Intrinsically disordered proteins : A challenge for structural biology.
Around 35% of the human proteome does not fold into stable three-dimensional structures but are either fully disordered, or contain disordered regions of significant length. The inherent flexibility of this class of proteins is essential for their function in a vast range of biomolecular processes such as molecular recognition, signal transduction and transcription and replication. Intrinsically disordered proteins (IDPs) also appear to play major roles in important human diseases such as neurodegenerative disorders and cancer. Despite the importance of characterizing this class of proteins, standard, single conformer-based approaches to structure determination necessarily fail to adequately describe such highly flexible systems. It has therefore become essential to develop new tools for characterizing their rapidly fluctuating conformational behaviour.
Our group is currently developing methods to determine local and long-range structural behaviour in IDPs from experimental NMR data. We are using NMR and complementary biophysical techniques such as molecular simulation and small angle scattering to study in fine detail the presence of local conformational preferences in a number of biologically and medically important IDPs that have until recently remained inaccessible to structural biology. In particular, we have developed the ASTEROIDS (A Selection Tool for Ensemble Representations Of Intrinsically Disordered States) algorithm that selects representative structural ensembles of IDPs on the basis of complementary experimental data sets following the evolution scheme of a genetic algorithm (Figure 1).
Figure 1. Defining representative conformational ensembles of IDPs on the basis of experimental NMR data using the ASTEROIDS approach. Initially a large pool of structures is generated that represents the entire conformational space available to the protein under investigation. Experimental data such as chemical shifts, residual dipolar couplings, paramagnetic relaxation enhancements and small angle X-ray scattering (SAXS) are exploited in a second step to refine this conformational space by selecting sub-ensembles that agree with the experimental data.
We have applied these approaches to study the role of intrinsic disorder in the transcription and replication machinery of measles virus. The genome of measles virus is encapsidated by multiple copies of the nucleoprotein (N), forming helical nucleocapsids of molecular mass approaching 150 Megadalton. N consists of two domains, NCORE (residues 1-400) and the intrinsically disordered C-terminal domain NTAIL (residues 401-525). NTAIL is essential for transcription and replication of the virus via interaction with the phosphoprotein P of the viral polymerase complex. We have reported the first in situ structural characterization of NTAIL in the context of the entire N-RNA nucleocapsid. Using solution NMR spectroscopy, small angle scattering, and electron microscopy, we demonstrate that NTAIL is highly flexible in intact nucleocapsids and we present a model in which the first 50 disordered amino acids of NTAIL are conformationally restricted as the chain escapes to the outside of the nucleocapsid via the interstitial space between successive NCORE helical turns (Figure 2). The model provides a structural framework for understanding the role of NTAIL in the initiation of viral transcription and replication.
Figure 2. Characterization of intrinsic disorder in the measles virus nucleoprotein (N). (A) Superposition of HSQC spectra from isolated NTAIL (blue) and the nucleocapsids (red) showing that NTAIL remains locally disordered in situ. (B) Electron microscopy of measles virus nucleocapsids. (C) Small angle X-ray scattering of intact (red) and cleaved (no NTAIL) (blue) nucleocapsids. (D) Model of NTAIL in the nucleocapsids. NTAIL (red) exfiltrates from the inside to the outside of the nucleocapsids between successive NCORE lobes (green and blue).
In the group, we study a number of IDPs and their complexes with physiological partner proteins. Currently, we study the role of intrinsic disorder in:
- Mitogen-activated protein kinase (MAPK) cell signalling pathways
- IDPs involved in neurodegenerative diseases such as alpha-synuclein and tau
M.R. Jensen, M. Zweckstetter, J.R. Huang and M. Blackledge
Exploring free-energy landscapes of intrinsically disordered proteins at atomic resolution using NMR spectroscopy.
Chem. Rev. 114, 6632-6660 (2014).
M. Schwalbe, V. Ozenne, S. Bibow, M. Jaremko , L. Jaremko, M. Gajda, M.R. Jensen, J. Biernat, S. Becker, E. Mandelkow, M. Zweckstetter* and M. Blackledge*
Predictive atomic resolution descriptions of intrinsically disordered hTau40 and alpha-synuclein in solution from NMR and small angle scattering.
Structure 22, 238-249 (2014).
G. Communie, J. Habchi, F. Yabukarski, D. Blocquel, R. Schneider, N. Tarbouriech, N. Papageorgiou, R.W. Ruigrok, M. Jamin, M.R. Jensen*, S. Longhi* and M. Blackledge
Atomic resolution description of the interaction between the nucleoprotein and phosphoprotein of Hendra virus.
PLoS Pathog. 9, e1003631 (2013).
V. Ozenne, R. Schneider, M. Yao, J.R. Huang, L. Salmon, M. Zweckstetter, M.R. Jensen and M. Blackledge
Mapping the potential energy landscape of intrinsically disordered proteins at amino acid resolution.
J. Am. Chem. Soc. 134, 15138-15148 (2012).
M. R. Jensen, G. Communie, E. A. Ribeiro, N. Martinez, A. Desfosses, L. Salmon, L. Mollica, F. Gabel, M. Jamin, S. Longhi, R. W. H. Ruigrok and M. Blackledge
Intrinsic disorder in measles virus nucleocapsids.
Proc. Natl. Acad. Sci. (U.S.A.) 108, 9839-9844 (2011).
M. R. Jensen*, L. Salmon, G. Nodet and M. Blackledge*
Defining conformational ensembles of intrinsically disordered and partially folded proteins directly from chemical shifts.
J. Am. Chem. Soc. 132, 1270-1272 (2010).