Quantitative fragmentomics allow affinity mapping of interactomes

Abstract

Human protein networks have been widely explored but most binding affinities remain unknown, hindering quantitative interactome-function studies. Yet interactomes rely on minimal interacting fragments displaying quantifiable affinities. Here we measured the affinities of 65,000 interactions involving PDZ domains and their target PDZ-binding motifs (PBM) within a human interactome region particularly relevant for viral infection and cancer. We calculate interactomic distances, identify hot spots for viral interference, generate binding profiles and specificity logos, and explain selected cases by crystallographic studies. Mass spectrometry experiments on cell extracts and literature surveys show that quantitative fragmentomics effectively complement protein interactomics by providing affinities and completeness of coverage, putting a full human interactome affinity survey within realistic reach. Finally, we show that interactome hijacking by the viral PBM of human papillomavirus (HPV) E6 oncoprotein deeply impacts the host cell proteome way beyond immediate E6 binders, illustrating the complex system-wide relationship between interactome and function.

A scalable strategy to solve structures of PDZ domains and their complexes

“…Similarly to the divergent PDZ folds, unexpected binding modes, such as that observed in the case of PDZD7_3, may often appear among the 10 6 putative complexes in the PDZ-PBM interactome. Systematic structural exploration of this interactomic space, either experimentally or by means of predictions, will complement the rapidly growing biochemical survey of interactomes in order to better understand how specificity and function emerge from the PDZ-PBM network (Tonikian et al, 2008;Stiffler et al, 2007;Gogl et al, 2021). One additional approach in our toolbox could be the ANXA2-fusion strategy, which could be used for the rapid determination of a large number of PDZdomain and PDZ-PBM complex structures, as well as structures from other domain families, as we have exemplified here and previously.…”

“…The structures of ANXA2 long -fused MAGI1_2, SYNJ2BP, SNX27 and SNTB1 have been described elsewhere in detail (Gogl et al, 2018(Gogl et al, , 2020(Gogl et al, , 2021. They, together with DLG1_2, SNTG1, SNTG2 and LRRC7, display the well known PDZ fold (Lee & Zheng, 2010; Fig.…”

“…Using the ANXA2 short strategy, we solved the structures of the second PDZ domain of DLG1_2, SNTG2, SNX27 and SYNJ2BP. Using the ANXA2 long strategy, we solved the structures of SNTB1, SNX27 and SYNJ2BP (Gogl et al, 2021). In addition, the ANXA2 long strategy was also successfully used with MAGI1_2, yet we never fused this domain to ANXA2 short (Gogl et al, 2018(Gogl et al, , 2020(Gogl et al, , 2021.…”

“…These diverse functions of PDZ domains are achieved through their interactions with the C-terminal tails of their partner proteins. Around 4000 putative PDZ-binding motifs can be identified in the human proteome (PBMs, collectively called as the 'PBMome'; Gogl et al, 2021). Together with the PDZome, they define a large interaction space representing about 10 6 potential PDZ-PBM interactions to explore.…”

“…Therefore, despite years of effort, the PDZ structural landscape is not yet fully explored. Moreover, we recently explored $5% of the steady-state dissociation constants of this interactomic space and found that >20% of all tested interactions show experimentally quantifiable binding at a dissociation-constant threshold of $300 mM (Gogl et al, 2021). Thus, the PDZ-PBM interactome still represents a plethora of unexplored targets for future structural investigation (>10 5 interactions with relatively strong affinities for the 266 human PDZ domains).…”