Exploring protein-mediated compaction of DNA by coarse-grained simulations and unsupervised learning
(2024)
Protein-DNA interactions and protein-mediated DNA compaction play key roles in a range of biological processes. The length scales typically involved in DNA bending, bridging, looping, and compaction (1 kbp) are challenging to address experimentally or by all-atom molecular dynamics simulations, making coarse-grained simulations a natural approach. Here, we present a simple and generic coarse-grained model for DNA-protein and protein-protein interactions and investigate the role of the latter in the protein-induced compaction of DNA. Our approach models the DNA as a discrete worm-like chain. The proteins are treated in the grand canonical ensemble, and the protein-DNA binding strength is taken from experimental measurements. Protein-DNA interactions are modeled as an isotropic binding potential with an imposed binding valency without specific assumptions about the binding geometry. To systematically and quantitatively classify DNA-protein complexes, we present an unsupervised machine learning pipeline that receives a large set of structural order parameters as input, reduces the dimensionality via principal-component analysis, and groups the results using a Gaussian mixture model. We apply our method to recent data on the compaction of viral genome-length DNA by HIV integrase and find that protein-protein interactions are critical to the formation of looped intermediate structures seen experimentally. Our methodology is broadly applicable to DNA-binding proteins and protein-induced DNA compaction and provides a systematic and semi-quantitative approach for analyzing their mesoscale complexes.
The human immunodeficiency virus (HIV) infects non-dividing cells and its genome must be compacted to enter the cell nucleus. Here, we show that the viral enzyme integrase (IN) compacts HIV DNA mimetics in vitro. Under physiological conditions, IN-compacted genomes are consistent in size with those found for pre-integration complexes in infected cells. Compaction occurs in two stages: first IN tetramers bridge DNA strands and assemble into “rosette” structures that consist of a nucleo-protein core and extruding bare DNA. In a second stage, the extruding DNA loops condense onto the rosette core to form a disordered and viscoelastic outer layer. Notably, the core complex is susceptible towards IN inhibitors, whereas the diffuse outer layer is not. Together, our data suggest that IN has a structural role in viral DNA compaction and raise the possibility to develop inhibitors that target IN-DNA interactions in disordered condensates.
Single-molecule force spectroscopy (SMFS) techniques play a pivotal role in unraveling the mechanics and conformational transitions of biological macromolecules under external forces. Among these techniques, multiplexed magnetic tweezers (MT) are particularly well suited to probe very small forces, pN, critical for studying noncovalent interactions and regulatory conformational changes at the single-molecule level. However, to apply and measure such small forces, a reliable and accurate force-calibration procedure is crucial. Here, we introduce a new approach to calibrate MT based on thermal motion using the Hadamard variance (HV). To test our method, we perform bead-tether Brownian dynamics simulations that mimic our experimental system and compare the performance of the HV method against two established techniques: power spectral density (PSD) and Allan variance (AV) analyses. Our analysis includes an assessment of each method’s ability to mitigate common sources of additive noise, such as white and pink noise, as well as drift, which often complicate experimental data analysis. We find that the HV method exhibits overall similar or higher precision and accuracy, yielding lower force estimation errors across a wide range of signal-to-noise ratios (SNRs) and drift speeds compared with the PSD and AV methods. Notably, the HV method remains robust against drift, maintaining consistent uncertainty levels across the entire studied SNR and drift speed spectrum. We also explore the HV method using experimental MT data, where we find overall smaller force estimation errors compared with PSD and AV approaches. Overall, the HV method offers a robust method for achieving sub-pN resolution and precision in multiplexed MT measurements. Its potential extends to other SMFS techniques, presenting exciting opportunities for advancing our understanding of mechanosensitivity and force generation in biological systems. To make our methods widely accessible to the research community, we provide a well-documented Python implementation of the HV method as an extension to the Tweezepy package.
