Modification of the sugar specificity of a plant lectin: structural studies on a point mutant of Erythrina corallodendron lectin

A mutant of Erythrina corallodendron lectin was generated with the aim of enhancing its affinity for N-acetylgalactosamine. A tyrosine residue close to the binding site of the lectin was mutated to a glycine in order to facilitate stronger interactions between the acetamido group of the sugar and the lectin which were prevented by the side chain of the tyrosine in the wild-type lectin. The crystal structures of this Y106G mutant lectin in complex with galactose and N-acetylgalactosamine have been determined. A structural rationale has been provided for the differences in the relative binding affinities of the wild-type and mutant lectins towards the two sugars based on the structures. A hydrogen bond between the O6 atom of the sugars and the variable loop of the carbohydrate-binding site of the lectin is lost in the mutant complexes owing to a conformational change in the loop. This loss is compensated by an additional hydrogen bond that is formed between the acetamido group of the sugar and the mutant lectin in the complex with N-acetylgalactosamine, resulting in a higher affinity of the mutant lectin for N-acetylgalactosamine compared with that for galactose, in contrast to the almost equal affinity of the wild-type lectin for the two sugars. The structure of a complex of the mutant with a citrate ion bound at the carbohydrate-binding site that was obtained while attempting to crystallize the complexes with sugars is also presented

Heteromeric p97/p97R155C complexes induce dominant negative changes in wild-type and autophagy 9-deficient Dictyostelium strains.

Heterozygous mutations in the human VCP (p97) gene cause autosomal-dominant IBMPFD (inclusion body myopathy with early onset Paget's disease of bone and frontotemporal dementia), ALS14 (amyotrophic lateral sclerosis with or without frontotemporal dementia) and HSP (hereditary spastic paraplegia). Most prevalent is the R155C point mutation. We studied the function of p97 in the social amoeba Dictyostelium discoideum and have generated strains that ectopically express wild-type (p97) or mutant p97 (p97(R155C)) fused to RFP in AX2 wild-type and autophagy 9 knock-out (ATG9(KO)) cells. Native gel electrophoresis showed that both p97 and p97(R155C) assemble into hexamers. Co-immunoprecipitation studies revealed that endogenous p97 and p97(R155C)-RFP form heteromers. The mutant strains displayed changes in cell growth, phototaxis, development, proteasomal activity, ubiquitinylated proteins, and ATG8(LC3) indicating mis-regulation of multiple essential cellular processes. Additionally, immunofluorescence analysis revealed an increase of protein aggregates in ATG9(KO)/p97(R155C)-RFP and ATG9(KO) cells. They were positive for ubiquitin in both strains, however, solely immunoreactive for p97 in the ATG9(KO) mutant. A major finding is that the expression of p97(R155C)-RFP in the ATG9(KO) strain partially or fully rescued the pleiotropic phenotype. We also observed dose-dependent effects of p97 on several cellular processes. Based on findings in the single versus the double mutants we propose a novel mode of p97 interaction with the core autophagy protein ATG9 which is based on mutual inhibition

Simplified overview of p97 mutation and dose dependent effects.

<p>Simplified overview of p97 mutation and dose dependent effects.</p

Cell growth in shaking culture and on <i>Klebsiella aerogenes</i> are altered in mutant strains expressing p97-RFP or p97^R155C-RFP.

<p>(A) Strains expressing p97-RFP or p97^R155C-RFP display specific growth defects in shaking culture. Please note the logarithmic scale of the y-axis. (B) Growth on <i>K. aerogenes</i> lawns. Mutation specific and dose dependent effects are seen in both wild-type and ATG9^KO strains. Growth of AX2 on day 5 was set to 100%.</p

Predicted and observed experimental outcomes of the expression of p97 and p97^R155C in AX2 wild-type and ATG9^KO cells.

<p>The table summarizes the predicted changes based on the model of p97 and ATG9 interaction and mutual inhibition as illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046879#pone-0046879-g009" target="_blank">Fig. 9</a>. Experimental results in black; model validity in green; “no” and “no” with uppercase “1)”, see Discussion section; “ref value”, changes were separately compared to wild-type and ATG9^KO backgrounds; ↑, ↓, →, increase, decrease or no change with respect to the reference value. Inverse, inverted mutation specific effect. Rescue, partial or full rescue of the ATG9^KO phenotype.</p

Subcellular localization of p97 and its co-localization with ubiquitin in wild-type and mutant strains.

<p>(<b>A</b>) Visualization of subcellular localization of p97 in AX2 wild-type cells with polyclonal antibodies p97_8_6841 directed against amino acids 23–73 and p97_9_6574 directed against amino acids 254–310. (<b>B</b>) Subcellular localization of p97 (<b>left panel</b>) using the p97_8_6841 polyclonal antibody and ubiquitin (<b>middle panel</b>) using the P4D1 monoclonal antibody (NEB, Germany) in AX2 wild-type cells and mutant strains. Merged images and DAPI staining to visualize nuclei (<b>right panel</b>). <b>Upper row</b>: AX2 wild-type cells; <b>middle row</b>: ATG9^KO mutant; <b>bottom row</b>: ATG9^KO/p97^R155C-RFP double mutant. Please note that ubiquitin positive protein aggregates frequently co-localize with p97 in the ATG9^KO mutant (arrows) but not in the ATG9^KO/p97^R155C-RFP double mutant (double arrowheads). The ATG9^KO mutant also contains protein aggregates that are either positive for p97 (arrowhead) or ubiquitin (double arrowhead). Cells were fixed with cold methanol and stained with the indicated antibodies. Scale bars are 10 µm and 2 µm in inset.</p

Sequence identity and sequence similarity of p97 from different organisms.

<p>Sequence identity, left, and sequence similarity, right, was determined by aligning the corresponding protein sequences using BLAST align program (bl2seq) at the NCBI. Percentage values are given. Hs: <i>Homo sapiens</i>, <i>Mm: Mus musculus, Rn: Rattus norwegicus, Xl: Xenopus laevis, Sc: S. cerevisiae, Dd: D. discoideum.</i></p