High resolution structural models of Ribosome nascent chain complexes restrained by experimental NMR data

As understanding of the ways in which the complex cellular environment affects the in vivo folding of proteins improves, improved methods for their study are required. It is possible to produce limited quantities of ribosome-nascent chain complexes (RNCs) and techniques for gathering data about them are improving, but no single technique provides all the information required to understand folding of nascent proteins on the ribosome and there are still significant data that cannot be obtained experimentally. In particular, while NMR chemical shift and residual dipolar couplings may be recorded, the samples are of too low concentration and stability to conduct the most informative NOESY experiments that are traditionally used for revealing atomic-resolution structure. Recently, the ability to use chemical shifts to reveal structural details and dynamic properties of small proteins has been developed. By simulating multiple molecules and predicting the average chemical shift of the ensemble, the simulation may be restrained to conform to the experimentally measured data, making testable predictions about the atomic-resolution dynamic properties of the molecule. By adapting these methods to the macromolecular RNC structures it is theorized that the limited chemical shift data available may be used to provide structural details of the protein as it emerges from a ribosome. This, however, is faced by many challenges, including the ability to simulate such large number of atoms in a suitable timescale and applying the restraints to the nascent chain alone. The thesis presented describes the development of computational techniques to characterize RNCs, including the concepts and challenges faced, the chemical-shift restrained simulation of nascent chains alone, the development of techniques to perform chemical-shift restrained molecular dynamics simulations of the RNCs and the application of these techniques to a model system

Triphenilphosphonium Analogs of Chloramphenicol as Dual-Acting Antimicrobial and Antiproliferating Agents

Activitat antibiòtica; Ribosoma bacterià; Simulacions de dinàmica molecularActividad antibiótica; Ribosoma bacteriano; Simulaciones de dinámica molecularAntibiotic activity; Bacterial ribosome; Molecular dynamics simulationsIn the current work, in continuation of our recent research, we synthesized and studied new chimeric compounds, including the ribosome-targeting antibiotic chloramphenicol (CHL) and the membrane-penetrating cation triphenylphosphonium (TPP), which are linked by alkyl groups of different lengths. Using various biochemical assays, we showed that these CAM-Cn-TPP compounds bind to the bacterial ribosome, inhibit protein synthesis in vitro and in vivo in a way similar to that of the parent CHL, and significantly reduce membrane potential. Similar to CAM-C4-TPP, the mode of action of CAM-C10-TPP and CAM-C14-TPP in bacterial ribosomes differs from that of CHL. By simulating the dynamics of CAM-Cn-TPP complexes with bacterial ribosomes, we proposed a possible explanation for the specificity of the action of these analogs in the translation process. CAM-C10-TPP and CAM-C14-TPP more strongly inhibit the growth of the Gram-positive bacteria, as compared to CHL, and suppress some CHL-resistant bacterial strains. Thus, we have shown that TPP derivatives of CHL are dual-acting compounds targeting both the ribosomes and cellular membranes of bacteria. The TPP fragment of CAM-Cn-TPP compounds has an inhibitory effect on bacteria. Moreover, since the mitochondria of eukaryotic cells possess qualities similar to those of their prokaryotic ancestors, we demonstrate the possibility of targeting chemoresistant cancer cells with these compounds.This research was funded by RFBR [grants 20-04-00873 to N.V.S. (synthesis of analogs, binding assays, in vitro translation), 20-015-00537 to P.A.N. (potential measurement, screening of TolC-containing transporters), and 20-54-76002 to I.A.O. (toeprinting and in vitro translation)], President grant MD 2626.2021.1.4 to I.A.O. (bacteria inhibition assays), grants from the Instituto de Salud Carlos III: PI17/02087 to A.L. (cancer cell proliferation assays) by the Ministry of Science and Higher Education of the Russian Federation [grant FENU-2020-0019 to G.I.M. (molecular dynamics simulations)] and by the Government of the Russian Federation [No. AAAA-A17-117120570004-6 to A.A.B.]

ATP-induced asymmetric pre-protein folding as a driver of protein translocation through the Sec machinery

Funding: Royal Society for a University Research Fellowship; Wellcome Multi-User Equipment Grant (099149/Z/12/Z) (JEL).Transport of proteins across membranes is a fundamental process, achieved in every cell by the 'Sec' translocon. In prokaryotes, SecYEG associates with the motor ATPase SecA to carry out translocation for pre-protein secretion. Previously, we proposed a Brownian ratchet model for transport, whereby the free energy of ATP-turnover favours the directional diffusion of the polypeptide [Allen et al. eLife 2016]. Here, we show that ATP enhances this process by modulating secondary structure formation within the translocating protein. A combination of molecular simulation with hydrogen-deuterium-exchange mass spectrometry and electron paramagnetic resonance spectroscopy reveal an asymmetry across the membrane: ATP induced conformational changes in the cytosolic cavity promote unfolded pre-protein structure, while the exterior cavity favours its formation. This ability to exploit structure within a pre-protein is an unexplored area of protein transport, which may apply to other protein transporters, such as those of the endoplasmic reticulum and mitochondria.Publisher PDFPeer reviewe

ATP-induced asymmetric pre-protein folding as a driver of protein translocation through the Sec machinery

Funding: Royal Society for a University Research Fellowship; Wellcome Multi-User Equipment Grant (099149/Z/12/Z) (JEL).Transport of proteins across membranes is a fundamental process, achieved in every cell by the 'Sec' translocon. In prokaryotes, SecYEG associates with the motor ATPase SecA to carry out translocation for pre-protein secretion. Previously, we proposed a Brownian ratchet model for transport, whereby the free energy of ATP-turnover favours the directional diffusion of the polypeptide [Allen et al. eLife 2016]. Here, we show that ATP enhances this process by modulating secondary structure formation within the translocating protein. A combination of molecular simulation with hydrogen-deuterium-exchange mass spectrometry and electron paramagnetic resonance spectroscopy reveal an asymmetry across the membrane: ATP induced conformational changes in the cytosolic cavity promote unfolded pre-protein structure, while the exterior cavity favours its formation. This ability to exploit structure within a pre-protein is an unexplored area of protein transport, which may apply to other protein transporters, such as those of the endoplasmic reticulum and mitochondria.Publisher PDFPeer reviewe

RNA Structural Dynamics As Captured by Molecular Simulations: A Comprehensive Overview

With both catalytic and genetic functions, ribonucleic acid (RNA) is perhaps the most pluripotent chemical species in molecular biology, and its functions are intimately linked to its structure and dynamics. Computer simulations, and in particular atomistic molecular dynamics (MD), allow structural dynamics of biomolecular systems to be investigated with unprecedented temporal and spatial resolution. We here provide a comprehensive overview of the fast-developing field of MD simulations of RNA molecules. We begin with an in-depth, evaluatory coverage of the most fundamental methodological challenges that set the basis for the future development of the field, in particular, the current developments and inherent physical limitations of the atomistic force fields and the recent advances in a broad spectrum of enhanced sampling methods. We also survey the closely related field of coarse-grained modeling of RNA systems. After dealing with the methodological aspects, we provide an exhaustive overview of the available RNA simulation literature, ranging from studies of the smallest RNA oligonucleotides to investigations of the entire ribosome. Our review encompasses tetranucleotides, tetraloops, a number of small RNA motifs, A-helix RNA, kissing-loop complexes, the TAR RNA element, the decoding center and other important regions of the ribosome, as well as assorted others systems. Extended sections are devoted to RNA-ion interactions, ribozymes, riboswitches, and protein/RNA complexes. Our overview is written for as broad of an audience as possible, aiming to provide a much-needed interdisciplinary bridge between computation and experiment, together with a perspective on the future of the field

Modeling RNA, protein, and synthetic molecules using coarse-grained and all-atom representations

The aim of computational chemistry is to depict and understand the dynamics and interactions of molecular systems. In addition to increased comprehension in the physical and life sciences, this insight yields important applications to therapeutic design and materials science. In computational chemistry, molecules can be modeled in a number of representations depending on the molecular system and phenomena of interest. In this work, both simplified, coarse-grained representations and all-atom representations are used to model the interactions of RNA, cucurbituril host-guest chemistry, and cadmium selenide quantum dot binding to the Src homology 3 domain. For RNA, a coarse-grained model was developed termed RACER (RnA CoarsE-gRained) to accurately predict RNA structure and folding free energy. After optimization to statistical potentials, RACER accurately predicted the structures of 14 RNAs with an average 4.15Å root mean square deviation (RMSD) to the experimental structure. Further, RACER captured the sequence-specific variation in folding free energy for a set of 6 RNA hairpins and 5 RNA duplexes, with a R² correlation of 0.96 to experiment. The binding free energies of a cucurbituril host with 14 guests were computed using a polarizable force field and the free energy techniques of Bennett acceptance ratio and the orthogonal space random walk. The polarizable force field captured binding accurately, yet unexpectedly, the orthogonal space random walk method converged slowly, albeit at still reduced computational expense to the Bennett acceptance ratio. Lastly, the nanotoxicity effects of trioctylphosphine oxide coated cadmium selenide quantum dots are investigated with the model Src homology 3 protein domain in complex with its native proline rich motif ligand. With increasing quantum dot concentration, there is an increasing preference for the quantum dots to bind to the proline rich motif active site, inhibiting Src homology 3 function.Biomedical Engineerin

Hallmarks of cotranslational protein complex assembly and its relationship with the dominant-negative effect

Proteins carry out most of the biochemical phenomena necessary for life as we know it. The majority of proteins do not function alone in the cell, but are instead subunits that assemble into complexes with copies of themselves and other proteins. For decades, due to limited evidence to support otherwise, the textbook model was that subunits have to be fully synthesised before they diffuse away and collide randomly with their partners to from a complex. More recently, however, increasing evidence has accumulated, revealing that this model is incomplete. We now understand that many subunits begin the assembly process during their translation on the ribosome. This phenomenon has important implications for the structure, function, and evolution of protein complexes, as well as for the understanding and the prediction of the mechanisms by which genetic mutations cause disease. The first chapter provides an overview of our current understanding of how and why proteins assemble into complexes. Two classes of complexes are discussed: homomers, which consist of genetically identical copies of a protein and exhibit structural symmetry, and heteromers, which involve the assembly of non-identical proteins and are more common in human cells. I review the historical experiments that contributed to the discovery of cotranslational assembly, including recent breakthroughs that have made its proteome-wide detection possible, which is of tremendous value to this thesis. I provide an overview of genetic mutations in the context of human disease, as the present work has considerable clinical applications beyond its contribution to fundamental biology. In the second chapter, I investigate the properties of subunit interfaces that influence cotranslational assembly using a combination of proteomic, structural, and computational approaches. I show that cotranslational assembly is particularly common between subunits that form large intermolecular interfaces. To test whether large interfaces have evolved to promote cotranslational assembly, as opposed to cotranslational assembly being a non-adaptive consequence of large interfaces, I compare the sizes of first and last translated interfaces of heteromeric subunits in the proteomes of three evolutionary distant species. This analysis reveals that N-terminal interfaces, on average, tend to be larger than C-terminal interfaces. Notably, the trend is significant in ancient subunits or those organised into operons in bacteria, suggesting that large N-terminal interfaces may have been selected for to seed the assembly pathway cotranslationally. The third chapter explores an important hypothesis regarding cotranslational assembly: can it counter the dominant-negative effect, whereby the co-assembly of mutant and wild-type subunits impairs the activity of a protein complex? First, I show that cotranslationally assembling subunits are much less likely to be associated with autosomal dominant relative to recessive disorders. Second, I observe that subunits with dominant-negative disease mutations are significantly depleted in cotranslational assembly compared to those associated with loss-of-function mutations. Additionally, I find that complexes with known dominant-negative effects tend to expose their interfaces later during translation, lessening the likelihood of cotranslational assembly. Altogether, I find strong support for the hypothesis that the allele-specific nature of cotranslational assembly can buffer the effect of certain dominant mutations. In the fourth chapter, I synthesize the hallmarks of cotranslational assembly and discuss their mechanistic interpretations, highlighting the differences between neutralist and selectionist perspectives regarding their functional importance. Finally, in the fifth chapter, by combining a diverse range of gene-level features, I train a computational model for predicting proteins likely to be associated with non-loss-of-function (non-LOF) disease mechanisms, with the aim of accelerating the discovery of novel disease variants. I first generate a model that utilizes protein complex structural data and showcase its ability to detect properties explicitly absent from the model but are linked to proteins that give rise to non-LOF disease mechanisms. Although the results reflect the idea that LOF and non-LOF mechanisms can be captured at the protein-level, the predictor is strongly limited by the availability of protein complex structural data. Due to this limitation, I introduce a new model architecture with a spectrum of surrogate features, notably excluding those based on experimental protein complex structure data. The resulting models enable the estimation of probabilities for a protein exhibiting loss-of-function, gain-of-function, and dominant-negative molecular disease mechanisms across the entire proteome. In preliminary results, I demonstrate the practical applications of these models, including the prioritization of mutations with non-LOF-like properties in population genetic data and the detection of cryptic de novo dominant-negative mutations in developmental disorders. This thesis offers fresh insights into the molecular and evolutionary aspects of cotranslational assembly and its role in human disease

Functionally Relevant Macromolecular Interactions of Disordered Proteins

Disordered proteins are relatively recent newcomers in protein science. They were first described in detail by Wright and Dyson, in their J. Mol. Biol. paper in 1999. First, it was generally thought for more than a decade that disordered proteins or disordered parts of proteins have different amino acid compositions than folded proteins, and various prediction methods were developed based on this principle. These methods were suitable for distinguishing between the disordered (unstructured) and structured proteins known at that time. In addition, they could predict the site where a folded protein binds to the disordered part of a protein, shaping the latter into a well-defined 3D structure. Recently, however, evidence has emerged for a new type of disordered protein family whose members can undergo coupled folding and binding without the involvement of any folded proteins. Instead, they interact with each other, stabilizing their structure via “mutual synergistic folding” and, surprisingly, they exhibit the same residue composition as the folded protein. Increasingly more examples have been found where disordered proteins interact with non-protein macromolecules, adding to the already large variety of protein–protein interactions. There is also a very new phenomenon when proteins are involved in phase separation, which can represent a weak but functionally important macromolecular interaction. These phenomena are presented and discussed in the chapters of this book