The Emerging Disorder-Function Paradigm

It’s rare to find a paper that connects all of the diverse areas of research of OPIG, but “The rules of disorder or why disorder rules” by Gsponer and Babu (2009) is one such paper. Protein folding, protein-protein interaction networks, protein loops (Schlessinger et al., 2007), and drug discovery all play a part in this story. What’s great about this paper is that it gives numerous examples of proteins and the evidence supporting that they are partially or completely unstructured. These are the so-called intrinsically unstructured proteins or IUPs, although more recently they are also being referred to as intrinsically disordered proteins, or IDPs. Intrinsically disordered regions (IDRs) “are polypeptide segments that do not contain sufficient hydrophobic amino acids to mediate co-operative folding” (Babu, 2016).

Such proteins contradict the classic “lock and key” hypothesis of Fischer, and challenge the prevalent notion exemplified by Anfinsen’s hypothesis (Anfinsen, 1973) that under normal physiological conditions, all proteins fold into a single 3D shape determined by their amino acid sequence and having the minimum Gibbs free energy. IDPs clearly demonstrate that function can follow both form and lack of form. From the abstract,

The finding that a large fraction of proteins (over 30%) in eukaryotic cells lack a unique three-dimensional structure but are functional has forced the scientific community to review its understanding of the structure–function paradigm. The involvement of many of these intrinsically unstructured proteins (IUPs) in intracellular signalling and regulatory processes as well as their central positioning (as interaction hubs) in recently mapped protein interaction networks is particularly intriguing. Here, we review the functional and structural properties of IUPs such as (i) their facilitated regulation via diverse post- translational modifications of specific amino acids (ii) scaffolding and recruitment of different binding partners in space and time via the ‘‘fly-casting’’ mechanism, through peptide motifs and by coupling folding with binding and (iii) conformational variability and adaptability. All of these properties allow these proteins to hold key positions in cellular organisation and regulation which in turn make them tractable as drug targets. In addition, we discuss how such properties, individually and in combination, facilitate combinatorial regulation and re-use of the same component in multiple biological processes.

Amino acid composition is believed to be one of the indicators of unstructured regions in a protein: a prevalence of polar and charged amino acids and a lack of hydrophobic amino acids create the conditions to frustrate the formation of a single stable fold: no stable hydrophobic core can emerge, and the repulsion of like charges and numerous interactions with water ensure a single conformation is not selected.

The lack of structure and high conformational flexibility for such disordered regions facilitates access by post-translational machinery to molecular recognition features (MoRFs) and short peptide motifs, and these PTMs (post-translational modifications) themselves modify the biophysical properties which in turn can affect their ability to fold. Another advantage of IDPs over structured proteins is a much larger interaction surface that facilitates the initial contact with binding partners. There are also examples of intrinsically disordered protein regions that fold only upon encountering their binding partners, and some that act as “scaffolding” to help multimeric proteins to assemble, such as ALL-1 involved in transcriptional regulation, and BRCA1, which regulates the formation of a large complex involved in DNA repair.

Fig. 1. Properties of IUPs that are pivotal to their central role in protein–protein interaction (PPI) networks (shown in the center with nodes representing proteins and links representing physical interactions). Hub proteins in PPI networks are enriched in intrinsically unstructured segments. The specific properties of these unstructured segments facilitate interactions with many diverse partners: (A) conformational flexibility facilitates access to enzymes and effectors that introduce or remove post-translational modifi- cations and read the post-translational code, respectively. Specific post-translational modifications permit combinatorial regulation and the re-use of the same components in multiple biological processes. (B) The availability of molecular recognition features and short linear peptide motifs within the unstructured segments enables the fishing for (‘‘fly- casting’’) and gathering of different partners. The interaction specificity provided by these motifs is essential for the precise assembly of macromolecular machines in space and time. (C) Conformational variability enables a nearly perfect moulding to fit the binding surfaces of very diverse partners. Context-dependent folding of an intrinsically unstructured segment can be used to activate or inhibit signalling processes that can have completely orthogonal outcomes.

Fig. 1. Properties of IUPs that are pivotal to their central role in protein–protein interaction (PPI) networks (shown in the center with nodes representing proteins and links representing physical interactions). Hub proteins in PPI networks are enriched in intrinsically unstructured segments. The specific properties of these unstructured segments facilitate interactions with many diverse partners: (A) conformational flexibility facilitates access to enzymes and effectors that introduce or remove post-translational modifications and read the post-translational code, respectively. Specific post-translational modifications permit combinatorial regulation and the re-use of the same components in multiple biological processes. (B) The availability of molecular recognition features and short linear peptide motifs within the unstructured segments enables the fishing for (‘‘fly-casting’’) and gathering of different partners. The interaction specificity provided by these motifs is essential for the precise assembly of macromolecular machines in space and time. (C) Conformational variability enables a nearly perfect moulding to fit the binding surfaces of very diverse partners. Context-dependent folding of an intrinsically unstructured segment can be used to activate or inhibit signalling processes that can have completely orthogonal outcomes.

Another key concept covered by this Gspner & Babu is fly-casting:

The fly-casting model postulates that IUPs have a greater ‘‘capture radius’’ and increased intermolecular association rates because they are less compact than structured proteins… A binding site attached to a flexible chain could be compared to a hook on a fishing rod, which allows for the sampling of large solution volumes and the ‘‘fishing’’ of targets therein. A good example of this involves the membrane-embedded, voltage-activated potassium channel (Kv), which mediates the generation and shaping of action potentials in neuronal cells.

Often the experimental evidence cited in this review has come from the efforts of Jane Dyson and Peter Wright at The Scripps Research Institute, who have been working on IDPs since the 1990s (Wright & Dyson, 2015), and who recently announced “The Human Dark Proteome” initiative“, which aims to understand and characterize the estimated 30% of the human proteome that lack a single 3D fold. Diseases such as cancer, diabetes, infectious disease, cardiovascular disease, and neurodegenerative disorders, and even some viruses, involve either IDPs or proteins with IDRs. In Parkinson’s disease, mutations within the IDR of α-synuclein increase its propensity for aggregation; it “exhibits characteristics of a partially folded protein when bound to a micelle” as in PDB entry 2KKW (Rao et al.):

Meanwhile, in another PDB entry, 2N0A, for exactly the same protein its authors point out that “misfolded α-synuclein amyloid fibrils are the principal components of Lewy bodies and neurites, hallmarks of Parkinson’s disease (PD)”, and go on to present “a high-resolution structure of an α-synuclein fibril, in a form that induces robust pathology in primary neuronal culture”:

DOI: 10.1038/nsmb.3194

DOI: 10.1038/nsmb.3194

After X-ray crystallography has just celebrated just over a century since the Nobel prize awarded to the Braggs, with the recent rise of high-resolution cryo-EM and its ability to reveal multiple conformational states of molecular machines, and the continuing development of ever more sophisticated NMR techniques, it is time to revise our picture of proteins as static molecular shapes, and embrace their structural complexity and nuance.

References

Anfinsen, C. B. (1973). “Principles that govern the folding of protein chains.” Science, 181(4096): 223-230.

Babu, M. M. (2016). “The contribution of intrinsically disordered regions to protein function, cellular complexity, and human disease.” Biochem Soc Trans, 44(5): 1185-1200. 10.1042/BST20160172.

Huang, J., S. Rauscher, G. Nawrocki, T. Ran, M. Feig, B. L. de Groot, H. Grubmuller and A. D. MacKerell, Jr. (2016). “CHARMM36m: an improved force field for folded and intrinsically disordered proteins.” Nat Methods. 10.1038/nmeth.4067.

Gsponer, J. and M. M. Babu (2009). “The rules of disorder or why disorder rules.” Prog Biophys Mol Biol, 99: 94-103. 10.1016/j.pbiomolbio.2009.03.001.

Rao, J. N., C. C. Jao, B. G. Hegde, R. Langen and T. S. Ulmer (2010). “A combinatorial NMR and EPR approach for evaluating the structural ensemble of partially folded proteins.” J Am Chem Soc, 132(25): 8657-8668. 10.1021/ja100646t.

Schlessinger, A., J. Liu and B. Rost (2007). “Natively unstructured loops differ from other loops.” PLoS Comput Biol, 3(7): e140. 10.1371/journal.pcbi.0030140.

Tuttle, M. D., G. Comellas, A. J. Nieuwkoop, D. J. Covell, D. A. Berthold, K. D. Kloepper, J. M. Courtney, J. K. Kim, A. M. Barclay, A. Kendall, W. Wan, G. Stubbs, C. D. Schwieters, V. M. Lee, J. M. George and C. M. Rienstra (2016). “Solid-state NMR structure of a pathogenic fibril of full-length human alpha-synuclein.” Nat Struct Mol Biol, 23(5): 409-415. 10.1038/nsmb.3194.

Wright, P. E. and H. J. Dyson (2015). “Intrinsically disordered proteins in cellular signalling and regulation.” Nat Rev Mol Cell Biol, 16(1): 18-29. 10.1038/nrm3920.