Plant polygalacturonase structures specify enzyme dynamics and processivities to fine-tune cell wall pectins

Abstract

Polygalacturonases (PGs) fine-tune pectins to modulate cell wall chemistry and mechanics, impacting plant development. The large number of PGs encoded in plant genomes leads to questions on the diversity and specificity of distinct isozymes. Herein, we report the crystal structures of 2 Arabidopsis thaliana PGs, POLYGALACTURONASE LATERAL ROOT (PGLR), and ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE2 (ADPG2), which are coexpressed during root development. We first determined the amino acid variations and steric clashes that explain the absence of inhibition of the plant PGs by endogenous PG-inhibiting proteins (PGIPs). Although their beta helix folds are highly similar, PGLR and ADPG2 subsites in the substrate binding groove are occupied by divergent amino acids. By combining molecular dynamic simulations, analysis of enzyme kinetics, and hydrolysis products, we showed that these structural differences translated into distinct enzyme-substrate dynamics and enzyme processivities: ADPG2 showed greater substrate fluctuations with hydrolysis products, oligogalacturonides (OGs), with a degree of polymerization (DP) of ≤4, while the DP of OGs generated by PGLR was between 5 and 9. Using the Arabidopsis root as a developmental model, exogenous application of purified enzymes showed that the highly processive ADPG2 had major effects on both root cell elongation and cell adhesion. This work highlights the importance of PG processivity on pectin degradation regulating plant development.

Figure 1.

Structure comparison of PGLR and ADPG2 and identification of novel amino acids required for activity. A) Overall structure of PGLR and ADPG2 represented in ribbon diagrams which are colored in blue and brown, respectively. PGLR and ADPG2 active site amino acids are pink and green colored. β-sheets and turns are indicated by red and blue arrows. B) Ribbon representation of P. vulgaris PGIP2 (PvPGIP2, plum), PGLR (blue), ADPG2 (brown), and F. phyllophilum PG (FpPG1, green). C) Detailed representation of AA involved in PvPGIP2-FpPG1 interaction (PvPGIP2 amino acids in blue and FpPG1 amino acids in gray), with orange lines representing van der Waals contacts. Key AA (N121, A274) mediating the interaction in FpPG1 are absent in PGLR and ADPG2. AA that can hinder the PG-PGIP interaction are represented in pink (PGLR) and green (ADPG2). D) Superimposition of crystallized PvPGIP2 with models of Arabidopsis PGIP1 (AtPGIP1, orange) and PGIP2 (AtPGIP2, blue). E) Interactions of AtPGIP1 with PGLR and ADPG2. Amino acids of AtPGIP1 (yellow), PGLR (pink), and ADPG2 (green) included in clashes closer than 0.6 Å are shown. The red lines indicated atoms with an overlap of more than 0.6 Å.

Figure 2.

Structure of the PGLR-ADPG2 active site and binding groove. Role of H196 and 237 for PGLR activity. A) Active site of PGLR/ADPG2 highlighting absolutely conserved AA. D193/D219, D214/D240, and D215/D241 are AA involved in substrate hydrolysis. Black numbers indicate the subsites. B) Total PG activity of WT and mutated forms of PGLR (H196K, H237K, H196K/H237K, R271Q, and D217A) on PGA (blue bars), and MST analysis of the interaction between WT and mutated forms of PGLR using a substrate of DP12 and DM5 (black rhomboids). Values correspond to means ± Sd of 3 replicates. C) Structure of PGLR binding groove (subsite −5 to +5). D) Structure of ADPG2 binding grove (subsites −5 to +5). E) Sequence of the fully demethylesterified (pattern 1) or 60% methylesterified (pattern 2) decasaccharides simulated in complex with ADPG2 and PGLR. D: demethylesterified GalA, M: methylesterified GalA. F) Cross-section of the substrate binding groove highlighting the positions of H196 and H237, which are represented as orange spheres. Positively and negatively charged residues are shown in blue and red, respectively, while polar residues are shown in green and represented as sticks. G) PGLR in complex with a decasaccharide substrate, with insets showing the conformational ensembles of the substrate in complex with WT PGLR, H196K, and H237K, by reporting conformations obtained every 10 ns. H) Root mean square fluctuations (RMSF) of demethylesterified decasaccharide across the binding groove for WT PGLR and PGLR mutants.

Figure 3.

PGLR and ADPG2 show distinct substrate dynamics. A, B) Root mean square fluctuations (RMSF) of each monosaccharide bound across the binding groove of PGLR A) or ADPG2 B). In each panel, fully demethylesterified (pattern 1—cyan in A and orange in B) or 60% methylesterified decasaccharides (pattern 2—yellow in A and pink in B) are shown. C) Analysis of the contacts between PGLR or ADPG2 and substrates either fully demethylesterified (pattern 1) or characterized by 60% methylesterification (pattern 2).

Figure 4.

PGLR and ADPG2 release distinct OGs. A) Activity tests performed on PGA (DM 0) after 1 h digestion by ADPG2 and PGLR and by adding PGLR or ADPG2 for 1 h after a first digestion by PGLR. NaOAc (sodium acetate): negative control. B) Oligoprofiling of OGs released after 1 h digestion of PGA by PGLR (black) or ADPG2 (gray) at 40 °C, pH 5.2. C) Oligoprofiling of OGs after overnight digestion of pectins DM 20% to 34% by PGLR (black) or ADPG2 (gray) at 40 °C, pH 5.2. Inset: cumulative OGs released by PGLR and ADPG2 after overnight digestion on pectins DM 20% to 34% at 40 °C, pH 5.2. In all figures, values correspond to means ± Sd of 3 replicates. a, b, and **** indicate statistically significant difference, P < 0.001.

Figure 5.

PGLR and ADPG2 are active on root pectins and have distinct effects on root length and root cap. A) Oligoprofiling of OGs after digestion of root cell wall by PGLR (black) and ADPG2 (gray) at 40 °C, pH 5.2 after overnight digestion (inset: cumulative OGs released by PGLR (black) and ADPG2 (gray) after overnight digestion of root cell walls at 40 °C, pH 5.2). * indicates statistically significant difference, P < 0.05. B) Effects of the exogenous application of PGLR and ADPG2 on total root length of Arabidopsis seedlings. PGLR and ADPG2 were applied at isoactivities for 1 or 3 days on 6-day-old seedlings grown in liquid media. The value marked with * indicates statistically significant difference between controls and ADPG2 analyzed by the 1-way ANOVA with the Tukey multiple comparison test P < 0.0001. n ≥ 14, ns = nonsignificant. C) Root cell numbering using EGFP-LTI6b reporter lines. D) Effects of 3-day exogenous application of PGLR and ADPG2 on the cell length of the firsts 50 root cells of 7-day-old seedlings. N > 50. E) Effects of 3-day exogenous application of PGLR and ADPG2 on the root cap structure of 7-day-old seedlings (2 representative images per condition). Buffer (Ø Enz) was used as negative control. Scale bar represents 100 mm.

Figure 6.

Model of PGLR and ADPG2 processivity. A) PGLR shows low processive dynamics where enzyme–substrate association is followed by hydrolysis and dissociation of the substrate from the enzyme. This low processivity produces OGs of variable DPs. B) ADPG2 sliding motion after forming enzyme–substrate complex allows multiple substrate hydrolysis while staying attached to the substrate showing highly processive dynamics. Processive enzymes can produce small DP OGs. Galacturonic acid is yellow colored. Galacturonic acid reducing end is gray colored. PG subsites are indicated by numbers. Red triangle represents the hydrolysis site.