells were cultured in the presence of an antisense oligonucleotide (AS) against PCPE mRNA, the synthesis of noncollagenous protein as well as collagen was reduced compared to the cells cultured with addition of a nonsense oligonucleotide (NS). The extent of the reduction was similar in both syntheses. The total RNA content of the AS-treated cells and NS-treated cells did not differ. In the presence of actinomycin D, however, such total RNA content was decreased more rapidly in the AS-treated cells than in the NS-treated cells. PCPE may be involved in stabilization of RNA strands in noncollagenous protein synthesis as well as collagen synthesis.     

Nonspecific binding of lac repressor to DNA. I. An absorption and circular dichroism study

Nonspecific binding of lac repressor on DNA has been studied by absorption and circular dichroism (CD) spectroscopies. In a first step, the complex formation is accompanied by an absorption difference spectrum and a change of the CD signal of the DNA. The absorption difference spectrum is mainly due to a spectral change of the DNA. The variation of the CD signal has been analyzed according to a model calculation, which takes into account the fact that the excluded site is shorter than the perturbed site. We found that in this first step one repressor can bind every 14 +/- 2 base-pairs, whereas one repressor perturbs 22 +/- 2 base-pairs. In a second step, more repressor can bind on DNA, but without further change in the absorption and CD spectrum, indicating that another binding process occurs. The model calculation developed here is general for all binding processes inducing a perturbation over a length of DNA longer than that of the excluded site.     

Characterisation of cation binding and gelation of polyuronates by circular dichroism

The cation-induced gelation of alginates and pectins with various metal ions has been monitored by circular dichroism (c.d.), using a controlled diffusion technique to prepare homogeneous gels in situ. Spectral changes observed with Ca²⁺ are closely similar to those previously reported for Ca²⁺-induced dimerisation of alginate poly-l-guluronate and pectin poly-d-galacturonate chain-sequences in solution, and the magnitude of the c.d. change on gel formation is directly related to the proportion of these structural types present. It therefore appears that gel formation does not introduce optical artefacts such as have been reported for particulate systems or biological membranes. Similar spectral changes are observed on gelation of pectin with Sr²⁺, Ba²⁺, Cd²⁺, Ni²⁺, or Pb²⁺, but with minor alterations in the wavelength of maximum c.d. change. These subtle differences are interpreted as reflecting variation in binding-site geometry to accommodate ions of different size. Differences in c.d. behaviour with Mg²⁺, Ca²⁺, and Sr²⁺ are far greater for alginate than for pectin, consistent with the greater selectivity of ion-binding. Gelation of both alginate and pectin with Cu²⁺ is accompanied by spectral changes that are opposite in sign to those observed with other divalent cations, and span a much wider range of wavelengths. This suggests a different and less-specific binding mechanism, consistent with the known lack of selectivity of Cu²⁺ for different polyuronates. However, for alginate, there is also evidence of some specific interchain chelation. A minor enhancement of alginate c.d. in the presence of K⁺ ions is attributed to a decrease in charge density of the polymer chain by bound cations, with consequent increase in segment-segment association in solution. The sign and magnitude of this effect confirm the selectivity of polyuronates for divalent cation.     

Shape,conformation and stability of fibronectin fragments determined by electron microscopy,circular dichroism and ultracentrifugation

Human plasma fibronectin digested with cathepsin D yields a number of fragments: an N-terminal fibrin-binding 30K³ fragment, a gelatin-binding 40K fragment, a 70K fragment containing the 30K and 40K domains, a central 95/105K fragment and a heparin-binding 140K fragment. The 140K fragment is linked by disulphide bonds and forms the C-terminal ends of the fibronectin. Electron microscopy of rotary-shadowed specimens revealed the 40K, 70K and 95/105K fragments as thin rods (diameter about 2.2 nm) with lengths of 13, 25 and 25 nm. respectively. These dimensions agree with the distances between flexible, proteasesusceptible regions in individual fibronectin strands determined previously. The 140K fragment appeared as a V-shaped structure with two arms 21.5 nm long emerging at a preferred angle of about 70 °. The distribution function of this angle was identical to that observed for the angle between the two arms of intact fibronectin. The free energy required for a distortion of this angle by 60 ° was estimated to be 8 kJ/mol. Reduced and alkylated fibronectin was visualized as single strands (about 55 nm long) with kinks of variable angles corresponding to sites of increased flexibility. The hydrodynamic shapes of the fragments in solution were similar to the shapes observed by electron microscopy, indicating that the individual domains of fibronectin maintain their native structure after proteolytic separation. This was also demonstrated by circular dichroism (c.d.) studies. The c.d. spectrum and the thermal melting curve of native fibronectin were well represented by a linear combination of the contributions of the fragments, indicating conformational independence of the domains. The data support earlier findings that fibronectin consists of two thin strands (length about 60 nm), which are composed of several domains separated by flexible and protease-susceptible intervening regions. This very extended structure agrees with the small sedimentation constant found for fibronectin under conditions where electrostatic interactions between domains are repulsive or depressed. Probably because of such interactions the sedimentation constant is larger at neutral pH and low ionic strength, but even under these conditions a very asymmetric shape is still maintained.     

Sequential cleavage of type I procollagen by procollagen N-proteinase. An intermediate containing an uncleaved pro alpha 1(I) chain

The conversion of type I procollagen to type I collagen was studied by cleaving the protein with partically purified type I procollagen N-proteinase from chick embryos. Examination of the reaction products after incubation for varying times at 30 degrees C indicated that, during the initial stages of the reaction, pro alpha 1(I) and pro alpha 2(I) chains were cleaved at about the same rate. As a result, all the pro alpha 2(I) chains were converted to pC alpha 2(I) chains well before all the pro alpha 1 chains were cleaved. When the reaction products were examined by gel electrophoresis without reduction of interchain disulfide bonds, a distinct band of an intermediate was detected. The same intermediate was seen when the reaction was carried out at 35, 37, and 40 degrees C. The data established that over two-thirds of the type I procollagen was converted to the intermediate and that this intermediate was then slowly converted to the final product of pCcollagen. The kinetics for the reaction, however, did not fit a simple model for precursor-product relationship among substrate, intermediate, and product. Examination of the reaction products with a two-step gel procedure demonstrated that the intermediate consisted of three polypeptide chains in which the N propeptide was cleaved from one pro alpha 1 chain and one pro alpha 2(I) chain but the N propeptide was still present on one of the pro alpha 1(I) chains. In further experiments it was demonstrated that a similar intermediate was seen when a homotrimer of pro alpha 1(I) chains was partially cleaved by the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circular dichroism, kinetic and mass spectrometric studies of copper(I) and mercury(II) binding to metallothionein

The metalloprotein metallothionein (MT) is remarkable in its metal binding properties: for the mammalian protein, well-characterized species exist for metal to sulfur ratios of M7S20, M12S20, and M18S20, where M = Cd(II), Zn(II), Hg(II), Ag(I), Au(I), and Cu(I). Optical spectra in general, and circular dichroism (CD) and luminescence spectra in particular, provide rich detail of a complicated metal binding chemistry when metals are added directly to the metal-free or zinc-containing protein. CD spectral data unambiguously identify key metal to protein stoichiometric ratios that result in well-defined structures. Electrospray ionization-mass spectrometry data are reported for reactions in which Hg(II) binds to apo-MT 2A as previously described from CD data. Emission spectra in the 450-750 nm region have been reported for metallothioneins containing Ag(I), Au(I), and Cu(I). The luminescence of Cu-MT can also be detected directly from mammalian and yeast cells. We report both steady-state and new dynamic data for titrations of Zn-MT with Cu(I). Analysis of kinetic data for the addition of the first two Cu(I) atoms to Zn-MT indicates a first-order mechanism over a concentration range of 5-50 microM. Three-dimensional modeling was carried out using the results of the CD and EXAFS studies, model calculations for Zn7-MT, Hg7-MT, and Cu12-MT are described.     

Absolute configuration of an axially chiral sulfonate determined from its optical rotatory dispersion,electronic circular dichroism,and vibrational circular dichroism spectra

The absolute configuration (AC) of an axially chiral sulfonate (aCSO), 3,5‐dimethyl‐2‐(naphthalen‐1‐yl)‐6‐(naphthalen‐1‐yl)benzenesulfonate (labeled as aCSO5), was investigated using optical rotatory dispersion (ORD), electronic circular dichroism (ECD), and vibrational circular dichroism (VCD) spectroscopies. All three methods led to the same conclusion and the AC of aCSO5 is reliably determined to be (−)‐(aR , aR ), or conversely (+)‐(aS , aS ).