Inactivation of VHL by tumorigenic mutations that disrupt dynamic coupling of the pVHL·hypoxia-inducible transcription factor-1α complex

The von Hippel-Lindau (VHL) gene product, pVHL, targets the a subunit of the hypoxia-inducible transcription factor (HIF-α) for ubiquitin-dependent degradation. This tumor suppressor function is mediated by the α- and β-domains responsible for assembling the pVHL E3 ubiquitin ligase complex and for recognizing the prolyl-liydroxylated HIF-α, respectively. The molecular basis for a large number of tumor-derived mutations can be attributed to alterations that directly compromise the ability of pVHL to assemble the E3 or to contact the substrate. Here we describe a new mechanism of oncogenic inactivation by VHL missense mutations that lie in the L1 and L7 linker regions distal to the HIF-α-binding pocket. Employing molecular dynamics simulations, we show that the tumorigenic L1 loop mutation of Ser⁶⁵ to Leu, deficient in promoting the degradation of HIF-α, disrupts the coordination of internal motions of the pVHL·HIF-1α complex. Furthermore, we demonstrate that in addition to S65L, five other tumor-derived VHL mutations located within the L1 loop are each defective in mediating proteolysis of HIF-2α. Moreover, dynamic organization of pVHL-HIF-1α recognition is focally centered on Gln¹⁴⁵ within the L7 loop, and its tumorigenic mutant Q145H abolishes almost all of the correlated dynamic motions. Intriguingly, Q145H, whereas defective in targeting cellular HIF-α for degradation, had an attenuated hydroxylation dependence in binding to HIF-1α in vitro. Taken together, our results suggest that specific association between pVHL and the hydroxylated HIF-α requires both the L1 and L7 loops to coordinate dynamic coupling among distant pVHL regions, whose mutational disruption inactivates VHL and is hence responsible for tumorigenesis.