Insight into Malt1 activation mechanism through synergetic approach of AlphaFold, MD Simulation and NMR dynamic analyses

Abstract

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a central regulator of immune signalling, yet the conformational dynamics governing its activation remain poorly defined. Building on our earlier solution-state analysis of apo MALT1(PCASP-Ig3)339-719, which revealed domain flexibility, dynamic autoinhibition, and sensitivity to physiological ionic conditions, we combine NMR relaxation, molecular dynamics simulations, and ensemble modelling to delineate how solution environment reshapes its conformational landscape. Because most structural information derives from dimeric or inhibitor-bound states, the behaviour of monomeric, ligand-free MALT1 in physiological solution has remained unclear. Here, MD simulations show that low-salt conditions drive all trajectories toward a unified inactive-like ensemble, marked by inward rotation of W580 and coordinated rearrangements of Loop 2 and Loop 3, indicating that the inactive state is energetically favoured and its reactivation kinetically suppressed. Physiological ionic strength partially restores access to active-like loop motions, aligning with NMR evidence that sodium modulates catalytic readiness. In contrast, high-salt conditions rigidify the PCASP-Ig3 module, suppressing loop fluctuations and preventing active-inactive transitions, thereby strongly enriching the active-state population. Importantly, the combined MD-NMR analysis demonstrates that the NMR-initiated ensembles provide the most faithful representation of backbone and loop dynamics under low-salt conditions, capturing substrate-independent loop rearrangements, stable hydrophobic-core behaviour, and the intrinsic transitions that shape MALT1s conformational equilibrium. Together, these findings identify ionic strength as a key regulator of MALT1 conformational equilibria,, highlighting how loop dynamics and domain flexibility tune its proteolytic competence and providing a dynamic framework for future structure-based modulation of MALT1 activity.