Sourav Maity

The mature human immunodeficiency virus (HIV-1) capsid is composed of approximately 250 capsid protein hexamers, each containing a central pore delimited by a β-hairpin and an α-helix from each monomer. The cellular metabolite inositol hexakisphosphate (IP6) binds this pore and modulates translocation of nucleoside triphosphates into the capsid, where they fuel reverse transcription of the viral genome. However, the mechanism by which IP6 modulates the pore conformation is poorly understood. Averaged structural models of the HIV-1 capsid protein lattice have shed some light on this mechanism but lack information on the dynamic behavior of individual hexamers that could provide deeper mechanistic insights. Here we visualized in real time by high-speed atomic force microscopy the asynchronous motions of individual hexamer pores in the presence or absence of IP6, identifying dynamic transitions between two major states. Quantitative analysis enabled estimation of the IP6-induced pore conformations and populations. Constant-pH molecular dynamics simulations further reveal that IP6 induces contact rewiring at the pore entrance, stabilizing the open state through coordinated rearrangement of intersubunit interactions. Consistently, mechanical fatigue assays show that IP6 markedly enhances the resistance of the capsid lattice to mechanical fatigue, revealing its role as a mechanically stabilized nanomaterial. Together, these results establish IP6 as a proviral regulator that couples nanoscale pore dynamics, intersubunit contact reconfiguration, and mechanical resilience of the HIV-1 capsid, providing a unified mechanistic framework for its role in viral replication.

Alix is a key adaptor protein of the endosomal sorting complex required for transport (ESCRT) membrane remodeling machinery. Although Alix-mediated recruitment of the ESCRT-III subunit CHMP4 is well established, the molecular mechanisms underlying Alix activation and ESCRT-III polymerization remain poorly understood. Here, we present the crystal structure of the dimeric Alix V-domain (Alix-V) in complex with a llama nanobody. Dimerization is mediated by domain swapping, generating an X-shaped flexible conformation. We demonstrate that Alix forms dimers in vivo and provide evidence for the recruitment of dimeric Alix to plasma membrane repair sites and the cytokinetic midbody. Notably, a mutation disrupting Alix dimerization impairs plasma membrane repair. Furthermore, high-speed AFM experiments reveal that dimeric Alix, but not the monomeric form, nucleates CHMP4B filament polymerization. Our data establish that dimeric Alix is the active form responsible for nucleating two ESCRT-III CHMP4 filaments, whose geometry creates a platform for the recruitment of downstream ESCRT-III components required to assemble the active membrane remodeling and fission machinery.

The pyruvate dehydrogenase complex (PDHc) ¹ links glycolysis to the Krebs cycle by catalyzing the oxidative decarboxylation of pyruvate to acetyl-CoA and CO₂, a process essential for life ^2,3 . PDHc is formed by structural proteins (E3-binding protein, E3BP) ^4–7 , enzymatic subunits (E1, E2, E3) ^4,6 , and mobile lipoyl domains (LDs), the latter shuttling intermediates across active sites ^6,8 . Although numerous details regarding pyruvate oxidation steps have been elucidated ⁹ , the precise organization of the entire PDHc remains unknown due to its large size and dynamic heterogeneity. Here, we employ in silico , in vitro , and in situ methods to propose a multi-scale model of PDHc that includes approximately one million atoms and to visualize multiple conformational states. This model reveals a ∼40-50 nm nested shell structure, formed by flexible linkers that spatially coordinate the E1 and E3 enzyme complexes around the E2-E3BP core scaffold. This structure acts as a molecular sieve, selectively guiding lipoyl arms while maintaining enzyme positioning with sub-nm precision. During catalysis, the nested shell structure expands and adopts a mechanically reinforced state comparable in magnitude to viral assemblies ¹⁰ . Our findings provide structural context for the textbook “link reaction” ¹¹ , building on decades of biochemical knowledge, are transferable to functional aspects of other α-ketoacid dehydrogenase complexes, and, ultimately, expand our understanding of primary metabolism as a whole. Abstract Figure:

Amyloid fibril formation is a hallmark of various neurodegenerative diseases such as Huntington's (HD), Alzheimer's, and Parkinson's disease. The protein aggregation process involves slow nucleation events followed by rapid growth and elongation of formed fibrils. Understanding the pathways of amyloid formation is key to development of novel therapeutic agents that can interfere with the pathogenic protein misfolding events. Recent studies of aggregation by polypeptides from Alzheimer's and Huntington's disease have identified the importance of a poorly understood secondary nucleation process that may even be the dominant source of protein aggregate formation. Here, we focus on the polyglutamine-expansion disorder HD and employ mechanistic and structural studies to study different aspects of secondary nucleation in the aggregation of huntingtin Exon 1 (HttEx1). Notably, we apply high-speed atomic force microscopy (HS-AFM) to directly observe the process on the single-particle level and in real time. Our observations show unique features of the amyloid formation dynamics in real time, including secondary nucleation, elongation, and the formation of large bundles of fibrils as a result of nucleated branching. We examine the role of HttEx1 flanking segments during the aggregation process, revealing that the N-terminal Htt^NT segment exhibits a clear primary nucleation-aggregation-enhancing ability; however, it does not seem to induce or affect the secondary nucleation process. The obtained results illuminate the complex aggregation process of HttEx1 and have implications for attempts to inhibit or modulate it for therapeutic purposes.

Long double-stranded (ds) RNA in the cytosol acts as a potent inflammatory molecule recognized by the receptor MDA5, triggering the innate immune response. Mutations affecting MDA5 ATPase activity lead to severe pathological conditions. MDA5 nucleoprotein filament assembly-disassembly dynamic is proposed to regulate dsRNA recognition, though the exact mechanism remains unclear. Here, we employed magnetic tweezers to monitor the assembly and manipulate MDA5 filaments at the single dsRNA level. Following a slow nucleation event, MDA5 assembles cooperatively and directionally into (partial) filaments and utilizes ATP hydrolysis to compact dsRNA via single-stranded (ss) RNA loop extrusion, even against a significant opposing force. This compacted state is further stabilized by oligomerization of MDA5’s caspase recruitment domain (CARD) and requires high force to be disrupted. ssRNA gaps impaired compaction, suggesting a new mechanism for dsRNA recognition. We propose that MDA5 mediated dsRNA compaction captures viral dsRNA, preventing further usage for viral replication.

Antimicrobial resistance is one of the leading concerns in medical care. Here we resolve the functional mechanism of the antimicrobial action of the cationic tripeptide AMC-109 by combining high speed-atomic force microscopy, molecular dynamics, fluorescence assays, and lipidomic analysis. We show that AMC-109 activity on the negatively charged plasma membrane of Staphylococcus aureus consists of two crucial steps. First, AMC-109 self-assembles into stable aggregates with specificity for negatively charged membranes. Second, by incorporation into the S. aureus membrane the lateral membrane organization is affected, dissolving membrane nanodomains. Domain dissolution affects membrane functions such as protein sorting and cell wall synthesis, and is suggested to cause a loss of resistance of methicillin-resistant S. aureus (MRSA) to methicillin. As the AMC-109 mode of action is similar to the activity of the disinfectant benzalkonium chloride (BAK), a broad applicability, but with low cytotoxicity to human cells, is expected.

Artificial protein cages have great potential in a number of areas including cargo capture and delivery and as artificial vaccines. Here, we investigate an artificial protein cage whose assembly is triggered by gold nanoparticles. Using biochemical and biophysical methods we were able to determine both the mechanical properties and the gross compositional features of the cage which, combined with mathematical models and biophysical data, allowed the structure of the cage to be predicted. The accuracy of the overall geometrical prediction was confirmed by the cryo-EM structure determined to sub-5 Å resolution. This showed the cage to be nonregular but similar to a dodecahedron, being constructed from 12 11-membered rings. Surprisingly, the structure revealed that the cage also contained a single, small gold nanoparticle at each 3-fold axis meaning that each cage acts as a synthetic framework for regular arrangement of 20 gold nanoparticles in a three-dimensional lattice.

Single-molecule fluorescence optical tweezers studies combined with high-speed AFM reveal early events in the HBV life cycle.

Self-assembly features prominently in fields ranging from materials science to biophysical chemistry. Assembly pathways, often passing through transient intermediates, can control the outcome of assembly processes. Yet, the mechanisms of self-assembly remain largely obscure due to a lack of experimental tools for probing these pathways at the molecular level. Here, the self-assembly of self-replicators into fibers is visualized in real-time by high-speed atomic force microscopy (HS-AFM). Fiber growth requires the conversion of precursor molecules into six-membered macrocycles, which constitute the fibers. HS-AFM experiments, supported by molecular dynamics simulations, revealed that aggregates of precursor molecules accumulate at the sides of the fibers, which then diffuse to the fiber ends where growth takes place. This mechanism of precursor reservoir formation, followed by one-dimensional diffusion, which guides the precursor molecules to the sites of growth, reduces the entropic penalty associated with colocalizing precursors and growth sites and constitutes a new mechanism for supramolecular polymerization.