Structural Dynamics of E6AP E3 Ligase HECT Domain and Involvement of a Flexible Hinge Loop in the Ubiquitin Chain Synthesis Mechanism.

Ubiquitin (Ub) ligases E3 are important factors in selecting target proteins for ubiquitination and determining the type of polyubiquitin chains on the target proteins. In the HECT (homologous to E6AP C-terminus)-type E3 ligases, the HECT domain is composed of an N-lobe and a C-lobe that are connected by a flexible hinge loop. The large conformational rearrangement of the HECT domain via the flexible hinge loop is essential for the HECT-type E3-mediated Ub transfer from E2 to a target protein. However, detailed insights into the structural dynamics of the HECT domain remain unclear. Here, we provide the first direct demonstration of the structural dynamics of the HECT domain using high-speed atomic force microscopy at the nanoscale. We also found that the flexibility of the hinge loop has a great impact not only on its structural dynamics but also on the formation mechanism of free Ub chains.

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.

Simulation atomic force microscopy for atomic reconstruction of biomolecular structures from resolution-limited experimental images.

Atomic force microscopy (AFM) can visualize the dynamics of single biomolecules under near-physiological conditions. However, the scanning tip probes only the molecular surface with limited resolution, missing details required to fully deduce functional mechanisms from imaging alone. To overcome such drawbacks, we developed a computational framework to reconstruct 3D atomistic structures from AFM surface scans, employing simulation AFM and automatized fitting to experimental images. We provide applications to AFM images ranging from single molecular machines, protein filaments, to large-scale assemblies of 2D protein lattices, and demonstrate how the obtained full atomistic information advances the molecular understanding beyond the original topographic AFM image. We show that simulation AFM further allows for quantitative molecular feature assignment within measured AFM topographies. Implementation of the developed methods into the versatile interactive interface of the BioAFMviewer software, freely available at www.bioafmviewer.com, presents the opportunity for the broad Bio-AFM community to employ the enormous amount of existing structural and modeling data to facilitate the interpretation of resolution-limited AFM images.

BioAFMviewer: An interactive interface for simulated AFM scanning of biomolecular structures and dynamics.

We provide a stand-alone software, the BioAFMviewer, which transforms biomolecular structures into the graphical representation corresponding to the outcome of atomic force microscopy (AFM) experiments. The AFM graphics is obtained by performing simulated scanning over the molecular structure encoded in the corresponding PDB file. A versatile molecular viewer integrates the visualization of PDB structures and control over their orientation, while synchronized simulated scanning with variable spatial resolution and tip-shape geometry produces the corresponding AFM graphics. We demonstrate the applicability of the BioAFMviewer by comparing simulated AFM graphics to high-speed AFM observations of proteins. The software can furthermore process molecular movies of conformational motions, e.g. those obtained from servers which model functional transitions within a protein, and produce the corresponding simulated AFM movie. The BioAFMviewer software provides the platform to employ the plethora of structural and dynamical data of proteins in order to help in the interpretation of biomolecular AFM experiments.

Coarse-Grained Protein Dynamics Studies Using Elastic Network Models.

Elastic networks have been used as simple models of proteins to study their slow structural dynamics. They consist of point-like particles connected by linear Hookean springs and hence are convenient for linear normal mode analysis around a given reference structure. Furthermore, dynamic simulations using these models can provide new insights. As the computational cost associated with these models is considerably lower compared to that of all-atom models, they are also convenient for comparative studies between multiple protein structures. In this review, we introduce examples of coarse-grained molecular dynamics studies using elastic network models and their derivatives, focusing on the nonlinear phenomena, and discuss their applicability to large-scale macromolecular assemblies.

Designed Elastic Networks: Models of Complex Protein Machinery.

Recently, the design of mechanical networks with protein-inspired responses has become increasingly popular. Here, we review contributions which were motivated by studies of protein dynamics employing coarse-grained elastic network models. First, the concept of evolutionary optimization that we developed to design network structures which execute prescribed tasks is explained. We then review what presumably marks the origin of the idea to design complex functional networks which encode protein-inspired behavior, namely the design of an elastic network structure which emulates the cycles of ATP-powered conformational motion in protein machines. Two recent applications are reviewed. First, the construction of a model molecular motor, whose operation incorporates both the tight coupling power stroke as well as the loose coupling Brownian ratchet mechanism, is discussed. Second, the evolutionary design of network structures which encode optimal long-range communication between remote sites and represent mechanical models of allosteric proteins is presented. We discuss the prospects of designed protein-mimicking elastic networks as model systems to elucidate the design principles and functional signatures underlying the operation of complex protein machinery.

Design of Elastic Networks with Evolutionary Optimized Long-Range Communication as Mechanical Models of Allosteric Proteins.

Allosteric effects often underlie the activity of proteins, and elucidating generic design aspects and functional principles unique to allosteric phenomena represent a major challenge. Here an approach consisting of the in silico design of synthetic structures, which, as the principal element of allostery, encode dynamical long-range coupling among two sites, is presented. The structures are represented by elastic networks, similar to coarse-grained models of real proteins. A strategy of evolutionary optimization was implemented to iteratively improve allosteric coupling. In the designed structures, allosteric interactions were analyzed in terms of strain propagation, and simple pathways that emerged during evolution were identified as signatures through which long-range communication was established. Moreover, robustness of allosteric performance with respect to mutations was demonstrated. As it turned out, the designed prototype structures reveal dynamical properties resembling those found in real allosteric proteins. Hence, they may serve as toy models of complex allosteric systems, such as proteins. Application of the developed modeling scheme to the allosteric transition in the myosin V molecular motor was also demonstrated.

Deciphering Intrinsic Inter-subunit Couplings that Lead to Sequential Hydrolysis of F1-ATPase Ring.

Rotary sequential hydrolysis of the metabolic machine F1-ATPase is a prominent manifestation of high coordination among multiple chemical sites in ring-shaped molecular machines, and it is also functionally essential for F1 to tightly couple chemical reactions and central γ-shaft rotation. High-speed AFM experiments have identified that sequential hydrolysis is maintained in the F1 stator ring even in the absence of the γ-rotor. To explore the origins of intrinsic sequential performance, we computationally investigated essential inter-subunit couplings on the hexameric ring of mitochondrial and bacterial F1. We first reproduced in stochastic Monte Carlo simulations the experimentally determined sequential hydrolysis schemes by kinetically imposing inter-subunit couplings and following subsequent tri-site ATP hydrolysis cycles on the F1 ring. We found that the key couplings to support the sequential hydrolysis are those that accelerate neighbor-site ADP and Pi release upon a certain ATP binding or hydrolysis reaction. The kinetically identified couplings were then examined in atomistic molecular dynamics simulations at a coarse-grained level to reveal the underlying structural mechanisms. To do that, we enforced targeted conformational changes of ATP binding or hydrolysis to one chemical site on the F1 ring and monitored the ensuing conformational responses of the neighboring sites using structure-based simulations. Notably, we found asymmetrical neighbor-site opening that facilitates ADP release upon enforced ATP binding. We also captured a complete charge-hopping process of the Pi release subsequent to enforced ATP hydrolysis in the neighbor site, confirming recent single-molecule analyses with regard to the role of ATP hydrolysis in F1. Our studies therefore elucidate both the coordinated chemical kinetics and structural dynamics mechanisms underpinning the sequential operation of the F1 ring.

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.

Tracing entire operation cycles of molecular motor hepatitis C virus helicase in structurally resolved dynamical simulations.

Hepatitis C virus helicase is a molecular motor that splits duplex DNA while actively moving over it. An approximate coarse-grained dynamical description of this protein, including its interactions with DNA and ATP, is constructed. Using such a mechanical model, entire operation cycles of an important protein machine could be followed in structurally resolved dynamical simulations. Ratcheting inchworm translocation and spring-loaded DNA unwinding, suggested by experimental data, were reproduced. Thus, feasibility of coarse-grained simulations, bridging a gap between full molecular dynamics and reduced phenomenological theories of molecular motors, has been demonstrated.

Protein dynamics by the combination of high-speed AFM and computational modeling.

High-speed atomic force microscopy (HS-AFM) allows direct observation of biological molecules in dynamic action. However, HS-AFM has no atomic resolution. This article reviews recent progress of computational methods to infer high-resolution information, including the construction of 3D atomistic structures, from experimentally acquired resolution-limited HS-AFM images.

BioAFMviewer software for simulation atomic force microscopy of molecular structures and conformational dynamics.

Atomic force microscopy (AFM) and high-speed scanning have significantly advanced real time observation of biomolecular dynamics, with applications ranging from single molecules to the cellular level. To facilitate the interpretation of resolution-limited imaging, post-experimental computational analysis plays an increasingly important role to understand AFM measurements. Data-driven simulation of AFM, computationally emulating experimental scanning, and automatized fitting has recently elevated the understanding of measured AFM topographies by inferring the underlying full 3D atomistic structures. Providing an interactive user-friendly interface for simulation AFM, the BioAFMviewer software has become an established tool within the Bio-AFM community, with a plethora of applications demonstrating how the obtained full atomistic information advances molecular understanding beyond topographic imaging. This graphical review illustrates the BioAFMviewer capacities and further emphasizes the importance of simulation AFM to complement experimental observations.

Biophysical Reviews Issue Focus call: Computational biophysics of atomic force microscopy.

This Commentary describes an open call for submissions to an Issue Focus of the IUPAB Biophysical Reviews journal on the topic of the, 'Computational biophysics of atomic force microscopy'. The Issue Focus will be published in Volume 15 Issue 6 of Biophysical Reviews in late December of 2023. The submission deadline is September 1st of 2023. Interested parties are requested to contact the Special Issue Editors prior to submission.

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.

Predicting the placement of biomolecular structures on AFM substrates based on electrostatic interactions.

Atomic force microscopy (AFM) and high-speed AFM allow direct observation of biomolecular structures and their functional dynamics. Based on scanning the molecular surface of a sample deposited on a supporting substrate by a probing tip, topographic images of its dynamic shape are obtained. Critical to successful AFM observations is a balance between immobilization of the sample while avoiding too strong perturbations of its functional conformational dynamics. Since the sample placement on the supporting substrate cannot be directly controlled in experiments, the relative orientation is a priori unknown, and, due to limitations in the spatial resolution of images, difficult to infer from a posteriori analysis, thus hampering the interpretation of measurements. We present a method to predict the macromolecular placement of samples based on electrostatic interactions with the AFM substrate and demonstrate applications to HS-AFM observations of the Cas9 endonuclease, an aptamer-protein complex, the Monalysin protein, and the ClpB molecular chaperone. The model also allows predictions of imaging stability taking into account buffer conditions. We implemented the developed method within the freely available BioAFMviewer software package. Predictions based on available structural data can therefore be made even prior to an actual experiment, and the method can be applied for post-experimental analysis of AFM imaging data.

High-Speed Atomic Force Microscopy for Filming Protein Molecules in Dynamic Action.

Structural biology is currently undergoing a transformation into dynamic structural biology, which reveals the dynamic structure of proteins during their functional activity to better elucidate how they function. Among the various approaches in dynamic structural biology, high-speed atomic force microscopy (HS-AFM) is unique in the ability to film individual molecules in dynamic action, although only topographical information is acquirable. This review provides a guide to the use of HS-AFM for biomolecular imaging and showcases several examples, as well as providing information on up-to-date progress in HS-AFM technology. Finally, we discuss the future prospects of HS-AFM in the context of dynamic structural biology in the upcoming era. Expected final online publication date for the Annual Review of Biophysics, Volume 53 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Dynamics of Target DNA Binding and Cleavage by Staphylococcus aureus Cas9 as Revealed by High-Speed Atomic Force Microscopy.

Programmable DNA binding and cleavage by CRISPR-Cas9 has revolutionized the life sciences. However, the off-target cleavage observed in DNA sequences with some homology to the target still represents a major limitation for a more widespread use of Cas9 in biology and medicine. For this reason, complete understanding of the dynamics of DNA binding, interrogation and cleavage by Cas9 is crucial to improve the efficiency of genome editing. Here, we use high-speed atomic force microscopy (HS-AFM) to investigate Staphylococcus aureus Cas9 (SaCas9) and its dynamics of DNA binding and cleavage. Upon binding to single-guide RNA (sgRNA), SaCas9 forms a close bilobed structure that transiently and flexibly adopts also an open configuration. The SaCas9-mediated DNA cleavage is characterized by release of cleaved DNA and immediate dissociation, confirming that SaCas9 operates as a multiple turnover endonuclease. According to present knowledge, the process of searching for target DNA is mainly governed by three-dimensional diffusion. Independent HS-AFM experiments show a potential long-range attractive interaction between SaCas9-sgRNA and its target DNA. The interaction precedes the formation of the stable ternary complex and is observed exclusively in the vicinity of the protospacer-adjacent motif (PAM), up to distances of several nanometers. The direct visualization of the process by sequential topographic images suggests that SaCas9-sgRNA binds to the target sequence first, while the following binding of the PAM is accompanied by local DNA bending and formation of the stable complex. Collectively, our HS-AFM data reveal a potential and unexpected behavior of SaCas9 during the search for DNA targets.

Imaging single CaMKII holoenzymes at work by high-speed atomic force microscopy.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) plays a pivotal role in synaptic plasticity. It is a dodecameric serine/threonine kinase that has been highly conserved across metazoans for over a million years. Despite the extensive knowledge of the mechanisms underlying CaMKII activation, its behavior at the molecular level has remained unobserved. In this study, we used high-speed atomic force microscopy to visualize the activity-dependent structural dynamics of rat/hydra/C. elegans CaMKII with nanometer resolution. Our imaging results revealed that the dynamic behavior is dependent on CaM binding and subsequent pT286 phosphorylation. Among the species studies, only rat CaMKIIα with pT286/pT305/pT306 exhibited kinase domain oligomerization. Furthermore, we revealed that the sensitivity of CaMKII to PP2A in the three species differs, with rat, C. elegans, and hydra being less dephosphorylated in that order. The evolutionarily acquired features of mammalian CaMKIIα-specific structural arrangement and phosphatase tolerance may differentiate neuronal function between mammals and other species.

Importance of annexin V N-terminus for 2D crystal formation and quick purification protocol of recombinant annexin V.

Annexin V forms trimeric structures which further assemble into two-dimensional crystal (2D crystal) lattices on negatively charged phospholipid bilayer in a Ca2+-dependent manner. It is also known that annexin V 2D crystals show two types of symmetric patterns with six-fold symmetry (p6) and three-fold symmetry (p3). The p6 lattice also contains additional trimers in the gaps between the p6 axes, which are also referred to as non-p6 trimers because they do not participate in the formation of the p6 lattice. We here show that the annexin V N-terminal has significant influence on 2D crystal formation using high-speed atomic force microscopy (HS-AFM) observations. We also present a quick purification method to purify recombinant annexin V without any residual affinity tag after protein purification in ~3h.

Novel DNA Aptamer for CYP24A1 Inhibition with Enhanced Antiproliferative Activity in Cancer Cells.

Overexpression of the vitamin D3-inactivating enzyme CYP24A1 (cytochrome P450 family 24 subfamily and hereafter referred to as CYP24) can cause chronic kidney diseases, osteoporosis, and several types of cancers. Therefore, CYP24 inhibition has been considered a potential therapeutic approach. Vitamin D3 mimetics and small molecule inhibitors have been shown to be effective, but nonspecific binding, drug resistance, and potential toxicity limit their effectiveness. We have identified a novel 70-nt DNA aptamer-based inhibitor of CYP24 by utilizing the competition-based aptamer selection strategy, taking CYP24 as the positive target protein and CYP27B1 (the enzyme catalyzing active vitamin D3 production) as the countertarget protein. One of the identified aptamers, Apt-7, showed a 5.8-fold higher binding affinity with CYP24 than the similar competitor CYP27B1. Interestingly, Apt-7 selectively inhibited CYP24 (the relative CYP24 activity decreased by 39.1 ± 3% and showed almost no inhibition of CYP27B1). Furthermore, Apt-7 showed cellular internalization in CYP24-overexpressing A549 lung adenocarcinoma cells via endocytosis and induced endogenous CYP24 inhibition-based antiproliferative activity in cancer cells. We also employed high-speed atomic force microscopy experiments and molecular docking simulations to provide a single-molecule explanation of the aptamer-based CYP24 inhibition mechanism. The novel aptamer identified in this study presents an opportunity to generate a new probe for the recognition and inhibition of CYP24 for biomedical research and could assist in the diagnosis and treatment of cancer.