Idps Evade The Structure-function Paradigm

Folded proteins have a defined three dimensional structure determined solely by its primary sequence. This principle holds a central place in biology and is a direct predecessor of the so called ‘structure-function’ paradigm which suggests a well-defined 3D structure encodes a specific function and thus by extrapolation a well-defined structure is a necessity for biological functionality1. There has been substantial evidence for this dogma based on the ever increasing numbers of structure in Protein Data Bank (PDB). For example a striking structural similarity of enzymes and often correspondence of structural footprints with evolution of molecular function support this dogma2,3 .

However there has been increasing evidences that a significant number of proteins remain unfolded in physiological conditions4,5,6,7. Such proteins adopt a multitude of rapidly interconverting structures instead of one predominant structure in contrast to folded proteins. Such proteins or such regions in a protein are thus named intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs) respectively8,9. For sake of simplicity I will henceforth refer to both IDRs and IDPs as IDPs. After the human genome project advanced protein structure prediction algorithms10,11,12 soon led to the realization that a large part of the human proteins contains disordered region reaching up to ~40% 13. Protein synthesis is an energy expensive process for a living cell14 and the discussions above indeed prompt us to think if functionality was solely a fallout of structure why would a cell expend its resources into synthesizing IDPs; suggesting there must be ‘method in the madness’. It has been shown eukaryotes/complex life forms have significantly larger disorder in their proteome compared to elementary organisms such as bacteria which typically have less than 10% of disordered proteins15. This indicates IDPs might be crucial to several complex functionalities necessary for cellular function16. As is discussed below, an IDP has certain sequence idiosyncrasies that encodes disorder and subsequently a function. Hence IDPs evade the classical structure-function paradigm and an alternate paradigm has to be invoked to comprehend IDP function where the sequence encodes disorder which encodes function 17 (Fig 1.1).

Folded proteins have certain sequence characteristics that helps them attain a folded globular structure. These sequence features includes a certain fraction of hydrophobic residues that favor a formation a collapsed state where the hydrophobic side-chains are buried and allows secondary structure elements to form. Also there is a degree to which folded proteins can tolerate unbalanced charge residues as these drives expansion of the protein due to electrostatics. A comprehensive sequence analysis of numerous IDPs by Uversky et. al. showed a clear trend in the sequence composition of IDPs, IDPs systematically have more unbalanced charges and less hydrophobic residues compared to folded proteins18. This analysis even led to an empirical relation that leads to a clear separation of the sequence space into folded and unfolded regions. (Fig 1.2). This suggests the presence of a threshold point in terms of the mean hydrophobicity relative to mean net charge after which proteins fail to fold18.

IDPs are known to bind several biological targets. The binding interactions of IDPs classified into two categories which are: 1) Coupled folding-binding and 2) Fuzzy complex formation (Fig 1.3) 19,20,21,22,23. Coupled folding-binding mechanisms involve folding transition of the IDP where in the bound state the IDP adopts a folded structure. In such a binding mechanism the binding partner offers structure forming interactions which the IDP otherwise lacks allowing it to adopt a folded structure in the context of the bound complex. Coupled folding-binding mechanisms again come in two flavors; induced fit and conformational selection24. In case of an induced fit mechanism the entire disordered ensemble can bind the partner and the folding transition occurs after binding.

In contrast, for a conformational selection, a binding prone minor conformation in the disordered ensemble bearing significant resemblance to the bound state is selected out of equilibrium ensemble of structures by the binding partner. This causes the equilibrium to reestablish producing again some binding competent conformers which again binds the partner and the process continues populating the bound state. Thus for a conformational selection the folding or structural transition primarily happens prior to the binding event.

Fuzzy complexes are formed when the disordered ensemble retains its disorder after binding the partner, without undergoing any large conformational changes. In several cases these involves multivalent interactions between the IDPs and the partner proteins where multiple small binding epitopes on the IDP serves as points of anchorage with the binding partner 25,26. IDPs often contain short linear motifs (SLiMs) 27,28 which serves as binding epitopes to target and multiple copies of such SLiMs can be present allowing the IDP to engage in multivalent interactions with the partner; such multivalency leads to an overall increase in binding affinity but at the same time the small size of the epitopes allows the binding without any major conformational change of the disordered ensemble.

It has now been clear that IDPs acts as key players in cellular regulation and function. Thus it is fair to say IDPs form a cornerstone in eukaryotic cell biology as we know it.

Sequence analysis reveals a very high content of long disordered regions in proteins involved in transcription regulation like transcriptional factors, transcription co-activators and trans-activators, and chromosomal proteins like linker histones29,30,31. The disordered regions play many roles in such cases32. IDPs can constitute linker regions between recognition motifs/domains in molecules acting as molecular hubs and facilitate allosteric interactions between distant sites. One classic example is the CBP/p300 which harbors multiple sites for transcription factor separated by IDPs33. Such a scaffold offers the possibility of allosteric and cooperative interactions. Interestingly several of the transcription factors that bind CBP/p300 are themselves disordered as well33. IDPs due to their promiscuous binding behavior can also themselves act as nodal points in regulatory pathways and encode regulatory and signaling function; a prime example being the transcription factor p53 that forms a disordered hub having ~700 binding partners34. IDPs having large surfaces are susceptible to posttranslational modifications which happen to be a facile way to achieve dynamic regulation. Such regulatory mechanisms are widely seen in IDPs functioning at all stages of transcription regulation starting, ranging from transcription factors regulated by phosphorylation such as p53 to dynamic regulation of linker histones via post translational modifications in their disordered tails35, 36.

Owing to their disorder, physical chemistry of IDPs in solution, such as collapse, scaling and phase behavior, can be explained to a large extent, based on the basic tenets of polymer physics37. Hence like polymers in solution IDPs can undergo liquid-liquid phase separation forming a concentrated phase surrounded by a dilute phase (Fig 1.4)38. In the recent years the discovery that many cellular organelles such as stress granules, P-bodies, nucleoli, Cajal-bodies, etc. are formed by liquid-liquid phase separation often driven by IDPs have led to fundamental new insights in cellular organization.. In fact the permeability of the nuclear pore complex (NPC), is also hypothesized to be formed by phase separated IDPs (discussed in greater details in the following sections)39,40,41,42,43, 44. Thus IDPs play a crucial role in cellular organization by forming different membraneless organelles, at different physiological cues via liquid-liquid phase separation, which serves as crucibles for several biochemical reactions which are otherwise not feasible in dilute concentrations.

1.2 Nucleocytoplasmic transport and the nuclear pore complex

1.2.1 The nuclear pore complex: Structure, function and the nucleocytoplasmic transport pathway

A cornerstone of eukaryotic cell-biology is the compartmentalization of cellular components. A eukaryotic cell is primarily compartmentalized into two components the nucleus, which is a double membrane bound enclosure that harbors DNA/genetic information, surrounded by the cytosol, which constitutes an aqueous milieu containing the essential bio-molecules required for cellular function. Transport of molecules from the cytoplasm to the nucleus and vice-versa is crucial for cellular homeostasis. The nuclear envelope is decorated with several nuclear pore complexes (NPCs), which are giant macromolecular complexes that serve as the sole conduit for transport of molecules across the nuclear envelope between the nucleus and cytoplasm45.

The NPC with a size of 120 MDa is the largest macromolecular complex in the eukaryotic cell. Since long the NPC had been known in the literature to have a ring like architecture with an apparent 8 fold rotational symmetry46. Recent developments in cryo-EM have resulted in visualization of the NPC structure with unprecedented details47,48,49 . The basic framework of the NPC structure includes three rings the inner ring, the nuclear ring and the cytoplasmic ring. The inner ring lies at the juncture of the outer and inner nuclear membrane and anchors the nuclear and cytoplasmic rings towards the nuclear and cytoplasmic sides respectively. The nuclear and the cytoplasmic rings bear extensions known as the cytoplasmic filaments and the nuclear basket respectively. The NPC structure is formed by 30 different proteins known as nucleoporins (Nups) which are present in multiple copies; the copy numbers always being a multiple of 847,48. A striking feature of all cryo-EM maps of the NPC is big central hole, ~27 nm in diameter at the narrowest, which might mislead one to think that the NPC really constitutes a hole in the nuclear envelope allowing molecular exchange. In reality the apparent hole is filled with a very high density IDPs. About 1/3 of Nups bears IDP extensions and are known as FG-Nucleoporins (FG-Nups)50,51. These disordered FG-Nups extend from the NPC scaffold structure and forms the permeability barrier of the NPC.

The nucleocytoplasmic transport is a highly regulated process; the regulation being important for maintenance of cellular homeostasis. Key to the regulatory function of the NPC is the permeability barrier formed by the disordered FG-Nups. The NPC acts as a size selective filter that allows free passage of cargoes below a size threshold of ~40kDa. Above this size threshold transport across the NPC necessities recognition of the cargo by molecules known as nuclear transport receptors (NTRs) which chaperones the molecule across the NPC. Interaction between the disordered FG-Nups and the NTRs forms the basis of NTR aided nucleocytoplasmic transport of cargoes52.

While the translocation across the permeability barrier of the pore does not require energy but the directionality of export or import across the NPC is maintained by a RanGTP/GDP gradient formed across the NPC52. Ran is a small GTPase; RanGTP is in excess on the nuclear side and RanGDP on the cytoplasmic side. The NTRs depending on whether they are involved in import (importins) or export (exportins) are differentially regulated by RanGTP and this governs the directionality of the transport. Nucleocytoplasmic transport involves three key steps which are recognition of the cargo by NTR, translocation of the NTR/cargo complex across the permeability barrier and release of the cargo from the NTR on either the nuclear or cytoplasmic side.