Developing a molecular dynamics force field for both folded and disordered protein states

Molecular dynamics (MD) simulation is a valuable tool for characterizing the structural dynamics of folded proteins and should be similarly applicable to disordered proteins and proteins with both folded and disordered regions. It has been unclear, however, whether any physical model (force field) used in MD simulations accurately describes both folded and disordered proteins. Here, we select a benchmark set of 21 systems, including folded and disordered proteins, simulate these systems with six state-of-the-art force fields, and compare the results to over 9,000 available experimental data points. We find that none of the tested force fields simultaneously provided accurate descriptions of folded proteins, of the dimensions of disordered proteins, and of the secondary structure propensities of disordered proteins. Guided by simulation results on a subset of our benchmark, however, we modified parameters of one force field, achieving excellent agreement with experiment for disordered proteins, while maintaining state-of-the-art accuracy for folded proteins. The resulting force field, a99SB-disp, should thus greatly expand the range of biological systems amenable to MD simulation. A similar approach could be taken to improve other force fields

Thermal Adaptation of Conformational Dynamics in Ribonuclease H

The relationship between inherent internal conformational processes and enzymatic activity or thermodynamic stability of proteins has proven difficult to characterize. The study of homologous proteins with differing thermostabilities offers an especially useful approach for understanding the functional aspects of conformational dynamics. In particular, ribonuclease HI (RNase H), an 18 kD globular protein that hydrolyzes the RNA strand of RNA:DNA hybrid substrates, has been extensively studied by NMR spectroscopy to characterize the differences in dynamics between homologs from the mesophilic organism E. coli and the thermophilic organism T. thermophilus. Herein, molecular dynamics simulations are reported for five homologous RNase H proteins of varying thermostabilities and enzymatic activities from organisms of markedly different preferred growth temperatures. For the E. coli and T. thermophilus proteins, strong agreement is obtained between simulated and experimental values for NMR order parameters and for dynamically averaged chemical shifts, suggesting that these simulations can be a productive platform for predicting the effects of individual amino acid residues on dynamic behavior. Analyses of the simulations reveal that a single residue differentiates between two different and otherwise conserved dynamic processes in a region of the protein known to form part of the substrate-binding interface. Additional key residues within these two categories are identified through the temperature-dependence of these conformational processes

International Ocean Discovery Program Expedition 393 Preliminary Report South Atlantic Transect 2

The South Atlantic Transect (SAT) is a multidisciplinary scientific ocean drilling experiment designed to investigate the evolution of the oceanic crust and overlying sediments across the western flank of the Mid-Atlantic Ridge. This project comprises four International Ocean Discovery Program expeditions: fully staffed Expeditions 390 and 393 (April–August 2022) built on engineering preparations during Expeditions 390C and 395E that took place without science parties during the height of the Coronavirus Disease 2019 (COVID-19) pandemic. Through operations along a crustal flow line at ~31°S, the SAT recovered complete sedimentary sections and the upper ~40–340 m of the underlying ocean crust formed at a slow to intermediate spreading rate at the Mid-Atlantic Ridge over the past ~61 My. The sediments along this transect were originally spot cored more than 50 y ago during Deep Sea Drilling Project Leg 3 (December 1968–January 1969) to help verify the theories of seafloor spreading and plate tectonics. The SAT expeditions targeted six primary sites on 7, 15, 31, 49, and 61 Ma ocean crust that fill critical gaps in our sampling of intact in situ ocean crust with regards to crustal age, spreading rate, and sediment thickness. Drilling these sites was required to investigate the history, duration, and intensity of the low-temperature hydrothermal interactions between the aging ocean crust and the evolving South Atlantic Ocean. This knowledge will improve the quantification of past hydrothermal contributions to global biogeochemical cycles and help develop a predictive understanding of the impacts of variable hydrothermal processes and exchanges. Samples from the transect of the previously unexplored sediment- and basalt-hosted deep biosphere beneath the South Atlantic Gyre are essential to refine global biomass estimates and examine microbial ecosystems’ responses to variable conditions in a low-energy gyre and aging ocean crust. The transect is located near World Ocean Circulation Experiment Line A10, which provides a baseline for records of carbonate chemistry and deepwater mass properties across the western South Atlantic through key Cenozoic intervals of elevated atmospheric CO2 and rapid climate change. Reconstruction of the history of the deep western boundary current and deepwater formation in the Atlantic basins will yield crucial data to test hypotheses regarding the role of evolving thermohaline circulation patterns in climate change and the effects of tectonic gateways and climate on ocean acidification. During engineering Expeditions 390C and 395E, a single hole was cored through the sediment cover and into the uppermost rocks of the ocean crust with the advanced piston corer (APC) and extended core barrel (XCB) systems at five of the six primary proposed SAT sites. Reentry systems with casing were then installed either into basement or within 10 m of basement at each of those five sites. Expedition 390 (7 April–7 June 2022) conducted operations at three of the SAT sites, recovering 700 m of core (77%) over 30.3 days of on-site operations. Sediment coring, basement coring, and wireline logging were conducted at two sites on 61 Ma crust (Sites U1556 and U1557), and sediment coring was completed at the 7 Ma Site U1559. Expedition 393 operated at four sites, drilling in 12 holes to complete this initial phase of the SAT. Complete sedimentary sections were collected at Sites U1558, U1583, and U1560 on 49, 31, and 15 Ma crust, respectively, and together with 257.7 m of sediments cored during earlier operations, more than 600 m of sediments was characterized. The uppermost ocean crust was drilled at Sites U1558, U1560, and U1583 with good penetration (~130 to ~204 meters subbasement), but at the youngest ~7 Ma Site U1559, only ~43 m of basement penetration was achieved in this initial attempt. Geophysical wireline logs were aquired at Sites U1583 and U1560. Expeditions 390 and 393 established legacy sites available for future deepening and downhole basement hydrothermal and microbiological experiments at Sites U1557, U1560, and U1559 on 61, 15, and 7 Ma crust, respectively

Rational optimization of a transcription factor activation domain inhibitor

Transcription factors are among the most attractive therapeutic targets but are considered largely 'undruggable' in part due to the intrinsically disordered nature of their activation domains. Here we show that the aromatic character of the activation domain of the androgen receptor, a therapeutic target for castration-resistant prostate cancer, is key for its activity as transcription factor, allowing it to translocate to the nucleus and partition into transcriptional condensates upon activation by androgens. On the basis of our understanding of the interactions stabilizing such condensates and of the structure that the domain adopts upon condensation, we optimized the structure of a small-molecule inhibitor previously identified by phenotypic screening. The optimized compounds had more affinity for their target, inhibited androgen-receptor-dependent transcriptional programs, and had an antitumorigenic effect in models of castration-resistant prostate cancer in cells and in vivo. These results suggest that it is possible to rationally optimize, and potentially even to design, small molecules that target the activation domains of oncogenic transcription factors

A practical guide to the simultaneous determination of protein structure and dynamics using metainference

Accurate protein structural ensembles can be determined with metainference, a Bayesian inference method that integrates experimental information with prior knowledge of the system and deals with all sources of uncertainty and errors as well as with system heterogeneity. Furthermore, metainference can be implemented using the metadynamics approach, which enables the computational study of complex biological systems requiring extensive conformational sampling. In this chapter, we provide a step-by-step guide to perform and analyse metadynamic metainference simulations using the ISDB module of the open-source PLUMED library, as well as a series of practical tips to avoid common mistakes. Specifically, we will guide the reader in the process of learning how to model the structural ensemble of a small disordered peptide by combining state-of-the-art molecular mechanics force fields with nuclear magnetic resonance data, including chemical shifts, scalar couplings and residual dipolar couplings.Comment: 49 pages, 9 figure

Small Molecules Targeting the Disordered Transactivation Domain of the Androgen Receptor Induce the Formation of Collapsed Helical States

AbstractCastration-resistant prostate cancer (CRPC) is a lethal condition suffered by ∼35% of prostate cancer patients who become resistant to existing FDA-approved drugs. Small molecules that target the intrinsically disordered N-terminal domain of the androgen receptor (AR-NTD) have shown promise in circumventing CPRC drug-resistance. A prodrug of one such compound, EPI-002, entered human trials in 2015 but was discontinued after phase I due to poor potency. The compound EPI-7170 was subsequently found to have improved potency, and a related compound entered human trials in 2020. NMR measurements have localized the strongest effects of these compounds to a transiently helical region of the disordered AR-NTD but no detailed structural or mechanistic rationale exists to explain their affinity to this region or the comparative potency of EPI-7170. Here, we utilize all-atom molecular dynamics simulations to elucidate the binding mechanisms of the small molecules EPI-002 and EPI-7170 to the disordered AR-NTD. We observe that both compounds induce the formation of collapsed helical states in the Tau-5 transactivation domain and that these bound states consist of heterogenous ensembles of interconverting binding modes. We find that EPI-7170 has a higher affinity to Tau-5 than EPI-002 and that the EPI-7170 bound ensemble contains a substantially higher population of collapsed helical states than the bound ensemble of EPI-002. We identify a network of interactions in the EPI-7170 bound ensemble that stabilize collapsed helical conformations. Our results provide atomically detailed binding mechanisms for EPI compounds consistent with NMR experiments that will prove useful for drug discovery for CRPC.SummaryIntrinsically disordered proteins (IDPs), which do not fold into a well-defined three-dimensional structure under physiological conditions, are implicated in many human diseases. Such proteins are difficult to characterize at an atomic level and are extremely challenging drug targets. Small molecules that target a disordered domain of the androgen receptor have entered human trials for the treatment of castration-resistant prostate cancer, but no structural or mechanistic rationale exists to explain their inhibition mechanisms or relative potencies. Here, we utilize molecular dynamics computer simulations to elucidate atomically detailed binding mechanisms of these compounds and understand their inhibition mechanisms. Our results suggest strategies for developing more potent androgen receptor inhibitors and general strategies for IDP drug design.</jats:sec

Determination of Protein Structures in the Solid State from NMR Chemical Shifts

SummarySolid-state NMR spectroscopy does not require proteins to form crystalline or soluble samples and can thus be applied under a variety of conditions, including precipitates, gels, and microcrystals. It has recently been shown that NMR chemical shifts can be used to determine the structures of the native states of proteins in solution. By considering the cases of two proteins, GB1 and SH3, we provide an initial demonstration here that this type of approach can be extended to the use of solid-state NMR chemical shifts to obtain protein structures in the solid state without the need for measuring interatomic distances