Structural Characterization of the Unligated and Choline-bound Forms of the Major Pneumococcal Autolysin LytA Amidase

Abstract

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.

Pneumococcal Virulence Factors: Structure and Function

“…The dimerization involves primarily the C-terminal part of the molecule, with preferential binding of two choline molecules to the dimer. On the other hand, saturation of the low-affinity sites requires a choline concentration similar to those necessary for inhibition of this amidase in in vivo assays (100). The presence of these two choline binding sites, together with inducible dimerization, might play an essential role in LytA cellular targeting by causing a preferential location of the enzyme at the sites of its action on the cell wall.…”

“…However, two modes of choline binding to the C-terminal module have been detected; one is low affinity and another is high affinity. Saturation of choline binding to the high-affinity sites induces dimerization and subsequent increase of the affinity for the substrate (100,159). The dimerization involves primarily the C-terminal part of the molecule, with preferential binding of two choline molecules to the dimer.…”

Pathogenesis of pneumococcal infection

“…The secondary structure of autolysin contains 19% á-helix, 47% â-sheet, 23% turns and 11% irregular structures [59]. The far UV spectrum of the protein is governed by chiral contributions from aromatic residues clustered in the COOH domain.…”

“…The addition of choline modi®es the far UV spectrum by alteration of the environment of the aromatic clusters, but the secondary structure remains unaltered. Dimerisation appears to occur through co-operation between the low-af®nity choline binding sites [59].…”

Construction of a Chimeric Thermostable Pyrophosphatase To Facilitate Its Purification and Immobilization by Using the Choline-Binding Tag

“…This domain is able to specifically bind to choline or its structural analogues (e.g., diethylaminoethanol or DEAE), allowing its purification by affinity chromatography in a single step using choline-or DEAE-containing supports (25). The ChBD has been studied extensively by biophysical and biochemical techniques (15), and its three-dimensional structure was recently elucidated (3). The ChBD has a novel solenoid fold consisting exclusively of ␤ hairpins that stack to form a left-handed superhelix, known as a ␤ spiral staircase.…”