Biophysical basis of the binding of WWOX tumor suppressor to WBP1 and WBP2 adaptors.
The WW-containing oxidoreductase (WWOX) tumor suppressor participates in a diverse array of cellular activities by virtue of its ability to recognize WW-binding protein 1 (WBP1) and WW-binding protein 2 (WBP2) signaling adaptors among a wide variety of other ligands. Herein, using a multitude of biophysical techniques, we provide evidence that while the WW1 domain of WWOX binds to PPXY motifs within WBP1 and WBP2 in a physiologically relevant manner, the WW2 domain exhibits no affinity toward any of these PPXY motifs. Importantly, our data suggest that while R25/W44 residues located within the binding pocket of a triple-stranded β-fold of WW1 domain are critical for the recognition of PPXY ligands, they are replaced by the chemically distinct E66/Y85 duo at structurally equivalent positions within the WW2 domain, thereby accounting for its failure to bind PPXY ligands. Predictably, not only does the introduction of E66R/Y85W double substitution within the WW2 domain result in gain of function but the resulting engineered domain, hereinafter referred to as WW2_RW, also appears to be a much stronger binding partner of WBP1 and WBP2 than the wild-type WW1 domain. We also show that while the WW1 domain is structurally disordered and folds upon ligand binding, the WW2 domain not only adopts a fully structured conformation but also aids stabilization and ligand binding to WW1 domain. This salient observation implies that the WW2 domain likely serves as a chaperone to augment the physiological function of WW1 domain within WWOX. Collectively, our study lays the groundwork for understanding the molecular basis of a key protein-protein interaction pertinent to human health and disease.
Department of Biochemistry and Molecular Biology, Leonard Miller School of Medicine, University of Miami, Miami, FL 33136, USA.
Caleb B
CB
McDonald
Laura
L
Buffa
Tomer
T
Bar-Mag
Zaidoun
Z
Salah
Vikas
V
Bhat
David C
DC
Mikles
Brian J
BJ
Deegan
Kenneth L
KL
Seldeen
Arun
A
Malhotra
Marius
M
Sudol
Rami I
RI
Aqeilan
Zafar
Z
Nawaz
Amjad
A
Farooq
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D001703:Biophysics; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000737:chemistry* / Q000378:metabolism*; D000074584:WW Domain-Containing Oxidoreductase
D048868:Adaptor Proteins, Signal Transducing; D008024:Ligands; D025521:Tumor Suppressor Proteins; D010088:Oxidoreductases; D000074584:WW Domain-Containing Oxidoreductase; C486071:WWOX protein, human