New opportunities in integrative structural modeling.

Integrative structural modeling enables structure determination of macromolecules and their complexes by integrating data from multiple sources. It has been successfully used to characterize macromolecular structures when a single structural biology technique was insufficient. Recent developments in cellular structural biology, including in-cell cryo-electron tomography and artificial intelligence-based structure prediction, have created new opportunities for integrative structural modeling. Here, we will review these opportunities along with the latest developments in integrative modeling methods and their applications. We also highlight open challenges and directions for further development.

IMProv: A Resource for Cross-link-Driven Structure Modeling that Accommodates Protein Dynamics.

Proteomics methodology has expanded to include protein structural analysis, primarily through cross-linking mass spectrometry (XL-MS) and hydrogen-deuterium exchange mass spectrometry (HX-MS). However, while the structural proteomics community has effective tools for primary data analysis, there is a need for structure modeling pipelines that are accessible to the proteomics specialist. Integrative structural biology requires the aggregation of multiple distinct types of data to generate models that satisfy all inputs. Here, we describe IMProv, an app in the Mass Spec Studio that combines XL-MS data with other structural data, such as cryo-EM densities and crystallographic structures, for integrative structure modeling on high-performance computing platforms. The resource provides an easily deployed bundle that includes the open-source Integrative Modeling Platform program (IMP) and its dependencies. IMProv also provides functionality to adjust cross-link distance restraints according to the underlying dynamics of cross-linked sites, as characterized by HX-MS. A dynamics-driven conditioning of restraint values can improve structure modeling precision, as illustrated by an integrative structure of the five-membered Polycomb Repressive Complex 2. IMProv is extensible to additional types of data.

Structure of the mycobacterial ESX-5 type VII secretion system pore complex.

The ESX-5 type VII secretion system is a membrane-spanning protein complex key to the virulence of mycobacterial pathogens. However, the overall architecture of the fully assembled translocation machinery and the composition of the central secretion pore have remained unknown. Here, we present the high-resolution structure of the 2.1-megadalton ESX-5 core complex. Our structure captured a dynamic, secretion-competent conformation of the pore within a well-defined transmembrane section, sandwiched between two flexible protein layers at the cytosolic entrance and the periplasmic exit. We propose that this flexibility endows the ESX-5 machinery with large conformational plasticity required to accommodate targeted protein secretion. Compared to known secretion systems, a highly dynamic state of the pore may represent a fundamental principle of bacterial secretion machineries.

The substrate specificity of the human TRAPPII complex's Rab-guanine nucleotide exchange factor activity.

The TRAnsport Protein Particle (TRAPP) complexes act as Guanine nucleotide exchange factors (GEFs) for Rab GTPases, which are master regulators of membrane trafficking in eukaryotic cells. In metazoans, there are two large multi-protein TRAPP complexes: TRAPPII and TRAPPIII, with the TRAPPII complex able to activate both Rab1 and Rab11. Here we present detailed biochemical characterisation of Rab-GEF specificity of the human TRAPPII complex, and molecular insight into Rab binding. GEF assays of the TRAPPII complex against a panel of 20 different Rab GTPases revealed GEF activity on Rab43 and Rab19. Electron microscopy and chemical cross-linking revealed the architecture of mammalian TRAPPII. Hydrogen deuterium exchange MS showed that Rab1, Rab11 and Rab43 share a conserved binding interface. Clinical mutations in Rab11, and phosphomimics of Rab43, showed decreased TRAPPII GEF mediated exchange. Finally, we designed a Rab11 mutation that maintained TRAPPII-mediated GEF activity while decreasing activity of the Rab11-GEF SH3BP5, providing a tool to dissect Rab11 signalling. Overall, our results provide insight into the GTPase specificity of TRAPPII, and how clinical mutations disrupt this regulation.

Harmonizing structural mass spectrometry analyses in the mass spec studio.

Structural Mass Spectrometry (SMS) provides a comprehensive toolbox for the analysis of protein structure and function. It offers multiple sources of structural information that are increasingly useful for integrative structural modeling of complex protein systems. As MS-based structural workflows scale to larger systems, consistent and coherent data interpretation resources are needed to better support modeling. Unlike the proteomics community, practitioners of SMS lack adequate computational tools. Here, we review new developments in the Mass Spec Studio: an expandable ecosystem of workflows for the analysis of complementary SMS techniques with linkages to modeling. Current functionality in the Studio (version 2) supports three major SMS workflows (crosslinking, hydrogen/deuterium exchange and covalent labelling) and two pipelines for structural modeling, with a special focus on data integration. The Mass Spec Studio is an architecture focused on rapid and robust extension of functionality by a community of developers. SIGNIFICANCE: This review surveys the new data analysis capabilities within the Mass Spec Studio, a rich framework for rapid software development specifically targeting the community of structural proteomics and structural mass spectrometry. Updates to crosslinking, hydrogen/deuterium-exchange and covalent labeling apps are provided as well as a utility for translating such analyses into restraints that support integrative structural modeling. These new capabilities, together with the underlying design tools and content, provide the community with a wealth of resources to tackle complex structural problem and design new approaches to data analysis.

Correlation between Labeling Yield and Surface Accessibility in Covalent Labeling Mass Spectrometry.

The functional properties of a protein are strongly influenced by its topography, or the solvent-facing contour map of its surface. Together with crosslinking, covalent labeling mass spectrometry (CL-MS) has the potential to contribute topographical data through the measurement of surface accessibility. However, recent efforts to correlate measures of surface accessibility with labeling yield have been met with mixed success. Most applications of CL-MS involve differential analysis of protein interactions (i.e., footprinting experiments) where such inconsistencies have limited effect. Extending CL-MS into structural analysis requires an improved evaluation of the relationship between labeling and surface exposure. In this study, we applied recently developed diazirine reagents to obtain deep coverage of the large motor domain of Eg5 (a mitotic kinesin), and together with computational methods we correlated labeling yields with accessibility data in a number of ways. We observe that correlations can indeed be seen at a local structural level, but these correlations do not extend across the structure. The lack of correlation arises from the influence of protein dynamics and chemical composition on reagent partitioning and, thus, also on labeling yield. We conclude that our use of CL-MS data should be considered in light of "chemical accessibility" rather than "solvent accessibility" and suggest that CL-MS data would be a useful tool in the fundamental study of protein-solute interactions.

Quantitative Analysis of Protein Covalent Labeling Mass Spectrometry Data in the Mass Spec Studio.

Covalent labeling with mass spectrometry (CL-MS) provides a direct measure of the chemical and structural features of proteins with the potential for resolution at the amino-acid level. Unfortunately, most applications of CL-MS are limited to narrowly defined differential analyses, where small numbers of residues are compared between two or more protein states. Extending the utility of high-resolution CL-MS for structure-based applications requires more robust computational routines and the development of methodology capable of reporting of labeling yield accurately. Here, we provide a substantial improvement in the analysis of CL-MS data with the development of an extended plug-in built within the Mass Spec Studio development framework (MSS-CLEAN). All elements of data analysis-from database search to site-resolved and normalized labeling output-are accommodated, as illustrated through the nonselective labeling of the human kinesin Eg5 with photoconverted 3,3'-azibutan-1-ol. In developing the new features within the CL-MS plug-in, we identified additional complexities associated with the application of CL reagents, arising primarily from digestion-induced bias in yield measurements and ambiguities in site localization. A strategy is presented involving the use of redundant site labeling data from overlapping peptides, the imputation of missing data, and a normalization routine to determine relative protection factors. These elements together provide for a robust structural interpretation of CL-MS/MS data while minimizing the over-reporting of labeling site resolution. Finally, to minimize bias, we recommend that digestion strategies for the generation of useful overlapping peptides involve the application of complementary enzymes that drive digestion to completion.

Photo-Cross-Linking Mass Spectrometry and Integrative Modeling Enables Rapid Screening of Antigen Interactions Involving Bacterial Transferrin Receptors.

Structure-based approaches to the delineation of immunogens for vaccine development have a throughput requirement that is difficult to meet in practice with conventional methods of structure determination. Here we present a strategy for rapid and accurate structure generation in support of antigen engineering programs. The approach is developed around the modeling of interactions between host transferrin (Tf) and the bacterial vaccine target transferrin binding protein B (TbpB) from Gram-negative pathogens such as Neisseria meningitidis. Using an approach based solely on cross-linking mass spectrometry (XL-MS) data, monomeric structural models, and the Integrative Modeling Platform (IMP), we demonstrate that converged representations of the Tf:TbpB interactions can be returned that accurately reflect the binding interface and the relative orientation of the monomeric units, with the capacity to scale to the analysis of interactions from any number of additional strains. We show that a key element to accurate modeling involves the application of hetero-bifunctional cross-linkers incorporating fast-acting photoactivatable diazirines coupled with conventional amine-targeting N-hydroxysuccinimide esters, and we demonstrate that conventional homo-bifunctional reagents used in cross-linking kinetically trap dynamic states in the ensemble. Therefore, the application of both classes of cross-linker provides an opportunity to empirically detect protein dynamics during integrative structural modeling.

Simultaneous Proteoform Analysis of Histones H3 and H4 with a Simplified Middle-Down Proteomics Method.

Dynamic post-translational modifications of histones regulate transcriptional gene expression in eukaryotes. Unique combinations of modifications, almost exclusively displayed at the flexible N-terminal tails on histones, create distributions of proteoforms that need to be characterized in order to understand the complexity of gene regulation and how aberrant modification patterns influence disease. Although mass spectrometry is a preferred method for the analysis of histone modifications, information is lost when using conventional trypsin-based histone methods. Newer "middle-down" protocols may retain a greater fraction of the full proteoform distribution. We describe a strategy for the simultaneous characterization of histones H3 and H4 with near-complete retention of proteoform distributions, using a conventional proteomics liquid chromatography-tandem mass spectrometry (LC-MS/MS) configuration. The selective prolyl endoprotease neprosin generates convenient peptide lengths for retention and dispersion of modified H3 and H4 peptides on reversed-phase chromatography, offering an alternative to the hydrophilic interaction liquid chromatography typically used in middle-down methods. No chemical derivatizations are required, presenting a significant advantage over the trypsin-based protocol. Over 200 proteoforms can be readily profiled in a single analysis of histones from HeLa S3 cells. An in-gel digestion protocol provides additional options for effective histone analysis.

Amino Acid Insertion Frequencies Arising from Photoproducts Generated Using Aliphatic Diazirines.

Mapping proteins with chemical reagents and mass spectrometry can generate a measure of accessible surface area, which in turn can be used to support the modeling and refinement of protein structures. Photolytically generated carbenes are a promising class of reagent for this purpose. Substituent effects appear to influence surface mapping properties, allowing for a useful measure of design control. However, to use carbene labeling data in a quantitative manner for modeling activities, we require a better understanding of their inherent amino acid reactivity, so that incorporation data can be normalized. The current study presents an analysis of the amino acid insertion frequency of aliphatic carbenes generated by the photolysis of three different diazirines: 3,3'-azibutyl-1-ammonium, 3,3'-azibutan-1-ol, and 4,4'-azipentan-1-oate. Leveraging an improved photolysis system for single-shot labeling of sub-microliter frozen samples, we used EThCD to localize insertion products in a large population of labeled peptides. Counting statistics were drawn from data-dependent LC-MS2 experiments and used to estimate the frequencies of insertion as a function of amino acid. We observed labeling of all 20 amino acids over a remarkably narrow range of insertion frequencies. However, the nature of the substituent could influence relative insertion frequencies, within a general preference for larger polar amino acids. We confirm a large (6-fold) increase in labeling yield when carbenes were photogenerated in the solid phase (77 K) relative to the liquid phase (293 K), and we suggest that carbene labeling should always be conducted in the frozen state to avoid information loss in surface mapping experiments. Graphical Abstract ᅟ.