Hidden structural states of proteins revealed by conformer selection
@NatureComms
1. The paper introduces AISAR (AI SAmpling with NMR Recall selection), a computational–experimental workflow that finds alternative protein conformations by selecting from AI-sampled structures using NOESY recall and other NMR observables, rather than enforcing traditional distance restraints.
2. Key motivation: conventional restraint-based NMR can “pin” dynamic proteins into compromised single structures when NOEs arise from multiple fast-exchanging states; AISAR aims to avoid these distortions by using conformer selection instead of restraint satisfaction.
3. AISAR pipeline: generate diverse conformers with AlphaFold2-based sampling (AFsample), score each model with a Bayesian-like metric combining (i) min–max scaled NOESY recall pTM (global fold reliability) and (ii) agreement between per-residue pLDDT and chemical-shift-derived flexibility (RCI), then cluster, select candidate states, and assemble multi-state ensembles guided by RMSF agreement with RCI.
4. Validation centerpiece: NOESY “Double Recall” analysis identifies subsets of NOESY peaks uniquely explained by each state (state-specific NOE support), providing evidence for multiple conformations in dynamic equilibrium rather than model overfitting.
5. On Gaussia luciferase (GLuc), AISAR identifies two dominant, interconverting states (<~1 ms lifetimes) with large rearrangements of two “lids” (H5/H6 loop and C-terminal broken helix H10/H11), switching between closed vs open conformations that reshape binding pockets and expose cryptic cavities.
6. Functional structural insight for GLuc: opening/closing modulates accessibility and environments of two catalytic residues, Arg76 and Arg147, positioned in two pockets; pocket volumes shift from small/variable to larger and include emergence of a large cryptic pocket (~600–800 ų). The two-state model also aligns with reported cooperativity in substrate binding and with NMR relaxation evidence of exchange.
7. GLuc NOE evidence: Double Recall finds ~158 NOEs unique to state 1 and ~175 unique to state 2; the combined two-state ensemble explains ~218 NOESY peaks (including many long-range peaks) inconsistent with a prior single-state NMR model, supporting genuine multi-state behavior.
8. On the tumor suppressor CDK2AP1 (dimeric 61–115 fragment), AISAR finds two closely related states in equilibrium distinguished by switching between inter-chain vs intra-chain Cys105 sulfur–π interactions with Tyr63, yielding state-dependent cryptic pocket remodeling not resolved by conventional single-state NMR or standard AF2 inference.
9. CDK2AP1 experimental link: despite high spectral degeneracy, Double Recall identifies small sets of state-specific NOEs (~22 unique to state 1; ~23 unique to state 2). Peak doubling for Thr109/Glu110 is consistent with slow exchange between a major (~80%) and minor (~20%) state, plausibly driven by differing ring-current environments near Tyr63.
10. Broader message: AISAR can return a restraint-independent single-state structure when appropriate (demonstrated on integrase DNA-binding domain 2KOB), but can also expose hidden conformational heterogeneity and cryptic pockets in ordered proteins—especially where AF2 confidence is high yet NMR dynamics (RCI/relaxation) indicate additional states.
💻Code:
github.com/MontelioneLab/AIS…
📜Paper:
doi.org/10.1038/s41467-026-7…
#NMR #ProteinDynamics #StructuralBiology #AlphaFold #ComputationalBiology #BayesianMethods #ConformationalEnsembles #DrugDiscovery #CrypticPockets