In vivo protein complex topologies: sights through a cross-linking lens

Abstract

Proteins are a remarkable class of molecules that exhibit wide diversity of shapes or topological features that underpin protein interactions and give rise to biological function. In addition to quantitation of abundance levels of proteins in biological systems under a variety of conditions, the field of proteome research has as a primary mission the assignment of function for proteins and if possible, illumination of factors that enable function. For many years, chemical cross-linking methods have been used to provide structural data on single purified proteins and purified protein complexes. However, these methods also offer the alluring possibility to extend capabilities to complex biological samples such as cell lysates or intact living cells where proteins may exhibit native topological features that do not exist in purified form. Recent efforts are beginning to provide glimpses of protein complexes and topologies in cells that suggest continued development will yield novel capabilities to view functional topological features of many proteins and complexes as they exist in cells, tissues, or other complex samples. This review will describe rationale, challenges, and a few success stories along the path of development of cross-linking technologies for measurement of in vivo protein interaction topologies.

FIGURE 1

A) Side-view of annotated crystal structure image of the E. coli periplasmic chaperone, Skp illustrating the observed intramolecular in vivo cross-link identified between lysine 77 and lysine 89. B) Alternate side view of crystal structure illustrating position of lysine 77 on the top subunit in this structure. The third subunit, here shown in green, was disordered in crystal structure analysis near the “forceps” tip and lysine 77 was not resolved in this subunit. C) Zoomed view of region encompassing both lysine 77 residues in two resolved subunits. These two lysine amino acids were observed from in vivo cross-linking indicating these sites were close to one another in cells.

FIGURE 1

A) Side-view of annotated crystal structure image of the E. coli periplasmic chaperone, Skp illustrating the observed intramolecular in vivo cross-link identified between lysine 77 and lysine 89. B) Alternate side view of crystal structure illustrating position of lysine 77 on the top subunit in this structure. The third subunit, here shown in green, was disordered in crystal structure analysis near the “forceps” tip and lysine 77 was not resolved in this subunit. C) Zoomed view of region encompassing both lysine 77 residues in two resolved subunits. These two lysine amino acids were observed from in vivo cross-linking indicating these sites were close to one another in cells.

FIGURE 1

A) Side-view of annotated crystal structure image of the E. coli periplasmic chaperone, Skp illustrating the observed intramolecular in vivo cross-link identified between lysine 77 and lysine 89. B) Alternate side view of crystal structure illustrating position of lysine 77 on the top subunit in this structure. The third subunit, here shown in green, was disordered in crystal structure analysis near the “forceps” tip and lysine 77 was not resolved in this subunit. C) Zoomed view of region encompassing both lysine 77 residues in two resolved subunits. These two lysine amino acids were observed from in vivo cross-linking indicating these sites were close to one another in cells.

FIGURE 2

A) Annotate crystal structure image of the E. coli metabolic protein, Tryptophanase, tnaA, annotated to illustrate several cross-link sites observed in cells. B) Close up view of cross-linked sites between lysine 115 and lysine 406 and 459.

FIGURE 2

A) Annotate crystal structure image of the E. coli metabolic protein, Tryptophanase, tnaA, annotated to illustrate several cross-link sites observed in cells. B) Close up view of cross-linked sites between lysine 115 and lysine 406 and 459.

FIGURE 3

Annotated crystal structure image of E. coli metabolic protein, Glyceraldehyde 3- phosphate dehydrogenase (GAPDH) illustrating in vivo cross-linked sites. Even though GAPDH contains more than 25 lysine residues, all observed cross-linked sites appear near the NAD+ cofactor binding pocket and are associated with regions of high predicted disorder.