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

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.