The Transcriptional Repressor RYBP Is a Natively Unfolded Protein Which Folds upon Binding to DNA

Abstract

RYBP (Ring1A and YY1 binding protein) is a zinc finger protein with an essential role during embryonic development, which binds transcriptional factors, Polycomb products, and mediators of apoptosis, suggesting roles in, apparently, unrelated functions. To investigate mechanisms underlying its association with functionally diverse partners, we set out to study its structural properties using a number of biophysical (fluorescence, circular dichroism, Fourier transform infrared, and NMR spectroscopies) and hydrodynamic (analytical ultracentrifugation, DOSY-NMR, and gel filtration chromatography) techniques. We find RYBP to be a noncompact protein with little residual secondary structure, lacking a well-defined tertiary structure. These observations are also supported by theoretical calculations using neural networks and pairwise energy content, suggesting that RYBP is a natively unfolded protein. In addition, structural studies on its binding to the C-terminal region of the Polycomb protein Ring1B or to DNA show conformational changes in the complexed RYBP, consistent with the acquisition of a folded structure. The data provide a structural explanation for RYBP engagement in functionally unrelated pathways by means of its assembly into various macromolecular complexes as an unstructured protein with the ability to acquire a well-structured fold due to its association with different partners.

Intrinsically disordered chromatin protein NUPR1 binds to the C-terminal region of Polycomb RING1B

“…NUPR1 is not the sole IDP capable of interacting with C-RING1B, because also RYBP protein binds to it (19,20,24,25). The main physicochemical difference between RYBP and NUPR1 is that the former self-associates (both are IDPs, DNA-binding proteins with a high isoelectric point).…”

“…The X-ray structure of the monomers of C-RING1B resembles that of a ubiquitin module, and it serves to interact with CBX proteins (18). Furthermore, we have also shown that C-RING1B is capable of interacting with RING1 and YY1 binding protein (RYBP) (19,20), which is a highly basic, oligomeric, intrinsically disordered protein (IDP) that interacts with DNA (19) and other proteins involved in apoptosis (21-23). The CBX proteins binding to C-RING1B undergo a tightening in their structures (24), and their presence hampers RYBP binding (25).…”

The regulatory factor SipA is a highly stable β‐II class protein with a SH3 fold

“…Since the correlation time, s c , is s c % 1 T 2 [13], and, roughly, the M is twice the s c , and thus M can be estimated. Experiments were carried out at 90 and 180 lM of protein concentration, and no variation was observed in the measured T 2 -time (data not shown).…”

A polycomb protein RYBP/DEDAF in vivo funkcióinak vizsgálata transzgenikus módszerekkel

“…Így az úgynevezett "moonlighting" fehérjék családjába tartozik, melyekre jellemző hogy egynél több, egymástól eltérő funkciót képesek ellátni [5]. Ennek megfelelően, az Rybp/DEDAF protein -amellett hogy maga is transzkripciós regulátor és polycomb protein -in vitro kölcsönhatásba lép számos, változatos biológiai funkcióval bíró fehérjével, úgymint DNS kötő (YY1), kromatin módosító (Brg1), proapoptotikus (pro-caspase-8), egyéb polycomb (Ring1A, Ring1B, M33, mPC2) vagy például a sejtek proliferációjában és differenciációjában fontos szabályzó funkcióval bíró Myc-Mad-Max hálózat tagjaival (Mxi1).…”

“…A két fejérje domén szerkezete is hasonló: azok N-terminális régiót kivéve, ahol a cink-finger domének találhatók, egy szerin-gazdag rész húzódik amely nem mutat homológiát más ismert fehérje doménekkel és nagyfokú rendezetlenséget muatat (IDP; intrinsically disordered proteins) [4,5,9]. …”