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In the non-homologous end-joining (NHEJ) of a DNA double-strand break, DNA ends are bound and protected by DNA-PK, which synapses across the break to tether the broken ends and initiate repair. There is little clarity surrounding the nature of the synaptic complex and the mechanism governing the transition to repair. We report an integrative structure of the synaptic complex at a precision of 13.5 Å, revealing a symmetric head-to-head arrangement with a large offset in the DNA ends and an extensive end-protection mechanism involving a previously uncharacterized plug domain. Hydrogen/deuterium exchange mass spectrometry identifies an allosteric pathway connecting DNA end-binding with the kinase domain that places DNA-PK under tension in the kinase-active state. We present a model for the transition from end-protection to repair, where the synaptic complex supports hierarchical processing of the ends and scaffold assembly, requiring displacement of the catalytic subunit and tension release through kinase activity.

Structures of biomolecular systems are increasingly computed by integrative modeling. In this approach, a structural model is constructed by combining information from multiple sources, including varied experimental methods and prior models. In 2019, a Workshop was held as a Biophysical Society Satellite Meeting to assess progress and discuss further requirements for archiving integrative structures. The primary goal of the Workshop was to build consensus for addressing the challenges involved in creating common data standards, building methods for federated data exchange, and developing mechanisms for validating integrative structures. The summary of the Workshop and the recommendations that emerged are presented here.

X-ray crystallography and electron microscopy maps resolved to 3-8 Å are generally sufficient for tracing the path of the polypeptide chain in space, while often insufficient for unambiguously registering the sequence on the path (i.e., threading). Frequently, however, additional information is available from other biophysical experiments, physical principles, statistical analyses, and other prior models. Here, we formulate an integrative approach for sequence assignment to a partial backbone model as an optimization problem, which requires three main components: the representation of the system, the scoring function, and the optimization method. The method is implemented in the open source Integrative Modeling Platform (IMP) (https://integrativemodeling.org), allowing a number of different terms in the scoring function. We apply this method to localizing the sequence assignment within a 199-residue disordered region of three structured and sequence unassigned helices in the DNA-PKcs crystallographic structure, using chemical crosslinks, hydrogen deuterium exchange, and sequence connectivity. The resulting ensemble of threading models provides two major solutions, one of which suggests that the crucial ABCDE cluster of phosphorylation sites cannot undergo intra-molecular autophosphorylation without a conformational rearrangement. The ensemble of solutions embodies the most accurate and precise sequence threading given the available information.

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. Our study therefore builds the framework for establishing best practice guidelines to conduct cross-linking experiments, perform data analysis, and define reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.

Non-homologous end joining (NHEJ) repairs DNA double strand breaks in non-cycling eukaryotic cells. NHEJ relies on polynucleotide kinase/phosphatase (PNKP), which generates 5΄-phosphate/3΄-hydroxyl DNA termini that are critical for ligation by the NHEJ DNA ligase, LigIV. PNKP and LigIV require the NHEJ scaffolding protein, XRCC4. The PNKP FHA domain binds to the CK2-phosphorylated XRCC4 C-terminal tail, while LigIV uses its tandem BRCT repeats to bind the XRCC4 coiled-coil. Yet, the assembled PNKP-XRCC4-LigIV complex remains uncharacterized. Here, we report purification and characterization of a recombinant PNKP-XRCC4-LigIV complex. We show that the stable binding of PNKP in this complex requires XRCC4 phosphorylation and that only one PNKP protomer binds per XRCC4 dimer. Small angle X-ray scattering (SAXS) reveals a flexible multi-state complex that suggests that both the PNKP FHA and catalytic domains contact the XRCC4 coiled-coil and LigIV BRCT repeats. Hydrogen-deuterium exchange indicates protection of a surface on the PNKP phosphatase domain that may contact XRCC4-LigIV. A mutation on this surface (E326K) causes the hereditary neuro-developmental disorder, MCSZ. This mutation impairs PNKP recruitment to damaged DNA in human cells and provides a possible disease mechanism. Together, this work unveils multipoint contacts between PNKP and XRCC4-LigIV that regulate PNKP recruitment and activity within NHEJ.

Amide hydrogen/deuterium exchange (H/DX) measurements by mass spectrometry provide a powerful tool for probing the structure and dynamics of proteins. In order to extend such measurements to complex multiprotein systems, new methods for delivering higher sensitivity and sequence coverage are required. In this study, we investigated the utility of tandem mass spectrometry (MS/MS) for providing an alternative to the conventional MS mode of operation applied to bottom-up H/DX experiments. Specifically, we aimed to determine whether differential deuteration measurements of collisionally induced dissociation (CID)-generated product ions can serve as effective surrogates for their corresponding intact peptide, thus providing an additional dimension for analysis. Replicate deuterium measurements of calmodulin (in its apo and holo forms) were obtained from peptic digests in both the MS and MS/MS domains and studied as a function of % deuteration, fragment ion selection, and contaminant level. We show that successful acquisition of MS/MS data for deuterated peptides requires controlled expansion of the isotopic envelope by limiting the range of deuterium label applied in the exchange-in reaction ([less than or equal to] 50%) and that automation of ion selection via data-dependent acquisition is ultimately dependent upon peak detection algorithms. Upon full transmission of the isotopic envelope, fragment data demonstrate that all ions, with the exception of neutral loss fragment ions, return deuteration ratios reflecting the apo/holo transition that ate in general agreement with values obtained from the corresponding precursor ions. The agreement is limited primarily by the ion statistics for each fragment, as the base peak in the MS/MS spectra provided the best correlation regardless of its m/z. We highlight that the freedom to select the base peak as a surrogate for the precursor ion derives from extensive H/D scrambling inducible under conventional CID conditions. When spectral interference prohibits conventional H/DX-MS measurements, we further show that the surrogate approach recovers accurate and precise per-peptide deuteration levels. Thus, a generalized strategy is presented in which CID-based automated H/DX-MS/MS acquisition can be used to extend measurements to complex protein systems, exceeding the peptide capacity of conventional H/DX-MS alone.

Hydrogen/deuterium exchange (H/DX) mass spectrometry (MS) is increasingly applied to problems in protein structural biology in order to map protein dynamics and identify sites of interactions. In theory, an MS-based readout of deuterium label incorporation can overcome the concentration, size, purity, and complexity limitations inherent in NMR-based measurements of exchange; however, in practice, these advantages are reduced due to spectral interference and dilution of the sample in deuterium oxide ([D.sub.2]O). In this study, we demonstrate that popular H/DX labeling strategies aggravate the interference problem and that significant recovery of spectral capacity may be achieved with a 'minimalist' strategy. Simulations of peptide deuteration justify large reductions in the level of [D.sub.2]O used in labeling experiments, as well as reduced numbers of peaks used in making relative labeling measurements between biochemical states of a protein. To demonstrate the utility of a minimalist approach, calmodulin was interrogated in a bottom-up H/DX-MS workflow, and sensitivity to the addition of [Ca.sup.2+] as a structural perturbation was measured as a function of % [D.sub.2]O and the number of peaks used in quantitating deuteration level. It is shown that high sensitivity to change is preserved with deuteration levels of 5.0 [+ or -] 1.1 (apt-CaM) and 1.4 [+ or -] 1.3% (holt-CaM) using 10% [D.sub.2]O in the labeling experiment. Further, only two peaks of a peptide peak distribution are needed to sensitively monitor changes in protein structure, dynamics, or both.

Proteins visualized by 2,2,2-trichloroethanol (TCE) on two-dimensional electrophoresis gels are efficiently identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and MS/ MS. In a previous study, a method was developed that placed TCE in the polyacrylamide gel so that protein bands can be visualized without staining in less than 5 min. A visible fluorophore is generated by reaction of TCE with tryptophan that allows for protein visualization. In this study, MALDI-TOF MS and LC--MS/MS are used to identify randomly selected Escherichia coli proteins. The identification of TCE visualized proteins is compared to the identification of Coomassie brilliant blue (CBB) stained proteins from two-dimensional gel electrophoresis of E. coli proteins. This study demonstrated that TCE visualized proteins are compatible with protein identification by MALDI-TOF peptide mass fingerprinting. For 10 randomly selected spots, TCE visualization lead to statistically significant identification of 5 proteins and CBB visualization lead to identification of 6 proteins. TCE visualized proteins are also shown to be well suited for protein identification using LC--MS/MS. In 16 spots selected for MS/MS analysis, TCE samples lead to the identification of 79 peptides; while CBB samples lead to the identification of 65 peptides. TCE samples also supported the identification of more proteins. The low stoichiometry of labeling of tryptophan residues does not require inclusion of this modification for database searches. In addition to being a rapid visualization technique compatible with MS, TCE visualization utilizes rapid washing conditions for sample preparation of proteins spots excised from polyacrylamide gels.

Measuring the statistical distribution of deuterium incorporated into enzymatically derived peptide fragments provides a valuable dimension to hydrogen/deuterium exchange mass spectrometry data. In this paper, we will discuss our improvement to the linear least-squares method for determining this distribution, through the addition of 'zeroes' to the end of the deuterated isotopic envelope, to partially compensate for data truncation due to finite instrumental signal-to-noise ratios. The value of the distribution is demonstrated in a simple experimental example, where the linearity between average deuteration and percent [D.sub.2]O used to label test peptides hides a more complex relationship between the site-labeling probability. and the total number of sites. This method offers the opportunity to resolve cases where a single peptide experiences distinct, independent biochemical states with each bearing a unique average deuteration; this can occur when a protein is modified to substoichiometric levels. From the experimentally determined distribution of a heterogeneously deuterated peptide, it was possible to extract the average deuteration of each component of the mixture.

This work presents new frontal affinity chromatography (FAC) methodologies for high-throughput screening of compound libraries, designed to increase screening rates and improve sensitivity and ruggedness in performance. A FAC column constructed around the enzyme N-acetylglucosaminyltransferase V (GnT-V) was implemented in the identification of potential enzyme inhibitors from two libraries of trisaccharides. Effluent from the FAC column was fractionated, sequentially processed via LC/MS, and referenced to a similar analysis through a control FAC column lacking the enzyme. The resulting multidimensional data sets were compared across corresponding sample and control fractions to identify binders, in a semiautomated approach. A strong binder in the protonated form at m/z 795 was identified from the first library of 81 compounds, exhibiting an estimated [K.sub.d] value of 0.3 [micro]M. Other binders yielded [K.sub.d] values ranging from 0.35 to 3.35 [micro]M. To demonstrate the improvement in performance of this FAC-LC/MS approach over the conventional online FAC/MS approach, 15 compounds from this library were blended with a second library of 1000 synthetic trisaccharides and screened against GnT-V. All ligands in the 15-compound set were identified in this larger screen, and no ligands of greater affinity than compound 1 were found. Our results show that FAC-LC/MS is a reliable method for screening large compound libraries directly and useful for large-scale ligand discovery initiatives.

Typical liquid- or gel-based protein separations require enzymatic digestion as an important first step in generating protein identifications. Traditional protocols involve long-term proteolytic digestion of the separated protein, often leading to sample loss and reduced sensitivity. Previously, we presented a rapid method of proteolytic digestion that showed excellent digestion of resistant and low concentrations of protein without requiring reduction and alkylation. Here, we demonstrate on-line, real-time tryptic digestion in conjunction with reversed-phase protein separation. The studies were aimed at optimizing pH and ionic strength and the size of the digestion element, to produce maximal protein digestion with minimal effects on chromatographic integrity. Upon establishing optimal conditions, the digestion element was attached downstream from a capillary C4 reversed-phase column. A four-protein mixture was processed through the combined system, and the resulting peptides were analyzed on-line by electrospray mass spectrometry. Extracted ion chromatograms for protein chromatography based on peptide elution were generated. These were shown to emulate ion chromatograms produced in a subsequent run without the digestion element, based on protein elution. The methodology will enable rapid and sensitive analysis of liquid-based protein separations using the power of bottom-up proteomics methodologies.