Biomolecular simulations: From dynamics and mechanisms to computational assays of biological activity

Biomolecular simulation is increasingly central to understanding and designing biological molecules and their interactions. Detailed, physics‐based simulation methods are demonstrating rapidly growing impact in areas as diverse as biocatalysis, drug delivery, biomaterials, biotechnology, and drug design. Simulations offer the potential of uniquely detailed, atomic‐level insight into mechanisms, dynamics, and processes, as well as increasingly accurate predictions of molecular properties. Simulations can now be used as computational assays of biological activity, for example, in predictions of drug resistance. Methodological and algorithmic developments, combined with advances in computational hardware, are transforming the scope and range of calculations. Different types of methods are required for different types of problem. Accurate methods and extensive simulations promise quantitative comparison with experiments across biochemistry. Atomistic simulations can now access experimentally relevant timescales for large systems, leading to a fertile interplay of experiment and theory and offering unprecedented opportunities for validating and developing models. Coarse‐grained methods allow studies on larger length‐ and timescales, and theoretical developments are bringing electronic structure calculations into new regimes. Multiscale methods are another key focus for development, combining different levels of theory to increase accuracy, aiming to connect chemical and molecular changes to macroscopic observables. In this review, we outline biomolecular simulation methods and highlight examples of its application to investigate questions in biology. This article is categorized under: Molecular and Statistical Mechanics > Molecular Dynamics and Monte‐Carlo Methods Structure and Mechanism > Computational Biochemistry and Biophysics Molecular and Statistical Mechanics > Free Energy Method

Biological Systems Workbook: Data modelling and simulations at molecular level

Nowadays, there are huge quantities of data surrounding the different fields of biology derived from experiments and theoretical simulations, where results are often stored in biological databases that are growing at a vertiginous rate every year. Therefore, there is an increasing research interest in the application of mathematical and physical models able to produce reliable predictions and explanations to understand and rationalize that information. All these investigations are helping to overcome biological questions pushing forward in the solution of problems faced by our society. In this Biological Systems Workbook, we aim to introduce the basic pieces allowing life to take place, from the 3D structural point of view. We will start learning how to look at the 3D structure of molecules from studying small organic molecules used as drugs. Meanwhile, we will learn some methods that help us to generate models of these structures. Then we will move to more complex natural organic molecules as lipid or carbohydrates, learning how to estimate and reproduce their dynamics. Later, we will revise the structure of more complex macromolecules as proteins or DNA. Along this process, we will refer to different computational tools and databases that will help us to search, analyze and model the different molecular systems studied in this course

STRUCTURE DETERMINATION OF HETEROGENEOUS BIOLOGICAL SPECIMENS IN CROWDED ENVIRONMENTS

The central dogma of molecular biology describes a strictly linear flow of genetic information stored in DNA transferred through RNA and translated into protein products. In the “post-genomic era” however, it is evident that abundant information flows from protein to protein and even protein back to DNA. The field of Structural Biology seeks to understand how the spatial and temporal organization of that information is stored and transmitted via the three-dimensional structure and dynamics of biological macromolecules. X-ray crystallography, nuclear magnetic resonance, and single particle cryo-electron microscopy (cryo-EM) are the primary techniques available to the structural biologist to deduce structure and dynamics at or near atomic resolutions. These tools are generally limited to the study of stable molecules that can be purified biochemically. Other approaches, like super-resolution light microscopy and cryo-electron tomography (cryo-ET), are amenable to the study of more labile macromolecular complexes or those found in situ; however, they are limited to resolutions of tens of nanometers. Improving the resolving capability of cryo-ET with sub-tomogram averaging to routinely reach beyond 10 Å is the primary goal of this work. My unique contribution to the field of structural biology is a suite of software tools called emClarity (enhanced macromolecular classification and alignment for high-resolution in situ tomography) which allows scientists with minimal computational background to probe the structural states of conformationally variable molecules present in complex and crowded environments

Towards bottom-up reconstitution of a functional FtsZ-based cell division machinery

Synthetic biology aims at the understanding of living organisms through an engineering perspective, with the goal of improving or creating new biological systems. The prospect of building a synthetic cell focuses on producing life from basic elements by combining synthetic and/or organic cellular components in a bottom-up manner. To create a synthetic cell, the minimal functions of life are required and cell-free synthetic biology offers a suitable framework for understanding biological processes outside the inherently noisy environment of cells. A synthetic cell is expected to exhibit characteristics of a living cell, such as fundamental metabolism, proliferation, and communication. The bottom-up approach utilizes a wide range of in vitro tools/technologies such as biomimetic membranes, protein reconstitution, cell-free expression reactions, and microfluidics. As tools, they enable the thorough characterization of functional modules such as metabolism, replication, and cell division. The ultimate goal is to integrate these modules to construct a predictable, customizable, and controllable entity. Among the functional modules of living organisms, cell division stands out as a hallmark feature. The machinery of division has evolved into a highly organized set of proteins with the aim of accurately splitting a mother cell into two daughter cells, while preserving the genetic information and cellular integrity. In the case of bacteria, and more concretely Escherichia coli, cell division is mediated by the divisome, a contractile ring consisting of a multiprotein complex that precisely assembles at midcell. At the center of this machinery is the essential FtsZ protein, which is able to polymerize and form the FtsZ-ring. This ring is key to the process, serving as a scaffold for the divisome and driving the division process. However, the molecular details of how the ring is functionally assembled, stabilized, and positioned are still not well understood. Therefore, the aim of this thesis is to develop and expand the knowledge about the molecular mechanism of the FtsZ-ring assembly and its function as a potential primary component in the minimal division machinery of synthetic cells. To this end, and following a bottom-up approach, we conducted assays based on the in vitro reconstitution of FtsZ in cellular mimic environments using lipid vesicles. This allows the characterization of FtsZ’s behavior and functionalities in environments that are similar to a potential synthetic cell. Firstly, we designed a microfluidic device to deform lipid vesicles into bacterial rod-shaped compartments to analyze the effect of different geometries and membrane tension on FtsZ. We found that FtsZ filaments align with the shorter axis of the rod-shaped vesicles and reorganize into cone-like structures when the membrane tension is lowered, causing membrane deformations. This suggests that there is a geometry and tension-dependent mechanism in the assembly of FtsZ structures on membranes. Secondly, we designed an in vitro reconstitution assay based on soft lipid tubes pulled from FtsZ-decorated vesicles using optical tweezers. We observed the transformation of lipid tubes into 3D spring-like structures, where the GTPase activity of FtsZ drives spring compression likely through torsional stress. This allowed us to gain mechanistic insights into the molecular dynamics behind the force generated by FtsZ filaments. Thirdly, we studied the spatiotemporal localization of the division ring by co-reconstituting FtsZ inside lipid vesicles with the MinCDE system, which is involved in positioning the divisome in vivo, and FtsA, the natural tether of FtsZ to the membrane. We achieved the assembly, placement, and onset of constriction of a minimal division ring inside lipid vesicles using two different approaches: purified components or cell-free expression of the MinCDE, FtsA, and FtsZ proteins. This represents a significant advance towards the in vitro reconstitution of functional modules in a synthetic cell and expands our understanding of the molecular mechanism underlying the spatiotemporal organization of the FtsZ-ring. Lastly, we employed biochemical studies combined with cryo-ET visualization to characterize the stabilization of the division ring and the crosslinking of FtsZ filaments by ZapD, a protein known as one of the stabilizers of the divisome. We observed the formation of toroidal structures in solution that are assembled by short FtsZ filaments connected by ZapD and have bacterial size. Their characterization in 3D brings valuable structural information about the FtsZ-ring and its functional stabilization, which is important for its further reconstitution in minimal systems. In conclusion, this thesis provides important insights into the molecular dynamics of the central protein of division in E. coli and most bacteria, addressing its activity on the membrane, mechanism of force constriction, spatiotemporal localization and stabilization of the FtsZ-ring. Furthermore, we demonstrate significant advancements towards the implementation of FtsZ-based division systems in minimal synthetic cells using a bottom-up approach

Mechanics and Dynamics of Nanosized Protein Cages

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Física de la Materia Condensada. Fecha de lectura: 18-02-2016Esta tesis tiene embargado el acceso al texto completo hasta el 18-08-201

Fitting small molecules to cryo-electron microscopy data

Recent innovations in the field of cryogenic-electron microscopy (cryo-EM) has enabled the visualisation of biological systems at atomic resolutions that rival that of X-ray crystallography. This is increasing the relevance of cryo-EM in the field of drug discovery, as it is now possible to solve high-resolution structures of biological complexes that may not have been amenable to crystallisation [1] and also in a more “native-like” state. However, it is not always possible to obtain structures to atomic resolutions with cryo-EM, currently only 16.28 % of structures deposited in the electron microscopy database [2] are at resolutions better than 3.0 Å, with the majority (45.05 %) at resolutions between 3.0 and 4.0 Å (correct as of December 2021). A vast body of work has been conducted with the aim of fitting biological macromolecules into cryo-EM at various resolutions [3–6]. However significantly less has been reported regarding the fitting of small molecules into cryo-EM maps. The work presented in this thesis aimed at developing methodologies that enable the fitting of small molecules to cryo-EM maps at resolutions from near atomic to 4.5Å. First, I used a fitting methodology that utilised consensus docking [7] in conjugation with a local difference mapping technique [8] to model the complex of the Eg5 kinesin motor domain with a novel inhibitor (GSK-1) in the presence of tubulin, into a 3.8 Å cryo-EM map (Chapter 2). The arrangement of structural elements within the protein allowed inferences to be made as to the mechanism of action of the drug [9]. Next, I present a new empirical molecular docking score for identifying correct ligand conformations within protein ligand complexes (Chapter 4). This score was integrated with goodness-of-fit scores commonly used for assessing the fit of biological molecules to cryo-EM maps [10]. Furthermore, we assessed the utility of this integrated score for fitting small molecules using simulated full maps and density difference maps (Chapter 4). This integrated score was then developed into a full methodology for fitting small molecules into cryo-EM maps, where its effectiveness was evaluated with experimental data at high (≤ 3.0 Å) and low (3.0 to 4.5 Å) resolution (Chapter 5). The accurate identification of protein ligand interactions from atomic models is an important consideration for drug discovery. To this end, a new software is presented that predicts protein ligand interactions using geometric parameters (Chapter 3). This software was benchmark using 35 high resolution protein-ligand complexes and compared to current state-of-the-art available software [11, 12]. Finally, I present the refined protein model of a Torpedo nicotinic acetylcholine receptor including the MX helix in a 6.6 Å cryo-EM map (Chapter 6). A combination of fitting software and bioinformatics identified the position of the MX helix relative to the cellular membrane. Our investigation suggested that the MX may function to entrap cholesterol, imposing rigidity to the receptor around the narrowest point of the central pore