A Bivalent Dissectional Analysis of the High-Affinity Interactions between Cdc42 and the Cdc42/Rac Interactive Binding Domains of Signaling Kinases in <i>Candida albicans</i>
Bibliographic record
Abstract
The small GTPase Cdc42, a member of the highly conserved Rho family of intracellular GTPases, communicates with downstream signaling proteins via high-affinity interactions with the consensus Cdc42/Rac interactive binding (CRIB) polypeptide sequence. Previous biochemical and structural studies show that the CRIB motif itself is insufficient for high-affinity binding to Cdc42 but requires the sequence segment C-terminal to the CRIB motif for enhanced affinity. In this study, we have investigated the high-affinity (K(d) in units of nanomolar) associations of two highly homologous extended CRIB domains (eCRIBs) from the PAK kinases, Cla4 and Cst20, with Cdc42 from Candida albicans. (1)H-(15)N NMR heteronuclear NOE data of the eCRIB polypeptides in complex with Candida Cdc42 (CaCdc42) indicate that both eCRIB peptides have approximately two binding loci for CaCdc42. When each of the two eCRIB peptides is dissected into two fragments, the N-terminal fragments containing the minimal CRIB motif (mCRIB), mCla4 and mCst20, have relatively high binding affinities with dissociation constants (K(d)) of 4.2 and 0.43 microM, respectively. On the other hand, the C-terminal fragments (cCRIB), cCla4 and cCst20, exhibit significantly lower affinities for their binding to CaCdc42. The cCla4 peptide binds to CaCdc42 with a sub-millimolar K(d) of 275 microM, and the cCst20 peptide shows an even lower binding affinity (K(d) = 1160 microM). Cross-titration experiments with the cognate fragments show that the binding affinity of cCst20 is enhanced approximately 5.5-fold (K(d) = 207 microM) in the presence of saturating amounts of mCst20, and vice versa. No such effect is observed for the binding of cCla4 and mCla4. These results suggest that the Cdc42-CRIB system can be represented by a "dual recognition" model for protein-protein interactions [Kleanthous, C., et al. (1998) Mol. Microbiol. 28, 227-233], following much the same mechanisms of multivalent molecular interactions [Song, J., and Ni, F. (1998) Biochem. Cell Biol. 76, 177-188; Mammen, M., et al. (1998) Angew Chem., Int. Ed. 37, 2754-2794]. The bivalent modeling of linked peptide fragments shows that the binding of eCla4 follows a simple additivity/avidity model, while binding of eCst20 appears to have a more complex mechanism involving cooperative effects. The differential binding mechanisms between closely related eCRIB polypeptides and CaCdc42 provide a new molecular basis for understanding kinase activation and for the design of antifungal agents targeting the large protein interaction interfaces engaged by the fungal GTPase.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.000 | 0.001 |
| 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 teacher head, 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".