Elucidating the higher-order structure of biopolymers by structural probing and mass spectrometry: MS3D

Abstract

Chemical probing represents a very versatile alternative for studying the structure and dynamics of substrates that are intractable by established high-resolution techniques. The implementation of MS-based strategies for the characterization of probing products has not only extended the range of applicability to virtually all types of biopolymers but has also paved the way for the introduction of new reagents that would not have been viable with traditional analytical platforms. As the availability of probing data is steadily increasing on the wings of the development of dedicated interpretation aids, powerful computational approaches have been explored to enable the effective utilization of such information to generate valid molecular models. This combination of factors has contributed to making the possibility of obtaining actual 3D structures by MS-based technologies (MS3D) a reality. Although approaches for achieving structure determination of unknown targets or assessing the dynamics of known structures may share similar reagents and development trajectories, they clearly involve distinctive experimental strategies, analytical concerns and interpretation paradigms. This Perspective offers a commentary on methods aimed at obtaining distance constraints for the modeling of full-fledged structures while highlighting common elements, salient distinctions and complementary capabilities exhibited by methods used in dynamics studies. We discuss critical factors to be addressed for completing effective structural determinations and expose possible pitfalls of chemical methods. We survey programs developed for facilitating the interpretation of experimental data and discuss possible computational strategies for translating sparse spatial constraints into all-atom models. Examples are provided to illustrate how the concerted application of very diverse probing techniques can lead to the solution of actual biological systems.

Figure 1

General operations and strategies followed to complete MS-based structural probing. In typical MS3D workflows, the substrate of interest is treated with the selected probe. The products can undergo purification/enrichment procedures, or can be analyzed directly by mass spectrometry, according to bottom up or top-down strategies (see text).

Figure 2

Multiple stages of tandem MS for the characterization of mono-methylated adduct of the RNA oligonucleotide SL3: (a) products obtained by isolation/activation of monomethyl SL3 (MS², precursor ion m/z 1617.02); (b) isolation/activation of the first-generation fragment produced by loss of methylguanine (MS³, m/z 1617.02 → m/z 1576.55 → products); (c) isolation/activation of the second-generation fragment induced by consecutive losses of methylguanine and guanine (MS⁴, m/z 1617.02 → m/z 1576.55 → m/z 1538.70 → products). All precursor ions selected at each stage had a 4– charge state. Reproduced with permission from reference .

Figure 3

Schematic overview of a 2D chromatography setup for the automated enrichment of peptide-RNA conjugates derived by complete hydrolysis of protein-RNA complexes irradiated with UV light. Interactions between phosphate groups and TiO₂ stationary phase are responsible for the selective retention of nucleic acid components. Hetero-conjugate enrichment is achieved through a multistep process involving the concerted utilization of appropriate C18 traps. Adapted with permission from reference .

Figure 4

Monitoring myoglobin probing with diethylpyrocarbonate (DEPC): (a) representative ESI-MS spectrum; (b) dose response plot. [P]₀ and [P] are the initial and final concentration of unmodified protein inferred from the corresponding peak areas. [X]₀ and [X] are the DEPC concentrations. The observed deviation from linearity suggests a protein structural change due to the modification. Adapted with permission from reference .

Figure 5

Structure of common bifunctional N-hydroxysuccinimide esters targeting amino groups: disuccimimidyl tartarate (DST); disuccinimidyl suberate (DSS); disuccinimidyl glutarate (DSG); bis(2 [succinimidooxycarbonyloxy]ethyl)sulfone (BSOCOES); ethyleneglycol bis-(succinimidylsuccinate) (EGS); and bis(sulfosuccinimidyl)adipate (BSSA). *Reported N-N distances were obtained from Pierce reference sheets. **Average N-N distances and N-N distance range distributions were obtained from reference .

Figure 6

Model of the calmodulin-Munc13 peptide complex based on crosslinking and photoaffintiy labeling (PAL). Munc13 (13-1) peptide (blue) structure was predicted and modeled as an α-helix, and calmodulin (CaM, grey) was modeled after multiple CaM structures in the PDB. The complex was created using PatchDock and refined using ROSETTADock based on PAL of CaM residues M122, M124 (green) and 13-1 residue W7 (green), and crosslinks between13-1 residue K13 (purple) with CaM lysines (yellow, and red). Adapted with permission from reference .

Figure 7

(a) The model of T. aquaticus Ffh-FtsY NG complex was created by docking the apo-NG domains of Ffh (green) and FtsY (blue) according to crosslinking constraints. The MS3D model overlays perfectly with the crystal structure (grey) of the complex. (b) The structure of S. solfataricus SRP54 (gray) was superimposed with T. aquaticus Ffh (green) to generate a model for the Ffh_FtsY complex including the M domain. Relative positions of T. aquaticus lysine residues in the SRP54 M domain are mapped (green). These residues formed crosslinks with residues G(−3) and K62 of FtsY (magenta line), suggesting a close proximity of the M domain to the Ffh-FtsY complex interface. Adapted with permission from reference .

Figure 8

(a) MS3D model of the ~100 kDa ANXA2/P11 complex. The location of lysine residues involved in intra- and inter-protein crosslinking of the p11 dimer and full-length ANXA2 are marked as blue and grey spheres. (b) These crosslinks were used for docking calculations that provided a hetero-octameric A2t complex consisting of four ANXA2 (dark green and blue) located on the periphery connected by two p11 dimers (red and light green) in the center. Adapted with permission from reference .

Figure 9

(a) MS3D model of the full-length Ψ-RNA generated using footprinting and crosslinking data. High-resolution structures for the discrete stem-loop domains SL1 (red), SL2 (green), SL3 (blue) and SL4(yellow) were employed as building blocks. Linker regions (orange) were generated de novo. (b) Details of H-bonding and interactions between SL1 and SL4 domains in the observed GNRA-tetraloop interaction. Adapted with permission from reference .