In Situ Structural Restraints from Cross-Linking Mass Spectrometry in Human Mitochondria.

Petra S J Ryl,Lisa Budzinski,Marta M L Mendes,Michael Bohlke-Schneider,Ludwig Sinn,Juri Rappsilber,Swantje Lenz,Marchel Stuiver,Francis J O’Reilly,Lutz Fischer

doi:10.1021/acs.jproteome.9b00541

Abstract

The field of structural biology is increasingly focusing on studying proteins in situ, i.e., in their greater biological context. Cross-linking mass spectrometry (CLMS) is contributing to this effort, typically through the use of mass spectrometry (MS)-cleavable cross-linkers. Here, we apply the popular noncleavable cross-linker disuccinimidyl suberate (DSS) to human mitochondria and identify 5518 distance restraints between protein residues. Each distance restraint on proteins or their interactions provides structural information within mitochondria. Comparing these restraints to protein data bank (PDB)-deposited structures and comparative models reveals novel protein conformations. Our data suggest, among others, substrates and protein flexibility of mitochondrial heat shock proteins. Through this study, we bring forward two central points for the progression of CLMS towards large-scale in situ structural biology: First, clustered conflicts of cross-link data reveal in situ protein conformation states in contrast to error-rich individual conflicts. Second, noncleavable cross-linkers are compatible with proteome-wide studies.

Highlights

Mitochondria are complex organelles that fulfill a wide set of essential cellular functions including energy metabolism in all eukaryotic cells.[1]
Human mitochondria have 1157 proteins currently annotated in MitoCarta 2.0;6 for fewer than 300 of these, we found structures deposited in the protein data bank (PDB), often only covering fragments of the proteins
Cross-linking mass spectrometry (CLMS) is a technique that can provide in situ middle-resolution structural information for individual multiprotein complexes and can be scaled up to more complex samples such as entire organelles[17] or bacterial cells.[18−20] Distance restraints are generated by identifying which residues were cross-linked in a protein or between two interacting proteins and considering the length of the most extended conformation of the cross-linking reagent

Summary

■ INTRODUCTION

Mitochondria are complex organelles that fulfill a wide set of essential cellular functions including energy metabolism in all eukaryotic cells.[1]. Complex biological samples could only be tackled by the use of cross-linkers that cleave in the mass spectrometer[19−23] or by the use of an isotope-labeled cross-linker, which create a special isotope pattern to aid in identifying cross-linked peptides.[24,25] Two recent studies investigated murine mitochondria using MS-cleavable cross-linkers and reported 187622 and 277923 cross-linked residue pairs (excluding ambiguous crosslinks, where one of the cross-linked peptides could have come from a number of proteins), respectively These studies focused on the discovery of protein−protein interactions (PPIs) and partially on in situ protein structure analysis, while possible gains of systematic analysis of protein flexibility have been less explored. Due to the expectation that cross-linked peptides are overall larger than linear peptides, we selected only early SEC fractions for MS acquisitions This entire workflow resulted in 88 different SCX−SD−SEC fractions, which were evaporated completely and resuspended in 4 μL of 0.1% (v/v) FA. To account for the peptide sequence between the domains, we imposed an upper limit of 35 Å

■ RESULTS AND DISCUSSION

■ CONCLUSIONS

■ ACKNOWLEDGMENTS

■ REFERENCES