Cross-linking measurements of in vivo protein complex topologies

Abstract

Identification and measurement of in vivo protein interactions pose critical challenges in the goal to understand biological systems. The measurement of structures and topologies of proteins and protein complexes as they exist in cells is particularly challenging, yet critically important to improve understanding of biological function because proteins exert their intended function only through the structures and interactions they exhibit in vivo. In the present study, protein interactions in E. coli cells were identified in our unbiased cross-linking approach, yielding the first in vivo topological data on many interactions and the largest set of identified in vivo cross-linked peptides produced to date. These data show excellent agreement with protein and complex crystal structures where available. Furthermore, our unbiased data provide novel in vivo topological information that can impact understanding of biological function, even for cases where high resolution structures are not yet available.

Fig. 1.

A, Structure of the PIR cross-linker. Dotted lines indicate mass spectrometry labile bonds. B, Cleavage of the PIR cross-linker. C, Mass relationships of the cross-linked products.

Fig. 2.

Experimental scheme. A, Sample preparation scheme for cross-linking experiments. B, Demonstration of LC-MS experiment with alternating In Source Collision Induced Dissociation (ISCID) voltage settings. The cross-linked complexes remain intact in the precursor scan (ISCID voltage 0V). ISCID scan at 80V results in cleavage of the labile bonds in the cross-linker and release of reporter and peptides. Cross-linked pairs were identified by searching for PIR mass relationships. C, A separate LC-MS/MS experiment was used to target released peptides for MS/MS analysis at specific mass and retention times. Database search with Mascot was used to identify their sequences of released peptides.

Fig. 3.

Cross-linked relationships shown on the protein complex crystallography structures. A, Skp trimeric complex (PDB entry 1SG2). Red: lysine 85; Yellow: lysine 97. B, KduI dimeric complex (1XRU). Red: lysine 13. C, Lpp multimeric model (2GUV). Red: lysine 75. D, FkpA dimeric complex (1Q6H). Red: lysine 110; Yellow: lysine 85. E, The docking result of FkpA dimer and 30S ribosome (2QAL) based on the PIR cross-linking result. Blue: FkpA dimer; Green: 30S ribosomal protein S6 (RpsF); Yellow: Ribosomal RNA proximal to RpsF. Remainder of ribosome is not shown.

Fig. 4.

Prediction of disordered regions and binding sites in E. coli. A) Prediction of disorder in GapA; B), Prediction of disorder in OmpA; and C) Prediction of binding regions in OmpA. Highly disordered regions are generally associated with prediction of 0.5 or greater. Identified cross-linked sites are indicated and appear in regions of higher predicted disorder. Blue and red from prediction algorithms VLXT and CAN_XT, respectively, that use various sequence lengths and datasets for training as described on website: www.pondr.com. Potential binding sites are predicted with tools provided on ‘http://anchor.enzim.hu/’. The shaded areas are predicted binding regions.

Fig. 5.

OmpA dimeric model and mass spectra of the homodimer cross-linking relationship. A, Precursor scan showing the homodimer cross-linked complex. Inset: isotopic distribution of the parent ion. B, ISCID scan showing the reporter and the released peptide. C, MS/MS spectrum of the OmpA released peptide with the sequence and assignment of fragments. D, OmpA monomer model. Red: Lysine 213; Yellow: Lysine 294; Orange: Lysine 338. E, A dimeric structural model from docking results (viewing angle: top-down). Red: Lysine 213; Yellow: Lysine 294; Orange: Lysine 338. The distances between each lysine are labeled in red.