Structural analysis of Brucella abortus RicA substitutions that do not impair interaction with human Rab2 GTPase
Bibliographic record
Abstract
BACKGROUND: Protein-protein interactions are at the basis of many cellular processes, and they are also involved in the interaction between pathogens and their host(s). Many intracellular pathogenic bacteria translocate proteins called effectors into the cytoplasm of the infected host cell, and these effectors can interact with one or several host protein(s). An effector named RicA was recently reported in Brucella abortus to specifically interact with human Rab2 and to affect intracellular trafficking of this pathogen. RESULTS: In order to identify regions of the RicA protein involved in the interaction with Rab2, RicA was subjected to extensive random mutagenesis using error prone polymerase chain reaction. The resulting allele library was selected by the yeast two-hybrid assay for Rab2-interacting clones that were isolated and sequenced, following the "absence of interference" approach. A tridimensional model of RicA structure was used to position the substitutions that did not affect RicA-Rab2 interaction, giving a "negative image" of the putative interaction region. Since RicA is a bacterial conserved protein, RicA homologs were also tested against Rab2 in a yeast two-hybrid assay, and the C. crescentus homolog of RicA was found to interact with human Rab2. Analysis of the RicA structural model suggested that regions involved in the folding of the "beta helix" or an exposed loop with the IGFP sequence could also be involved in the interaction with Rab2. Extensive mutagenesis of the IGFP loop suggested that loss of interaction with Rab2 was correlated with insolubility of the mutated RicA, showing that "absence of interference" approach also generates surfaces that could be necessary for folding. CONCLUSION: Extensive analysis of substitutions in RicA unveiled two structural elements on the surface of RicA, the most exposed β-sheet and the IGFP loop, which could be involved in the interaction with Rab2 and protein folding. Our analysis of mutants in the IGFP loop suggests that, at least for some mono-domain proteins such as RicA, protein interaction analysis using allele libraries could be complicated by the dual effect of many substitutions affecting both folding and protein-protein interaction.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".