High-resolution structural characterization of Noxa, an intrinsically disordered protein, by microsecond molecular dynamics simulations

Abstract

High-resolution characterization of the structure and dynamics of intrinsically disordered proteins (IDPs) remains a challenging task. Consequently, a detailed understanding of the structural and functional features of IDPs remains limited, as very few full-length disordered proteins have been structurally characterized. We have performed microsecond-long molecular dynamics (MD) simulations of Noxa, the smallest member of the large Bcl-2 family of apoptosis regulating proteins, to characterize in atomic-level detail the structural features of a disordered protein. A 2.5-μs MD simulation starting from an unfolded state of the protein revealed the formation of a central antiparallel β-sheet structure flanked by two disordered segments at the N- and C-terminal ends. This topology is in reasonable agreement with protein disorder predictions and available experimental data. We show that this fold plays an essential role in the intracellular function and regulation of Noxa. We demonstrate that unbiased MD simulations in combination with a modern force field reveal structural and functional features of disordered proteins at atomic-level resolution.

Structural Mechanism for Regulation of Bcl-2 protein Noxa by phosphorylation

“…4 ), indicating that Noxa is intrinsically disordered, in agreement with circular dichroism experiments 5 . However, MD simulations suggest that the structural ensemble of unphosphorylated Noxa does not consist of several dissimilar conformations that exchange rapidly in the microsecond time scale, as shown previously for other disordered proteins in solution 9 10 . Instead, Noxa folds into a relatively well-defined structure in solution, which consists of a β-loop-β motif (residues Ala19 to Lys41) flanked by two disordered segments at the N-terminus (residues Met1-Pro18) and the C-terminus (residues Asn44-Thr54), shown in Fig.…”

“…The MD simulations in our study suggested that human Noxa is intrinsically disordered but rather than assuming several conformations, as is the case with other disordered proteins 9 , Noxa appears to fold into a β-loop-β motif that encompasses the hydrophobic BH3 domain and is flanked by disordered N- and C-terminal segments 10 . While the amplitude of the backbone dynamics of the N-terminus showed a substantial decrease upon phosphorylation of Ser13, there was no detectable change in the backbone dynamics of the BH3 domain surrounding residue Phe32, suggesting that the decreased flexibility of the N-terminus was the result of the salt-bridge network around the phosphorylation site, rather than a well-defined secondary structure.…”

“…Analysis of the time-dependent changes of the secondary structure revealed that the initial structure of unphosphorylated Noxa, consisting of a random coil and two helical segments at the N- and C-terminus of the protein, folds into a stable β-loop-β motif in less than a microsecond ( Fig. 4a,c , also described in 10 ). Furthermore, we found that the β-loop-β motif of unphosphorylated Noxa remained stable for the rest of the simulation period.…”

Site-directed spectroscopy of cardiac myosin-binding protein C reveals effects of phosphorylation on protein structural dynamics

“…These results also are consistent with findings that the MyBP-C motif is intrinsically disordered (15,16) and that phosphorylation elicits a structural change to a more compact tertiary protein structure (22). To confirm further the nature of the motif's intrinsic disorder and the specific structural changes associated with the unphosphorylated and phosphorylated states, we next harnessed recent advances in MD simulations (23,24) and applied these approaches to cMyBP-C.…”

“…A complete description of the construction of the motif and MD methods is supplied in the SI Appendix, SI Text. This in silico approach has been useful in previous studies of muscle protein structural dynamics to complement studies of muscle contraction (23) and of other intrinsically disordered proteins (24). Here, we performed two independent MD simulations to determine the localized effects of cMyBP-C phosphorylation (Figs.…”