Structure of a Plant Cell Wall Fragment Complexed to Pectate Lyase C

Abstract

The three-dimensional structure of a complex between the pectate lyase C (PelC) R218K mutant and a plant cell wall fragment has been determined by x-ray diffraction techniques to a resolution of 2.2 A and refined to a crystallographic R factor of 18.6%. The oligosaccharide substrate, alpha-D-GalpA-([1-->4]-alpha-D-GalpA)3-(1-->4)-D-GalpA , is composed of five galacturonopyranose units (D-GalpA) linked by alpha-(1-->4) glycosidic bonds. PelC is secreted by the plant pathogen Erwinia chrysanthemi and degrades the pectate component of plant cell walls in soft rot diseases. The substrate has been trapped in crystals by using the inactive R218K mutant. Four of the five saccharide units of the substrate are well ordered and represent an atomic view of the pectate component in plant cell walls. The conformation of the pectate fragment is a mix of 21 and 31 right-handed helices. The substrate binds in a cleft, interacting primarily with positively charged groups: either lysine or arginine amino acids on PelC or the four Ca2+ ions found in the complex. The observed protein-oligosaccharide interactions provide a functional explanation for many of the invariant and conserved amino acids in the pectate lyase family of proteins. Because the R218K PelC-galacturonopentaose complex represents an intermediate in the reaction pathway, the structure also reveals important details regarding the enzymatic mechanism. Notably, the results suggest that an arginine, which is invariant in the pectate lyase superfamily, is the amino acid that initiates proton abstraction during the beta elimination cleavage of polygalacturonic acid.

Characterization and Implications of Ca2+Binding to Pectate Lyase C

“…No positive electron density, consistent with partially occupied Ca 2ϩ ions, is present at any other site at which the exogenous Ca 2ϩ concentration is 30 mM or less. This observation is consistent with the R218-(Ca 2ϩ ) 4 -pentaGalpA complex structure in which the other Ca 2ϩ ions coordinate to only one or two amino acids, suggesting that the affinity of these Ca 2ϩ ions for the protein may be very weak (17).…”

“…The present research does not address whether the affinity of Ca 2ϩ for PelC increases in the presence of the substrate. Such a question is difficult to answer because additional Ca 2ϩ ions are bound at the PelC-oligosaccharide interface (17).…”

“…strain KSM-P15 pectate lyase (16) reveal details of a Ca 2ϩ binding site at the same relative location in each structure. Another structure, that of a complex between an inactive PelC 1 mutant and a pentagalacturonate substrate, has revealed three additional Ca 2ϩ sites linking the substrate to the protein (17). Although the PelC R218K-(Ca 2ϩ ) 4 -pentaGalpA structure provides many insights into the pectate lyase cleavage mechanism, the structure complicates questions regarding the function of the Ca 2ϩ ions.…”

Structural requirements of endo polygalacturonase for the interaction with PGIP (polygalacturonase-inhibiting protein)

“…1b). This finding is of some interest in view of the polyanionic nature of the substrate and the fact that bacterial pectate lyases and PGs are generally richer in basic amino acids (34,41). It is well known that fungal PGs interact with and are inhibited by plant inhibitors named PGIPs.…”

Crystal Structure of Jun a 1, the Major Cedar Pollen Allergen from Juniperus ashei, Reveals a Parallel β-Helical Core

“…The salt bridge between Asp 5 in the N-terminal helix-turnhelix loop and His 165 on T1.5 of the ␤-helix aids in holding the N-terminal helix-turn-helix loop in place and shields the vWiDH sequence, which is always present in the Pnl and Pel and proposed to be an enzymatically active site (20), from the solvent (Table II). In addition, the salt bridge between Asp 177 and His 203 traverses the presumed substrate binding groove in the vicinity of the putative active site Arg 229 .…”