Chromatin remodeling complexes like BAF finely regulate transcriptional programs by working in concert with transcription factors. However, evidence is lacking as to whether TFs interact directly with BAF and if so, what the mechanistic and structural principles governing these critical interactions are. Here, we establish direct engagement between a crucial and therapeutically relevant full-length human TF, PU.1 ( SPI1 ), and BAF. Within this 1MDa+ complex, we precisely map the binding site of PU.1 to a YEATS-like domain on BAF60A and elucidate the structure of the PU.1-BAF60A complex. This work reveals that upon binding to BAF, a disordered region within the TF adopts a helical conformation, and that disruption of this functionally critical interface via knockdown abrogates the ability of PU.1 to rescue cell viability. To explore the druggability of TF-BAF protein-protein interactions (PPIs), we conducted a high-throughput screen that identified small molecules capable of disrupting the PU.1-BAF60A PPI by binding to BAF60A. Co-crystal structures reveal distinct compound binding modes that converge on a critical PU.1-BAF60A interaction hotspot. These findings define, for the first time, the structural interface between a human TF and a chromatin remodeling complex and establish a platform that enables the targeting of these interactions, a novel mechanism in cancer therapeutics. ### Competing Interest Statement The authors have declared no competing interest.

Deposits of misfolded tau proteins are leading indicators of cognitive decline in Alzheimer’s disease (AD), and our recent data implicate distinctly misfolded conformers of the tau protein with high seeding potency in rapid progression. We considered prion-like templated propagation of misfolding in neurons as an underlying mechanism and derived sensitive conformational assays to test this concept and identify critical structural drivers. Using novel photochemical hydroxylation monitored with a panel of Europium-labeled monoclonal antibodies, we investigated the structural organization of different microtubule binding domains (MTBDs) in brain-derived tau conformers in AD with different progression rates. We analyzed the impact of structural organization of different MTBDs on seeding potency in vitro and in primary neurons, and on the propagation rate of tau misfolding, compartmentalization, cytotoxicity, and calcium homeostasis in neuronally differentiated SH-SY5Y cells. Within the extensive inter-individual structural variability in all MTBDs and C-terminal tails, the most significant driver of seeding potency and propagation of tau protein misfolding in both in vitro seeding assays and in neuronal cultures was the structural exposure of the fourth MTBD (R4). In contrast, the major driver of calcium influx induced in neurons by the accumulation of misfolded tau was the structural exposure of the R1 domain. The data provide compelling evidence for a major diversity in the structural organization of MTBDs of misfolded AD brain-derived tau protein and implicate the structural exposure of distinct domains in different pathogenetic steps of AD — R4 tau domain in progression rate, and R1 domain in variable synaptic toxicity of misfolded tau, and thus in cognitive decline.

Reactive oxygen species play a crucial role in cellular processes, but their effects on protein structure and function in vivo remain challenging to study. Here, we present an approach using synchrotron-based X-ray footprinting methods to probe protein structure, via quantitative LC-coupled mass spectrometry of methionine oxidation (MSOx) in live E. coli. A label-free proteomic analysis identified 2104 proteins from E. coli, with 465 proteins exhibiting MSOx modifications distributed across multiple cellular compartments. Changes in MSOx modification with increasing X-ray dose revealed a correlation between rates of modification and solvent-accessible surface area in vivo for selected proteins responsive to exposure, providing a direct probe of protein structure and its conformational plasticity in the cell. The approach developed here offers a unique in-cell quantitative readout of methionine oxidation and solvent accessibility through radiolytic hydroxyl radical labeling. With this method, the landscape of methionine oxidation in E. coli can be mapped, providing insights into protein behavior under oxidative stress. It represents a first step in developing radiolysis and E. coli as platforms for in vivo protein structure assessment. The potential applications in drug discovery, protein engineering, and systems biology of protein conformations are considerable.

The BRG-/BRM-associated factor (BAF) chromatin remodeling complex is a central actor in transcription. One mechanism by which BAF affects gene expression is via its various histone mark readers, including double plant homeodomains (DPF), located in the BAF45D subunit. DPF domains recognize lysine acetyl and acylations, including crotonylation, localized at promoters and enhancers. Despite a significant degree of conservation between DPF domains, attempts to crystallize BAF45D with a crotonylated histone 3 peptide (H3K14Cr) were unsuccessful. In addition, recent cryoEM and modeled structures failed to define the Req domain of BAF45D, which is responsible for reading lysine modifications. Thus, the precise mechanism of crotonyl group recognition and binding by BAF45D within the BAF complex remains unclear. We turned to protein footprinting mass spectrometry to map the binding interface between H3K14Cr and BAF45D. This technique is able to demarcate protein-binding interfaces by modifying surface-accessible residues and is not limited by protein size or composition. Experiments performed in the isolated DPF domain of BAF45D (BAF45DDPF)-delineated H3K14Cr peptide binding across the PHD1 and PHD2 pockets. We observed markedly similar effects on the BAF45D subunit when assessing H3K14Cr binding in the purified full BAF complex. The ATPase motor, BRM, also displayed H3K14Cr-protected peptides in two separate domains that were subsequently evaluated in direct binding assays. These data confirm the BAF45D-crotonylamide interaction within its obligate complex and are the first to demonstrate H3K14Cr direct binding to BRM.

In higher eukaryotes, a single DOT1 histone H3 lysine 79 (H3K79) methyltransferase processively produces H3K79me2/me3 through histone H2B mono-ubiquitin interaction, while the kinetoplastid Trypanosoma brucei di-methyltransferase DOT1A and tri-methyltransferase DOT1B efficiently methylate the homologous H3K76 without H2B mono-ubiquitination. Based on structural and biochemical analyses of DOT1A, we identify key residues in the methyltransferase motifs VI and X for efficient ubiquitin-independent H3K76 methylation in kinetoplastids. Substitution of a basic to an acidic residue within motif VI (Gx 6 K ) is essential to stabilize the DOT1A enzyme-substrate complex, while substitution of the motif X sequence VYGE by CAKS renders a rigid active-site loop flexible, implying a distinct mechanism of substrate recognition. We further reveal distinct methylation kinetics and substrate preferences of DOT1A (H3K76me0) and DOT1B (DOT1A products H3K76me1/me2) in vitro, determined by a Ser and Ala residue within motif IV, respectively, enabling DOT1A and DOT1B to mediate efficient H3K76 tri-methylation non-processively but cooperatively, and suggesting why kinetoplastids have evolved two DOT1 enzymes.

Hydroxyl radical-based protein footprinting (HRPF) coupled with mass spectrometry is a valuable medium-resolution technique in structural biology, facilitating the assessment of protein structure and molecular-level interactions in solution conditions. In HRPF with X-rays (XFP), hydroxyl radicals generated by water radiolysis covalently label multiple amino acid (AA) side chains. However, HRPF technologies face challenges in achieving their full potential due to the broad (>103) dynamic range of AA reactivity with •OH and difficulty in detecting slightly modified residues, most notably in peptides with highly reactive residues like methionine, or where all residues have low •OH reactivities. To overcome this limitation, we developed a multiplex labeling chemistry that utilizes both CF3 radicals (•CF3) produced from a trifluoromethylation (TFM) reagent and OH radicals (•OH), under controlled and optimized radiolysis doses generated by X-rays. We optimized the dual •CF3/•OH chemistry using model peptides and proteins, thereby extending the existing •OH labeling platform to incorporate simultaneous •CF3 labeling. We labeled >50% of the protein sequence and >80% of protein solvent-accessible AAs via multiplex TFM labeling resulting in high-resolution footprinting, primarily by enhancing the labeling of AAs with low •OH reactivity via the •CF3 channel, while labeling moderate and highly •OH-reactive AAs in both •CF3 and •OH channels. Moreover, the low reactivity of methionine with •CF3 enabled the detection and quantification of additional AAs labeled by •CF3 within methionine-containing peptides. Finally, we found that the solvent accessibility of protein AAs directly correlated with •CF3 labeling, demonstrating that multiplex TFM labeling enables a high-resolution assessment of molecular interactions for enhanced HRPF.

Hydroxyl radical based protein footprinting (HRPF) coupled with mass spectrometry is a valuable medium-resolution technique in structural biology, facilitating the assessment of protein structure and molecular-level interactions in a wide range of solution conditions. In hydroxyl radical protein footprinting with X-rays (XFP), hydroxyl radicals (•OH) generated by water radiolysis covalently label multiple amino acid (AA) side chains simultaneously. However, HRPF technologies faces challenges in achieving their full potential due to the broad (>103) dynamic range of AA’s reactivity to •OH and the difficulty to detect slightly modified residues, particularly in peptides with highly reactive residues like methionine-containing peptides and in peptides containing all low reactive residues. To overcome this limitation, we developed a synchrotron-based multiplex labeling chemistry that utilizes CF3 radicals (•CF3) produced from a trifluoromethylation (TFM) reagent under controlled and optimized •OH doses generated by X-rays. We optimized the dual •CF3/•OH chemistry in this TFM labeling approach using six model peptides and lysozyme, there-by extending the existing •OH labeling platform with simultaneous •CF3 labeling. This optimization led to a two-fold increase in labeled AAs in multiplex TFM labeling, primarily by labeling to a greater degree AAs with low •OH reactivity via the •CF3 channel, while moderate and highly •OH reactive AAs were labeled in both •CF3 and •OH channels. Importantly, the low reactivity of methionine to •CF3 enabled the detection and quantification of additional AAs labeled by •CF3 across methionine-containing pep-tides. Consistent with observations in model peptides and protein, we observed a balanced dual •CF3/•OH chemistry and more uniform labeling of residues in both •CF3/•OH channels optimizing protein footprinting. Furthermore, the solvent accessibility of lysozyme residues directly correlated with •CF3 labeling demonstrating that multiplex labeling enables a high-resolution assessment of molecular interactions for enhanced HRPF.

Hydroxyl radical protein footprinting (HRPF) using synchrotron X-ray radiation and mass spectrometry is a well-validated structural biology method that is providing critical insights into macromolecular dynamics. Numerous alternative sources for HRPF such as laser photolysis and plasma irradiation complement synchrotron-based HRPF. A recently developed commercially available instrument based on flash lamp photolysis, the Fox® system, enables access to laboratory benchtop HRPF. Here, we evaluate the feasibility of standardizing HRPF experiments in-house with a benchtop Fox® instrument as a precursor to synchrotron-based X-ray footprinting at the NSLS-II XFP beamline. Using lactate oxidase enzyme (LOx) as a model system, we carried out hydroxyl radical (•OH) labeling experiments using both instruments, followed by nanoLC-MS/MS bottom-up peptide mass mapping. Experiments were performed with high glucose concentrations to mimic highly scavenging conditions in biological buffers and human clinical samples, where less •OH are available for reaction with the biomolecule(s) of interest. The performance of the Fox® and XFP HRPF methods was compared, and we found that tuning •OH dosage enabled an optimum labeling coverage for both setups under physiologically relevant highly scavenging conditions. Our study demonstrates the complementarity of Fox® and XFP labeling approaches, showing that benchtop instruments such as Fox® photolysis system can increase throughput and accessibility of HRPF technology.

Hydroxyl Radical Protein Footprinting (HRPF) monitors macromolecular structure and dynamics by utilizing hydroxyl radicals to probe the solvent-accessible side chains of proteins. Hydroxyl radicals form irreversible covalent bonds with protein side chains based on their solvent accessibility and intrinsic reactivity. Following labeling, bottom-up proteomics which involves protease digestion and liquid chromatography (LC-MS/MS) coupled with mass spectrometry, is routinely employed to detect and quantify the modified protein side chains. The HRPF technique has been a breakthrough in the field of structural biology, enabling the assessment of structures and interrelationships between proteins, protein-drug complexes or such macromolecular mixtures. It is now being extended to complex applications such as in-cell and in-vivo studies. This perspective focuses on detailing aspects of peptide separations technology in HRPF, with a particular emphasis on chromatography. The discussion further encompasses the HRPF methodology, its current limitations, recent developments, and proposed ideas for future developments for selected research fields.

Neuropeptide Y (NPY) signals important stress and mood phenotypes in the brain. Peptide biological recognition elements (BREs) based on phage display can be used in hormone biosensors for stress and weight management. We employed protein footprinting mass spectrometry (PF-MS) to probe NPY structure and provide readout of NPY residues responsive to BRE binding. Three complementary PF-MS approaches using hydroxyl radical, trifluoromethyl radical, and carboxyl chemistries were applied independently.

<p>Antibody information and primer sequences.</p>

15-prostaglandin dehydrogenase (15-PGDH) is a negative regulator of tissue stem cells that acts via enzymatic activity of oxidizing and degrading PGE2, and related eicosanoids, that support stem cells during tissue repair. Indeed, inhibiting 15-PGDH markedly accelerates tissue repair in multiple organs. Here we have used cryo-electron microscopy to solve the solution structure of native 15-PGDH and of 15-PGDH individually complexed with two distinct chemical inhibitors. These structures identify key 15-PGDH residues that mediate binding to both classes of inhibitors. Moreover, we identify a dynamic 15-PGDH lid domain that closes around the inhibitors, and that is likely fundamental to the physiologic 15-PGDH enzymatic mechanism. We furthermore identify two key residues, F185 and Y217, that act as hinges to regulate lid closing, and which both inhibitors exploit to capture the lid in the closed conformation, thus explaining their sub-nanomolar binding affinities. These findings provide the basis for further development of 15-PGDH targeted drugs as therapeutics for regenerative medicine.

Hydroxyl radical protein footprinting (HRPF) using synchrotron radiation is a well-validated method to assess protein structure in the native solution state. In this method, X-ray radiolysis of water generates hydroxyl radicals that can react with solvent accessible side chains of proteins, with mass spectrometry used to detect the resulting labeled products. An ideal footprinting dose provides sufficient labeling to measure the structure but not so much as to influence the results. The optimization of hydroxyl radical dose is typically performed using an indirect Alexa488 fluorescence assay sensitive to hydroxyl radical concentration, but full evaluation of the experiment's outcome relies upon bottom-up liquid chromatography mass spectrometry (LC-MS) measurements to directly determine sites and extent of oxidative labeling at the peptide and protein level. A direct evaluation of the extent of labeling to provide direct and absolute measurements of dose and "safe" dose ranges in terms of, for example, average numbers of labels per protein, would provide immediate feedback on experimental outcomes prior to embarking on detailed LC-MS analyses. To this end, we describe an approach to integrate intact MS screening of labeled samples immediately following exposure, along with metrics to quantify the extent of observed labeling from the intact mass spectra. Intact MS results on the model protein lysozyme were evaluated in the context of Alexa488 assay results and a bottom-up LC-MS analysis of the same samples. This approach provides a basis for placing delivered hydroxyl radical dose metrics on firmer technical grounds for synchrotron X-ray footprinting of proteins, with explicit parameters to increase the likelihood of a productive experimental outcome. Further, the method directs approaches to provide absolute and direct dosimetry for all types of labeling for protein footprinting.

Supplemental Figure 1. Comparison of the anti-proliferative activity of SMAP across a panel of cell lines. Supplemental Figure 2. The chemical structure of SMAPs utilized in the studies detailed in the manuscript. Supplemental Figure 3. The effects of SMAPs on cell proliferation and cell survival. Supplemental Figure 4. Control studies for co-immunoprecipitation experiments. Supplemental Figure 5. Activity of SMAPs in vivo with the correlation of anti-tumor activity with SMAP-2 exposure in serum of treated mice.

Table S1. KSEA Analysis and the Mean FC Method in LNCaP treated with SMAP. Table S2. ANOVA with multiple comparisons and Dunnett''s post-hoc test for SMAP treated LNCaP colony formation assay. Table S3. ANOVA with multiple comparisons and Dunnett''s post-hoc test for SMAP treated 22Rv1 colony formation assay. Table S4. ANOVA with multiple comparisons and Dunnett''s post-hoc test for Annexin V staining of LNCap and 22Rv1 cells. Table S5. PPI of the phosphoproteomics dataset. Table S6. Kinase Substrate Database for LNCaP treated with SMAP. Table S7. ANOVA with multiple comparisons and Tukey''s post-hoc test for SMAP treatment in LNCaP cells phosphorylated to total AR ratio protein. Table S8. ANOVA for qRT-PCR RNA for AR targets in LNCaP and 22Rv1 cells in presence of SMAPs. Table S9. ANOVA with multiple comparisons and Dunnett''s post-hoc test for LNCaP and 22Rv1 cells treated with SMAP, bortezomib, and combination. Table S10. ANOVA with multiple comparisons and Tukey''s post-hoc test for relative AR protein expression in LNCaP- Small T stably expressing lines treated with SMAP. Table S11. ANOVA with multiple comparisons and Tukey''s post-hoc test for relative AR protein expression in LNCaP cells in FBS or CSS in presence or absence of R1881. Table S12. ANOVA for qRT-PCR RNA AR targets statistical analysis for LNCaP cells in FBS or CSS in presence or absence of R1881. Table S13. ANOVA with multiple comparisons and Dunnett''s post-hoc test for LnCaP/AR xenograft treatment study: vehicle control, SMAP 400mg/kg, SMAP 100mg/kg and MDV3100 100mg/kg. Table S14. ANOVA with multiple comparisons and Dunnett''s post-hoc test for castrated LNCaP/AR tumor volumes for individual treatments groups: Vehicle control, SMAP-2 100mg/kg and SMAP-2 30mg/kg. Table S15. ANOVA with multiple comparisons and Dunnett''s post-hoc test for TUNEL positive staining quantification from castrated LNCaP/AR tumors from individual treatments groups: Vehicle control, SMAP-2 30mg/kg and SMAP-2 100mg/kg. Table S16. ANOVA with multiple comparisons and Dunnett''s post-hoc test for PCNA positive staining quantification from castrated LNCaP/AR for individual treatments groups: Vehicle control, SMAP-2 100mg/kg and SMAP-2 30mg/kg.

<p>STR DNA Profiling for Cell Line Authentication</p>

Protein footprinting with mass spectrometry is an established structural biology technique for mapping solvent accessibility and assessing molecular-level interactions of proteins. In hydroxyl radical protein footprinting (HRPF), hydroxyl (OH) radicals generated by water radiolysis or other methods covalently label protein side chains. Because of the wide dynamic range of OH reactivity, not all side chains are easily detected in a single experiment. Novel reagent development and the use of radical chain reactions for labeling, including trifluoromethyl radicals, is a potential approach to normalize the labeling across a diverse set of residues. HRPF in the presence of a trifluoromethylation reagent under the right conditions could provide a "one-pot" reaction for multiplex labeling of protein side chains. Toward this goal, we have systematically evaluated amino acid labeling with the recently investigated Langlois' reagent (LR) activated by X-ray-mediated water radiolysis, followed by three different mass spectrometry methods. We compared the reactivity of CF3 and OH radical labeling for all 20 protein side chains in a competition-free environment. We found that all 20 amino acids exhibited CF3 or OH labeling in LR. Our investigations provide the evidence and knowledge set to perfect hydroxyl radical-activated trifluoromethyl chemistry as "one-pot" reaction for multiplex labeling of protein side chains to achieve higher resolution in HRPF.

Endothelial hemoglobin (Hb)α regulates endothelial nitric oxide synthase (eNOS) biochemistry. We hypothesized that Hb could also be expressed and biochemically active in the ciliated human airway epithelium. Primary human airway epithelial cells, cultured at air–liquid interface (ALI), were obtained by clinical airway brushings or from explanted lungs. Human airway Hb mRNA data were from publically available databases; or from RT-PCR. Hb proteins were identified by immunoprecipitation, immunoblot, immunohistochemistry, immunofluorescence and liquid chromatography- mass spectrometry. Viral vectors were used to alter Hbβ expression. Heme and nitrogen oxides were measured colorimetrically. Hb mRNA was expressed in human ciliated epithelial cells. Heme proteins (Hbα, β, and δ) were detected in ALI cultures by several methods. Higher levels of airway epithelial Hbβ gene expression were associated with lower FEV 1 in asthma. Both Hbβ knockdown and overexpression affected cell morphology. Hbβ and eNOS were apically colocalized. Binding heme with CO decreased extracellular accumulation of nitrogen oxides. Human airway epithelial cells express Hb. Higher levels of Hbβ gene expression were associated with airflow obstruction. Hbβ and eNOS were colocalized in ciliated cells, and heme affected oxidation of the NOS product. Epithelial Hb expression may be relevant to human airways diseases.