Structure of PITPbeta in complex with phosphatidylcholine: comparison of structure and lipid transfer to other PITP isoforms.

Phosphatidylinositol transfer protein (PITP) is a ubiquitous eukaryotic protein that preferentially binds either phosphatidylinositol or phosphatidylcholine and catalyzes the exchange of these lipids between membranes. Mammalian cytosolic PITPs include the ubiquitously expressed PITPalpha and PITPbeta isoforms (269-270 residues). The crystal structure of rat PITPbeta complexed to dioleoylphosphatidylcholine was determined to 2.18 A resolution with molecular replacement using rat PITPalpha (77% sequence identify) as the phasing model. A structure comparison of the alpha and beta isoforms reveals minimal differences in protein conformation, differences in acyl conformation in the two isoforms, and remarkable conservation of solvent structure around the bound lipid. A comparison of transfer activity by human and rat PITPs, using small unilamellar vesicles with carefully controlled phospholipid composition, indicates that the beta isoforms have minimal differences in transfer preference between PtdIns and PtdCho when donor vesicles contain predominantly PtdCho. When PtdCho and PtdIns are present in equivalent concentrations in donor vesicles, PtdIns transfer occurs at approximately 3-fold the rate of PtdCho. The rat PITPbeta isoform clearly has the most diminished transfer rate of the four proteins studied. With the two rat isoforms, site-directed mutations of two locations within the lipid binding cavity that possess differing biochemical properties were characterized: I84alpha/F83beta and F225alpha/L224beta. The 225/224 locus is more critical in determining substrate specificity. Following the mutation of this locus to the other amino acid, the PtdCho transfer specific activity became PITPalpha (F225L) approximately PITPbeta and PITPbeta (L224F) approximately PITPalpha. The 225alpha/224beta locus plays a modest role in the specificity of both isoforms toward CerPCho.

Structure of pectate lyase A: comparison to other isoforms.

Pectate lyase A is a virulence factor secreted by the plant-pathogenic bacteria Erwinia chrysanthemi. The enzyme cleaves the glycosidic bond of pectate polymers by a calcium-dependent beta-elimination mechanism. The crystal structure of pectate lyase A from E. chrysanthemi EC16 has been determined in two crystal forms, monoclinic C2 to 1.8 A and rhombohedral R3 to 2.1 A. The protein structure is compared with two other pectate lyase isoforms from E. chrysanthemi EC16, pectate lyase C and pectate lyase E. Pectate lyase A is unique as it is the only acidic pectate lyase and has end products that are significantly more varied in length in comparison to those of the other four major pectate lyase isozymes. Differences and similarities in polypeptide trace, size and volume of the active-site groove and surface electrostatics are discussed.

Effect of mutations in the T1.5 loop of pectate lyase A from Erwinia chrysanthemi EC16.

Pectate lyase A (PelA) is a pectate-degrading enzyme secreted by plant pathogens. PelA from Erwinia chrysanthemi has 61% amino-acid identity and a conserved structural similarity to pectate lyase E (PelE). Although similar in structure and sequence, the enzymatic characteristics of PelA differ from those for PelE. A structural alignment of PelA and PelE reveals differences in the T1.5 loop. The sequence of the T1.5 loop in PelA was mutated to the homologous sequence in PelE. The crystal structure of the PelA T1.5 mutant has been solved to 1.6 and 2.9 A resolution. The enzymatic and structural properties of the T1.5 mutant are discussed.