Aug 2026· Biochemistry· Vol 65 17, pp.
2728-2735
· 0 citations· 23 references
Medicine
Abstract
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.
Spns transporters are a mechanistically distinct branch of the major facilitator superfamily that regulate lipid transport, lysosomal homeostasis, immunity and disease, yet how the conserved Spns fold integrates protonation, substrate binding and alternating access to support chemically and directionally diverse transport activities remains unresolved. Here, we combine DEER spectroscopy in lipid nanodiscs with DEER- and AlphaFold-guided modeling to define the conformational landscape of the Mycobacterium smegmatis homolog MsSpns. Protonation shifts MsSpns toward an inward-facing state, whereas deprotonation favors a broader outward-facing ensemble through coordinated remodeling of intracellular and extracellular gates. These transitions are governed by membrane-embedded protonation switches and proton-sensing networks, while the substrate-binding cavity shows distinct proton sensitivity and weaker cooperativity. Hydrophilic cationic substrates, capreomycin and ethidium bromide, stabilize the outward-facing state, consistent with efflux antiport, whereas lipophilic compounds, including rifampicin, epicholesterol and selected phospholipids, favor the inward-facing state, suggesting uptake or allosteric stabilization. Thus, conserved proton-coupling elements can power opposing transport modes, revealing the mechanistic versatility of the Spns fold and its therapeutic potential.
Reza Dastvan, Samantha Gies, Kevin L. Jagessar et al.· Research Square· 0 citations
Dynamic allostery is established as the mechanism of N-terminal activation in this subclass of serine proteases in a pathogenic methicillin-resistant S. aureus strain and mutational perturbation of this dynamic network modulates substrate engagement and catalytic activity in a manner consistent with dynamic control of binding competence.
Eunjeong Lee, J. Redzic, Samrat Sarkar et al.· Science Advances· 0 citations
Ring-like AAA+ (ATPases Associated with diverse cellular Activities) biological machines mediate protein remodeling to assist a broad range of essential cellular functions. The nucleotide-dependent remodeling action involves intra- and inter-ring allosteric communication to generate mechanical force applied onto the substrate by a set of loops that protrude into the central channel. In this study, we probe these allosteric mechanisms through a comparative study of the katanin, a microtubule severing protein including a clade 3 AAA domain, and the double-ring ClpB, a protein disaggregase including both a clade 3 and a clade 5 AAA domain. Our molecular dynamics simulations, combined with machine learning and bioinformatic analysis, reveal both similar mechanisms involving the clade 3 domain and ClpB-specific ones involving communication with the clade 5 domain. We find that both nucleotide and substrate polypeptide binding restrict the conformational landscape sampled by katanin and ClpB, with ligand-specific conformations observed in the latter case. Allosteric contributions of secondary structure elements, ranked by using SHapley Additive exPlanations analysis in machine learning approaches and binary classification of features in ligand states, highlight the important role of regions adjacent to the nucleotide-binding site and the pore loops. Amino acid-level analysis of the allosteric paths reveals that intra-ring cooperativity modulates long-distance communication within the AAA+ protomers.
Acetyl-coenzyme A synthetases convert ATP, acetate, and coenzyme A (CoA) into acetyl-CoA, a central metabolite that fuels lipid biosynthesis and regulates protein and RNA acetylation. ACS enzymes contain N- and C-terminal domains that coordinate a two-step ping-pong mechanism involving sequential adenylation and thioester formation at the interdomain interface. How domain motions coordinate these chemical steps remains unclear. Here, we report single-particle cryo-electron microscopy structures of Schizosaccharomyces pombe ACSA captured in apo, pre-adenylation, intermediate, and product states. These structures reveal ligand-dependent reorganization of the C-terminal domain: apo and pre-adenylation forms display increased conformational heterogeneity, whereas intermediate- and product-bound states adopt ordered conformations compatible with catalysis. Structure-guided mutagenesis and in vitro activity assays, together with sequence conservation, support the functional importance and evolutionary conservation of the observed conformational transitions across ACS homologs. These findings establish a ligand-coupled interdomain rearrangement mechanism underlying catalysis by ACS enzymes and a structural framework for inhibitor development.
Meng Li, Ming-Yang Zhou, R. Marmorstein· Structure· 0 citations
Biomolecular condensates formed by multidomain proteins are increasingly linked to disease and are emerging targets for chemical modulation. Yet most molecular models emphasize intrinsically disordered regions (IDRs), whereas many available ligands bind folded-domain pockets. How IDR modifications and folded-domain ligands are integrated to control condensate organization remains unclear. Here, we address this problem using histone deacetylase 6 (HDAC6), a disease-associated deacetylase whose S22 phosphorylation and catalytic-pocket ligands differentially regulate condensation. Using atomistic-informed Martini 3 coarse-grained simulations, we show that S22 phosphorylation enhances phase separation of the N-terminal IDR1 without compacting isolated chains. Instead, phosphorylation creates a phospho-S22/D26-centered anionic contact node that strengthens dense-phase interactions with Arg/Lys-rich patches. The HDAC6 ligands Nexturastat A and HPOB remodel this IDR network through distinct charged-contact mechanisms: Nexturastat A acts as a focused competitor for cationic patches, whereas HPOB permits partial compensation through alternative charged contacts. In full-length phospho-HDAC6, the assembled network is not IDR1-dominated but is organized by FD3, the C-terminal ZnF-UBP domain. Pocket-bound ligands redirect this folded-domain network from FD3–FD3 association toward pocket–FD3 contacts, with stronger and more persistent remodeling by Nexturastat A. These results reveal a two-layer regulatory mechanism in which phosphorylation rewires IDR electrostatics while pocket-binding ligands redirect folded-domain interactions, suggesting a strategy for modulating multidomain condensates through ligand-induced contact-network remodeling.
Trehalose-6-phosphate (T6P) homeostasis is a critical determinant of pathogen survival, positioning T6P phosphatase (TPP) as a high-priority antimicrobial target. Despite its significance, the development of universal TPP inhibitors has remained elusive. Here, we report the biochemical and structural characterization of Dermatophilus congolensis TPP (Dcon-TPP), which reveals a moderate catalytic turnover consistent with other bacterial orthologs. Our 2.9 Å crystal structure uncovers an unprecedented wide-open conformation in a state of functional disassembly, with the catalytic machinery sequestered across a ∼26 Å spatial gap. Comparative analysis reveals a remarkable ∼40 Å trajectory and 79° global rotation of the cap domain-the largest conformational rearrangement reported for the HAD superfamily to date. Our structure reveals that this extreme plasticity is linked to structural instability within the β8-strand, which likely acts as a mechanical latch to enable massive domain sweeps via a lever-arm mechanism. This inherent flexibility imposes a substantial entropic penalty on active-site pre-organization, providing a structural rationale for the modest catalytic efficiency shared by Group 3 TPPs. By elucidating this unique molecular switch, our findings suggest a cryptic allosteric site that can be targeted to lock the enzyme in an inert state, offering a promising strategy to overcome long-standing challenges in TPP inhibitor design.
San Kim, Karthik Rajan Rajamanickam, Han-Gyeol Woo et al.· Biochemical and Biophysical...· 0 citations
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