Observing Dynamic Conformational Changes within the Coiled-Coil Domain of Different Laminin Isoforms Using High-Speed Atomic Force Microscopy.

Laminins are trimeric glycoproteins with important roles in cell-matrix adhesion and tissue organization. The laminin α, ß, and γ-chains have short N-terminal arms, while their C-termini are connected via a triple coiled-coil domain, giving the laminin molecule a well-characterized cross-shaped morphology as a result. The C-terminus of laminin alpha chains contains additional globular laminin G-like (LG) domains with important roles in mediating cell adhesion. Dynamic conformational changes of different laminin domains have been implicated in regulating laminin function, but so far have not been analyzed at the single-molecule level. High-speed atomic force microscopy (HS-AFM) is a unique tool for visualizing such dynamic conformational changes under physiological conditions at sub-second temporal resolution. After optimizing surface immobilization and imaging conditions, we characterized the ultrastructure of laminin-111 and laminin-332 using HS-AFM timelapse imaging. While laminin-111 features a stable S-shaped coiled-coil domain displaying little conformational rearrangement, laminin-332 coiled-coil domains undergo rapid switching between straight and bent conformations around a defined central molecular hinge. Complementing the experimental AFM data with AlphaFold-based coiled-coil structure prediction enabled us to pinpoint the position of the hinge region, as well as to identify potential molecular rearrangement processes permitting hinge flexibility. Coarse-grained molecular dynamics simulations provide further support for a spatially defined kinking mechanism in the laminin-332 coiled-coil domain. Finally, we observed the dynamic rearrangement of the C-terminal LG domains of laminin-111 and laminin-332, switching them between compact and open conformations. Thus, HS-AFM can directly visualize molecular rearrangement processes within different laminin isoforms and provide dynamic structural insight not available from other microscopy techniques.

Structural heterogeneity of the ion and lipid channel TMEM16F.

Transmembrane protein 16 F (TMEM16F) is a Ca2+-activated homodimer which functions as an ion channel and a phospholipid scramblase. Despite the availability of several TMEM16F cryogenic electron microscopy (cryo-EM) structures, the mechanism of activation and substrate translocation remains controversial, possibly due to restrictions in the accessible protein conformational space. In this study, we use atomic force microscopy under physiological conditions to reveal a range of structurally and mechanically diverse TMEM16F assemblies, characterized by variable inter-subunit dimerization interfaces and protomer orientations, which have escaped prior cryo-EM studies. Furthermore, we find that Ca2+-induced activation is associated to stepwise changes in the pore region that affect the mechanical properties of transmembrane helices TM3, TM4 and TM6. Our direct observation of membrane remodelling in response to Ca2+ binding along with additional electrophysiological analysis, relate this structural multiplicity of TMEM16F to lipid and ion permeation processes. These results thus demonstrate how conformational heterogeneity of TMEM16F directly contributes to its diverse physiological functions.

Computational biophysics of atomic force microscopy-an IUPAB-sponsored workshop.

Atomic Force Microscopy (AFM) is a structural determination technique that involves 'prodding' surfaces with a nanometer sized needle with concomitant measurement of the resisting force. Due to its ability to interrogate the nanometer-to-micrometer size range, AFM is especially suited to the structural analysis of everything from biopolymers to cells and, as such, has become an important biophysical method. As AFM was only invented in 1986 it is relatively less scientifically developed than other structural techniques, such as NMR, X-ray crystallography and electron microscopy, that have a longer history of usage. In September of 2022 the first workshop of its kind was held to examine modern computational methods useful for simulating and analysing bioAFM experiments. Sponsored by a small IUPAB workshop grant, the three day meeting was of the hybrid (joint online /in person) type and had presenting participants based in Australia, UK, Finland, Thailand, South Korea, Vietnam and Japan. Each invited speaker was asked to deliver a lecture composed of half educational material (pitched at the level of an advanced postgraduate student) and half cutting edge research material (gathered from their own studies). IUPAB funds were used to invite young researchers (postgraduate students and early career scientists) from both within Japan and countries in the near asian region who had an interest in learning about the theoretical and experimental basis of the AFM technique. This Editorial describes the workshop and introduces the written contributions from the invited lecturers.