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

Mechanistic Insights into Autoinhibition of the Human Flippase ATP8B1

P4-ATPases are lipid flippases that maintain membrane phospholipid asymmetry by transporting specific phospholipids from the exoplasmic to the cytosolic leaflet, an essential process for membrane integrity, trafficking and signaling. Several P4-ATPases are tightly regulated by autoinhibitory N- and C-terminal extensions, yet the molecular basis of this regulation remains incompletely understood. Here, we investigated the autoinhibition mechanism of the human flippase ATP8B1 using trans-inhibition assays with synthetic peptides derived from its C-terminal tail. Using purified C-terminally truncated ATP8B1-CDC50A, we systematically assessed the inhibitory properties of peptides corresponding to distinct segments of the C-terminus. We show that the distal disordered region of the C-terminal tail significantly contributes to autoinhibition, likely through transient interactions with the cytosolic domains. We further identify a critical interaction between R1228 in the C-terminal tail and E219 in the A-domain, whose disruption markedly reduces inhibitory potency. In addition, we demonstrate that a minimal peptide spanning residues 1216-1228, which bridges the A- and N-domains in the autoinhibited conformation, is sufficient to inhibit ATPase activity. Together, these results refine the molecular description of ATP8B1 autoinhibition, open the way for structure-based activation strategies and provide insight into conserved regulatory mechanisms among P4-ATPases.

The Gordian Knot Enhances Ubiquitin Binding in UCH-L1

Substrate biasing in UCHL5 proteoforms

Proteolytic deubiquitinating enzymes bridge a gap in substrate recognition through complex regulatory mechanisms. A growing portion of these are accomplished through proteoforms that uniquely control association and diverse sets of cleavage capabilities that relay distinct physiological outcomes. This study describes substrate biasing governed by UCHL5 proteoforms. It demonstrates that N-terminal ubiquitination activates the enzyme towards monoubiquitin substrates, a feature that is conserved across UCHL5 homologs. Crystallographic and spectroscopic data suggest that the N-terminal ubiquitin binds intramolecularly in an allosteric binding site and inhibits branched chain substrate cleavage. Association with Rpn13/Adrm1 relieves this inhibition and reestablishes its ability to debranch, potentially controlling nonspecific debranching compared to retention of needed activity on the 26S proteasome. Collectively, this study describes the molecular basis for substrate selectivity in a deubiquitinating enzyme, an unexplored area in the enzymes that counteract ubiquitin E3 ligases.

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.

A Charge-Encoded Rheostat Permits Helix Nucleation but Limits Propagation in Skp1

The findings suggest that charged- hydrophobic-charged sequence patterning can encode conditional, context-dependent structure as a general organisational principle in intrinsically disordered proteomes.

Structural and Functional Interrogation of Active Streptococcus pneumoniae Sortase A

Sortase A (SrtA) enzymes covalently anchor surface proteins to Gram-positive bacterial cell walls, promoting colonization and virulence. In Streptococcus pneumoniae, previous studies identified both a domain-swapped dimer and an active refolded monomer, but the active enzyme has not been characterized at the structural and residue-specific level. Here, we performed quantitative proteomic comparisons of wild-type and SrtA knockout strains that confirmed the loss of multiple LPxTG-containing virulence factors, including ZmpB, NanA, and IgA1 protease, consistent with an essential role for SrtA in surface protein anchoring. To enable mechanistic studies, we established a biochemical framework to produce monomeric Streptococcus pneumoniae SrtA by refolding and developed a gel-based assay using recombinant substrates to monitor catalytic activity. The refolded monomer, but not the swapped dimer, catalyzed cleavage and transpeptidation of a canonical LPxTG substrate in a metal-independent manner under the conditions examined. We further report high-resolution NMR backbone assignments for the active monomer and identify substrate-induced chemical shift perturbations that localize to the active site. Together, these findings provide an integrated proteomic, biochemical, and NMR characterization of monomeric, catalytically active Streptococcus pneumoniae SrtA and reveal residue-specific interactions with a canonical LPNTG recognition peptide.