Development of an enabling platform biotechnology for the production of proteins.

Protein-based drugs are a mainstay of modern medicine. In contrast to antibodies, most of these need highly individualized production processes which often limits their development. Here, we develop an immunoglobulin domain tag (i-Tag), which can be fused to any protein of interest. This tag is made of a linear arrangement of antibody light chain constant domains. It enhances expression as well as secretion of the fusion partner and allows for simple purification of several structurally and functionally distinct fusion proteins. Furthermore, it improves the biophysical characteristics of most fusion proteins tested, is inert, and does not compromise the fusion partners' functionality. Taken together, the i-Tag should facilitate the development of biopharmaceuticals and diagnostic proteins otherwise lacking a common structural element.

Modeling of the human interleukin 12:receptor complex allows to engineer attenuated cytokine variants.

Interleukin 12 (IL-12) plays major roles in immune defense against intracellular pathogens. By activating T cells and increasing antigen presentation, it is also a very potent anti-tumor molecule. Strong immune activation and systemic toxicity, however, so far limit its potential therapeutic use. Building on recent experimental structures of IL-12 related cytokine:receptor complexes, we here provide a high-resolution computational model of the human IL-12:receptor complex. We design attenuated IL-12 variants with lower receptor binding affinities based on molecular dynamics simulations, and subsequently validate them experimentally. These variants show reduced activation of natural killer cells while maintaining T cell activation. This immunological signature is important to develop IL-12 for cancer treatment, where natural killer cells contribute to severe side-effects. Taken together, our study provides detailed insights into structure and dynamics of the human IL-12:receptor complex and leverages them for engineering attenuated variants to elicit fewer side-effects while maintaining relevant biological activity.

Coupling of conformation and CPD damage in nucleosomal DNA.

UV-light can cause photodimerization and hence damages in DNA. Most frequent are cyclobutane pyrimidine dimer (CPD) damages, which predominantly form at TpT (thymine-thymine) steps. It is well known that CPD damage probability is different for single-stranded or double stranded DNA and depends on the sequence context. However, DNA deformation due to packing in nucleosomes can also influence CPD formation. Quantum mechanical calculations and Molecular Dynamics simulations indicate little CPD damage probability for DNA's equilibrium structure. We find that DNA needs to be deformed in a specific way to allow the HOMO → LUMO transition required for CPD damage formation. The simulation studies further show that the periodic CPD damage patterns measured in chromosomes and nucleosomes can be directly explained by the periodic deformation pattern of the DNA in the nucleosome complex. It supports previous findings on characteristic deformation patterns found in experimental nucleosome structures that relate to CPD damage formation. The result may have important implications for our understanding of UV-induced DNA mutations in human cancers.

Toward Force Fields with Improved Base Stacking Descriptions.

Recent DNA force fields indicate good performance in describing flexibility and structural stability of double-stranded B-DNA. However, it is not clear how accurately base stacking interactions are represented that are critical for simulating structure formation processes and conformational changes. Based on the equilibrium nucleoside association and base pair nicking, we find that the recent Tumuc1 force field improves the description of base stacking compared to previous state-of-the-art force fields. Nevertheless, base pair stacking is still overstabilized compared to experiment. We propose a rapid method to reweight calculated free energies of stacking upon force field modifications in order to generate improved parameters. A decrease of the Lennard-Jones attraction between nucleo-bases alone appears insufficient; however, adjustments in the partial charge distribution on base atoms could help to further improve the force field description of base stacking.

The development of nucleic acids force fields: From an unchallenged past to a competitive future.

Molecular dynamics simulations have strongly matured as a method to study biomolecular processes. Their validity, however, is determined by the accuracy of the underlying force fields that describe the forces between all atoms. In this article, we review the development of nucleic acids force fields. We describe the early attempts in the 1990s and emphasize their strong influence on recent force fields. State-of-the-art force fields still use the same Lennard-Jones parameters derived 25 years ago in spite of the fact that these parameters were in general not fitted for nucleic acids. In addition, electrostatic parameters also are deprecated, which may explain some of the current force field deficiencies. We compare different force fields for various systems and discuss new tests of the recently developed Tumuc1 force field. The OL-force fields and Tumuc1 are arguably the best force fields to describe the DNA double helix. However, no force field is flawless. In particular, the description of sugar-puckering remains a problem for nucleic acids force fields. Future refinements are required, so we review methods for force field refinement and give an outlook to the future of force fields.

Orientation Dependence of DNA Blunt-End Stacking Studied by Free-Energy Simulations.

DNA blunt ends can associate mediated by stacking interactions between the terminal base pairs that form blunt ends. The blunt end association plays a role in DNA repair and recombination processes and can also be of importance for the design of DNA-based nano-materials. Its function depends on the sequence and on the geometric arrangement that leads to stable interaction. For a stacked state, the relative orientation (twisting) of the base pairs is important. Molecular dynamics and advanced sampling simulations were used to calculate free energy change associated with twist changes of the stacked blunt-end base pairs. The calculations reproduce blunt stacking arrangements found in crystal structures of DNA oligonucleotides as free energy minima. To elucidate the physical origin of the stabilization of certain angular arrangements, the interactions between backbone atoms in the blunt-end stack were switched off in additional free energy calculations. It allows us to decipher the contributions to stacking stabilization due to the nucleobases and the backbone and to analyze the sequence dependence of the angular stacking preferences. Good qualitative agreement was also found for the comparison with quantum mechanical calculations. The results may help in the design of novel DNA-based materials.

Tumuc1: A New Accurate DNA Force Field Consistent with High-Level Quantum Chemistry.

An accurate molecular mechanics force field forms the basis of Molecular Dynamics simulations to obtain a realistic view of the structure and dynamics of biomolecules such as DNA. Although frequently updated to improve agreement with available experimental data, DNA force fields still rely in part on parameters introduced more than 20 years ago. We have developed an entirely new DNA force field, Tumuc1, derived from quantum mechanical calculations to obtain a consistent set of bonded parameters and partial atomic charges. The performance of the force field was extensively tested on a variety of DNA molecules. It excels in accuracy of B-DNA simulations but also performs very well on other types of DNA structures and structure formation processes such as hairpin folding, duplex formation, and dynamics of DNA-protein complexes. It can complement existing force fields in order to provide an increasingly accurate description of the structure and dynamics of DNA during simulation studies.

Accurate modeling of DNA conformational flexibility by a multivariate Ising model.

The sequence-dependent structure and deformability of DNA play a major role for binding of proteins and regulation of gene expression. So far, most efforts to model DNA flexibility are based on unimodal harmonic stiffness models at base-pair resolution. However, multimodal behavior due to distinct conformational substates also contributes significantly to the conformational flexibility of DNA. Moreover, these local substates are correlated to their nearest-neighbor substates. A description for DNA elasticity which includes both multimodality and nearest-neighbor coupling has remained a challenge, which we solve by combining our multivariate harmonic approximation with an Ising model for the substates. In a series of applications to DNA fluctuations and protein-DNA complexes, we demonstrate substantial improvements over the unimodal stiffness model. Furthermore, our multivariate Ising model reveals a mechanical destabilization for adenine (A)-tracts to undergo nucleosome formation. Our approach offers a wide range of applications to determine sequence-dependent deformation energies of DNA and to investigate indirect readout contributions to protein-DNA recognition.

Targeting Telomeres: Molecular Dynamics and Free Energy Simulation of Gold-Carbene Binding to DNA.

DNA sequences in regulatory regions and in telomers at the ends of chromosomes frequently contain tandem repeats of guanine nucleotides that can form stacked structures stabilized by Hoogsten pairing and centrally bound monovalent cations. The replication and elongation of telomeres requires the disruption of these G-quadruplex structures. Hence, drug molecules such as gold (Au)-carbene that stabilize G-quadruplexes may also interfere with the elongation of telomeres and, in turn, could be used to control cell replication and growth. To better understand the molecular mechanism of Au-carbene binding to G-quadruplexes, we employed molecular dynamics simulations and free energy simulations. Whereas very restricted mobility of two Au-carbene ligands was found upon binding as a doublet to one side of the G-quadruplex, much larger translational and orientational mobility was observed for a single Au-carbene binding at the second G-quadruplex surface. Comparative simulations on duplex DNA in the presence of Au-carbene ligands indicates a preference for the minor groove and weaker unspecific and more salt-dependent binding than to the G-quadruplex surface. Analysis of energetic contributions reveals a dominance of nonpolar and van der Waals interactions to drive binding. The simulations can also be helpful for proposing possible modifications that could improve Au-carbene affinity and specificity for G-quadruplex binding.

How global DNA unwinding causes non-uniform stress distribution and melting of DNA.

DNA unwinding is an important process that controls binding of proteins, gene expression and melting of double-stranded DNA. In a series of all-atom MD simulations on two DNA molecules containing a transcription start TATA-box sequence we demonstrate that application of a global restraint on the DNA twisting dramatically changes the coupling between helical parameters and the distribution of deformation energy along the sequence. Whereas only short range nearest-neighbor coupling is observed in the relaxed case, long-range coupling is induced in the globally restrained case. With increased overall unwinding the elastic deformation energy is strongly non-uniformly distributed resulting ultimately in a local melting transition of only the TATA box segment during the simulations. The deformation energy tends to be stored more in cytidine/guanine rich regions associated with a change in conformational substate distribution. Upon TATA box melting the deformation energy is largely absorbed by the melting bubble with the rest of the sequences relaxing back to near B-form. The simulations allow us to characterize the structural changes and the propagation of the elastic energy but also to calculate the associated free energy change upon DNA unwinding up to DNA melting. Finally, we design an Ising model for predicting the local melting transition based on empirical parameters. The direct comparison with the atomistic MD simulations indicates a remarkably good agreement for the predicted necessary torsional stress to induce a melting transition, for the position and length of the melted region and for the calculated associated free energy change between both approaches.

How methyl-sugar interactions determine DNA structure and flexibility.

The sequence dependent structure and flexibility of the DNA double helix is of key importance for gene expression and DNA packing and it can be modulated by DNA modifications. The presence of a C5'-methyl group in thymine or the frequent C5'-methylated-cytosine affects the DNA fine structure, however, the underlying mechanism and steric origins have remained largely unexplained. Employing Molecular Dynamics free energy simulations that allow switching on or off interactions with the methyl groups in several DNA sequences, we systematically identified the physical origin of the coupling between methyl groups and DNA backbone fine structure. Whereas methyl-solvent and methyl-nucleobase interactions were found to be of minor importance, the methyl group interaction with the 5' neighboring sugar was identified as main cause for influencing the population of backbone substates. The sterical methyl sugar clash prevents the formation of unconventional stabilizing hydrogen bonds between nucleobase and backbone. The technique was also used to study the contribution of methyl groups to DNA flexibility and served to explain why the presence of methyl sugar clashes in thymine and methyl-cytosine can result in an overall local increase of DNA flexibility.

Unwinding Induced Melting of Double-Stranded DNA Studied by Free Energy Simulations.

DNA unwinding plays a major role in many biological processes, such as replication, transcription, and repair. It can lead to local melting and strand separation and can serve as a key mechanism to promote access to the separate strands of a double-stranded DNA. While DNA unwinding has been investigated extensively by DNA cyclization and single-molecule studies on a length-scale of kilo base pairs, it is neither fully understood at the base pair level nor at the level of molecular interactions. By employing a torque acting on the termini of DNA oligonucleotides during molecular dynamics free energy simulations, we locally unwind the central part of a DNA beyond an elastic (harmonic) regime. The simulations reproduce experimental results on the twist elasticity in the harmonic regime (characterized by a mostly quadratic free energy change with respect to changes in twist) and a deformation up to 7° was found as a limit of the harmonic response. Beyond this limit the free energy increase per twist change dropped dramatically coupled to local base pair disruptions and significant deformation of the nucleic acid backbone structure. Restriction of the DNA bending flexibility resulted in a stiffer harmonic response and an earlier onset of the anharmonic response. Whereas local melting with a complete disruption of base pairing and flipping of nucleotides was observed in case of an AT rich central segment strong backbone changes and changes in the stacking arrangements were observed in case of a GC rich segment. Unrestrained MD simulations starting from locally melted DNA reformed regular B-DNA after 50-300 ns simulation time. The simulations may have important implications for understanding DNA recognition processes coupled with significant structural alterations.

Explaining the striking difference in twist-stretch coupling between DNA and RNA: A comparative molecular dynamics analysis.

Double stranded helical DNA and RNA are flexible molecules that can undergo global conformational fluctuations. Their bending, twisting and stretching deformabilities are of similar magnitude. However, recent single-molecule experiments revealed a striking qualitative difference indicating an opposite sign for the twist-stretch couplings of dsDNA and dsRNA [Lipfert et al. 2014. Proc. Natl. Acad. Sci. U.S.A. 111, 15408] that is not explained by existing models. Employing unconstrained Molecular Dynamics (MD) simulations we are able to reproduce the qualitatively different twist-stretch coupling for dsDNA and dsRNA in semi-quantitative agreement with experiment. Similar results are also found in simulations that include an external torque to induce over- or unwinding of DNA and RNA. Detailed analysis of the helical deformations coupled to twist indicate that the interplay of helical rise, base pair inclination and displacement from the helix axis upon twist changes are responsible for the different twist-stretch correlations. Overwinding of RNA results in more compact conformations with a narrower major groove and consequently reduced helical extension. Overwinding of DNA decreases the size of the minor groove and the resulting positive base pair inclination leads to a slender and more extended helical structure.