Fusion-associated carcinomas of the breast: Diagnostic, prognostic, and therapeutic significance.

Recurrent gene fusions comprise a class of viable genetic targets in solid tumors that have culminated several recent breakthrough cancer therapies. Their role in breast cancer, however, remains largely underappreciated due to the complexity of genomic rearrangements in breast malignancy. Just recently, we and others have identified several recurrent gene fusions in breast cancer with important clinical and biological implications. Examples of the most significant recurrent gene fusions to date include (1) ESR1::CCDC170 gene fusions in luminal B and endocrine-resistant breast cancer that exert oncogenic function via modulating the HER2/HER3/SRC Proto-Oncogene (SRC) complex, (2) ESR1 exon 6 fusions in metastatic disease that drive estrogen-independent estrogen-receptor transcriptional activity, (3) BCL2L14::ETV6 fusions in a more aggressive form of the triple-negative subtype that prime epithelial-mesenchymal transition and endow paclitaxel resistance, (4) the ETV6::NTRK3 fusion in secretory breast carcinoma that constitutively activates NTRK3 kinase, (5) the oncogenic MYB-NFIB fusion as a genetic driver underpinning adenoid cystic carcinomas of the breast that activates MYB Proto-Oncogene (MYB) pathway, and (6) the NOTCH/microtubule-associated serine-threonine (MAST) kinase gene fusions that activate NOTCH and MAST signaling. Importantly, these fusions are enriched in more aggressive and lethal breast cancer presentations and appear to confer therapeutic resistance. Thus, these gene fusions could be utilized as genetic biomarkers to identify patients who require more intensive treatment and surveillance. In addition, kinase fusions are currently being evaluated in breast cancer clinical trials and ongoing mechanistic investigation is exposing therapeutic vulnerabilities in patients with fusion-positive disease.

Integrative structural modeling of a multidomain polo-like kinase.

Polo-like kinase 1 (PLK1) is a key regulator and coordinator for mitotic signaling that contains two major functional units of a kinase domain (KD) and a polo-box domain (PBD). While individual domain structures of the KD and the PBD are known, how they interact and assemble into a functional complex remains an open question. The structural model from the KD-PBD-Map205PBM heterotrimeric crystal structure of zebrafish PLK1 represents a major step in understanding the KD and the PBD interactions. However, how these two domains interact when connected by a linker in the full length PLK1 needs further investigation. By integrating different sources of structural data from small-angle X-ray scattering, hydroxyl radical protein footprinting, and computational sampling, here we report an overall architecture for PLK1 multidomain assembly between the KD and the PBD. Our model revealed that the KD uses its C-lobe to interact with the PBD via the site near the phosphopeptide binding site in its auto-inhibitory state in solution. Disruption of this auto-inhibition via site-directed mutagenesis at the KD-PBD interface increases its kinase activity, supporting the functional role of KD-PBD interactions predicted for regulating the PLK1 kinase function. Our results indicate that the full length human PLK1 takes dynamic structures with a variety of domain-domain interfaces in solution.

Cyclin-dependent kinase 7 (CDK7)-mediated phosphorylation of the CDK9 activation loop promotes P-TEFb assembly with Tat and proviral HIV reactivation.

The HIV trans-activator Tat recruits the host transcription elongation factor P-TEFb to stimulate proviral transcription. Phosphorylation of Thr-186 on the activation loop (T-loop) of cyclin-dependent kinase 9 (CDK9) is essential for its kinase activity and assembly of CDK9 and cyclin T1 (CycT1) to form functional P-TEFb. Phosphorylation of a second highly conserved T-loop site, Ser-175, alters the competitive binding of Tat and the host recruitment factor bromodomain containing 4 (BRD4) to P-TEFb. Here, we investigated the intracellular mechanisms that regulate these key phosphorylation events required for HIV transcription. Molecular dynamics simulations revealed that the CDK9/CycT1 interface is stabilized by intramolecular hydrogen bonding of pThr-186 by an arginine triad and Glu-96 of CycT1. Arginine triad substitutions that disrupted CDK9/CycT1 assembly accumulated Thr-186-dephosphorylated CDK9 associated with the cytoplasmic Hsp90/Cdc37 chaperone. The Hsp90/Cdc37/CDK9 complex was also present in resting T cells, which lack CycT1. Hsp90 inhibition in primary T cells blocked P-TEFb assembly, disrupted Thr-186 phosphorylation, and suppressed proviral reactivation. The selective CDK7 inhibitor THZ1 blocked CDK9 phosphorylation at Ser-175, and in vitro kinase assays confirmed that CDK7 activity is principally responsible for Ser-175 phosphorylation. Mutation of Ser-175 to Lys had no effect on CDK9 kinase activity or P-TEFb assembly but strongly suppressed both HIV expression and BRD4 binding. We conclude that the transfer of CDK9 from the Hsp90/Cdc37 complex induced by Thr-186 phosphorylation is a key step in P-TEFb biogenesis. Furthermore, we demonstrate that CDK7-mediated Ser-175 phosphorylation is a downstream nuclear event essential for facilitating CDK9 T-loop interactions with Tat.

Charge Neutralization Drives the Shape Reconfiguration of DNA Nanotubes.

Reconfiguration of membrane protein channels for gated transport is highly regulated under physiological conditions. However, a mechanistic understanding of such channels remains challenging owing to the difficulty in probing subtle gating-associated structural changes. Herein, we show that charge neutralization can drive the shape reconfiguration of a biomimetic 6-helix bundle DNA nanotube (6HB). Specifically, 6HB adopts a compact state when its charge is neutralized by Mg2+ ; whereas Na+ switches it to the expanded state, as revealed by MD simulations, small-angle X-ray scattering (SAXS), and FRET characterization. Furthermore, partial neutralization of the DNA backbone charges by chemical modification renders 6HB compact and insensitive to ions, suggesting an interplay between electrostatic and hydrophobic forces in the channels. This system provides a platform for understanding the structure-function relationship of biological channels and designing rules for the shape control of DNA nanostructures in biomedical applications.

Quantitative protein topography analysis and high-resolution structure prediction using hydroxyl radical labeling and tandem-ion mass spectrometry (MS).

Hydroxyl radical footprinting based MS for protein structure assessment has the goal of understanding ligand induced conformational changes and macromolecular interactions, for example, protein tertiary and quaternary structure, but the structural resolution provided by typical peptide-level quantification is limiting. In this work, we present experimental strategies using tandem-MS fragmentation to increase the spatial resolution of the technique to the single residue level to provide a high precision tool for molecular biophysics research. Overall, in this study we demonstrated an eightfold increase in structural resolution compared with peptide level assessments. In addition, to provide a quantitative analysis of residue based solvent accessibility and protein topography as a basis for high-resolution structure prediction; we illustrate strategies of data transformation using the relative reactivity of side chains as a normalization strategy and predict side-chain surface area from the footprinting data. We tested the methods by examination of Ca(+2)-calmodulin showing highly significant correlations between surface area and side-chain contact predictions for individual side chains and the crystal structure. Tandem ion based hydroxyl radical footprinting-MS provides quantitative high-resolution protein topology information in solution that can fill existing gaps in structure determination for large proteins and macromolecular complexes.

A Practical Guide to iSPOT Modeling: An Integrative Structural Biology Platform.

Integrative structure modeling is an emerging method for structural determination of protein-protein complexes that are challenging for conventional structural techniques. Here, we provide a practical protocol for implementing our integrated iSPOT platform by integrating three different biophysical techniques: small-angle X-ray scattering (SAXS), hydroxyl radical footprinting, and computational docking simulations. Specifically, individual techniques are described from experimental and/or computational perspectives, and complementary structural information from these different techniques are integrated for accurate characterization of the structures of large protein-protein complexes.

Theoretical modeling of multiprotein complexes by iSPOT: Integration of small-angle X-ray scattering, hydroxyl radical footprinting, and computational docking.

Structural determination of protein-protein complexes such as multidomain nuclear receptors has been challenging for high-resolution structural techniques. Here, we present a combined use of multiple biophysical methods, termed iSPOT, an integration of shape information from small-angle X-ray scattering (SAXS), protection factors probed by hydroxyl radical footprinting, and a large series of computationally docked conformations from rigid-body or molecular dynamics (MD) simulations. Specifically tested on two model systems, the power of iSPOT is demonstrated to accurately predict the structures of a large protein-protein complex (TGFß-FKBP12) and a multidomain nuclear receptor homodimer (HNF-4α), based on the structures of individual components of the complexes. Although neither SAXS nor footprinting alone can yield an unambiguous picture for each complex, the combination of both, seamlessly integrated in iSPOT, narrows down the best-fit structures that are about 3.2Å and 4.2Å in RMSD from their corresponding crystal structures, respectively. Furthermore, this proof-of-principle study based on the data synthetically derived from available crystal structures shows that the iSPOT-using either rigid-body or MD-based flexible docking-is capable of overcoming the shortcomings of standalone computational methods, especially for HNF-4α. By taking advantage of the integration of SAXS-based shape information and footprinting-based protection/accessibility as well as computational docking, this iSPOT platform is set to be a powerful approach towards accurate integrated modeling of many challenging multiprotein complexes.

Quantitative mapping of protein structure by hydroxyl radical footprinting-mediated structural mass spectrometry: a protection factor analysis.

Measurements from hydroxyl radical footprinting (HRF) provide rich information about the solvent accessibility of amino acid side chains of a protein. Traditional HRF data analyses focus on comparing the difference in the modification/footprinting rate of a specific site to infer structural changes across two protein states, e.g., between a free and ligand-bound state. However, the rate information itself is not fully used for the purpose of comparing different protein sites within a protein on an absolute scale. To provide such a cross-site comparison, we present a new, to our knowledge, data analysis algorithm to convert the measured footprinting rate constant to a protection factor (PF) by taking into account the known intrinsic reactivity of amino acid side chain. To examine the extent to which PFs can be used for structural interpretation, this PF analysis is applied to three model systems where radiolytic footprinting data are reported in the literature. By visualizing structures colored with the PF values for individual peptides, a rational view of the structural features of various protein sites regarding their solvent accessibility is revealed, where high-PF regions are buried and low-PF regions are more exposed to the solvent. Furthermore, a detailed analysis correlating solvent accessibility and local structural contacts for gelsolin shows a statistically significant agreement between PF values and various structure measures, demonstrating that the PFs derived from this PF analysis readily explain fundamental HRF rate measurements. We also tested this PF analysis on alternative, chemical-based HRF data, showing improved correlations of structural properties of a model protein barstar compared to examining HRF rate data alone. Together, this PF analysis not only permits a novel, to our knowledge, approach of mapping protein structures by using footprinting data, but also elevates the use of HRF measurements from a qualitative, cross-state comparison to a quantitative, cross-site assessment of protein structures in the context of individual conformational states of interest.

Identification of a novel LXXLL motif in α-actinin 4-spliced isoform that is critical for its interaction with estrogen receptor α and co-activators.

α-Actinins (ACTNs) are a family of proteins cross-linking actin filaments that maintain cytoskeletal organization and cell motility. Recently, it has also become clear that ACTN4 can function in the nucleus. In this report, we found that ACTN4 (full length) and its spliced isoform ACTN4 (Iso) possess an unusual LXXLL nuclear receptor interacting motif. Both ACTN4 (full length) and ACTN4 (Iso) potentiate basal transcription activity and directly interact with estrogen receptor α, although ACTN4 (Iso) binds ERα more strongly. We have also found that both ACTN4 (full length) and ACTN4 (Iso) interact with the ligand-independent and the ligand-dependent activation domains of estrogen receptor α. Although ACTN4 (Iso) interacts efficiently with transcriptional co-activators such as p300/CBP-associated factor (PCAF) and steroid receptor co-activator 1 (SRC-1), the full length ACTN4 protein either does not or does so weakly. More importantly, the flanking sequences of the LXXLL motif are important not only for interacting with nuclear receptors but also for the association with co-activators. Taken together, we have identified a novel extended LXXLL motif that is critical for interactions with both receptors and co-activators. This motif functions more efficiently in a spliced isoform of ACTN4 than it does in the full-length protein.

Cross-talk between the ligand- and DNA-binding domains of estrogen receptor.

Estrogen receptor alpha (ERα) is a hormone-responsive transcription factor that contains several discrete functional domains, including a ligand-binding domain (LBD) and a DNA-binding domain (DBD). Despite a wealth of knowledge about the behaviors of individual domains, the molecular mechanisms of cross-talk between LBD and DBD during signal transduction from hormone to DNA-binding of ERα remain elusive. Here, we apply a multiscale approach combining coarse-grained (CG) and atomistically detailed simulations to characterize this cross-talk mechanism via an investigation of the ERα conformational landscape. First, a CG model of ERα is built based on crystal structures of individual LBDs and DBDs, with more emphasis on their interdomain interactions. Second, molecular dynamics simulations are implemented and enhanced sampling is achieved via the "push-pull-release" strategy in the search for different LBD-DBD orientations. Third, multiple energetically stable ERα conformations are identified on the landscape. A key finding is that estradiol-bound LBDs utilize the well-described activation helix H12 to pack and stabilize LBD-DBD interactions. Our results suggest that the estradiol-bound LBDs can serve as a scaffold to position and stabilize the DBD-DNA complex, consistent with experimental observations of enhanced DNA binding with the LBD. Final assessment using atomic-level simulations shows that these CG-predicted models are significantly stable within a 15-ns simulation window and that specific pairs of lysine residues in close proximity at the domain interfaces could serve as candidate sites for chemical cross-linking studies. Together, these simulation results provide a molecular view of the role of ERα domain interactions in response to hormone binding.

Fast-SAXS-pro: a unified approach to computing SAXS profiles of DNA, RNA, protein, and their complexes.

A generalized method, termed Fast-SAXS-pro, for computing small angle x-ray scattering (SAXS) profiles of proteins, nucleic acids, and their complexes is presented. First, effective coarse-grained structure factors of DNA nucleotides are derived using a simplified two-particle-per-nucleotide representation. Second, SAXS data of a 18-bp double-stranded DNA are measured and used for the calibration of the scattering contribution from excess electron density in the DNA solvation layer. Additional test on a 25-bp DNA duplex validates this SAXS computational method and suggests that DNA has a different contribution from its hydration surface to the total scattering compared to RNA and protein. To account for such a difference, a sigmoidal function is implemented for the treatment of non-uniform electron density across the surface of a protein/nucleic-acid complex. This treatment allows differential scattering from the solvation layer surrounding protein/nucleic-acid complexes. Finally, the applications of this Fast-SAXS-pro method are demonstrated for protein/DNA and protein/RNA complexes.

Multidomain assembled states of Hck tyrosine kinase in solution.

An approach combining small-angle X-ray solution scattering (SAXS) data with coarse-grained (CG) simulations is developed to characterize the assembly states of Hck, a member of the Src-family kinases, under various conditions in solution. First, a basis set comprising a small number of assembly states is generated from extensive CG simulations. Second, a theoretical SAXS profile for each state in the basis set is computed by using the Fast-SAXS method. Finally, the relative population of the different assembly states is determined via a Bayesian-based Monte Carlo procedure seeking to optimize the theoretical scattering profiles against experimental SAXS data. The study establishes the concept of basis-set supported SAXS (BSS-SAXS) reconstruction combining computational and experimental techniques. Here, BSS-SAXS reconstruction is used to reveal the structural organization of Hck in solution and the different shifts in the equilibrium population of assembly states upon the binding of different signaling peptides.

Biophysical and Integrative Characterization of Protein Intrinsic Disorder as a Prime Target for Drug Discovery.

Protein intrinsic disorder is increasingly recognized for its biological and disease-driven functions. However, it represents significant challenges for biophysical studies due to its high conformational flexibility. In addressing these challenges, we highlight the complementary and distinct capabilities of a range of experimental and computational methods and further describe integrative strategies available for combining these techniques. Integrative biophysics methods provide valuable insights into the sequence-structure-function relationship of disordered proteins, setting the stage for protein intrinsic disorder to become a promising target for drug discovery. Finally, we briefly summarize recent advances in the development of new small molecule inhibitors targeting the disordered N-terminal domains of three vital transcription factors.

Coarse-grained simulations of protein-protein association: an energy landscape perspective.

Understanding protein-protein association is crucial in revealing the molecular basis of many biological processes. Here, we describe a theoretical simulation pipeline to study protein-protein association from an energy landscape perspective. First, a coarse-grained model is implemented and its applications are demonstrated via molecular dynamics simulations for several protein complexes. Second, an enhanced search method is used to efficiently sample a broad range of protein conformations. Third, multiple conformations are identified and clustered from simulation data and further projected on a three-dimensional globe specifying protein orientations and interacting energies. Results from several complexes indicate that the crystal-like conformation is favorable on the energy landscape even if the landscape is relatively rugged with metastable conformations. A closer examination on molecular forces shows that the formation of associated protein complexes can be primarily electrostatics-driven, hydrophobics-driven, or a combination of both in stabilizing specific binding interfaces. Taken together, these results suggest that the coarse-grained simulations and analyses provide an alternative toolset to study protein-protein association occurring in functional biomolecular complexes.

Mapping the conformational transition in Src activation by cumulating the information from multiple molecular dynamics trajectories.

The Src-family kinases are allosteric enzymes that play a key role in the regulation of cell growth and proliferation. In response to cellular signals, they undergo large conformational changes to switch between distinct inactive and active states. A computational strategy for characterizing the conformational transition pathway is presented to bridge the inactive and active states of the catalytic domain of Hck. The information from a large number (78) of independent all-atom molecular dynamics trajectories with explicit solvent is combined together to assemble a connectivity map of the conformational transition. Two intermediate states along the activation pathways are identified, and their structural features are characterized. A coarse free-energy landscape is built in terms of the collective motions corresponding to the opening of the activation loop (A-loop) and the rotation of the alphaC helix. This landscape shows that the protein can adopt a multitude of conformations in which the A-loop is partially open, while the alphaC helix remains in the orientation characteristic of the inactive conformation. The complete transition leading to the active conformation requires a concerted movement involving further opening of the A-loop, the relative alignment of N-lobe and C-lobe, and the rotation of the alphaC helix needed to recruit the residues necessary for catalysis in the active site. The analysis leads to a dynamic view of the full-length kinase activation, whereby transitions of the catalytic domain to intermediate configurations with a partially open A-loop are permitted, even while the SH2-SH3 clamp remains fully engaged. These transitions would render Y416 available for the transphosphorylation event that ultimately locks down the active state. The results provide a broad framework for picturing the conformational transitions leading to kinase activation.

Incorporation of D2O-Induced Fluorine Chemical Shift Perturbations into Ensemble-Structure Characterization of the ERalpha Disordered Region.

Structural characterization of intrinsically disordered proteins (IDPs) requires a concerted effort between experiments and computations by accounting for their conformational heterogeneity. Given the diversity of experimental tools providing local and global structural information, constructing an experimental restraint-satisfying structural ensemble remains challenging. Here, we use the disordered N-terminal domain (NTD) of the estrogen receptor alpha (ERalpha) as a model system to combine existing small-angle X-ray scattering (SAXS) and hydroxyl radical protein footprinting (HRPF) data and newly acquired solvent accessibility data via D2O-induced fluorine chemical shifting (DFCS) measurements. A new set of DFCS data for the solvent exposure of a set of 12 amino acid positions were added to complement previously acquired HRPF measurements for the solvent exposure of the other 16 nonoverlapping amino acids, thereby improving the NTD ensemble characterization considerably. We also found that while choosing an initial ensemble of structures generated from a different atomic-level force field or sampling/modeling method can lead to distinct contact maps even when the same sets of experimental measurements were used for ensemble-fitting, comparative analyses from these initial ensembles reveal commonly recurring structural features in their ensemble-averaged contact map. Specifically, nonlocal or long-range transient interactions were found consistently between the N-terminal segments and the central region, sufficient to mediate the conformational ensemble and regulate how the NTD interacts with its coactivator proteins.

Glycine substitution in SH3-SH2 connector of Hck tyrosine kinase causes population shift from assembled to disassembled state.

A combination of small angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations based on a coarse grained model is used to examine the effect of glycine substitutions in the short connector between the SH3 and SH2 domains of Hck, a member of the Src-family kinases. It has been shown previously that the activity of cSrc kinase is upregulated by substitution of 3 residues by glycine in the SH3-SH2 connector. Here, analysis of SAXS data indicates that the population of Hck in the disassembled state increases from 25% in the wild type kinase to 76% in the glycine mutant. This is consistent with the results of free energy perturbation calculations showing that the mutation in the connector shifts the equilibrium from the assembled to the disassembled state. This study supports the notion that the SH3-SH2 connector helps to regulate the activity of tyrosine kinases by shifting the population of the active state of the multidomain protein independent of C-terminal phosphorylation.

Protein Footprinting: Auxiliary Engine to Power the Structural Biology Revolution.

Structural biology is entering an exciting time where many new high-resolution structures of large complexes and membrane proteins are determined regularly. These advances have been driven by over fifteen years of technology advancements, first in macromolecular crystallography, and recently in Cryo-electron microscopy. These structures are allowing detailed questions about functional mechanisms of the structures, and the biology enabled by these structures, to be addressed for the first time. At the same time, mass spectrometry technologies for protein structure analysis, "footprinting" studies, have improved their sensitivity and resolution dramatically and can provide detailed sub-peptide and residue level information for validating structures and interactions or understanding the dynamics of structures in the context of ligand binding or assembly. In this perspective, we review the use of protein footprinting to extend our understanding of macromolecular systems, particularly for systems challenging for analysis by other techniques, such as intrinsically disordered proteins, amyloidogenic proteins, and other proteins/complexes so far recalcitrant to existing methods. We also illustrate how the availability of high-resolution structural information can be a foundation for a suite of hybrid approaches to divine structure-function relationships beyond what individual techniques can deliver.

A rapid coarse residue-based computational method for x-ray solution scattering characterization of protein folds and multiple conformational states of large protein complexes.

We present a coarse residue-based computational method to rapidly compute the solution scattering profile from a protein with dynamical fluctuations. The method is built upon a coarse-grained (CG) representation of the protein. This CG representation takes advantage of the intrinsic low-resolution and CG nature of solution scattering data. It allows rapid scattering determination from a large number of conformations that can be extracted from CG simulations to obtain scattering characterization of protein conformations. The method includes several important elements, effective residue structure factors derived from the Protein Data Bank, explicit treatment of water molecules in the hydration layer at the surface of the protein, and an ensemble average of scattering from a variety of appropriate conformations to account for macromolecular flexibility. This simplified method is calibrated and illustrated to accurately reproduce the experimental scattering curve of Hen egg white lysozyme. We then illustrated the applications of this CG method by computing the solution scattering patterns of several representative protein folds and multiple conformational states. The results suggest that solution scattering data, when combined with the reliable computational method that we developed, show great potential for a better structural description of multidomain complexes in different functional states, and for recognizing structural folds when sequence similarity to a protein of known structure is low.

Atomistic view of the conformational activation of Src kinase using the string method with swarms-of-trajectories.

The inactive-to-active conformational transition of the catalytic domain of human c-Src tyrosine kinase is characterized using the string method with swarms-of-trajectories with all-atom explicit solvent molecular dynamics simulations. The activation process occurs in two main steps in which the activation loop (A-loop) opens first, followed by the rotation of the alphaC helix. The computed potential of mean force energy along the activation pathway displays a local minimum, which allows the identification of an intermediate state. These results show that the string method with swarms-of-trajectories is an effective technique to characterize complex and slow conformational transitions in large biomolecular systems.