A functional role for intrinsic disorder in the tau-tubulin complex Ana M. Meloa,b, Juliana Coraorc,1,2, Garrett Alpha-Cobba,1, Shana Elbaum-Garfinklea,1,3, Abhinav Nathd, and Elizabeth Rhoadesb,4 aDepartment of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520; bDepartment of Chemistry, University of Pennsylvania, Philadelphia, PA 19104; cDepartment of Physics, Yale University, New Haven, CT 06520; and dDepartment of Medicinal Chemistry, University of Washington, Seattle, WA 98195-7610 Edited by Gregory A. Petsko, Weill Cornell Medical College, New York, NY, and approved November 3, 2016 (received for review June 23, 2016) Tau is an intrinsically disordered protein with an important role microtubules (23, 24), suggesting possible mechanisms for the in maintaining the dynamic instability of neuronal microtubules. loss of microtubule stability. Despite intensive study, a detailed understanding of the func- Tau consists of a microtubule binding region (MTBR) composed tional mechanism of tau is lacking. Here, we address this of imperfect repeats R1–R4 (25), a flanking basic proline-rich re- deficiency by using intramolecular single-molecule Förster Reso- gion (P1 and P2) that enhances microtubule binding and assembly nance Energy Transfer (smFRET) to characterize the conforma- (9), and an N-terminal projection domain with putative roles in tional ensemble of tau bound to soluble tubulin heterodimers. microtubule spacing (26), membrane anchoring (27), and tau di- Tau adopts an open conformation on binding tubulin, in which merization (28) (Fig. 1). Alternative splicing results in the expres- the long-range contacts between both termini and the microtu- sion of six isoforms of tau in the adult human brain, with up to two bule binding region that characterize its compact solution struc- N-terminal inserts and three or four repeats in the MTBR. Each ture are diminished. Moreover, the individual repeats within the repeat consists of an interrepeat or linker region and the conserved microtubule binding region that directly interface with tubulin sequence. Contrasting models of tau in complex with microtubules expand to accommodate tubulin binding, despite a lack of exten- suggest binding on the outer surface either along or across proto- sion in the overall dimensions of this region. These results sug- filaments (29, 30) or inside the microtubule lumen (31). Both the gest that the disordered nature of tau provides the significant negatively charged C-terminal tails of tubulin (32) and the interface flexibility required to allow for local changes in conformation α β while preserving global features. The tubulin-associated confor- between / -subunits (33) have been identified as important for tau mational ensemble is distinct from its aggregation-prone one, high- binding. Although significant work has focused on this interaction, lighting differences between functional and dysfunctional states relatively little is known about the structural features of tau when of tau. Using constraints derived from our measurements, we con- associated with tubulin or microtubules. This lack of detail is, in struct a model of tubulin-bound tau, which draws attention to the part, because tau is intrinsically disordered in solution (34, 35) and importance of the role of tau’s conformational plasticity in function. appears to remain largely disordered even on binding, making its structural characterization challenging. single-molecule FRET | intrinsically disordered proteins | microtubule- Single-molecule Förster resonance energy transfer (smFRET) is associated protein | tauopathies | Alzheimer’s disease a powerful approach for probing conformations of large, dynamic proteins, including tau (22, 36). In addition to providing insight into icrotubules play an important role in a host of critical cellular structural features of such proteins, the picomolar concentrations of Mprocesses, including positioning of organelles, maintaining cell shape, axonal transport, and cell division (1–3). Their constit- Significance uent subunits, α/β-tubulin heterodimers, polymerize head to tail to form linear protofilaments that subsequently associate laterally into Tau is a neuronal microtubule-associated protein linked to nu- microtubules. Microtubules are dynamic structures with biological merous neurodegenerative disorders, including Alzheimer’sdis- functions that are dependent on their ability to grow and shrink in a ease. Several lines of evidence support tau aggregation as well temporally and spatially controlled manner in response to cellular as loss of its native interactions with microtubules as contrib- signals. Critical to microtubule function is that they undergo non- uting to pathology. Here, we explored the largely overlooked equilibrium phases of elongation and depolymerization, termed first step of microtubule assembly, namely the interaction of dynamic instability (4). Given the importance of dynamic instability to tau with soluble tubulin heterodimers. Using single-molecule microtubule function, it is highly regulated by a family of nucleotide- Förster Resonance Energy Transfer (smFRET), we determine the independent microtubule-associated proteins (5). topological features of tau in complex with tubulin. Our results Tau is a microtubule-associated protein first characterized as contrast differences in tau isoforms and underscore the im- a promoter of microtubule assembly both in vitro (6) and in vivo portance of conformational flexibility in tau function. (7). Numerous studies have helped provide details on its role in – Author contributions: A.M.M., J.C., G.A.-C., S.E.-G., and E.R. designed research; A.M.M., modulating microtubule growth dynamics (8 11). Tau is found J.C., G.A.-C., and S.E.-G. performed research; A.M.M., J.C., G.A.-C., S.E.-G., A.N., and E.R. primarily in the axons of neurons (12, 13). It is thought that tau analyzed data; A.N. carried out simulations; and A.M.M. and E.R. wrote the paper. may have an important role in regulating axonal transport (14, 15), The authors declare no conflict of interest. and it has been reported to inhibit the activity of several micro- – This article is a PNAS Direct Submission. tubule motor proteins (16 18). Aggregation of tau is linked to a 1J.C., G.A.-C., and S.E.-G. contributed equally to this work. number of neurodegenerative disorders termed tauopathies, 2Present address: Harvard Medical School, Cambridge, MA 02115. among them Alzheimer’s disease (reviewed in ref. 19). Addi- 3Present address: Department of Chemical and Bioengineering, Princeton University, tionally, the loss of native, functional interactions between tau Princeton, NJ 08544. and microtubules and subsequent destabilization of microtu- 4To whom correspondence should be addressed. Email: [email protected]. bules are thought to contribute to pathology (20, 21). Mutations edu. linked to disease have been observed to enhance binding of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tau to soluble tubulin (22) as well as disrupt interactions with 1073/pnas.1610137113/-/DCSupplemental. 14336–14341 | PNAS | December 13, 2016 | vol. 113 | no. 50 www.pnas.org/cgi/doi/10.1073/pnas.1610137113 Downloaded by guest on October 2, 2021 (tau2N4R17/244)(Fig.1C) and the C terminus and MTBR (tau2N4R354/433)(Fig.1D). Importantly, these results show that the surprisingly close contact between the N and C termini of tau that is observed in solution (36, 37) is lost on binding to tubulin (Fig. 1A). Similarly, both termini, which also exhibit a relatively close interaction with the MTBR in solution (36), extend away from this central domain in the tubulin-bound state (Fig. 1 C and D), re- vealing that tau adopts an overall open structure under these conditions. Subsequently, the loss of these long-range interactions exposes the MTBR and its flanking proline-rich region, promoting binding to tubulin. Local Rearrangements Throughout the MTBR. Because the MTBR directly interfaces with tubulin, constructs spanning this region and overlapping all four repeats within this region of tau2N4R were measured (Fig. 2 A–F). The construct tau2N4R244/354 spans nearly the entire MTBR; it shows only a small change in ETeff on binding to tubulin (Fig. 2F). The lack of change is striking for two reasons. The first is that our previous smFRET measurements of the K16 fragment of tau (consisting of the proline-rich region and MTBR) labeled at the same positions found an expansion of this region on Fig. 1. Schematic of tau2N4R (upper schematic) and tau2N3R (lower sche- ΔET ∼ matic). The functional domains of tau indicated are the projection domain, binding to tubulin (for tauK16, eff 0.07 with the addition of the proline-rich region, and the MTBR. Alternative splicing results into six tubulin) (22). However, in solution, this region of tau is more different tau isoforms (spliced regions marked with dashed lines). Tau2N3R compact in the protein fragment than in the full-length protein is lacking R2. The residues labeled for smFRET measurements are indicated [for tauK16244/354, ETeff = 0.40 (22); for tau2N4R244/354, ETeff = below the schematic, with all numbering is based on tau2N4R. (A–D) smFRET 0.32 ± 0.006]. The tubulin-bound conformations of both K16 and histograms are in the absence (gray) and presence of 10 μM tubulin (blue tau2N4R are comparable (ETeff ∼ 0.33), suggesting that the pre- and red for tau2N4R and tau2N3R, respectively). (A–C) For constructs that viously observed expansion for tauK16244/354