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The authors contributed several interesting and surprising findings by characterizing the competition between HIF-1? and CITED2 by NMR spectroscopy. The 1H- 15N HSQC spectrum of both 15N TAZ1 bound to HIF-1? and CITED2 have different resonances allowing site-specific depiction of competitors. 15N TAZ1 was shown to bind with equal binding affinities (Dissociation constant, kd = 10±2 nM) for HIF-1? and CITED2. The observation of only cross peaks for CITED2-TAZ1 complex when equimolar concentration (1:1:1) of HIF-1? and CITED2 was mixed suggests a co-operative mechanism CITED2 displacement of HIF-1?.


Fluorescence anisotropy competition assays complemented the NMR results indicating unlabelled CITED2 is extremely efficient in displacing HIF-1? from labelled HIF-1?-TAZ1 complex with 50-fold smaller disassociation constant than for the binary TAZ1:CITED2 complex. The equal binding affinities of HIF-1? and CITED2 to TAZ1 were obtained from Bio-layer interferometry experiments. 

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The rates of displacement of HIF-1? by CITED2 were further measured using time-resolved fluorescence measurements. CITED2 displaces labelled HIF-1? from TAZ1 within 10 seconds whereas unlabelled HIF-1? requires 200 seconds for complete displacement which was measured using manual mixing experiments. These experiments strengthen the view of a transient ternary intermediate from which HIF-1? gets displaced by CITED2 in a kinetically driven process in picoseconds-nanosecond timescales.


Measurements of the {1H}-15N heteronuclear nuclear overhauser effect (NOE) for the transactivation domains of HIF-1? (776-826) and CITED2 (216-269) report the conformation of a highly flexible and disordered region encompassing ?A Helix with the LPQL motif and an extremely ordered ?A Helix with LPEL motif respectively. The disordered region attains well-ordered and dynamic contacts upon TAZ1 binding. An anchored region (?B and ?C) of HIF-1? still allows binding of ?A helix of CITED2, dynamically displacing the disordered ?A helix of HIF-1?. Simultaneous binding of HIF-1? and CITED2 to TAZ1 domain facilitated through this LP(Q/E)L site suggests a reasonable mechanism of enhanced dissociation of HIF-1? by CITED2.


Binding studies of truncated CITED2 peptide (?A helix without LPEL motif) and truncated HIF-1? (without ?A helix and LPQL motif) with reduced binding affinities (kd=36±10 mM and kd=1.9±0.1 mM respectively) reinforce the importance of LP(Q/E)L motif. NMR studies of truncated mutants did not show this decline in affinity for binding.

 NMR titration experiments provided direct validation of ternary complex formation with the chemical shift changes observed in the 15N TAZ1 residues located in the binding sites of CITED2. Negligible chemical shift changes observed when HIF-1? added to CITED2-bound TAZ1 intensifies the view of CITED2 displaces HIF-1? but vice-versa is not true.