Surface Accessibility and Dynamics of Macromolecular Assemblies Probed by Covalent Labeling Mass Spectrometry and Integrative Modeling.

Carla Schmidt,Franca Fraternali,Andy M Lau,Jamie A Macpherson,Argyris Politis,Ken Wei Tan

doi:10.1021/acs.analchem.6b02875

Abstract

Mass spectrometry (MS) has become an indispensable tool for investigating the architectures and dynamics of macromolecular assemblies. Here we show that covalent labeling of solvent accessible residues followed by their MS-based identification yields modeling restraints that allow mapping the location and orientation of subunits within protein assemblies. Together with complementary restraints derived from cross-linking and native MS, we built native-like models of four heterocomplexes with known subunit structures and compared them with available X-ray crystal structures. The results demonstrated that covalent labeling followed by MS markedly increased the predictive power of the integrative modeling strategy enabling more accurate protein assembly models. We applied this strategy to the F-type ATP synthase from spinach chloroplasts (cATPase) providing a structural basis for its function as a nanomotor. By subjecting the models generated by our restraint-based strategy to molecular dynamics (MD) simulations, we revealed the conformational states of the peripheral stalk and assigned flexible regions in the enzyme. Our strategy can readily incorporate complementary chemical labeling strategies and we anticipate that it will be applicable to many other systems providing new insights into the structure and function of protein complexes.

Highlights

Mass spectrometry (MS) is an emerging technique in biophysics, and in the last two decades, it has gained in importance when studying the structure and dynamics of macromolecular protein assemblies.[1]
By subjecting the models generated by our restraint-based strategy to molecular dynamics (MD) simulations, we revealed the conformational states of the peripheral stalk and assigned flexible regions in the enzyme
We assessed the predictive power of our integrative models for of ε, δ, and γ subunits were utilized as method for three-dimensional protein modeling based on previously described.[12] structural MS restraints on four protein complexes previously

Summary

■ RESULTS AND DISCUSSION

Integrating Covalent Labeling into Computational the distance estimated from the missing residues. To investigate the merit of modeling restraints in predicting the correct structure of the three training complexes (tryptophan synthase, CPS and RvB1/2), we determined receiver-operating characteristic (ROC) plots for the MS techniques employed (Figures 2D and S7) This enabled us to test the ability of our method in generating correct model structures on systems with diverse topological features that include symmetry, ring-like geometries and heteromeric subunits. To study the accuracy and precision of SASA restraint from covalent labeling followed by MS, we projected the labeled serines, threonines, tyrosines and histidines on the crystal structures of tryptophan synthase and the cATPase head and examined their SASA (Figure 4). This is consistent with the twisting motion of the catalytic head of an A-ATPase proposed previously[60] and may be related to the intermediate states of rotary ATPases during ATP synthesis.[68,69]

■ CONCLUSIONS

■ ACKNOWLEDGMENTS

■ REFERENCES