Kinetics of the multitasking high-affinity Win binding site of WDR5 in restricted and unrestricted conditions

Imran, Ali Shariq; Moyer, Brandon S.; Canning, Ashley J.; Kalina, Dan; Duncan, T. M.; Moody, Kelsey; Wolfe, Aaron; Cosgrove, Michael S.; Movileanu, Liviu

doi:10.1042/bcj20210253

Cited by 8 publications

(27 citation statements)

References 96 publications

(123 reference statements)

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“…In a recent study 42 , we have shown that the k on values of Win motif peptides with WDR5 are influenced by their tethering on a sensor surface. In addition, the partitioning of MLL4 Win into the WDR5 cavity significantly reduces the k on due to the entropic penalty of this process 32 .…”

Section: Resultsmentioning

confidence: 97%

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Disentangling the recognition complexity of a protein hub using a nanopore

et al. 2022

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WD40 repeat proteins are frequently involved in processing cell signaling and scaffolding large multi-subunit machineries. Despite their significance in physiological and disease-like conditions, their reversible interactions with other proteins remain modestly examined. Here, we show the development and validation of a protein nanopore for the detection and quantification of WD40 repeat protein 5 (WDR5), a chromatin-associated hub involved in epigenetic regulation of histone methylation. Our nanopore sensor is equipped with a 14-residue Win motif of mixed lineage leukemia 4 methyltransferase (MLL4Win), a WDR5 ligand. Our approach reveals a broad dynamic range of MLL4Win-WDR5 interactions and three distant subpopulations of binding events, representing three modes of protein recognition. The three binding events are confirmed as specific interactions using a weakly binding WDR5 derivative and various environmental contexts. These outcomes demonstrate the substantial sensitivity of our nanopore sensor, which can be utilized in protein analytics.

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Section: Resultsmentioning

confidence: 97%

“…These experiments were executed using an Octet Red384 instrument (FortéBio, Fremont, CA) 42 . MLL4 Win was biotinylated and amidated at the N and C terminus, respectively.…”

Section: Methodsmentioning

confidence: 99%

Disentangling the recognition complexity of a protein hub using a nanopore

et al. 2022

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“…This outcome confirms our previous results, indicating that measured interactions are stronger in unrestricted conditions than those corresponding to restrained conditions ( Table S11 ). 44 Because these WDR5 mutants have been examined using BLI and FP, we can also compare these approaches qualitatively. 65 For example, using BLI, we can determine the kinetic fingerprint of these interactions.…”

Section: Resultsmentioning

confidence: 99%

“…Human WDR5 (UniProtKB—P61964; WDR5_HUMAN) and its mutants were expressed and purified as described previously. 22 , 23 , 44 …”

Section: Methodsmentioning

confidence: 99%

“… 15 , 22 , 23 SET1 Win ligands recapitulate the native interactions of the six SET1 proteins with WDR5 through the Win binding site. 35 , 36 Therefore, we utilized the benefit of biolayer interferometry (BLI) 44 , 58 − 60 for high-throughput settings and immobilized these SET1 Win ligands onto the sensor surface. In this way, we probed the real-time kinetics and dynamics of their interactions with a subset of these WDR5 mutants, whose missense alterations are located within and around the Win binding site.…”

Section: Introductionmentioning

confidence: 99%

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Convergent Alterations of a Protein Hub Produce Divergent Effects within a Binding Site

Imran

Moyer²,

Kalina

et al. 2022

ACS Chem. Biol.

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Progress in tumor sequencing and cancer databases has created an enormous amount of information that scientists struggle to sift through. While several research groups have created computational methods to analyze these databases, much work still remains in distinguishing key implications of pathogenic mutations. Here, we describe an approach to identify and evaluate somatic cancer mutations of WD40 repeat protein 5 (WDR5), a chromatin-associated protein hub. This multitasking protein maintains the functional integrity of large multi-subunit enzymatic complexes of the six human SET1 methyltransferases. Remarkably, the somatic cancer mutations of WDR5 preferentially distribute within and around an essential cavity, which hosts the WDR5 interaction (Win) binding site. Hence, we assessed the real-time binding kinetics of the interactions of key clustered WDR5 mutants with the Win motif peptide ligands of the SET1 family members (SET1 Win ). Our measurements highlight that this subset of mutants exhibits divergent perturbations in the kinetics and strength of interactions not only relative to those of the native WDR5 but also among various SET1 Win ligands. These outcomes could form a fundamental basis for future drug discovery and other developments in medical biotechnology.

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