Abstract
Characterization of protein interaction domains is crucial for understanding protein functions. Here we combine cross-linking mass spectrometry (XL-MS) with deletion analysis to accurately locate minimal protein interaction domains. As a proof of concept, we investigated in detail the binding interfaces of two protein assemblies: the complex formed by MICAL3, ELKS and Rab8A, which is involved in exocytosis, and the complex of SLAIN2, CLASP2 and ch-TOG, which controls microtubule dynamics. We found that XL-MS provides valuable information to efficiently guide the design of protein fragments that are essential for protein interaction. However, we also observed a number of cross-links between polypeptide regions that were dispensable for complex formation, especially among intrinsically disordered sequences. Collectively, our results indicate that XL-MS, which renders distance restrains of linked residue pairs, accelerates the characterization of protein binding regions in combination with other biochemical approaches.
Highlights
Proteins are the primary effectors of the cell
We focused on two protein complexes of various nature, namely, a complex formed by MICAL3, ELKS and Rab8A, involved in exocytotic vesicle trafficking[20], and a complex of CLASP2, SLAIN2, and ch-TOG, important for the regulation of microtubule plus end dynamics[21]
The first protein complex we investigated is formed by MICAL3, ELKS and Rab8A
Summary
Proteins are the primary effectors of the cell. They execute a plethora of cellular processes, which depend on protein-protein interactions (PPIs) responsible for formation of stable protein complexes and dynamic interaction networks[1,2]. Numerous techniques have been introduced to determine protein interaction domains, among which affinity purification combined with deletion analysis (generation of deletion mutants) is one the most popular methods[8,9]. Protein complexes were purified from cell lysates using the biotin (Bio) tag[25] and thereafter subjected to XL-MS experiments to determine amino acid residues that are in close proximity. These residue-to-residue connectivities were used to guide the design of protein truncations and deletion mutants, thereby confirming minimal binding domains by affinity purification-based assays (Fig. 1)
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