Autotransporter folding avoids a kinetic trap during vectorial translocation across the bacterial outer membrane

Opposing Effects of Periplasmic Chaperones on Protein Folding.

Protein translocation across the bacterial SecYEG channel involves mechanical constraints arising from ATP-driven SecA activity, geometric confinement within the translocon, and folding of the emerging polypeptide on the periplasmic side. Periplasmic chaperones assist substrate maturation during this process, but how they influence protein folding under force remains poorly understood. Using protein L as a model two-state substrate, we applied physiologically relevant force pulses using custom-built single-molecule magnetic tweezers to examine how bacterial periplasmic chaperones modulate folding under tension. To isolate the direct effects of individual chaperones, these experiments were performed in the absence of SecA and the SecYEG translocon. We show that the periplasmic chaperones PpiD and DsbC increase folding probability and accelerate refolding under force, while having minimal effect on unfolding kinetics. In contrast, Spy and Skp reduce folding probability and suppress refolding, consistent with holdase-like behaviour. These observations show that distinct classes of periplasmic chaperones differentially modulate folding probability and refolding kinetics under mechanical force. Increased folding probability correspondingly enhances the expected mechanical work output of substrate folding under force. Together, our findings establish a quantitative framework for investigating how bacterial periplasmic chaperones modulate protein folding under controlled mechanical conditions.

Thermodynamic Profiling of Glucose Translocation in POPC embedded GLUT-4 protein models

The facilitative transport of hexose through the GLUcose Transporter type 4 (GLUT4) is essential for cellular metabolism and is regulated by allosteric nucleotide interactions. In this study, we conducted a comparative biophysical analysis of glucose translocation using both the native Cryo-EM structure (7WSN) and the AI-predicted AlphaFold model using Steered Molecular Dynamics (SMD) at physiological (310.15 K) and attenuated (303.15 K) temperatures with two different allosteric modulators, ATP and ADP. While the AlphaFold model managed to capture baseline static topologies, dynamic profiling revealed that it intrinsically over-optimizes for static stability, immediately collapsing into a sterically occluded, hyper-packed artifact. Translocation through this constricted geometry forces severe steric solvent exclusion, abruptly stripping the substrate’s hydration shell and resulting in immediate and immense thermodynamic friction (>300 kJ/mol). Under ATP-bound physiological strain, this hyper-bonded structural clamp prevents functional relaxation, ultimately inducing a catastrophic kinematic failure. Furthermore, ensemble Dynamic Cross-Correlation Matrix (DCCM) analysis demonstrates that the AI-predicted model suffers from persistent rigid-body locking, forcing the transmembrane domain to behave as a kinetically trapped conformation. In contrast, the functionally hydrated Cryo-EM architecture actively maintains a dual-gating pore and successfully isolates the exact mechanical lever driving transport: a native Proline hinge (PRO379) that seamlessly routes allosteric signals from the nucleotide anchor to the pore gate. Within the AlphaFold model, this critical allosteric wiring is disrupted, forcing mechanical coupling through an unphysiological, hyper-correlated aromatic pathway. These findings yield novel atomistic insights into GLUT4 gating mechanics, while definitively establishing that rigid AI-generated architectures lack the essential internal free volume and conformational plasticity required to sustain accurate thermodynamic profiling of dynamic membrane transporters.

N-terminal processing unlocks global dynamics for substrate engagement in Spl proteases

Staphylococcus aureus secretes a family of serine protease–like enzymes (SplA to SplF) that resemble eukaryotic granzymes, yet the mechanism by which amino-terminal processing activates this subclass has remained unresolved. Structural studies show insertion of the processed amino terminus without detectable changes in active-site geometry, creating a longstanding paradox as to how catalytic competence is achieved. Here, we identify SplB as the most highly expressed member of this family in a pathogenic methicillin-resistant S. aureus strain and use it to define the basis of activation. Solution nuclear magnetic resonance spectroscopy shows that precise amino-terminal processing triggers a long-range allosteric network coupling the amino terminus to the active site ∼20 angstroms away, unlocking global microsecond-to-millisecond dynamics that enable substrate engagement. Molecular dynamics simulations reveal the conformational ensembles underlying these motions. Last, mutational perturbation of this dynamic network modulates substrate engagement and catalytic activity in a manner consistent with dynamic control of binding competence. Together, these findings establish dynamic allostery as the mechanism of N-terminal activation in this subclass of serine proteases.

Intrinsic flexibility of Type VII secretion central pore is required for substrate translocation

Structural determinants of endopilus assembly, stability, and functional specificity in bacterial type II secretion.

Key determinants of secretion specificity and endopilus stability are identified, revealing how minor sequence variations in conserved nanomachines drive functional adaptation to diverse environments.

Chemical Pathway–Dependent Structural Reorganization at a Hinge Microenvironment in the AcrA Adaptor Protein

Adaptor protein AcrA plays a central role in the assembly and function of tripartite multidrug efflux pumps in Gram-negative bacteria, yet how its structural organization responds to coupled chemical perturbations rather than solely to equilibrium conditions remains unclear. Residues near His285 define a hinge microenvironment linking the lipoyl and β-barrel domains, suggesting a site for chemically sensitive structural modulation. Here, site-directed spin labeling combined with continuous-wave electron paramagnetic resonance spectroscopy was used to examine AcrA under an Mg2+-driven perturbation that simultaneously alters proton availability. Mg2+ addition produced spectral broadening at residue 62 that was fully reversed by spin dilution, indicating increased interspin proximity without changes in intrinsic side-chain dynamics. In contrast, direct acidification to a comparable bulk pH in the absence of Mg2+ did not reproduce this behavior. Structural mapping places residue 62 in proximity to the His285-centered hinge region, suggesting that coupled changes in protonation and metal coordination bias local interaction networks and modulate interdomain organization. These findings demonstrate that equivalent bulk conditions can mask distinct molecular states and identify chemical pathways as an important determinant of AcrA structural dynamics.