Do sequence repeats play an equivalent role in the choline-binding module of pneumococcal LytA amidase?

LytA amidase breaks down the N-acetylmuramoyl-l-alanine bonds in the peptidoglycan backbone of Streptococcus pneumoniae. Its polypeptide chain has two modules: the NH(2)-terminal module, responsible for the catalytic activity, and the COOH-terminal module, constructed by six tandem repeats of 20 or 21 amino acids (p1-p6) and a short COOH-terminal tail. The polypeptide chain must contain at least four repeats to efficiently anchor the autolysin to the choline residues of the cell wall. Nevertheless, the catalytic efficiency decreases by 90% upon deletion of the final tail. The structural implications of deleting step by step the two last (p5 and p6) repeats and the final COOH-tail and their effects on choline-amidase interactions have been examined by comparing four truncated mutants with LytA amidase by means of different techniques. Removal of this region has minor effects on secondary structure content but significantly affects the stability of native conformations. The last 11 amino acids and the p5 repeat stabilize the COOH-terminal module; each increases the module transition temperature by about 6 degrees C. Moreover, the p5 motif also seems to participate, in a choline-dependent way, in the stabilization of the NH(2)-terminal module. The effects of choline binding on the thermal stability profile of the mutant lacking the p5 repeat might reflect a cooperative pathway providing molecular communication between the choline-binding module and the NH(2)-terminal region. The three sequence motives favor the choline-amidase interaction, but the tail is an essential factor in the monomer <--> dimer self-association equilibrium of LytA and its regulation by choline. The final tail is required for preferential interaction of choline with LytA dimers and for the existence of different sets of choline-binding sites. The p6 repeat scarcely affects the amidase stability but could provide the proper three-dimensional orientation of the final tail.

Structural characterization of the unligated and choline-bound forms of the major pneumococcal autolysin LytA amidase. Conformational transitions induced by temperature.

The secondary and tertiary structures of the choline-dependent major pneumococcal autolysin LytA amidase and of its COOH-terminal domain, C-LytA, have been investigated by circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy. Deconvolution analysis shows that the far-UV CD spectrum of both proteins is governed by chiral contributions, ascribed to aromatic residue clusters contained in the COOH-terminal module. The secondary structure of LytA, determined from the FTIR spectral features of the amide I' band, results in 19% of alpha-helix and tight loops, 47% of beta-sheets, 23% of turns, and 11% of irregular structures. Similar values are obtained for C-LytA. The addition of choline significantly modifies the far- and near-UV CD spectra of LytA and C-LytA. These changes are attributed to alterations in the environment of their aromatic clusters, since the FTIR spectra indicate that the secondary structure is essentially unaffected. CD choline titration curves at different wavelengths show the existence of two types of binding sites/subunit. Data analysis assuming protein dimerization upon saturation of the high affinity sites reveals positive cooperativity between the low affinity sites. Thermal denaturation of both proteins occurs with the formation of unfolding intermediates and the presence of residual secondary structure in the final denatured state. The irreversibility of the thermal denaturation of LytA and C-LytA results from the collapse of the polypeptide chain into intermolecular extended structures. At saturating concentrations, choline prevents the formation of these structures in the isolated COOH-terminal module.

Analysis of the structural organization and thermal stability of two spermadhesins. Calorimetric, circular dichroic and Fourier-transform infrared spectroscopic studies.

The CUB domain is a widespread 110-amino-acid module found in functionally diverse, often developmentally regulated proteins, for which an antiparallel beta-barrel topology similar to that in immunoglobulin V domains has been predicted. Spermadhesins have been proposed as a subgroup of this protein family built up by a single CUB domain architecture. To test the proposed structural model, we have analyzed the structural organization of two members of the spermadhesin protein family, porcine seminal plasma proteins I/II (PSP-I/PSP-II) heterodimer and bovine acidic seminal fluid protein (aSFP) homodimer, using differential scanning calorimetry, far-ultraviolet circular dichroism and Fourier-transform infrared spectroscopy. Thermal unfolding of PSP-I/PSP-II and aSFP were irreversible and followed a one-step process with transition temperatures (Tm) of 60.5 degrees C and 78.6 degrees C, respectively. The calorimetric enthalpy changes (delta Hcat) of thermal denaturation were 439 kJ/mol for PSP-I/PSP-II and 660 kJ/mol for aSFP dimer. Analysis of the calorimetric curves of PSP-I/PSP-II showed that the entire dimer constituted the cooperative unfolding unit. Fourier-transform infrared spectroscopy and deconvolution of circular dichroic spectra using a convex constraint analysis indicated that beta-structure and turns are the major structural element of both PSP-I/PSP-II (53% of beta-sheet, 21% of turns) and aSFP (44% of beta-sheet, 36% of turns), and that the porcine and the bovine proteins contain little, if any, alpha-helical structure. Taken together, our results indicate that the porcine and the bovine spermadhesin molecules are probably all-beta-structure proteins, and would support a beta-barrel topology like that predicted for the CUB domain. Other beta-structure folds, such as the Greek-key pattern characteristic of many carbohydrate-binding protein domains cannot be eliminated. Finally, the same combination of biophysical techniques was used to characterize the residual secondary structure of thermally denatured forms of PSP-I/PSP-II and aSFP, and to emphasize the aggregation tendency of these forms.

In search of a correlation between the electron affinity and the radiosensitizing activity of some glyoxylic compounds.

The electron affinity of some glyoxylic compounds with radiosensitizing properties was evaluated using different approaches. The half-wave reduction potentials were measured by polarography, and the molecular structures in solution were determined by NMR spectroscopy. The electron affinity of the unhydrated and hydrated molecules was theoretically evaluated by means of a semi-empirical quantum mechanical procedure, the MNDO. The possible correlations between these values and the radiosensitizing efficiency of these glyoxylic compounds is discussed.

The in vitro interaction with DNA of several hypoxic radiosensitizers.

The five 5-nitroimidazole derivatives and the four glyoxylic compounds tested in this paper for their interaction with DNA with and without irradiation had previously been reported to act as radiosensitizers at a cellular level. Our aim was to find if the radiosensitizing activity of these products could be due to their interaction with DNA. For this purpose the formation of a complex was tested by comparing the spectrophotometrical absorbances of (DNA-product) solutions with those of DNA and of the products, at the absorption maxima of the products. None of the products formed in vitro a complex with DNA. The irradiation of product solutions with and without DNA induced a molecular degradation of the product which was linearly correlated with the radiation dose. The G values characterizing this radiodegradation showed that the presence of DNA did not modify the radiosensitivity of the products, neither in aerobic nor under hypoxic irradiation conditions. These results proved that the radiosensitizing properties of these compounds cannot be attributed to their chemical interaction with DNA.