Ahmad Reza Mehdipour

The cell wall is a complex structure that protects bacteria from environmental threats. Phosphocholine-containing teichoic acids are key cell wall biopolymers critical for host colonization, immune evasion, competence, and persistence in Streptococcus pneumoniae . The flippase TacF, a member of the multidrug/oligosaccharide-lipid/polysaccharide (MOP) superfamily, monitors the phosphocholine content of teichoic acids during transport, yet the underlying mechanism of this process remains unresolved. We present a cryo-EM structure of S. pneumoniae TacF in lipid nanodiscs. In vivo complementation assays and molecular dynamics simulations reveal key residues involved in teichoic acid recognition and transport, while coevolutionary and conservation analyses delineate common mechanistic elements among MOP flippases, indicating a shared mechanism for polyprenyl-diphosphate-linked oligosaccharide lipid transport. Our findings provide mechanistic insights into an essential flippase involved in S. pneumoniae pathogenesis and a potential drug target.

The cell wall is a complex structure that protects bacteria from environmental threats. Phosphocholine-containing teichoic acids are key cell wall biopolymers critical for host colonization, immune evasion, competence, and persistence in Streptococcus pneumoniae . The flippase TacF, a member of the multidrug/oligosaccharide-lipid/polysaccharide (MOP) superfamily, monitors the phosphocholine content of teichoic acids during transport, yet the underlying mechanism of this process remains unknown. We present a cryo-EM structure of S. pneumoniae TacF in lipid nanodiscs. In vivo complementation assays and molecular dynamics simulations reveal key residues involved in teichoic acid recognition and transport, while coevolutionary and conservation analyses delineate common mechanistic elements among MOP flippases, indicating a shared mechanism for polyprenyl-diphosphate-linked oligosaccharide lipid transport. Our findings provide mechanistic insights into an essential flippase involved in S. pneumoniae pathogenesis and a potential drug target.

Factor VIIa (FVIIa) catalyzes the first step of the blood coagulation cascade. The expected wide therapeutic window between antithrombotic efficacy and bleeding risk makes FVIIa an attractive drug target. However, no FVIIa inhibitors have reached the market so far, mostly due to poor oral bioavailability. To date, in all ligand‐bound X‐ray crystal structures of FVIIa, the binding pocket of FVIIa is in an active, open form. Here, we present an X‐ray crystal structure of the FVIIa–tissue factor complex with a bound oxazole‐based inhibitor at 1.9 Å resolution, with an extensively remodeled active site and a collapsed S1 pocket. Using collectively 0.17 ms of atomistic molecular dynamics simulations, we observed conformational transitions between the collapsed and open forms of the S1 pockets of FVIIa and 12 other serine peptidases out of 16 studied, indicating an equilibrium of open and collapsed states of the S1 pocket in FVIIa and the majority of the serine proteases studied. Therefore, our results point to a general S1 pocket plasticity, which provides the basis for a completely new way of inhibiting FVIIa and other serine proteases.

In eukaryotic cells, LINC complexes physically bridge the two nuclear membranes to cytoskeletal filaments, transmitting mechanical forces to the nucleus. Due to their dynamic and membrane-embedded nature, their architecture in native membranes has been hitherto elusive. Here, we combine in-situ electron cryo-tomography, AlphaFold predictions and molecular dynamics simulations to determine the sub-nanometer-resolution architecture of the trimeric ∼130 kDa SUN5-Nesprin3 LINC complex in human spermatozoa. The structure reveals a hexagonal lattice that stitches the nuclear envelope, creating a rigid interface that links the sperm nucleus to the flagellum. Our structure unveils a unique membrane-anchoring mechanism: a SUN5 KASH-lid β-hairpin and a Nesprin3 amphipathic helix insert into the outer nuclear membrane, leading to cooperative remodelling of the nuclear envelope shape by the extended LINC lattice, which results in flattening the caudal sperm nucleus. Our integrative data provides key insights into the architecture and role of a LINC complex in situ , revealing molecular details of a severe male infertility phenotype.

1 Summary: Iron acquisition is a crucial determinant of Mycobacterium tuberculosis (Mtb) survival and pathogenesis, as the bacterium must scavenge iron from the host environment during infection. To achieve this, Mtb synthesizes two structurally distinct siderophores, mycobactin (MBT) and carboxymycobactin (cMBT), efficiently chelate iron and facilitate its uptake. While MBT remains embedded in the membrane due to its hydrophobicity, cMBT is secreted to scavenge extracellular iron. Despite their biological importance, the dynamics of siderophore–membrane interactions remain poorly understood. In this study, we employ coarse-grained molecular dynamics simulations to investigate the spontaneous flip-flop of iron-bound MBT (Fe-MBT) across the mycobacterial membrane. Our results reveal that Fe-MBT flip-flop occurs on a significantly slower timescale compared to generic bacterial lipids, with rates nearly an order of magnitude lower. The lipid composition of the membrane plays a critical role in modulating this process, with mycobacterial membranes exhibiting a higher degree of lipid order and reduced Fe-MBT mobility compared to generic bacterial membranes. Furthermore, we show that flip-flop transitions are relatively fast, occurring within 100 ns and, in some cases, on the order of a few nanoseconds. Our findings contribute to a mechanistic understanding of siderophore dynamics in Mtb membranes and provide a foundation for future studies on iron acquisition and potential drug targeting strategies.

An increasing number of ligand-bound membrane protein structures reveal ligand-binding sites on the lipid-exposed surface of the protein within the membrane bilayer. Binding events to such sites have previously been studied using molecular dynamics (MD) simulations and experiments in cases such as calcium-gated potassium channels ¹ and sodium channels ² . The proposed binding mechanism is that these ligands partition into the membrane to gain access to their binding site. What is currently unavailable is what the thermodynamic and kinetic contributions of the ligand-membrane and ligand-protein interactions are to the overall binding event. Here, we used MD simulations and enhanced sampling methods to study the membrane partitioning of a DHP calcium channel antagonist, nifedipine, from the voltage-gated calcium channel Cav1.1. We present that drug-membrane interactions occur on a much faster timescale than the overall binding of nifedipine to Cav1.1.

1 Summary: The spike protein of SARS-CoV-2 is a highly flexible membrane receptor that triggers the translocation of the virus into cells by attaching to the human receptors. Like other type I membrane receptors, this protein has several extracellular domains connected by flexible hinges. The presence of these hinges results in high flexibility, which consequently results in challenges in defining the conformation of the protein. Here, We developed a new method to define the conformational space based on a few variables inspired by the robotic field’s methods to determine a robotic arm’s forward kinematics. Using newly performed atomistic molecular dynamics (MD) simulations and publicly available data, we found that the Denavit-Hartenberg (DH) parameters can reliably show the changes in the local conformation. Furthermore, the rotational and translational components of the homogenous transformation matrix constructed based on the DH parameters can identify the changes in the global conformation of the spike and also differentiate between the conformation with a similar position of the spike head, which other types of parameters, such as spherical coordinates, fail to distinguish between such conformations. Finally, the new method will be beneficial for looking at the conformational heterogeneity in all other type I membrane receptors.

Summary: Lysosomal degradation of macromolecules in lysosomes produces diverse small metabolites exported by specific transporters for reuse in biosynthetic pathways. Here, we deorphanized the Major Facilitator Superfamily Domain Containing 1 (MFSD1) protein, which forms a tight complex with the Glycosylated Lysosomal Membrane Protein (GLMP) in the lysosomal membrane. Untargeted metabolomics analysis of MFSD1-deficient mouse lysosomes revealed an increase in cationic dipeptides. Purified MFSD1 selectively bound diverse dipeptides, while electrophysiological, isotope tracer, and fluorescence-based studies in Xenopus oocytes and proteoliposomes showed that MFSD1/GLMP acts as a uniporter for cationic and neutral dipeptides. Cryo-EM structure of the dipeptide-bound MFSD1/GLMP complex in outward-open conformation characterized the heterodimer interface and, in combination with molecular dynamics simulations, provided a structural basis for its selectivity towards diverse dipeptides. Together, our data identify MFSD1 as a general lysosomal dipeptide uniporter, providing an alternative route to recycle lysosomal proteolysis products when lysosomal amino acid exporters are overloaded.