The Impact of Glycosylation on the Conformational Ensembles of β -, δ -, and γ Sarcoglycans

“…simulations, we used the standard input file for NPT simulations generated by CHARMM-GUI, with temperature control using the Langevin thermostat 27 with a friction coefficient of 1.0 ps − 1 and semi-isotropic pressure control using the Berendsen barostat 28 with a relaxation time of 1.0 ps.”

“22 The effects of long-range van der Waals interactions were estimated using a dispersion correction model.”

“14,15 To mimic the in vitro environment, all structures, whether as a monomeric or heterotrimeric system, were integrated into a DMPC bi-layer membrane (1,2-dimyristoyl-sn-glycero-3-phosphocholine).”

“17,18 Simulations were prepared with the CHARMM-GUI server, 19,20 and each subunit was oriented according to the Orientations of Proteins in Mem-branes (OPM) database, 21 with standard N-and C-terminal patches applied.”

“23,24 Then, 20 independent 100 ns production runs were performed using the GPU-accelerated CUDA 25 version of AMBER18, pmemd.”

“Guided by these homologs, the amino acid sequences of β -, δ -, and γ -sarcoglycans were extracted from UniProt 11 (see Table S1) and modeled as both monomers and a heterotrimer using AlphaFold 12,13 and homology modeling via the SWISS-MODEL server.”

“17,18 Simulations were prepared with the CHARMM-GUI server, 19,20 and each subunit was oriented according to the Orientations of Proteins in Mem-branes (OPM) database, 21 with standard N-and C-terminal patches applied.”

“17,18 Simulations were prepared with the CHARMM-GUI server, 19,20 and each subunit was oriented according to the Orientations of Proteins in Mem-branes (OPM) database, 21 with standard N-and C-terminal patches applied.”

“23,24 Then, 20 independent 100 ns production runs were performed using the GPU-accelerated CUDA 25 version of AMBER18, pmemd.”

“17,18 Simulations were prepared with the CHARMM-GUI server, 19,20 and each subunit was oriented according to the Orientations of Proteins in Mem-branes (OPM) database, 21 with standard N-and C-terminal patches applied.”

“35 Overall, glycosylation compacts the conformational space of γ -SG, stabilizing its native fold and supporting the structural integrity of the sarcoglycan complex.”

“39 Beyond dynamics, N-glycosylation promotes protein folding and stability by increasing rigidity and protecting against stress, with complex glycans capable of structurally “locking” protein domains into functional orientations.”

“…simulations, we used the standard input file for NPT simulations generated by CHARMM-GUI, with temperature control using the Langevin thermostat 27 with a friction coefficient of 1.0 ps − 1 and semi-isotropic pressure control using the Berendsen barostat 28 with a relaxation time of 1.0 ps.”

“38 Although crystallographic characterization of glycans is challenging, complementary approaches show that glycosylation on flexible loops is particularly sensitive to environmental stress and can strongly affect allosteric communication.”

“14,15 To mimic the in vitro environment, all structures, whether as a monomeric or heterotrimeric system, were integrated into a DMPC bi-layer membrane (1,2-dimyristoyl-sn-glycero-3-phosphocholine).”

“34 In this study, we aim to investigate the impact of glycosylation on the conformational ensemble of SG complex.”

“36,37 In the SG complex, contact loss is largely confined to regions near glycosylation sites and the C-terminus, likely arising from secondary structure changes or steric effects.”

“33 In the case of large glycoproteins like sarcoglycans, glycans typically influence local conformational preferences and dampen fluctuations.”

“Moreover, the SG complex itself likely mediates extracellular-to-intracellular signal transmission, 42–44 with assembled sub-units exhibiting distinct, more regulated flexibility compared to isolated monomers, leading to coordinated local and global structural responses.”

“4,5 These findings highlight the central role of the β – δ – γ core in complex stability and provide a rationale for focusing on this trimer in structural and mechanistic studies.”

“36,37 In the SG complex, contact loss is largely confined to regions near glycosylation sites and the C-terminus, likely arising from secondary structure changes or steric effects.”

“In this study, we employed size-and-shape space Gaussian Mixture Model (Shape-GMM) 29 to identify structural states (clusters) of SG proteins (individual subunits and complex) based on particle positions.”

“40,41 Consistent with these principles, our results indicate that glycosylation modulates conformational heterogeneity in individual sarcoglycan monomers, altering adaptability relevant to complex formation and stress response.”

“Altogether, these cooperative interactions maintain the cohesion of the β – δ – γ core, providing a scaffold for subsequent recruitment of α -SG. 6 Despite its central role, the contribution of glycosylation to sarcoglycan folding, stability, and assembly remains poorly defined.”

“8,9 How glycosylation modulates the conformational landscape and inter-subunit interactions of β -, δ -, and γ -SG remains largely unknown, representing a critical barrier to mechanistic understanding and therapeutic design.”

“10 These findings suggest that glycans can remodel local folding preferences and inter-subunit contacts, thereby influencing complex assembly and function.”

“40,41 Consistent with these principles, our results indicate that glycosylation modulates conformational heterogeneity in individual sarcoglycan monomers, altering adaptability relevant to complex formation and stress response.”

“16 Given this, SHIFTX2 30 was used to compute the back-bone and side chain 1 H and 15 N chemical shifts for γ -SG using the last 50ns of the trajectory from molecular dynamics simulations across all three replicas (Figure S1).”