Structural biology of intrinsically disordered proteins: Revisiting unsolved mysteries

Accepted Manuscript Structural Biology of Intrinsically Disordered Proteins: Revisiting Unsolved Mysteries Alexander B. Sigalov PII:

S0300-9084(16)30033-5

DOI:

10.1016/j.biochi.2016.03.006

Reference:

BIOCHI 4954

To appear in:

Biochimie

Received Date: 22 February 2016 Accepted Date: 17 March 2016

Please cite this article as: A.B. Sigalov, Structural Biology of Intrinsically Disordered Proteins: Revisiting Unsolved Mysteries, Biochimie (2016), doi: 10.1016/j.biochi.2016.03.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Mini-review Structural Biology of Intrinsically Disordered Proteins: Revisiting Unsolved

SignaBlok, Inc., Shrewsbury, MA, USA

*Corresponding author: Tel.: +1 203 505 3807

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E-mail address: [email protected]

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Alexander B. Sigalov

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Mysteries

Keywords: intrinsically unstructured proteins; T cell receptor; dimerization; protein-

Highlights:

Long-standing contradictions in the field of intrinsically disordered proteins are revisited.

Intrinsically disordered proteins demonstrate two different modes of lipid-binding

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protein interactions; lipid-protein interactions; no disorder-to-order transition

activity depending on the cell membrane model.

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Intrinsically disordered proteins can use "coupled binding and folding" or "binding

without folding" mechanisms upon interaction with their lipid and protein partners. •

Unusual biophysics of intrinsically disordered proteins contributes to drug discovery.

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Abstract

The emergence of intrinsically disordered proteins (IDPs) has challenged the classical

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protein structure-function paradigm by introducing a new paradigm of “coupled binding and folding”. This paradigm suggests that IDPs fold upon binding to their partners.

Further studies, however, revealed a novel and previously unrecognized phenomenon of

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"uncoupled binding and folding" suggesting that IDPs do not necessarily fold upon interaction with their lipid and protein partners. The complex and often unusual

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biophysics of IDPs makes structural characterization of these proteins and their complexes not only challenging but often resulting in opposite conclusions. For this reason, some crucial questions in this field remain unsolved for well over a decade. Considering an important role of IDPs in cellular regulation, signaling and control in

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health and disease, more efforts are needed to solve these mysteries. Here, I focus on two long-standing contradictions in the literature concerning dimerization and membranebinding activities of IDPs. Molecular explanation of these discrepancies is provided. I

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also demonstrate how resolution of these critical issues in the field of IDPs results in our expanded understanding of cell function and has multiple applications in biology and

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medicine.

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One of the tenets of modern structural biology is that the three-dimensional protein structure determines the protein function. However, over the last two decades or so, the emergence of intrinsically disordered proteins (IDPs), a class of proteins that fail to adopt

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a well-defined folded structure under physiological conditions, has challenged this notion [1-4]. Instead, studies of IDPs triggered the development of another paradigm known as "coupled binding and folding". This paradigm assumes that in order to perform their

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diverse biological functions, IDPs adopt folded structures upon binding to their targets

[5-7]. This subject has been previously addressed in many comprehensive reviews and

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primary research articles [5-11]. Recently, a new paradigm of "uncoupled binding and folding" has been introduced (reviewed in [9]). Intriguingly, structural studies of IDPs [12-20] reveal unusual biophysical phenomena that substantially complicate our understanding of the structural biology of IDPs. In addition, these studies raise several

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important questions concerning the structure of IDPs in their complexes with protein and lipid partners. Together, these findings suggest that the complex and unusual biophysics of IDPs makes structural characterization of these proteins and their complexes not only

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challenging but often resulting in opposite conclusions. Considering an important role of IDPs in cellular regulation, signaling and control in health and disease [1, 6, 8, 10, 21-

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36], more efforts are needed to solve outstanding contradictions in the field of IDPs. Here, in order to discuss two long-standing contradictions and discrepancies in the

literature concerning the structural features and the biophysical activities of IDPs, I will use an example of the cytoplasmic signaling domains of multichain immune recognition receptors (MIRRs) that represent a new family of IDPs [37-39]. Among the members of this family are the cytoplasmic domains of the T cell receptor (TCR) ζ and CD3ε

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signaling subunits (ζcyt and CD3εcyt, respectively) and the γ signaling subunit of FcεRI receptor (FcεRIγcyt) [9, 13, 19].

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1. Lipid-binding activity of IDPs: Do IDPs fold upon binding to the cell membrane?

Cytoplasmic intrinsically disordered regions (IDRs) of MIRR signaling subunits all

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are characterized by the presence of one or more copies of an immunoreceptor tyrosinebased activation motif (ITAM) that contains two tyrosine residues. Upon ligand binding,

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these residues become phosphorylated, staring the intracellular signaling cascade. Thus, intrinsically disordered ζcyt, CD3εcyt, and FcεRIγcyt are in a close proximity to the cell membrane and play a crucial role in immune signaling. This makes the question as to whether or not membrane binding of these IDPs promotes the folding of their ITAMs and

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thereby leads to inaccessibility of the ITAM tyrosines for phosphorylation of fundamental importance. However, not only detailed information regarding the lipid binding activity of ITAM-containing IDRs is still lacking but also all available data are

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strikingly contradictory (Fig. 1).

In studies by Aivazian and Stern from 2000 [19], the authors used a combination of

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fluorescence and circular dichroic (CD) spectroscopy and found that the ζcyt ITAMs undergo α-helical folding transition in the presence of acidic detergents and phospholipids: lysomyristoyl-phosphatidylglycerol (LMPG) micelles and dimyristoylphosphatidylglycerol (DMPG) vesicles, respectively. Using an in vitro kinase assay, the authors also demonstrated that the presence of LMPG micelles prevents ITAM phosphorylation. Based on these findings, the authors concluded that this lipid binding-

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dependent folding transition is a conformational switch that regulates TCR triggering in vivo [19]. Further structural studies performed using CD and multidimensional nuclear magnetic resonance (NMR) spectroscopy confirmed the formation of partial helices

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within the ζcyt ITAM motifs in the presence of LMPG micelles [20]. Later, in studies by Xu et al. [12], similar findings were reported for CD3εcyt. In these studies, DMPG

vesicles, bicelles composed of 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), and

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dihexanoyl-phosphatidylcholine (DHPC) were used to model the cell membrane. It has

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been found that similarly to ζcyt [19-20], CD3εcyt binds to acidic phospholipid molecules and that electrostatic interactions between the N-terminal basic residues of CD3εcyt and the polar lipid groups play an important role in this binding [12]. An in vitro kinase assay, CD and NMR spectroscopy revealed that upon binding to these molecules, the protein folds with partial helix formation within the CD3εcyt ITAM region and sequestration of

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two ITAM tyrosines into the hydrophobic lipid area, such that these residues become inaccessible for phosphorylation by tyrosine kinases [12]. Fluorescence resonance energy transfer experiments performed using Jurkat cells found a close interaction of CD3εcyt

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with the cell membrane [12]. Together, these findings led the authors to conclude that α-

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helical folding transition of the CD3εcyt ITAM region upon membrane binding of CD3εcyt regulates TCR triggering in vivo and that “sequestration of key tyrosines into the lipid bilayers represents a previously unrecognized mechanism for control of receptor activation” [12].

Thus, based on partial α-helix formation of the ITAM regions observed for both CD3εcyt [12] and ζcyt [19] upon binding to micelles, bicelles or model membranes, the socalled "conformational model of T cell activation" was suggested [12, 19, 40]. The model

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suggests that a conformational α-helical folding transition upon binding of ITAMcontaining IDRs to the cell membrane is a key regulator for TCR signaling in vivo. However, in 2006 [14], no disorder-to-order transition was found for ζcyt, CD3εcyt,

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and FcεRIγcyt upon binding to POPG vesicles, suggesting a principally different, "binding without folding" mode of their lipid binding activity (Fig. 1). These findings were

surprising because the stable POPG bilayers should better mimic the cell membrane than

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micelles, DMPG vesicles or bicelles used in other studies [12, 19-20]. In addition, while the clusters of N-terminal basic residues in CD3εcyt and ζcyt are important for binding of

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these IDPs to acidic phospholipid bilayers, the ITAM residues are not involved in these interactions [9, 12, 15]. These data indicate that the ITAM tyrosines in POPG-bound ζcyt, CD3εcyt, and FcεRIγcyt can remain accessible for phosphorylation. This questions a physiological role of the ITAM α-helical folding that was suggested for CD3εcyt [12] and

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ζcyt [19-20] in the relevant conformational models of T cell activation. In addition, the fully phosphorylated intrinsically disordered ζcyt has a net charge of -5.5 but still binds to acidic phospholipid bilayers with no disorder-to-order transition observed upon this

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binding [14]. This suggests that the clusters of basic residues rather than the overall net

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charge drive the interaction of fully phosphorylated ζcyt with POPG lipid bilayers. Together, these data [14] challenge one of the main assumptions of a conformation model that binding of ITAM-containing IDRs to the cell membrane affects ITAM phosphorylation. Surprisingly, despite the main results and conclusions of our studies [14] oppose the results, conclusions and interpretations reported in the studies by Aivazian and Stern [19] and by Xu et al. [12], our data were later largely misinterpreted

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and used by many authors as evidence to support the conformational models of receptor triggering [41-49]. Thus, most of the conclusions made in the studies by Aivazian and Stern [19] and by

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Xu et al. [12] are based on the assumption that lipid binding of CD3εcyt and ζcyt is

necessarily accompanied by α-helical folding of their ITAM regions, which should make the ITAM tyrosines inaccessible for phosphorylation. However, our current

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understanding suggests that first, there are two different mechanisms involved in this process: 1) electrostatic interactions between the negatively charged polar groups of

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acidic phospholipids and the clusters of positively charged CD3εcyt and ζcyt residues; and 2) hydrophobic interactions between the CD3εcyt and ζcyt ITAMs and lipid tails [14-15]. And most importantly, these two processes may or may not be coupled. Further, not only LMPG micelles [19] or DMPG vesicles [19] but also trifluoroethanol [50] can induce α-

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helical folding of the ζcyt ITAM regions. This clearly indicates that hydrophobic rather than electrostatic interactions are necessary to promote disorder-to-order transition. Similarly to ζcyt, α-helical structure formation within the CD3εcyt ITAM region can be

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induced by such principally different lipid systems as LMPG micelles, DMPG vesicles

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and POPG/DHPC bicelles [12]. In addition, in the study by Xu et al. [12], NMR findings revealed structural differences in the CD3εcyt α-helices induced by LMPG micelles or POPG/DHPC bicelles. This suggests that lipid binding mechanisms depend upon the lipid system used. Interestingly, the ITAM residues were found not to be important in mediating binding of CD3εcyt to acidic phospholipid bilayers [12]. This further confirms that binding of intrinsically disordered CD3εcyt and ζcyt to lipids and folding of these

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proteins upon this binding are mediated by different types of interactions and may not be necessarily coupled. Importantly, contradictory data from structural studies of lipid binding activity of

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immune signaling-related cytoplasmic IDRs [12, 14-15, 19] resulted in the open

discussion [51-52] whether these IDRs fold upon binding to the cell membrane in vivo, thereby making the ITAM tyrosines of TCR signaling subunits inaccessible to tyrosine

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kinases and preventing TCR activation as was suggested for CD3εcyt [12] and ζcyt [19].

Resolution of this contradiction is of both fundamental and clinical importance because if

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the "conformational switch" model of TCR triggering [12, 19, 40] is correct, we could in principle, control TCR-mediated T cell activation by modulating the lipid binding and folding activity of TCR-related cytoplasmic IDRs.

A molecular explanation for these contradictions and discrepancies was provided in

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our studies from 2009 and 2011 [9, 15] where we demonstrated that detergents and/or some lipid systems cannot be used as appropriate models to study IDP binding to the cell membrane. We found that depending on the model used, there are two different modes of

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binding of intrinsically disordered ζcyt, CD3εcyt, and FcRγcyt to acidic phospholipids (Fig. 1) [9, 15]. One mode, "coupled binding and folding", is characteristic for micelles and

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unstable lipid bilayers (for example, bilayers of DMPG vesicles that become unstable upon binding to IDP). In another mode, when IDP binds to stable lipid bilayers (for example, bilayers of POPG vesicles), binding of the protein is not accompanied by its folding. Further, while the initial binding driven by electrostatic interactions between basic amino acid stretches in the regions outside of the ITAMs and the polar heads of acidic phospholipids is necessary and involved in both interaction modes, the ITAM

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residues may (mode I) or may not (mode II) contribute to binding depending on the lipid model used. In the IDP/micelle systems (e.g., LMPG micelles), involvement of ITAMs in the IDP-lipid binding (mode I), driven by hydrophobic interactions between ITAMs and

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detergent tails promotes folding of ITAMs and makes the ITAM tyrosines inaccessible for phosphorylation [9, 15, 19-20]. In the IDP/vesicle systems (e.g., DMPG and POPG

vesicles), initial IDP binding driven by electrostatic interactions may (mode I) or may not

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(mode II) induce vesicle fusion and rupture, depending on the lipid bilayer integrity and stability (Fig. 1) [9, 15]. Disruption of the lipid bilayers results in hydrophobic

the ITAM α-helixes (Fig. 1).

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interactions between ITAMs and lipid tails that promote formation and stabilization of

Thus, these studies [9, 14-15] demonstrate, for the first time, that diametrically opposite results and conclusions can be obtained in the biophysical studies that address

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binding of proteins (especially, IDPs) to the cell membrane and its physiological relevance. Importantly, as shown [9, 14-15], the use of lipid vesicles of the same size and surface charge but with different lipid bilayer stability (e.g., small or large unilamellar

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vesicles of DMPG and POPG) may lead to opposite data. These findings not only highlight the importance of the cell membrane model used in protein-lipid binding studies

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but also underline that ensuring the integrity of model lipid bilayers upon protein binding is a critical and necessary step in these studies, especially in studies of IDPs. Instability of the cell membrane models used [12, 19-20] in combination with the data generated by using physiologically irrelevant α-helix promoters [50] suggest that α-helical folding of ITAMs observed in the presence of these agents [12, 19-20, 50] does not likely play a

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role in vivo. This also questions a physiological significance of a general "coupled binding and folding" paradigm in the context of binding of IDPs to their lipid partners. To summarize, in light of the discussion above, the choice of an appropriate model to

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mimic the cell membrane plays a crucial role when studying IDP-lipid interactions and

protein-lipid interactions in general. The studies also highlight how substantial is critical evaluation of the data accumulated to date in this field for our improved perception and

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understanding of fundamentally and clinically important transmembrane signal

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transduction events in vivo.

2. Dimerization of IDPs: Can IDPs dimerize and stay unfolded upon dimerization?

Biophysical evidences of the existence of specific homo-interactions between IDP

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molecules (Fig. 2) were first reported in 2004 [13], when the ITAM-containing members of immune signaling-related family of IDPs including ζcyt, CD3εcyt, CD3δcyt, CD3γcyt, Igαcyt, Igβcyt, and FcRγcyt, were all found to form specific homodimers under

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physiological conditions. This unusual phenomenon of IDP oligomerization is distinct from non-specific aggregation behavior seen in many systems (e.g., elastin [53]) and has

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been suggested to play an important role in cell signaling in health and disease [13, 39]. Later studies by independent groups [54-61] confirmed the existence of this phenomenon in other classes of IDPs and further supported its physiological relevance. CD and NMR studies surprisingly revealed that in contrast to the most of other dimeric IDPs [56, 58, 60-61], the immune signaling-related IDPs do not undergo a disorder-to-order structural transition upon dimerization [13-14, 17] suggesting the

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existence of an unprecedented and previously unrecognized phenomenon – specific interactions between disordered protein molecules, and introducing a paradigm of "binding without folding" for IDPs. In addition, similarly to the unphosphorylated ζcyt

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and FcRγcyt proteins, the fully phosphorylated protein species were also found to have a random-coil conformation in either monomeric or dimeric forms [13-14]. Finally, the

lack of secondary structure in IDP homodimers was later independently confirmed for

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other unrelated IDPs: the Aedes aegypti N-terminal domain of Ultraspiracle isoform B [59] and AT-Hook 2 (HMGA2), the mammalian high mobility group protein [57].

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However, despite the growing evidence that: 1) IDPs can dimerize [13, 17, 54-61] and 2) IDPs do not necessarily fold upon dimerization [13, 17, 57, 59], the existence of this unusual biophysical phenomenon is still questioned and debated today not only in reviews [62-63] but also in structural studies of IDPs [18]. Intriguingly, in the study by

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Norse and Mittag [18], the authors studied the immune signaling-related IDPs, ζcyt and CD3εcyt, similar to those studied earlier in our studies [13, 17] but came to the opposite conclusions that these IDPs do not form dimers (Fig. 2). While a detailed analysis of

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potential reasons for this discrepancy is outside the scope of this piece, this contradiction

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highlights how unusual the biophysics of IDPs is [9, 64-66] and how important it is to consider each study's methodology, data quality, and validity when interpreting the results of biophysical studies of IDPs. A specific example is the use of gel filtration (or size exclusion chromatography,

SEC) for characterizing the oligomeric state of immune signaling-related IDPs. In our gel filtration studies of ζcyt, CD3εcyt and other ITAM-containing IDPs [13], increasing protein concentration in the analyzed samples resulted in the gradual shift in retention

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volume (except of Igαcyt), which is characteristic for a fast dynamic equilibrium between monomeric and dimeric protein species. This chromatographic pattern is different from that observed with increasing Igαcyt concentration – the appearance of new peaks and

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changes in the intensity of peaks corresponding to monomeric and dimeric Igαcyt that

indicates a slow equilibrium between these species. Fast monomer-dimer equilibrium chromatographic patterns are rarely observed in gel filtration studies of oligomeric

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proteins. Thus, to further confirm the existence of such equilibrium, we cross-linked ζcyt to trap the ζcyt dimer and analyzed the obtained samples by gel filtration [13]. In contrast

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to the non-cross-linked protein, the gel filtration profile of the sample containing the ζcyt monomer and the cross-linked ζcyt dimer exhibited two separate peaks, with retention volumes that correspond to monomeric and dimeric forms of ζcyt. Interestingly, when studying the oligomeric state of ζcyt by using a Shodex KW-803

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gel filtration column and the chromatographic conditions similar to those used in our study [13]: 20 mM phosphate buffer (pH 7) and 150 mM NaCl, the authors [18] obtained gel filtration profiles that are characteristic for dimeric ζcyt species [13]. However, these

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data were compromised by other findings obtained under those SEC conditions (0 or 1 M

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NaCl) [18] that are inappropriate because of non-specific matrix-protein interactions and should be avoided in all SEC experiments in general and especially when studying IDPs. In summary, taken together, these findings illustrate how unusual and intriguing

structural features of IDP-IDP complexes can affect the results of biophysical studies and lead to opposite conclusions and interpretations. Nowadays, it becomes evident that dimerization of IDPs plays an important role in cell function. Thus, rigorous biophysical

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and biochemical approaches need to be developed in response to this trend to study complexes of IDP molecules.

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3. Conclusion

IDPs play an important role in cellular regulation, signaling and control in health and

multiple applications in biology and medicine.

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disease. Thus, our expanded understanding of structure and function of IDPs can have

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One example is the ability of immune signaling-related IDPs to form specific homodimers and higher-order homooligomers [9, 13, 17]. This phenomenon represents a missing piece to the cell receptor triggering puzzle, and provides the molecular basis for a new platform of transmembrane signal transduction, the Signaling Chain

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HOmoOLigomerization (SCHOOL) model [39, 67-68]. Interestingly, the SCHOOL model suggests that membrane binding of ITAM-containing ζcyt, CD3εcyt, and FcεRIγcyt does not affect phosphorylation of their ITAMs per se but rather prevents

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homooligomerization of these signaling chains within surface receptor clusters in resting cells and during diffusion-driven random receptor encounters in the cell membrane [15,

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33, 69].

Further, according to the SCHOOL platform, specific protein-protein interactions in

the cell membrane between the MIRR ligand-binding and signaling subunits are critical for signal transduction and, as such, represent universal therapeutic points of intervention [67, 70]. These transmembrane interactions can be specifically targeted by short synthetic peptides (SCHOOL peptides) that are designed in line with the SCHOOL platform-based

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strategy to inhibit multichain receptors. Importantly, the SCHOOL peptides can access their target site of action from both outside and inside the cell. Successful application of SCHOOL peptide technology both in vitro and in vivo [71-73] can stimulate the

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development of novel mechanism-based therapies. Finally, multiple and diverse receptors are involved in the pathogenesis of a variety of serious human diseases with unmet

clinical needs that include cancer, cardiovascular disease, sepsis, rheumatoid arthritis,

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retinopathy, and others. Thus, the SCHOOL platform in combination with the lessons

learned from viral pathogenesis [74-75] can accelerate significantly the advancement of

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novel therapeutic strategies for these disorders.

In summary, two long-standing contradictions in the field of IDPs concerning dimerization and membrane-binding activities of these proteins that are briefly reviewed and commented here highlight the unusual biophysics of IDPs and the challenges of

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structural characterization of IDPs and their complexes. Thus, more research efforts are needed to solve the existing mysteries in the field of IDPs and further evolve our

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understanding of an important physiological role of these proteins.

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Acknowledgments

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I thank Dr. Zu T. Shen for his help with writing the manuscript.

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Figures Legends

Fig. 1. Mode of binding of intrinsically disordered proteins to lipid bilayers depends on

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the cell membrane model. The use of inappropriate models results in physiologically irrelevant conclusions. Illustrated on the example of intrinsically disordered ζcyt that

contain three immunoreceptor tyrosine-based activation motifs (ITAMs). Binding of ζcyt

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to micelles and unstable lipid bilayers involves ITAMs and promotes helical folding of these domains mediated by hydrophobic interactions. No disorder-to-order structural

electrostatic interactions.

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transition of ITAMs is observed upon binding of ζcyt to stable lipid bilayers driven by

Fig. 2. Biophysical studies of dimerization of intrinsically disordered proteins may result

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to opposite conclusions and interpretations.

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