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.

Pal amidase, encoded by pneumococcal bacteriophage Dp-1, represents one step beyond in the modular evolution of pneumococcal murein hydrolases. It exhibits the choline-binding module attaching pneumococcal lysins to the cell wall, but the catalytic module is different from those present in the amidases coded by the host or other pneumococcal phages. Pal is also an effective antimicrobial agent against Streptococcus pneumoniae that may constitute an alternative to antibiotic prophylaxis. The structural implications of Pal singular structure and their effect on the choline-amidase interactions have been examined by means of several techniques. Pal stability is maximum around pH 8.0 (T m Х 50.2°C; ⌬H t ‫؍‬ 183 ؎ 4 kcal mol ؊1 ), and its constituting modules fold as two tight interacting cooperative units whose denaturation merges into a single process in the free amidase but may proceed as two well resolved events in the choline-bound state. Choline titration curves reflect low energy ligand-protein interactions and are compatible with two sets of sites. Choline binding strongly stabilizes the cell wall binding module, and the conformational stabilization is transmitted to the catalytic region. Moreover, the high proportion of aggregates formed by the unbound amidase together with choline preferential interaction with Pal dimers suggest the existence of marginally stable regions that would become stabilized through choline-protein interactions without significantly modifying Pal secondary structure. This structural rearrangement may underlie in vitro "conversion" of Pal from the low to the full activity form triggered by choline. The Pal catalytic module secondary structure could denote folding conservation within pneumococcal lytic amidases, but the number of functional choline binding sites is reduced (2-3 sites per monomer) when compared with pneumococcal LytA amidase (4 -5 sites per monomer) and displays different intermodular interactions.Dp-1, the first described pneumococcal bacteriophage, belongs to the Siphoviridae family (1). Its peptidoglycan-degrading enzyme, Pal, 1 was biochemically characterized as a cholinedependent amidase (2) synthesized as a low activity form that requires in vitro incubation with choline or choline-containing cell walls to achieve full activity (3), a process designated as "conversion." Pal shows the modular organization characteristic of pneumococcal cell wall lysins (3), but represents one step beyond in the modular evolution of pneumococcal murein hydrolases. Its N-terminal region has no similarity with the amidases coded by the host or other pneumococcal phages (4, 5), 2 but is highly similar to the murein hydrolase coded by Lactococcus phage BK5-T. 3 The C-terminal region comprises a choline binding module (ChBM) homologous to that found in the pneumococcal lytic system 4 (6) that attaches the enzyme to choline residues present in pneumococcal envelope (7). As in most pneumococcal lysins so far known, six repeats of about 20 amino acids (p1-p6) and a short C-terminal tail fo...

The CUB domain is a widespread 1 lO-amino-acid twdule lound in fiinclionally diverse, often developmentally regulated proteins, for which an antiparallel P-b:itrel topology similar to that in irriniunoflobulin V domains has been predicted. Spemxidhesins have becn proposed as a subgroup of this protein family built up by B single CUB domain architccturc. To tcst the proposed structural model, wc have analyzed the structural organkition o f two meinher.; of the spei-madhesin protein frimily, porcine seminal plasma proteins 1/11 (PSP-I/PSP-TI) heterodimer arid bovinc acidic seminal fluid protein (aSFP) horiiodimer, using differential scanning calorimetry, far-ultraviolet circular dichroism and ~;ourici-transforrn infrared spcctroscopy. Thermal unfolding of PSP-l/PSP-II and aSFP werc irreversible and follocvcd a onc-step process with transition temperatures (Tin) of 60.S"C and 78.6"C, rcspectively. The calorimctric cnthalpy changes (AH,,,,) of thermal denaturation werc 439 k.l/niol for PSP-I/PSP-I1 and 660 kJ/tnol for aSIT dimer. Analysis of the calorimetric curves of PSP-I/PSP-I1 showcd thut the cntire dimer constituted the cooperative unfolding unit. Fourier-transform infrared spectroscopy and deconvolution of circular dichroic spectra using ii convex constraint analysis indicated that [I-structurc arid turns are the miijor slructural element of both PSP-UPSP-I1 (53% of p-sheet, 21 %I of turns) and aSFP (44% of /I-shcet, 36% of turns), ant1 that the porcine and the bovine proteins contain little, if any, Ix-helical slructurc. Taken together, OLIT rcsults indicate that the porcine and the bovine spermadhesin molecules are probably all-/j-struclure proteins, and would support a /$-barrel topology like thal predicted for the CUB domain. Othcr p-structurc folds, such as the Greek-key pattern characteristic of inany carbohydrate-binding protein dotnains ciinnot be eliminated. Finally, the saiiic combination of biophysical tcchniques was used to characterize the residual secondary slructurc of thermally denatured forms of PSP-I/PSP-II and aSFP, and to emphustze the aggregation tendency of these lorms.Keywords: scminal plasrna proteins; spermadhesins; CUB domain; conformational analysis; calorimetry.The early events of niamtrialian fertilization involvc a coinplcx series of highly orchestrated interactions between complementary molecules located on the surface of homologous gametes. In mammiils, sperm-egg recognition m d binding i s mediated hy spermatozoa-associated carbohydrate-binding proteins and saccharide chains of the oocyte's cxtracellular matrix called thc zona pcllucida (reviewed 1 1 1 ) . A variety of sperm proteins with zona-pellucida-binding capability, both integral plasrnii membrane and peripherally associated proteins, have becn idcntified in dilferent nummalian species (see [2] and refcrences therein). This suggests that fertilization is probably mediated by multiple zona pellucida spermatozoon-receptor systems, which collectively dictate the species specificity of gamete interaction.Cr,rr~.sl)rttrdmrp t...

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