Solution NMR reveals micelle-stabilized α-helical segments flanking the RIPK1 RHIM amyloid core

Receptor Interacting Protein Kinase 1 (RIP1 or RIPK1) can transition from a monomeric state to amyloid assemblies during cell signaling via a conserved RIP homotypic interaction motif (RHIM). Through RHIM–RHIM interactions, RIPK1 forms both homomeric and heteromeric amyloids implicated in programmed cell death, but solution-state characterization of unassembled RIPK1 is hindered by rapid aggregation. Building on our recent RIPK1 fibril structure, we designed an aggregation-slowing mutant (N545D) and show that two segments within RIPK1’s disordered domain display nascent helical propensity under near-physiological pH conditions. SDS micelles further stabilize these helices and enable measurements on the wild-type sequence. Notably, analogous conditions do not reveal comparable α-helical populations in RIPK3, despite a conserved RHIM sequence and amyloid structure endpoint. • Solution NMR access to RIPK1 RHIM pre-assembly states is enabled by a structure-guided, slow-aggregating N545D variant at near-physiological pH. • SDS micelles suppress RIPK1 RHIM aggregation and stabilize two α-helical propensities flanking the disordered IQIG core (∼527–537 and ∼563–577), enabling measurements on wild-type sequence. • Micelle-stabilized helical tendencies are supported by chemical-shift–based ncSSP and backbone reporters (J-couplings/hNOE/R2eff). • Under analogous micellar conditions, RIPK3 does not show comparable α-helical populations, suggesting distinct pre-assembly conformational biases despite conserved RHIM amyloid endpoints.