Abstract
Ribosomal protein L11 (RPL11) has been shown to activate p53 by binding to MDM2 and negating its p53 suppression activity in response to ribosomal stress. Although a mutation at Cys-305 within the zinc finger domain of MDM2 has been shown to drastically impair MDM2 interaction with RPL11 and thus escapes the inhibition by this ribosomal protein, it still remains elusive whether RPL11 inactivates MDM2 via direct action on this zinc finger domain and what is the chemical nature of this specific interaction. To define the roles of the MDM2 zinc finger in association with RPL11, we conducted hydrogen-deuterium exchange mass spectrometry, computational modeling, circular dichroism, and mutational analyses of the zinc finger domain of MDM2 and human RPL11. Our study reveals that RPL11 forms a stable complex with MDM2 in vitro through direct contact with its zinc finger. This binding is disrupted by single mutations of non-cysteine amino acids within the zinc finger domain of MDM2. Basic residues in RPL11 are crucial for the stable binding and RPL11 suppression of MDM2 activity toward p53. These results provide the first line of evidence for the specific interaction between RPL11 and the zinc finger of MDM2 via hydrophilic residues as well as a molecular foundation for better understanding RPL11 inhibition of MDM2 function.
Highlights
Whether Ribosomal protein L11 (RPL11) directly binds to the zinc finger domain of MDM2 still remains elusive
Mutations, including E292A, D294A, T306A/S307A, or N320A, within the zinc finger domain, but not mutations at S290A or L325A, drastically disrupted the interaction of MDM2 with only RPL11 (Fig. 4A) but not RPL5 or RPL23 (Fig. 4B). These results unambiguously argue that the zinc finger domain is where RPL11, but not RPL5 or RPL23, physically contacts when associating with MDM2 and that acidic or polar residues, such as Glu-292, Asp-294, or Thr-306/Ser-307, within this domain are essential for this specific association
When examining the direct binding between L11 and the zinc finger domain of MDM2, we identified several noncysteine acidic or polar residues within the C4 zinc finger domain of MDM2 essential for binding to RPL11 and several basic residues within the MDM2-binding domain of RPL11 crucial for binding to MDM2. These findings suggest that the chemical nature of MDM2-RPL11 interaction must be electrostatic interactions, such as H-bond, between the two proteins
Summary
Whether RPL11 directly binds to the zinc finger domain of MDM2 still remains elusive. The RS-p53 pathway has been recently linked to human genetic disorders characteristic with bone marrow defects, such as the two types of myelodysplastic syndromes [47], 5q syndrome [33], and Diamond Blackfan anemia [48] In these diseases, haploinsufficiency caused by mutations of either RPS14 [49] or other ribosomal protein-encoding genes, such as RPS19 [50] or RPL5 [51], induces ribosomal stress and consequent p53 activation and p53-dependent apoptosis of developing blood cells in bone marrows, causing anemia of several blood types. We uncover several non-cysteine amino acids within the zinc finger and basic amino acids in MDM2-binding domain of RPL11, which are important for the specific interactions between the two proteins Mutations of these residues without altering the intact zinc finger motif impair their interaction in vitro and in cells and the ability of RPL11 to inactivate MDM2 and to induce p53 in cells
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