The sheet-to-helix transition is a potential gas-phase unfolding pathway for a multidomain protein CRM197

Abstract

Despite the expansive applications of gas-phase unfolding techniques, the molecular mechanism for the solvent-free forced unfolding pathway which substrate multidomain proteins usually adopt remains elusive at the secondary structure level. Herein, upon carefully selecting CRM₁₉₇ as a therapeutically-relevant model system containing multiple secondary structure-separated domains, we systematically examine its solvent-free unfolding pathway. Further-more, utilizing the hybrid of noncovalent chemical probing with niacinamide and ion mobility-mass spectrometry-guided all-atom molecular dynamics simulations, we map a nearly complete unfolding atlas for the conjugate vaccine carrier protein CRM₁₉₇ in a domain- and secondary structure-resolved manner. The totality of our data supports the preferential unfolding of the sheet-rich domain, indicating the dynamic transition from β-sheet to α-helices, and demonstrating that helices exhibit comparatively higher stability than β-sheets. We propose that this sheet-to-helix dynamic transition may be central to the gas-phase unfolding pathways of multidomain proteins, suggesting the need for systematic studies on additional multidomain protein systems.

Fig. 1.

CRM₁₉₇ unfolding as a function of cofactor NCA. (A) CRM₁₉₇ (PDB ID: 4ae0), (B) CRM₁₉₇-NCA complex (PDB ID: 4ae1), and (C) crystal structure for CRM₁₉₇-NCA complex showing the evidence of noncovalent specific interactions of NCA with catalytic domain D1, forming a large π-π conjugated hydrophobic pocket with surrounding amino acids such as H21, Y65 and F140 (the key amino acids surrounding NCA within a distance of less than 7.0 Å). (D) Representative native MS spectrum for CRM₁₉₇ and CRM₁₉₇-NCA complex (CRM: NCA = 1: 50). (E, F) CIU fingerprints for CRM₁₉₇ (15+) and CRM₁₉₇-NCA complex (15+). Theoretical CCS values were derived from both the trajectory method (TJM) and projection approximation (PA) method. (G) CIU50-based conformational stability comparison between CRM₁₉₇ and CRM₁₉₇-NCA complex. CIU50 data are derived from averaging CIU fingerprints of three independent measurements (Student’s t test, *P < 0.05, **P < 0.01).

Fig. 2.

Capturing sheet-to-helix dynamic transitions during CRM₁₉₇ and CRM₁₉₇-NCA unfolding in the absence of solvent. CCS changing curves for CRM₁₉₇ (A) and CRM₁₉₇-NCA (B) derived from temperature-jumping MD simulation from 300 to 750 K. (C) Representative models for CRM₁₉₇ unfolding intermediates. (D-F) Quantitative comparison of secondary structures of five representative unfolding features. (G-J) Overall occupancies of α-helix and β-sheet throughout the unfolding process for each domain. For CRM₁₉₇, the duration of gas-phase heating for F1, F2, F3/F4 and F5 was 0–180 ns, 190–420 ns, 420–504 ns and 504–1000 ns, respectively. For CRM₁₉₇-NCA, the duration of gas-phase heating for F1, F2, F3/F4 and F5 was 0–380 ns, 400–470 ns, 470–550 ns and 550–1000 ns, respectively. All data were obtained from three parallel simulations.