The Intrinsically Disordered RNR Inhibitor Sml1 Is a Dynamic Dimer

Abstract

Sml1 is a small ribonucleotide reductase (RNR) regulatory protein in Saccharomyces cerevisiae that binds to and inhibits RNR activation. NMR studies of 15N-labeled Sml1 (104 residues), as well as of a truncated variant (residues 50-104), have allowed characterization of their molecular properties. Sml1 belongs to the class of intrinsically disordered proteins with high degree of dynamics and very little stable structures. Earlier suggestions for a dimeric structure of Sml1 were confirmed and from translation diffusion NMR measurements a dimerization dissociation constant of 0.1 mM at 4 °C could be determined. The hydrodynamic radius for the monomeric form of Sml1 was determined to be 23.4 Å corresponding to a protein size in between a globular protein and a coil. The dimer formation results in a hydrodynamic radius of 34.4 Å. The observed chemical shifts showed in agreement with previous studies two segments with transient helical structure, residues 4-20 and 60-86, and relaxation studies clearly showed restricted motion in these segments. A spin label attached to C14 showed long range interactions with residues 60-70 and 85-95, suggesting that the N-terminal domain folds onto the C-terminal domain. Importantly, protease degradation studies combined with mass-spectrometry indicated that the N-terminal domain is degraded before the C-terminal region and thus may serve as a protection against proteolysis of the functionally important C-terminal region. The dimer formation was not associated with significant induction of structure, but was found to provide further protection against proteolysis. We propose that this molecular shielding and protection of vital functional structures from degradation by functionally unimportant sites may be a general attribute of other natively disordered proteins.

A soluble α-synuclein construct forms a dynamic tetramer

“…The data presented here suggest that αSyn is like many other proteins whose structure depends on subunit concentration and environmental factors (26). In vitro, and probably in vivo, an equilibrium exists between unfolded monomer, compact oligomer, and (perhaps) amyloid-forming species.…”

“…The formation of a secondary structure in the absence of lipids or micelles is likely in response to intersubunit hydrophobic interactions that drive oligomer formation, as has been observed for other intrinsically disordered proteins (26). Indeed, 1 H, 15 N-HSQC spectra of dilute (50 μM) αSyn construct show clear correlations only for the C-terminal residues, suggesting an increase in dynamic broadening due to an equilibrium between more compact and extended forms of the protein at low concentrations.…”

Temperature‐dependent structural changes in intrinsically disordered proteins: Formation of α‒helices or loss of polyproline II?

“…55,56 Therefore, a large number of theoretical 55,57 and experimental studies 14,18,[58][59][60][61] of IDPs has focused on identifying elements of partially formed secondary structure, particularly a-helices. In experimental studies trying to identify partially formed helices, it is desirable to work under conditions where the helical elements have the highest population.…”

“…12 Despite the structural decoupling between distant regions of IDPs, even highly disordered proteins have been found not to be elongated but still to form specific tertiary and even quaternary interactions. [13][14][15] The heterogeneous nature of IDPs means that their structure is best described by an ensemble of structures containing both long range and local interactions.…”

Biophysical characterization of the isolated C‐terminal region of the transient receptor potential vanilloid 1

“…It has been demonstrated that disrupting mutations in the TRP domain might have a high influence on the activation energy of channel gating, suggesting that TRP domain interactions between monomers are critical for channel gating [16]. Despite the intrinsically disordered structure of some proteins, they are still able of forming oligomers, such as the dynamic dimer formed by the small disordered ribonuclease reductase Sm11 [44], HIV-1 Vif [45], the disordered inhibitory domains of the coiled-coil dimeric kinases [46], the termini of coiled-coil dimeric proteins involved in signaling pathways [47], or the cytoplasmic domain of the f-chain of the T-cell receptor. This latter domain has been demonstrated to form homodimers without a disorder-order transition [48].…”