Phosphorylation compromises FAD binding and intracellular stability of wild-type and cancer-associated NQO1: Insights into flavo-proteome stability

Naturally-Occurring Rare Mutations Cause Mild to Catastrophic Effects in the Multifunctional and Cancer-Associated NQO1 Protein

“…11 of 31 states) [91]. It must be noted that all mutations studied in this work are located at least 15-20 Å from the TCS ( Figure 4A).…”

“…In particular, proteolysis by thermolysin typically provides information on the local conformational stability of the region surrounding the primary cleavage site for this protease (i.e., the thermolysin cleavage site (TCS)) located in the NTD of NQO1 holo ; it cleaves between S72-V73) [41]. For instance, the phosphomimetic mutation S82D locally destabilizes this region, accelerating 30-fold proteolysis by thermolysin of NQO1 holo (i.e., it causes 2.0 kcal•mol −1 of local destabilization in terms of ∆G prot as the mutational effect on the unfolding free energy between the native and cleavable states) [91]. It must be noted that all mutations studied in this work are located at least 15-20 Å from the TCS ( Figure 4A).…”

Targeting HIF-1α Function in Cancer through the Chaperone Action of NQO1

“…Phosphorylation of NQO1 at T128 by the kinase Akt results in polyubiquitination and degradation of NQO1 via the proteasome [89]. A phosphomimetic mutation on S82 showed reduced FAD binding affinity and consequent loss of intracellular protein stability, although the kinase involved in this phosphorylation event has not been identified yet [90]. Consequently, phosphorylation of NQO1 at different sites may have an impact on the intracellular stability of its protein partners through destabilization of NQO1.…”

“…Although no high resolution structural model is available for this state, recent kinetic studies using hydrogen-deuterium exchange mass spectrometry (HDXMS) have identified a minimal stable core that holds the protein dimer while most of the protein exists forming a highly dynamic structural ensemble, including the FAD and substrate binding sites in non-competent states for binding [129]. This remarkable conformational flexibility makes NQO1apo likely the most relevant state to understand the intracellular stability of NQO1, since the flexible CTD acts as initiation site for rapid degradation through ubiquitin-dependent and -independent proteasomal pathways [90,116]. It is plausible that this unstable and highly dynamic NQO1apo state is not capable of interacting with NQO1 protein partners, or at least, does so with lower affinity [78].…”

“…This seems to be paradoxical, because P187 is fully buried in the protein structure, close to the NQO1 dimer interface and belongs to the minimally stable core of NQO1apo, and thus, a mutation to serine should have catastrophic effects on protein structure and function [129,136]. Detailed biochemical, biophysical, computational and mutational studies have shown that the effects of p.P187S are pleiotropic in the structure, function and stability of NQO1apo and NQO1holo, highlighting the critical role of propagation of the local stability effects due to p.P187S to long distances in the protein structure and affecting the dynamics and stability of critical regions of NQO1 function [78,79,90,119,120,122,131,133,136,137]. These studies on p.P187S have also supported that different functional sites located far (over 10-20 Å ) in the structure are functionally and energetically coupled, and thus, we might modify the interaction of NQO1 with its partners by using an allosteric site (i.e.…”