Structure

Ribonucleotide reductases (RNRs) employ radical chemistry to generate deoxyribonucleotides required for DNA synthesis and repair. A notable feature of RNRs is half-site reactivity, where, despite the enzyme being a symmetric 2 dimer, only one active site is catalytically active at a time while the other remains in a "poised" state for substrate binding. This phenomenon is tightly linked to the asymmetric 2{beta}2 interaction required for radical transfer. Here, we determined cryo-EM structures of the -subunit in the apo and holo states, i.e., the complex bound to TTP (effector) and GDP (substrate). The structures reveal asymmetric binding of the effector TTP and the substrate GDP across the dimer, with concomitant stabilization of loops surrounding the ligand-binding site. Interestingly, this asymmetry leads to well-resolved N-terminal density for [~]150 residues in the substrate-bound subunit, but weak density for this region in the effector-bound monomer. N-terminal domains are unresolved in both monomers of the apo structure. Isothermal titration calorimetry supports asymmetric binding of pyrimidine effectors with micromolar affinities. Molecular dynamics simulations and three-dimensional variability analysis reveal synchronous motions of loop 2, which together with the N-terminal domain drive alternate opening and closing of the active sites in the two monomers. These conformational dynamics provide key insights into the mechanistic basis of half-site reactivity. Together, these findings provide new insights into the structural dynamics and thermodynamic principles governing regulation and half-site activity in Class Ib RNRs. Significance statementRibonucleotide reductases (RNRs) are essential enzymes that supply the building blocks required for DNA synthesis and repair, yet the structural basis of their half-site reactivity has remained unclear. Using cryo-electron microscopy, calorimetry, molecular dynamics simulations, and conformational variability analysis, we show that the catalytic -subunit of a Class Ib RNR exhibits asymmetric nucleotide binding and coordinated conformational dynamics between the two monomers. These motions drive alternating opening and closing of the active sites and are linked to differential stabilization of the N-terminal region. Our findings suggest that asymmetric conformational gating and N-terminal sampling regulate productive interaction with the radical-generating {beta}-subunit, providing a mechanistic framework for understanding half-site reactivity and allosteric regulation in RNRs.

Protein structure predictors achieve high single-state accuracy, but it remains unclear whether they can recover functionally relevant conformational ensembles or account for the presence of ligands and/or binding partners. Here, we benchmark AlphaFold3, Boltz-2, Chai-1, and BioEmu on four canonical multi-state proteins (Pf-MATE, LAO, SecA, and {beta}2AR), quantifying state bias and sampling breadth against experimental reference structures. Models frequently default to a dominant state represented in the PDB; small-molecule ligands have weak or inconsistent effects, while large protein partners drive clear conformational switching between states. Multiple sequence alignment (MSA)-based approaches (AF-Cluster and random subsampling) recapitulate similar biases, indicating that this behavior is not unique to newer architectures. These results underscore current limitations for multi-state protein structure prediction and structure-guided ligand discovery. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/737860v1_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@3bf389org.highwire.dtl.DTLVardef@1f1c436org.highwire.dtl.DTLVardef@188ea8aorg.highwire.dtl.DTLVardef@1de236e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Microtubules are dynamic filaments of tubulin heterodimers that comprise an essential part of the eukaryotic cytoskeleton1. The nucleotide state of tubulin controls microtubule dynamics: stable GTP-microtubules favor polymerization, whereas unstable GDP-microtubules drive depolymerization2. Anticancer compounds such as Taxol (paclitaxel) target microtubule dynamicity by preventing microtubule depolymerization3,4. Despite decades of work, the molecular basis of microtubule dynamics remains poorly defined. Using cryo-EM, we determined [~]2.2 [A] structures of human microtubules in GTP-like (GMPCPP) and GDP states. Comparison of these two states revealed switch-like structural changes as tubulins transition from the pre-hydrolysis (GMPCPP) to the post-hydrolysis (GDP) state. Additional structure determination of Taxol-bound microtubules at [~]2.2 [A] showed that Taxol binding converts the microtubule lattice into a pre-hydrolysis state by reversing the structural switches flipped during GTP hydrolysis. Focusing our analysis on the microtubule seam shows that the pre-hydrolysis conformation of GMPCPP or Taxol-GDP exhibits favorable lateral interactions at the seam, with lattice deformations clearly visible at the GDP seam. Together, our data show the existence of structural switches in tubulin that are coupled to the nucleotide state and are exploited by Taxol to stabilize microtubules into a pre-hydrolysis-like state. (191 words)

Peptidoglycan biogenesis requires membrane flippases to translocate lipid-linked precursors across the cytoplasmic membrane for processing (1). This essential step is mediated by MurJ, the lipid II flippase conserved across all peptidoglycan-producing bacteria (2). While MurJ from diderm bacteria has been structurally resolved in multiple conformational states (3-6), its monoderm homolog remains uncharacterized. Monoderm MurJ homologs exhibit substantial sequence divergence yet retain the same lipid II flipping function (7) and are promising antibiotic targets. Here we report structures of Staphylococcus aureus MurJ (SaMurJ) captured in both outward- and inward-facing conformations. These structures show that SaMurJ adopts the conserved MOP family fold and undergoes conformational transitions consistent with an alternating-access mechanism. Our findings reveal conserved and divergent features of MurJ between diderm and monoderm bacteria that are critical for lipid II flipping and provide a structural framework for probing substrate recognition and specific inhibition. Significance StatementThe growing global threat of antibiotic resistance and the limited development of new antibacterial therapies underscore the urgent need to identify and mechanistically characterize new antibiotic targets and mechanisms. MurJ is an essential membrane transporter required for cell wall biosynthesis and represents an attractive but unexplored antibiotic target. Here we determine the structures of MurJ from a clinically critical monoderm pathogen Staphylococcus aureus in key conformational states during its transport cycle. This work advances our understanding of an essential step in bacterial cell wall synthesis, reveals key distinctions between monoderm and diderm MurJ, and defines structural features that can be exploited for antibiotic discovery.

Upon RAS-driven membrane recruitment, RAF kinases ARAF, BRAF and CRAF are activated via formation of homo- or hetero-dimers to initiate signaling through the MAP kinase cascade. Although RAF heterodimers are important for both physiologic and oncogenic signaling, they have been little studied at a structural and biochemical level. Here we report the preparation, biochemical characterization, and the cryo-EM structure of a 14-3-3-bound BRAF/CRAF heterodimer complex. The heterodimer exhibited kinetic parameters and sensitivity to a panel of twelve structurally diverse RAF inhibitors that were closely similar to, or intermediate between, those of BRAF and CRAF homodimers. Cryo-EM structures of the heterodimer with and without MEK1 revealed an overall organization essentially identical to that of RAF homodimers, but with an asymmetric interaction in the MEK1-bound structure in which the BRAF N-terminal acidic (NtA) motif extends across the dimer interface to engage the CRAF RKTR motif. Mutagenesis of this interface unexpectedly revealed that replacing the acidic NtA sequence with a basic RARA sequence yields highly active RAF homodimers and heterodimers, demonstrating that negative charge in the NtA motif is not required for activity. Collectively, our findings suggest that the charge state of the NtA motif influences RAF activity through effects on local backbone dynamics and the stability of the inactive kinase conformation, rather than via stereospecific recognition across the dimer interface.

Fuzzy protein complexes, in which an intrinsically disordered protein (IDP) retains conformational disorder upon binding, pose a fundamental challenge for structure predictors trained on ordered systems, where crystal structures capture only the most ordered ensemble snapshot, making standard benchmarking metrics misleading. Here, we present the first systematic evaluation of AlphaFold3 (AF3), AlphaFold2-Multimer (AF2MM), Chai-1, and Boltz-2 on a curated dataset of fuzzy complexes from FuzDB, benchmarked against DockQ against PDB structures and NOE violation rates against manually curated BMRB restraint files, the first comprehensive collection of this kind. Across all four predictors, approximately 30% of NOE restraints were violated with nearly identical distributions regardless of predictor architecture or training data. DockQ scores fell uniformly in the Acceptable range, with AF3 marginally higher but showing equivalent NOE violation rates to the weakest-performing model. Ensemble-level analysis using a first-principles implementation of the Hadzi thermodynamic model revealed that AF3 uniquely achieves near-zero mean helicity bias versus systematic overconfidence in the other predictors, yet all four models show poor per-residue helicity correlation with thermodynamic expectations. DockQ rankings reflect training data similarity to crystal structures rather than physical accuracy, and no current predictor captures fuzzy complex ensemble behavior. The FuzzyBench-NOE dataset, comprising NOE restraint files, predicted structures, interface hotspot annotations, and Hadzi--DSSP analysis outputs, is released on Zenodo (https://doi.org/10.5281/zenodo.20470556).

The emergence of single-particle cryoEM as a powerful method for structure determination has in large part been fueled by its ability to resolve both single static structures and complex conformational landscapes. Indeed, modern approaches to the heterogeneous reconstruction task can resolve 100s-1,000s of different maps from a single cryoEM dataset. How accurate these algorithms are, however, has proven difficult to rigorously assess, due to a lack of suitable benchmark datasets containing both realistic noise features and ground-truth labels. To address this obstacle, we recently developed a series of benchmark datasets that leverage the targeting power of Cas9 and the programmable heterogeneity of DNA to newly offer access to ground-truth per-particle structural labels in real data. Here, we challenged two popular heterogeneous reconstruction algorithms with mixed particle stacks resampled in silico from these datasets, finding that existing approaches resolve the encoded heterogeneity with limited accuracy. In particular, in realistic particle stacks with complex, multi-scale, and multi-axis heterogeneity, we observed that reconstruction of encoded heterogeneity depended strongly on the application of prior information about where heterogeneity was expected, and that individual particle assignments were made with significant error even when the correct structural states were reconstructed. Both molecular breathing motions and data collection features, such as defocus and projection angle, contributed to the observed particle assignment error. These results highlight important shortcomings of existing heterogeneous reconstruction methods and suggest new avenues for method development in both data collection strategies and in heterogeneous classification and reconstruction algorithms.

Macromolecular crystallography is limited by the phase problem: diffraction experiments measure amplitudes but not the phases required to reconstruct electron density. Existing phasing routes usually seek enough continuous phase information for density modification and model building to converge. Here, we ask how much phase information can be discarded while preserving convergence. We analyzed 14,148 diffraction datasets from chiral crystals to characterize centric reflections in reciprocal-space asymmetric units. After conditioning by centric trace and, where required, index parity, the two theoretical symmetry-allowed phase values were populated near equally, close to 50:50, independent of space group, defining a compact symmetry scaffold. We then retained this exact scaffold while compressing reference acentric phases to a one-bit alphabet {0, {pi}}; as expected from their diffuse parent distribution, the assignments were also near-balanced. Although this binary representation, with fixed attenuation 2/{pi}, introduces large angular errors (mean of 52{degrees}), it frequently supported automated structure solution: in paired Phenix AutoBuild tests, 705 of 894 binary initializers met a conservative joint criterion of final Free R [≤] 30% and relative chain recovery [≥] 70%, within a 20.0-2.5 [A] resolution window. To rank candidate seeds without rebuilding, we developed a branch-balanced Basin Score from inexpensive density-modification and map-connectivity observables computed at 20.0-3.5 [A]. The empirical score quickly separates productive from unproductive initializers before AutoBuild. Controlled phase inversion shows that basin compatibility decays gradually and can reappear in an anti-phase-related branch, indicating that buildability is not confined to a single neighborhood around the reference phase set but extends to a much broader field. These results recast phase initialization as basin entry and support future symmetry-aware, binary phase-search strategies.

The human sodium-citrate cotransporter NaCT imports various tri- and dicarboxylates into the cell as TCA cycle intermediates. This substrate uptake process is driven by an inward sodium gradient. The protein is a member of the Divalent Anion-Sodium Symporter (DASS) family. Whereas extensive biochemical and structural studies have been carried out for NaCT, how the substrate binding and translocation is coupled to the sodium gradient remains unclear. Here using single particle cryo-electron microscopy, we determined the structures of the human NaCT protein in three states: sodium-free, in the presence of sodium, and sodium- and substrate-bound. These structures suggest a simultaneous binding mechanism for sodium-substrate coupling, distinct from the sequential binding, conformational selection mechanism previously observed for the bacterial DASS protein VcINDY.

The Rcs phosphorelay regulates gene expression in response to cell envelope stress and is critical for the virulence of pathogenic bacteria, including Klebsiella pneumoniae, due to its regulation of genes related to extracellular capsule, cell division, and motility. The RcsC histidine kinase, RcsD phosphotransfer protein and RcsB response regulator, which form the core of the Rcs phosphorelay, are negatively regulated by the unique inner membrane protein IgaA via interaction with RcsD. An outer membrane lipoprotein, RcsF, activates signaling by interaction with IgaA, but the precise activation mechanisms remain unclear. In this study, we determined the structures of IgaA and the IgaA/RcsF complex using Cryo-electron microscopy (Cryo-EM). We also determined the structures of RcsC and RcsD, which both form homodimers stabilized by hydrophobic interactions, creating ladder-like structures. Combining the Cryo-EM structures, AlphaFold3 structure predictions of IgaA/RcsD and RcsF/IgaA/RcsD, and genetic studies, we describe a model for how RcsF modifies the IgaA/RcsD interaction, lifting negative regulation and activating the Rcs phosphorelay. Our findings provide a high-resolution depiction of the Rcs stress response system and suggest potential targets for small molecule inhibitors.

Frontier Structural Biology methods are transitioning from analysis of reconstituted macromolecular complexes in vitro to imaging of macromolecular assemblies within the physiological confines of the cell. Preparation of samples for in situ cryoEM analysis requires FIB milling or ultramicrotome sectioning, laborious and technically challenging procedures that are low-throughput and require a high degree of technical skills. We have devised a simple approach for cryoEM of nuclear macromolecular complexes that preserves to a high degree their physiological environment while removing the need for thin sectioning of the sample. The method requires only the preparation of nuclear extracts without additional purification or enrichment steps. We applied the method to obtain a 2.3 [A] cryoEM structure of nucleosomes visualised directly in the nuclear lysate of human cells. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/732463v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@15f4785org.highwire.dtl.DTLVardef@506f84org.highwire.dtl.DTLVardef@c95ceaorg.highwire.dtl.DTLVardef@1f326da_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cryogenic-electron microscopy (cryo-EM) has revolutionized the field of protein structural biology. The structures of large membrane proteins are now routinely determined by cryo-EM to near atomic resolution. However, in the medium resolution range of cryo-EM maps (>[~]2 [A]), negatively charged sidechains of acidic residues are not well-resolved due to the negative electrostatic potential of the region. This may lead to incorrect sidechain models for residues like glutamic acid or aspartic acid that are central for proton transfer activity in various respiratory and photosynthetic enzymes. We previously proposed that the acidic residues with weak or non-existent cryo-EM density can be modeled to represent their low proton affinity conformations. Here, we tested this hypothesis on a larger data set of acidic amino acid residues in two high-resolution respiratory complex I structures. By using faster sidechain modeling and proton affinity prediction tools, we created a workflow that generates sidechain conformations of selected amino acid residues. We validated the sidechain conformation predictions by Q-score analysis and atomistic molecular dynamics simulations in different charged states. The proposed workflow provides a way to rapidly obtain sidechain conformations of acidic residues with weak cryo-EM densities and can be integrated into the existing cryo-EM modeling pipelines to speed up sidechain rotamer prediction.

Double-stranded DNA minicircles have been observed in a variety of biological settings and are also widely employed in biotechnology, therapeutic applications, and basic research. Here, we report a cryo-EM structure of a 95-basepair minicircle (dsMC95) at a 5.3 [A] resolution. dsMC95 forms a closed ring as designed and no local deformation is observed. The two DNA strands are fully resolved, with the major and minor grooves clearly distinguishable. Analysis reveals a nine-fold periodicity in the helical twist, which corresponds to approximately 10.56 base pairs per turn. Together with groove width analysis, the data indicate that dsMC95 maintains a B-DNA configuration. The dsMC95 ring exhibits an in-plane ellipticity of 1.13 and an out-of-plane displacement of 15{degrees}, with differences in out-of-plane displacements observed between the two half-segments. The dsMC95 structure, which is the only free DNA cryo-EM structure with a resolution better than 6 [A] to date, allows comparison to other structures to better understand DNA physical features such as bending. The findings advance our understanding of DNA structure under topological constraints and may inform studies of naturally occurring small circular DNA as well as the manipulation of DNA in nanotechnology applications.

Multiple peptide resistance factor (MprF) are bi-functional enzymes encoded by several bacterial species and carry out the transfer of an amino acid from a charged tRNA to the lipid head group and further translocate the lipid across the membrane. Biochemical studies have revealed that the soluble synthase domain generates specificity and the structures of MprF have defined the general architecture of these enzymes, and that they can exist in different oligomeric states. Here, we characterise the gene product of lpiA, a MprF homologue from Agrobacterium fabrum (formerly called A. tumefaciens strain C58), a microbe that is commonly used in plant molecular biology. Cryo-EM analysis of AfMprF reveals a dimeric structure both in detergent micelle and in lipid nanodisc, and similar in architecture to the homologous enzyme from related Rhizobium sp. We further analyse some conserved residues in the soluble domain and suggest that the sulphur-aromatic motifs play a key role in substrate binding. Similar architecture of enzymes in closely related bacterial species of Agrobacterium and Rhizobium hints an evolutionary relationship but the importance of these oligomeric states in vivo remains to be analysed.

Building and refining cryo-EM atomic models often requires long, project-specific workflows that combine map inspection, prior structural knowledge, restraints, refinement, validation and expert review. Existing programs perform many individual operations, but coordinating them across iterative model-building sessions remains manual and difficult to audit. We present StructAgent, a user-guided multi-agent resource for cryo-EM model building and refinement. StructAgent couples a domain agent for literature-grounded structural reasoning with an execution agent that runs local software, tracks state, recovers from failures and records provenance. Expert approval gates control major model-changing actions. In three case studies, StructAgent refitted a 64-chain proteasome from an earlier template, audited 530 ribosomal metal-ion sites and guided a chemically ambiguous ligand fit in a folate-metabolism enzyme from ongoing work. These demonstrations show that agentic orchestration can convert modeling intent into auditable, reviewable software workflows while preserving expert control and final scientific judgment.

Neuroendocrine cells communicate with other cells by releasing neurotransmitters or hormones by exocytosis, which involves SNARE-mediated fusion between secretory vesicles and the plasma membranes of the secreting cells. In neurons two plasma membrane SNARE proteins, Syntaxin-1a and SNAP25, join with the vesicle membrane SNARE protein Synaptobrevin-2 to form a four-helix bundle, which drives membrane fusion. The assembly of these SNAREs, which is highly orchestrated in cells, has been intensely studied in solution using fragments of the SNARE proteins without their transmembrane domains or lipid anchors. However, in cell and model membranes, Syntaxin and SNAP25 are known to oligomerize and cluster, and little is known about how clustering affects their incorporation into SNARE complexes. In cells, the SM protein Munc18 has been implicated in aiding secretory vesicle docking and facilitating SNARE complex assembly through its interactions with Syntaxin. To understand how Munc18 orchestrates SNARE complex assembly on membranes, we employed protein reconstitution in model membranes as well as biochemical and biophysical assays to show that lipid-dependent oligomerization of Syntaxin affects Munc18-Syntaxin binding and SNAP25 insertion into the plasma membrane acceptor SNARE complex. We showcase the consequences of the different modes of Munc18-Syntaxin and SNAP25 interaction on Syntaxins oligomerization and orientation relative to the membrane surface, as well as on docking and fusion of purified insulin granules. We also determined low-resolution structures by cryoEM in nanodiscs and on the surface of proteoliposomes of membrane-bound assembly states of Munc18/Syntaxin and Munc18/Syntaxin/SNAP25 complexes.

Multiple AI prediction tools for protein structural ensembles have recently been released, building on the much heralded advances from AlphaFold, large language models, and other machine-learning approaches. Here we report on a comparison of several tools (BioEmu, AFSample2, ESMFlow) using a small test set of proteins, including three which exhibit well-studied structural transitions. Overall, while the AI platforms generate structurally diverse ensembles with overlapping regions, each tool produces clearly distinct conformational distributions. Thus, it is impossible that all the tools generate ensembles of high biophysical quality, analogous to a Boltzmann distribution. Experimental structures are often, but not always, covered by the ensembles in dimensionally reduced spaces. In cases where point mutations are known experimentally to cause large structural shifts, the AI tools exhibit either small or negligible shifts. Although our current analysis cannot evaluate the absolute quality of an ensemble, and hence cannot identify a best-performing AI tool, the results suggest users pursuing downstream applications such as protein engineering or drug design should interpret these ensembles with caution.

The inhibitor of {kappa}B kinase (IKK) complex integrates diverse cellular inflammatory responses, and induces transcription factor NF-{kappa}B. The molecular mechanism by which IKK becomes catalytically active in response to signaling remains unclear despite structural knowledge of the individual IKK1/, IKK2/{beta}, and NEMO/IKK{gamma} protein components within its hetero-oligomeric assembly. Cryo-EM of the IKK2/{beta} homodimer bound to an associating NEMO/IKK{gamma} protein fragment, reveals multiple conformers. Mutual exclusivity of dimeric conformers, canonical versus alternate, is reflected in and dependent upon order-to-disorder transition of the canonical 6-helical bundle dimerization interface. Correlation of this unusual structural plasticity of IKK2/{beta} with its biochemical and cellular activities suggests mechanistic possibilities for how association with its partner scaffold protein NEMO/IKK{gamma} and polyubiquitin chains might dictate catalytic activation of IKK through distinct IKK2/{beta} conformers.

DnaK, a prokaryotic Hsp70 chaperone, plays a central role in proteostasis by restoring native structures to heat-denatured proteins in an ATP-hydrolysis-dependent manner. While structures of DnaK in complex with nucleotides, co-chaperones, and short peptides have been resolved, structures with larger, stably folded substrates--such as firefly luciferase (Fluc, 61 kDa)--are lacking, limiting mechanistic understanding of how DnaK refolds such proteins. Here, we generated models of the DnaK-Fluc complex using AlphaFold3 and evaluated their mechanistic relevance. In one of three major model clusters, Fluc is unexpectedly immobilized beneath the DnaK -helical lid against the nucleotide-binding domain (NBD), rather than interacting primarily with the substrate-binding domain {beta} (SBD{beta}), as commonly assumed. All-atom molecular dynamics simulations indicate that, in this configuration, the lid can engage a thermally destabilized Fluc helix (residues 405-411), which we recently identified as the first--and likely the only--helix to irreversibly melt at 42 {degrees}C. Upon binding, the lid forms extensive hydrogen-bonding interactions with the melted helix. These interactions persist during lid movement toward SBD{beta} (following ATP hydrolysis), enabling the lid to actively extract the helix from the Fluc surface. In contrast, simulations with the helix in its native folded state show that the lid cannot extract it, leaving the native structure unaffected. Equilibrium simulations further indicate that, once extracted and mechanically stretched, the melted helix can refold to its native conformation. Together, these findings suggest a revised mechanism for DnaK-mediated protein refolding, in which the -helical lid selectively recognizes structurally compromised segments, forms stabilizing hydrogen bonds, and--powered by ATP hydrolysis--mechanically pulls them away from the protein surface to facilitate their refolding. SIGNIFICANCEDnaK is a model chaperone, which can reactivate thermally denatured proteins. Over the span of 40 years, significant findings have been made about DnaKs structure, dynamics and interactions with its co-chaperones, the exact molecular mechanism by which DnaK refolds misfolded proteins remains a mystery. This work exploited Alphafold3 to generate atomistic models of complexes between DnaK and Firefly luciferase. Molecular dynamics simulations directly captured how DnaK may assist thermally denatured proteins by mechanically pulling out their misfolded helices. This study provides a new insight into the DnaK mechanism.

Gram-negative bacteria maintain an asymmetric outer membrane that protects cells from environmental stresses and antibiotics. The maintenance of lipid asymmetry (Mla) pathway contributes to outer membrane lipid homeostasis through phospholipid transport between the outer and inner membranes. Although the periplasmic lipid carrier MlaC is thought to transfer phospholipids to the inner membrane MlaFEDB transporter via the hexameric protein MlaD, the molecular mechanism underlying this process remains unclear. Here we show crystal structures of two distinct MlaC-MlaD complexes from Pseudomonas aeruginosa that reveal distinct conformational states of MlaC. In these structures, an ordered conformation of the C-terminal 8 helix of MlaC positions MlaC distally from the central pore of the MlaD hexamer and limits accessibility of the lipid-binding cavity, whereas partial disordering of the 8 helix allows closer association with the MlaD hexamer and increased exposure of the cavity. Structure-based biochemical analyses further demonstrate that the C-terminal region negatively regulates MlaC- MlaD interaction while stabilizing phospholipid binding. These findings identify the C-terminal 8 helix as a conformational switch that couples MlaC positioning with lipid cavity accessibility, providing structural insight into phospholipid transfer at the MlaC-MlaD interface.