The LC8 Recognition Motif Preferentially Samples Polyproline II

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The LC8 Recognition Motif Preferentially Samples Polyproline II Structure in its Free State Jessica Morgan, Malene Ringkjøbing Jensen, Valéry Ozenne, Martin Blackledge, and Elisar J Barbar Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00552 • Publication Date (Web): 09 Aug 2017 Downloaded from http://pubs.acs.org on August 17, 2017

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Biochemistry

The LC8 Recognition Motif Preferentially Samples Polyproline II Structure in its Free State

Jessica L. Morgan†, Malene Ringkjøbing Jensen‡, Valéry Ozenne‡X, Martin Blackledge‡, and Elisar Barbar*†

†

Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, United

States ‡

Institut de Biologie Structurale, Univ. Grenoble Alpes, CNRS, CEA, Grenoble, France

X

Current address: The Electrophysiology and Heart Modeling Institute, Hôpital Xavier Arnozan, Pessac,

France

*Corresponding author: Department of Biochemistry and Biophysics, Oregon State University, 2011 ALS, Corvallis, OR 97331. E-mail: [email protected]. Telephone: (541) 737-4143, Fax: (541) 737-0481.

Funding information: This work is supported by NSF MCB 1617019 to E.B., and ANR JCJC NMRSignal to M.R.J.

ACS Paragon Plus Environment

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ABBREVIATIONS LC8, the 10-kDa dynein light chain corresponding to gene Cdlc2; IC, 74-kDa dynein intermediate chain corresponding to gene Cdic2b; N–IC, IC residues 1–289; ICTL, IC residues 84–143; Tctex1, the 12-kDa dynein light chain protein corresponding to gene Dlc90F; IDP, intrinsically disordered protein; RDC, residual dipolar coupling; CS, chemical shift; FM, flexible-meccano; PPII, polyproline II or ‘poly-Lproline type II’ a.k.a. ‘left-handed 31-helix’.


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ABSTRACT LC8 is a ubiquitous hub protein that binds intrinsically disordered proteins and promotes their assembly into higher order complexes. A common feature among the over 100 essential LC8-binding proteins is that in the 10-12 amino acid recognition sequence there is a conserved QT motif but variable amino acids N- and C-terminal to the QT pair. The sequence diversity among LC8-binding partners implies that structural factors also contribute to specificity. To investigate whether one such factor is the transient secondary structure favored by an LC8-binding sequence, we report here a molecular ensemble description of ICTL, a domain of dynein intermediate chain that includes binding sites for light chains LC8 and Tctex1. NMR secondary chemical shifts and residual dipolar coupling (RDC) values combined with ensemble generation and selection algorithms indicate a deviation from statistical (random) coil behavior with elevated population of polyproline II (PPII) conformations for the ICTL regions that bind LC8 and Tctex1. Independent measurements of one- and three-bond scalar couplings confirm the PPII transient secondary structure propensity. Given that in the IC/Tctex1/LC8 ternary complex ICTL forms a β-strand at the interface of Tctex1 and of LC8, we hypothesize that a PPII conformation may facilitate its initial docking and insertion into the binding cleft of the β-sheet LC8 dimer interface. Molecular ensemble calculations for other intrinsically disordered LC8 binding partners also reveal PPII conformational sampling within and proximate to the LC8 recognition motifs, suggesting that a preference for a PPII conformation is general for LC8 binding partners.


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INTRODUCTION Intrinsically disordered proteins (IDPs) are involved in a vast array of cellular processes including molecular recognition, signal transduction, transcriptional regulation, cell cycle control, and assembly of multi-protein complexes1. The flexible character of IDPs facilitates their versatile binding interactions with myriad protein partners2, as well as their incorporation into multi-protein macromolecular assemblies3. One particular class of IDPs facilitates protein assembly by forming a polybivalent duplex scaffold as a platform for multiple binding partners4. In all cases currently known, the polybivalent IDP duplex scaffold consists of 2 copies of the IDP aligned in-parallel via symmetric binding interaction with multiple bivalent homodimeric proteins; though the number and types of bivalent protein binding partners may vary among IDP duplex scaffolds, they all share the common feature of being bridged by the dimeric dynein light chain LC8—a “hub” protein involved in binding myriad IDP partners5. Examples of polybivalent LC8/IDP duplex scaffolds include: the intermediate chain (IC) of the dynein cargo attachment subcomplex 6,7, Nup159 in the nuclear pore8, mitotic protein Chica9, the RNA-binding protein Swallow, and the zinc finger protein ASCIZ (reviewed in 4). In our ongoing efforts to characterize the molecular/structural determinants that drive LC8 recognition and binding, we focus on the structure of IC as the best-studied LC8 partner. Dynein IC (642 residues in Drosophila melanogaster) consists of two structurally and functionally distinct subdomains: a long, primarily disordered domain (‘N–IC’, residues 1–289) that is central to the cytoplasmic dynein ‘cargo attachment subcomplex’ and includes the binding sites for the three homodimeric dynein light chains 6,10,11, and the C-terminal domain (‘C–IC’, residues 290–642) that includes seven WD40 repeats predicted to fold into a toroidal β-propeller structure. Dynein N–IC is bridged by LC8 binding at IC residues 126–1356, forming an IDP duplex scaffold for the other homodimeric dynein light chains Tctex1 and LC7, with crystallographically-determined binding sites corresponding to IC residues 110–122 (Tctex16) and 221–258 (LC711) in Drosophila melanogaster. Two N–IC chains bind in-parallel to bivalent Tctex1 and LC8, with the 13- (Tctex1) or 10- (LC8) residue


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recognition sequence in each IC chain undergoing a disorder-to-order transition to extended β-strands that are each incorporated at the edges of anti-parallel β-sheets within the light chain dimer interface 6,12. All known LC8 binding partners have an intrinsically disordered ~10-residue recognition motif that often contains a conserved ‘QT’ (Glutamine–Threonine) pair of residues near the C-terminus5. A disorder-to-βstrand transition at the site of binding is the common theme among all known IDP/LC8 crystal structures4. Many IDPs exhibit functionally relevant regions of transient secondary structures that frequently mediate their interactions13, and these regions often bear strong resemblance to their bound-state forms14. Previous solution-state characterization of an IC chain peptide comprised of residues 84–143 (ICTL)— containing the binding sites for Tctex1 and LC8—evidenced high flexibility and a lack of secondary structure12. In order to determine whether there exists some propensity for secondary structure within the LC8 recognition sequence of apo IC (that might resemble the β-strand structure this segment adopts in the bound state), we here use the flexible-meccano/ASTEROIDS approach to develop a molecular ensemble description of apo ICTL based on NMR chemical shifts (CSs) and four types of residual dipolar couplings (RDCs) per residue. Contrary to expectation, the analysis reveals decreased conformational sampling of β-strand structure but increased polyproline II (PPII) conformational sampling (relative to the statistical coil description), most prominently within the LC8 recognition sequence of ICTL. Additional flexiblemeccano /ASTEROIDS molecular ensemble analysis of secondary structural propensities in the intrinsically disordered LC8 binding domains of human Chica, yeast Nup159, and yeast dynein IC proteins provides evidence for elevated sampling of PPII conformations in the LC8 binding domains, suggesting that enhanced PPII structure in the apo state constitutes another common feature of LC8 binding partners, and that transition from significant PPII conformation in the free state to a bound-state β-strand may be a common feature of IDP segments that undergo folding assembly into β-sheet complexes.


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EXPERIMENTAL PROCEDURES

Protein Preparation Isotopically labeled 15N and 13C,15N ICTL proteins were prepared using previously published protocols12. Protein concentrations were determined using sequence-based calculated molar extinction coefficients at 280 nm (ProtParam, http://expasy.org). NMR Sample Preparation and Experiments A 13C,15N-labeled ICTL sample was prepared at a concentration of 0.5 mM in 10 mM sodium phosphate (pH 6.5) with 50 mM NaCl, 1 mM NaN3, 10% 2H2O, a mixture of protease inhibitors (Roche Applied Science), and 1 mM 2,2-dimethyl-2-silapentane-5 sulfonic acid (DSS) for 1H chemical shift referencing. The protein sample was aligned in a liquid crystalline phase composed of poly-ethylene glycol (PEG) and 1-hexanol15. 1DN-HN, 1DCα-C’, 1DCα-Hα, and 4DHN-Hα(-1) RDCs were obtained using 3D BEST-type triple resonance experiments allowing for evolution of the different scalar couplings in the 13C dimension16 collected on a Varian 800 MHz instrument equipped with a cryogenic TXI triple-resonance probe at 20˚C. The isotropic reference sample of 13C,15N ICTL was prepared identically and the same experiments were performed on a Varian 600 MHz instrument equipped with a cryogenic TXI tripleresonance probe at 20˚C. 1

H–15N T1 and T2 relaxation times were determined from experiments17 recorded for 15N-labeled

ICTL at pH 6.5 and 20˚C. T1 experiments used a 1.5-second recycle delay, with relaxation delay times in the 0.05 to 1 sec range. T2 experiments used a 1.4-second recycle delay and relaxation delay times in the 15 to 139 millisecond range. Both T1 and T2 experiments were recorded with at least one redundant data point to aid estimation of experimental error. Steady-state 1H–15N heteronuclear NOEs17 were recorded at 20˚C, and experiments with proton saturation utilized a 3 second period of saturation and additional delay of 1.5 seconds. All relaxation experiments were collected on a 600 MHz Bruker DRX spectrometer. 3

α

J(HN–H ) coupling constants for apo ICTL at 20˚C were obtained from a three-dimensional HNHA18


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experiment with a dephasing/rephasing delay of 13.05 milliseconds, collected on a 500 MHz Bruker Ascend spectrometer. NMR Data Analysis NMR spectra were processed with NMRPipe19 and quantitatively analyzed using NMRView20. Chemical shift assignments for apo ICTL were as reported previously12 with revisions21. All RDC values were calculated as the difference between splittings measured for the aligned and isotropic phases of ICTL with a sign inversion for the 1DN-HN couplings to account for the negative gyromagnetic ratio of 15N. For all relaxation experiments, peak intensities were measured as the peak height and the associated error taken to be the spectral baseline noise as described previously12. Reduced spectral density mapping was performed on the NMR relaxation data using ‘Method 2’ as described by Farrow et. al., 199522 to yield values for spectral densities J(0), J(ωN), and J(0.87ωH); the micro- to millisecond chemical exchange α

contribution (Rex) to the transverse relaxation rate is omitted in this analysis. Secondary 3J(HN–H ) scalar α

coupling values were calculated as the difference between experimental 3J(HN–H ) scalar couplings and residue-specific random coil values23. One-bond 1JCαHα scalar couplings16 were used to obtain secondary 1

JCαHα scalar couplings as the difference between the observed and residue-specific random coil 1JCαHα

α β values24. Secondary chemical shifts (∆δ13C and ∆δ13C ) were calculated relative to random coil

chemical shifts corrected for temperature, pH, and for primary sequence25. Amino acid-specific correction factors to the random coil values for residues preceding Proline were also used26. Generation of Descriptive Conformational Ensembles from NMR Data. A conformational ensemble description of the ICTL protein was generated using the flexiblemeccano/ASTEROIDS CS-RDC approach27. Briefly, an initial pool of 20,000 conformers of ICTL was generated by flexible-meccano (FM) using a statistical coil model28,29. Chemical shifts were calculated for the residues of each member of the ensemble using the program SPARTA30 as previously described31. RDCs were calculated for each conformer in an ensemble using a local alignment window (LAW)32 of 15


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amino acid residues in length; the RDC was calculated for the central residue of each (sliding) LAW using an efficient in-house routine based on steric alignment. The resulting RDCs were multiplied by a generic hyperbolic cosine baseline curve, that assumes absence of highly populated long-range intramolecular contacts33,34, and a uniform scaling factor (to account for the degree of alignment) was applied to the entire predicted set of RDCs to best reproduce the experimental data. The genetic algorithm ASTEROIDS33 was used to select five equivalent conformational sub-ensembles in agreement with the α β experimental data (13C , 13C , 13C’, 15N, and 1HN chemical shifts and 1DN-HN, 1DCα-Hα, 1DCα-C’, and 4DHN-

Hα(-1)

RDCs). Ensemble-averaged chemical shifts and RDCs were also calculated as described above for a

reference statistical coil model ensemble of ICTL generated using flexible-meccano. In this case, the RDCs were calculated using a global alignment tensor and averaged over 50,000 conformations. Residue-specific backbone dihedral angle propensities were also calculated using the flexibleα β meccano/ASTEROIDS approach with ensemble selection based upon the 1HN, 13C , 13C , 13C’, and 15N

experimental NMR chemical shifts for ICTL, yeast (dynein) Pac11 residues 1–877, yeast nucleoporin Nup159 residues 1075–11788, and human Chica residues 410–4789. To quantify the sampling of conformational space for residues in a given ensemble, Ramachandran space is divided into four quadrants, defined as follows: αL, {φ > 0˚}; αR, {φ < 0˚, –120˚ < ψ < 50˚}; βP, {–100˚ < φ < 0˚, ψ > 50˚ or ψ < –120˚}; βS, {–180˚ < φ < –100˚, ψ > 50˚ or ψ < –120˚}. The populations of these quadrants are denoted as p(αL), p(αR), p(βP), and p(βS), respectively. Sequence-based Analysis. For the ICTL construct (D. melanogaster IC isoform 2, accession number AF 263371.1) a sequenced-based “bulkiness” function was calculated35, with bulkiness values averaged over a fiveresidue window size, and the result multiplied by a bell-shaped function with persistence length = 7,


8

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taking into account that Ser84 is not the true N-terminal residue in the ICTL construct used for NMR relaxation experiments.

RESULTS

Transient Secondary Structure in ICTL. Residues 84–143 of the D. melanogaster dynein intermediate chain (e.g. ‘ICTL’) include the Tctex1 and LC8 recognition sequences, and is primarily disordered as measured by UV-CD and limited α

NMR amide proton chemical shift dispersion12. Here we measure RDCs, chemical shifts, and 3J(HN–H ) α

α

and 1J(C –H ) scalar coupling constants to identify structures that deviate from random coil behavior.

For unfolded proteins partially oriented in aligning media, RDCs measured between different pairs of nuclei are sensitive probes of local conformational propensity and residual structure36. 1DHN (a.k.a. 1DN-HN), 1DCα-Hα, 1DCα-C’, and 4DHN-Hα(-1) RDCs were measured for ICTL aligned in liquid crystalline polyethylene glycol/alcohol lyotropic phase. 1DHN RDCs from ICTL (Figure 1A, left ordinate) globally follow the expected distribution of values for an unfolded protein or a random coil in stericallyaligning media, with predominately negative values throughout and a tapering of values toward zero at the chain termini, consistent with the bell-like shape and distribution predicted from random flight chain models37. However, a sign inversion (e.g. positive RDC values) occurs that spans residues 91–93; such positive RDC values can be qualitatively attributed to local helical or turn motifs 36,38. In addition, relatively larger negative amplitude RDCs occur in the stretch of residues 120–134 than in the rest of the protein; larger negative RDCs are associated with locally more extended conformations, such as β-strand or PPII structure. For ICTL, numerous deviations from values predicted (by flexible-meccano) for a


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random coil are found in the experimental 1DHN as well as in the other RDCs measured (1DCα-Hα, 1DCα-C’, and 4DHN-Hα(-1), Figure S1).

A

Tctex1

LC8

B

C

Figure 1. Residual secondary structure in ICTL. Structural information from experimental data collected for apo ICTL at pH 6.5 and 20˚C. (A) 1DNH RDCs measured in liquid crystalline aligning media α

(grey bars, left-hand ordinate) compared to secondary 3J(HN–H )-coupling values (thin black bars, rightα α β hand ordinate). (B) Deviations of the δC (grey bars, left ordinate) and (δC – δC ) (thin black bars, right ordinate) chemical shifts from values expected for a random coil ensemble of conformations25. (C) Plot of the differences between the observed and residue-specific random coil values for the 1JCαHα scalar coupling. Above, the crystallographically-determined regions of ICTL that bind to dynein light chains Tctex1 (residues 110–122) and LC8 (residues 126–135) are demarcated by vertical dashed lines; these segments of IC transition to β-strand structure in the bound state6.


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α The 13C secondary chemical shifts are the most reliable in distinguishing alpha-helical structure β from random coil, while secondary chemical shifts in 13C provide a more reliable means to distinguish α α β β-strand structure from random coil 27,39. The values of ∆δC and (∆δC – ∆δC ) (Figure 1B) have

magnitude of less than ± 1.5 ppm for all residues, indicating a lack of fully-formed secondary structure. Small positive values over a few consecutive residues (such as for residues 90–92 in ICTL) are indicative of transiently populated helical or turn conformations in the ensemble, while small negative values over a few consecutive residues have been suggested to be indicative of transient conformational sampling of PPII or β-strand structures40. The NMR scalar coupling constant 3JHNHα of a residue depends on its φ backbone torsion angle, providing discrimination between different regions of secondary structure in Ramachandran space; positive values of ∆3JHNHα (3JHNHα(experimental) – 3JHNHα(random coil)) on the order of +1.5 Hz correspond to full βstrand structure, while negative values on the order of –2.0 Hz correspond to full α-helical structure for most residue types41. Negative values of ∆3JHNHα are also consistent with PPII structure, with values generally ~0.6 Hz lower than those for random coil42. Secondary 3JHNHα values for ICTL (Figure 1A, right ordinate) are primarily of magnitude ±1 Hz or less, suggesting that any α-helical or β-strand structure in this protein is weakly populated within the ensemble. 1DHN RDCs (above) identified a region (residues 120–134) with relatively larger negative values, suggestive of locally more extended conformations such as β-strand or PPII; the predominance of small negative secondary chemical shift values measured for this same region is also consistent with transient PPII or β-strand structure. While the 1DHN RDCs and the two 13

C secondary chemical shifts taken alone do not reliably distinguish between these two types of

secondary structure in this particular case (given the above coarse-grained consideration and interpretation of parameter values), the small negative secondary 3JHNHα values provide the distinction of PPII rather than β-strand structure (for which positive values of ∆3JHNHα would be expected).


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The one-bond heteronuclear coupling constant 1JCαHα for a given amino acid residue is primarily determined by the ψ angle, and the φ angle to a lesser extent24. Positive values of ∆1JCαHα on the order of ~4–5 Hz are indicative of full α-helical structure24, while small negative values (on the order of ~ –0.6 Hz) are expected for extended β-strand structure. Positive deviations of 1JCαHα values from random coil on the order of ~1.1 Hz are expected for PPII structure42. Figure 1C shows the difference (∆1JCαHα) between observed and residue-specific random coil values24 for residues of ICTL. Given the nature of the experiment16, neither the C-terminal residue nor residues preceding Prolines (e.g. residues preceding Pro89, Pro107, and Pro121) can be observed. Also, additional splitting due to geminal Hα protons in Glycine hinders measurements for those particular residues. For the remaining residues, the majority of ∆1JCαHα values are positive and < 2 Hz in magnitude. Only a few residues show negative values but are not sequential, indicating absence of β-strand structure. The majority of values for ∆1JCαHα in ICTL therefore are intermediate between the values corresponding to a random coil and those expected for PPII structure. Ensemble of Structures Populated by ICTL in Solution. To obtain a quantitative measure of the ensemble distribution of secondary structures populated by ICTL in solution, we applied the flexible-meccano/ASTEROIDS approach27 with ensemble selection on the basis of agreement with the experimental RDCs (1DHN, 1DCα-Hα, 1DCα-C’, and 4DHN-Hα(-1)) and chemical α β shifts (CSs) (13C , 13C , 13C’, 15N, and 1HN) measured for ICTL. The back-calculated CS and RDC values

from the final ASTEROIDS-selected ensembles are in good agreement with the experimentally determined values (Figures S1 and S2). Populations of the different regions of Ramachandran space determined from the ensemble analysis of ICTL are presented in Figure 2. Most notably, the results indicate a general decreased sampling of backbone dihedral angles in the βS (beta-sheet) quadrant of Ramachandran space, compared to the statistical (random) coil description. This reduction in beta-sheet


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(βS) conformational sampling is largely coincident with increased βP (polyproline II) population, compared to the statistical coil description of ICTL. The population of the βP conformational region is most significantly and continuously elevated in residues 123–136 (on average ~20% higher than expected for random coil), which contains the LC8 binding site (IC residues 126–1356, sequence highlighted in green in Figure 2). In addition, small increased (above statistical coil) αR (right-handed α-helix) populations are seen for residues 91–94 and 110–113. In contrast to the other three quadrants, conformational sampling of the left-handed α-helix (αL) quadrant of Ramachandran space by ICTL does

p(αR)

p(βP)

p(βS)

not exhibit deviation from that expected for a statistical (random) coil.

p(αL)

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Figure 2. Conformational sampling of the ICTL ensemble. The ensemble conformational population distribution of ICTL among the four quadrants of Ramachandran space as defined in Experimental Procedures: β-sheet (βS), polyproline II (βP), right-handed α-helix (αR), and left-handed α-helix (αL); the population of these quadrants is denoted as p(βS), p(βP), p(αR), and p(αL), respectively. Population values were generated by application of the ASTEROIDS approach using experimental chemical shifts and RDCs collected for the protein. The population distributions for ICTL are shown in color for each quadrant of conformational space, while calculated distributions for a statistical coil model (from flexiblemeccano) with the same amino acid sequence as ICTL are shown in black. The amino acid sequence of the protein is shown, with the crystallographically-determined binding sites for dynein light chains Tctex1 and LC8 highlighted in yellow and green, respectively. Note that the comparison with the statistical coil model aids in clarification of detail that might otherwise be subsumed (or, masked) by the inherent amino acid-specific sampling of backbone dihedral angles.

Dynamical Characterization of ICTL. 15

N relaxation T1 values (Figure 3A) are predominately homogeneous throughout the chain and

range from 0.41 to 1.53 s (with an overall average value of 0.51 ± 0.15(1σ) s) with values increasingly elevated at the C-terminus; note that residue 84 in the ICTL construct used in relaxation experiments is preceded by non-native residues in the purification tag. Similarly, 15N T2 values (Figure 3B) are predominately homogeneous and range from 0.15 to 0.81 s (with overall average value of 0.19 ± 0.09(1σ) s). The 1H–15N steady-state heteronuclear NOEs range from +0.032 (residue Val112) to –4.680 (near the C-terminus), exhibiting greater heterogeneity along the sequence (Figure 3C) than seen for the T1 and T2 values. When measured at 20˚C and 14.1 T field strength (600 MHz 1H frequency), the 1H–15N NOE values are consistent with high flexibility and a lack of stable structure. The relaxation data were further analyzed using the reduced spectral density mapping approach22. Spectral density function values J(0), J(ωN), and J(0.87ωH) were calculated (Figure S3) and the ratios J(ωN)/J(0.87ωH) are shown for each residue of ICTL in Figure 3D (left ordinate). Differences in J(ωN)/J(0.87ωH) ratios are observed along the protein sequence, with values ranging from 0.62 (near the C-terminus) to 11.29 (in the interior of the protein sequence). Lower values of the J(ωN)/J(0.87ωH) ratio


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are correlated with increased flexibility on the pico- to nanosecond timescale. Decreasing below-average J(ωN)/J(0.87ωH) values for the C-terminal portion of ICTL are consistent with the increased flexibility expected for this terminal region. Regions with higher-than-average J(ωN)/J(0.87ωH) values are seen in the interior of the sequence, with localized maxima in regions containing Ser92; Leu110, Val112, and Tyr113; Lys123, Leu126, and Gln131. The rotational correlation time calculated from J(0) and J(ωN), is in the range of 10-12 ns ( closer to 12 in the three most ordered regions, and closer to 10 ns in the rest of the sequence). 122 126

110

84

Tctex1

T1 (seconds)

A

135

143

LC8

0.8 0.6 0.4 0.2 0

T2 (seconds)

B

84

90

100

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0.4 0.3 0.2 0.1 0

Isat / Iunsat

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-1

-2

-3

J(ω N) / J(0.87ω H) (ns/rad)

D

12 20

9

16 12

6

8

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"Bulkiness"

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4

0

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84

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110 120 Residue Number Residue Number

130

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Figure 3. Dynamics of ICTL. Plots of 15N longitudinal T1 (A) and transverse T2 (B) relaxation data recorded for 15N ICTL at pH 6.5 and 20˚C; T1 and T2 values greater than 0.9 and 0.4 seconds, respectively, corresponding to the C-terminal residues were truncated in these plots. (C) Steady state 1H–15N heteronuclear NOE values are shown as Isat/Iunsat; NOE values lower than negative 3 corresponding to the C-terminal residues were truncated in the plot. (D) The ratio of the spectral density functions J(ωN)/J(0.87ωH) for ICTL at 600 MHz are plotted for each residue (grey circles, left ordinate); the -1

horizontal dashed line indicates the average value of the ratio J(ωN)/J(0.87ωH) (7.17 ns•rad ) over all residues. The “bulkiness” function as defined by Zweckstetter and Blackledge35 is also shown in this plot (red line, right-hand ordinate). A diagram is shown (top) indicating the crystallographically-determined binding regions of the dynein light chains Tctex1 and LC8 in ICTL.

Secondary Structural Propensities in Other Intrinsically Disordered LC8 Binding Partners. In light of the secondary structural propensities obtained for ICTL from the detailed flexiblemeccano/ASTEROIDS ensemble calculations, we sought to estimate the secondary structural propensities (particularly for PPII) in the apo states of several other intrinsically disordered LC8 binding partners studied previously. Secondary structure populations were generated from flexible-meccanoASTEROIDS ensemble calculations with selection using NMR CS data (Figure S4) for the proteins: human Chica residues 410–4789, yeast nucleoporin Nup159 residues 1075–11788, and yeast Pac11 (ortholog of dynein IC) residues 1–877 (Figures 4 and S5)—all of which contain multiple demonstrated binding sites for LC8 or Dyn2 (the yeast ortholog of LC8) that are intrinsically disordered and form β-strands in the LC8(Dyn2) bound complexes (reviewed in 4).


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Figure 4. Conformational propensities in human Chica and yeast nucleoporin Nup159. Conformational population distribution among the four quadrants of Ramachandran space from ASTEROIDS-generated ensembles with selection on the basis of CSs for (A) human Chica residues 410– 4789 and (B) yeast Nup159 residues 1075–11788. Color scheme as for Figure 2. The ‘QT’ recognition motifs for LC8(Dyn2) in each protein are outlined in purple rectangular panels.

The human mitotic spindle-associated protein Chica:410–478 construct contains four QT


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recognition motifs for LC8 (Figure 4A, rectangular outlines) and is intrinsically disordered by UV-CD and NMR spectroscopic examination9. The flexible-meccano/ASTEROIDS molecular ensemble calculations reveal that sampling of the helical quadrants (αR and αL) of Ramachandran space does not exhibit noticeable deviation from the statistical (random) coil description, while there is a general decreased sampling of βS conformations and enhanced (~14% overall across the sequence) sampling of βP (PPII) conformations (Figure 4A). The βP population is most notably and continuously elevated (by ~20% relative to statistical coil) within the linker region between the Chica ‘QT3’ and ‘QT4’ motifs, and within ‘QT4’ itself where the population of βP even exceeds 70% in this non-Proline-containing segment. Yeast nucleoporin Nup159:1075–1178 contains six QT Dyn2(LC8) recognition motifs separated by short flexible linkers (Figure 4B, rectangular outlines) and is demonstrated by UV-CD and NMR experiments to be primarily disordered, lacking elements of fixed secondary structure8. As for the case of ICTL and Chica:410–478, ensemble calculations here demonstrate generally decreased sampling of βS conformations and increased PPII content relative to statistical coil. Short segments of small increased αR sampling are seen with QT1–2 and QT4. The yeast ortholog of dynein IC, Pac11:1–87 contains two QT Dyn2(LC8) recognition motifs separated by a 20-residue flexible linker (Figure S5). NMR experiments identify a strong single α-helix (SAH) domain in residues 1–24 and a nascent, more weakly-populated α-helix within residues 66–73 (just N-terminal to the second ‘QT’ recognition motif), with a predominance of disorder in the remaining segments of the protein7. Results of the ensemble calculations identify an N-terminal α-helix as being nearly 100% populated (residues ~2–20) and another α-helical segment (residues 65–70) with ~20% population above that predicted from the statistical coil model (Figure S5). Outside of the definite Nterminal α-helix, the analysis shows generally decreased β S and increased βP conformational sampling relative to the statistical coil.


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DISCUSSION We present here a structural characterization of the disordered polypeptide chain comprised of IC residues 84–143 (ICTL), containing the binding sites for the dynein light chains LC8 and its structural homolog Tctex1. Whereas a previous study evidenced a lack of secondary structure and high local mobility in this segment of IC12, the more detailed examination presented here indicates significant deviation from random coil conformations in the form of enhanced PPII sampling particularly for the region that binds LC8. We also report elevated PPII conformational propensity (relative to the statistical coil description) within and proximate to the LC8 recognition motifs of 3 other intrinsically disordered protein partners of LC8. The implications of substantial PPII conformations within the intrinsically disordered partners of the LC8 hub protein are discussed. ICTL Has Enhanced Polyproline II Structures Analysis of chemical shifts, scalar couplings, and RDCs for ICTL indicates a highly disordered and flexible polypeptide, lacking unique secondary structure, but also clearly shows deviations from random coil values, indicative of transient elements of secondary structure in specific regions of the protein. When deviations from expected random coil values are relatively small (compared to the values expected for more static, fully-formed secondary structure), these values correspond to potentially several different possible combinations of secondary structure conformational sampling. While deviation from random coil values indicates that sampling of Ramachandran space differs from that of a random coil distribution, it does not necessarily directly indicate the particular types and populations of conformations being sampled. A major advantage of the flexible-meccano/ASTEROIDS approach 29,33 is that it provides a quantitative mapping of Ramachandran space for each residue in terms of conformational propensities, through the combination of multiple experimental parameters whose backbone φ/ψ dihedral angle conformational dependencies are complementary in terms of mapping various regions of Ramachandran α β space. The combination of 1DHN RDCs with 13C , 13C , and 13C’ (with or without 15N and 1HN) chemical


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shifts (CSs) unambiguously distinguishes populations of βP (polyproline II), βS (beta-sheet), αR (righthanded α-helix), and αL (left-handed α-helix) regions of Ramachandran space27. Application of the flexible-meccano/ASTEROIDS approach shows that deviations from statistical coil behavior of the ICTL conformational ensembles occur as generally decreased βS sampling coupled with increased sampling of βP conformations and two short regions of consecutive residues with increased αR conformational sampling (Figure 2). We note that the observation of reduced beta-sheet (βS) conformational sampling that is largely coincident with increased βP (PPII) (relative to the statistical coil description of ICTL) is consistent with previous work on small model peptides that found an anticorrelation between PPII and β S propensities43. Additionally, our relaxation data reveal three regions (residues 91–98, 108–114, and 120–131) in ICTL with increased order relative to the rest of the chain (Figure 3D, left ordinate), correlating with the two segments containing increased αR conformation, and with a segment of increased PPII conformations (Figure 2). In this case, the ratio of spectral densities J(ωN)/J(0.87ωH) coincides with the relative levels of motions on the timescales of a few nanoseconds (~2 ns) vs. a few hundred picoseconds (~300 ps), respectively. Intra-chain backbone hydrogen bonds (as in α-helices) increase the ordering of backbone H– N vectors, resulting in decreased amounts of motions on the faster timescale of a few 100s of picoseconds (hence, larger J(ωN)/J(0.87ωH)). A larger J(ωN)/J(0.87ωH) ratio is also consistent with increased motions on the timescale of a few nanoseconds; recent critical assessment of dynamical modes contributing to NMR relaxation in IDPs proposed that nanosecond timescale motions report on diffusion within distinct Ramachandran basins (e.g. α-helical, β-strand, PPII)44. In contrast to the case for helices, the extended conformation of a PPII precludes formation of main-chain intramolecular hydrogen bonds45; however, the side chains of certain amino acids (Glutamine, Asparagine, Arginine, Lysine, Threonine, and Histidine) can participate in main-chain/side-chain interactions in PPII, donating or accepting hydrogen bonds from their side chain atoms to backbone carbonyl oxygens and nonprolyl amides46. Several of the above-listed


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residue types occur in ICTL, including the region of increased PPII structure near the C-terminus, potentially providing an explanation for the increased backbone ordering inferred from the relaxation data for this region. Alternatively, as noted by Cho et. al. (2007)35, for an unfolded protein, certain NMR observables (including relaxation measurements and RDCs) can exhibit noticeable variation in values along the polypeptide chain that can be attributed to and clearly predicted by amino acid ‘bulkiness’ (the ratio of the side chain volume to its length) without the need of invoking transient secondary or higher-order structure as an explanation; local steric interactions between side chains and the backbone can restrict motions on the pico- to nanosecond timescale. Side chain ‘bulkiness’ correlates with amino acid hydrophobicity47, and for unfolded apomyoglobin, for example, some of the regions observed to have more restricted backbone motions (as evidenced by locally elevated J(ωN) coincident with minima in J(0.87ωH)) were ascribed to local hydrophobic collapse rather than formation of any particular transient secondary structure48. However, for ICTL, local maxima in the ‘bulkiness’ function (Figure 3D, right ordinate) do not closely match the local maxima in the J(ωN)/J(0.87ωH) ratios (Figure 3D, left ordinate), in that there are regions (e.g. residues 92–94; residues 110, 112, and 113; and residues 126 and 131) with higher values of J(ωN)/J(0.87ωH) that are not accounted for by the ‘bulkiness’ criteria. This suggests that localized restrictions in the backbone motions may indeed correlate with local elements of transient secondary structure in these regions. Earlier work on the construct IC:1–143 shows slow hydrogen exchange for residues 86–98 and 105–13221, which correlate roughly with the ‘bulkiness’ profile within residues 84– 143 (Figure 3D), further confirming the restricted order for these residues. Proposed Relationship Between Enhanced Polyproline II Content in ICTL and Molecular Recognition. Given the substantial PPII conformations exhibited by the LC8 recognition site of apo IC, we propose that this conformational character may possibly facilitate the “docking” of this segment of IC on the LC8 surface prior to assumption of its final β-strand bound-state structure deep in the LC8 binding


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groove (Figure 5) (and likewise for Tctex1, though to a smaller extent). The importance of extended PPII structure in protein–protein recognition stems from its particular molecular architecture wherein the backbone carbonyl oxygen atoms and the amide N–H protons (of non-Proline residues), as well as both the hydrophobic and polar portions of residue side-chains, are free to participate in inter-molecular interactions across the binding interface 45,49. While a single, isolated β-strand may also possess some of these topological characteristics (e.g. unsatisfied backbone hydrogen bonds and exposure of side chain atoms), a PPII helix enjoys the advantage of potential intra-chain (e.g. side-chain/main-chain, discussed above) hydrogen bonds that can stabilize the unbound form. The preexistence of such ordering in the apo polypeptide backbone of a disordered segment can help decrease the entropic penalty that accompanies the folding of a disordered protein region upon binding.

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Figure 5. Hypothetical model for the role of PPII structure in IC docking with and binding to LC8. The model depicts ICTL with the crystallographically-determined Tctex1 and LC8 binding sites indicated in yellow (residues 110–122) and green (residues 126–135), respectively (I). ICTL exists as an ensemble of rapidly inter-converting conformers (I and II), with enhanced polyproline II (PPII) conformational


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sampling identified from ensemble calculations (Figure 2) for IC residues 123–136. The enhanced polyproline II conformational sampling by these residues may facilitate the initial docking of IC on its binding partner LC8 (purple) (III), followed by evolution of the complex to the bound state (IV), wherein some of these same IC residues transition to β-strand structure, with each IC chain contributing a sixth strand (shown as green cartoon arrow) to one of two β-sheets at the homodimeric interface of LC8. Included is a Ramachandran plot indicating the conformational regions corresponding to β-strand and polyproline II (PPII) structures. The IC•LC8 subcomplex moiety of the model in panel IV (including IC residues 127–135) is from a crystal structure (Protein Data Bank entry 2P2T). All molecular images, including apo LC8 (Protein Data Bank entry 3BRI) were generated using PyMOL (http://www.pymol.org). Fragments of IC presented in panels I–III are the structures of conformers present in a final ASTEROIDS-selected ensemble for ICTL. The bound-state IC•LC8 complex consists of two IC chains bound to dimeric LC8 though, for simplicity, the initial docking (III) and transition from PPII to β-strand LC8-bound structure (III to IV) are depicted for a single chain of IC.

With its three-fold symmetry and its high surface exposure of backbone and side-chain atoms, PPII structure can present different ‘faces’ to a potential binding partner45 which, coupled with the particular residues found in these LC8 recognition motifs (including the QT moiety), could contribute a degree of specificity to its initial docking on the surface of LC8. Examples of the rapid formation of initial transient encounter complexes followed by evolution of the flexible polypeptide chain from this intermediate stage to its final bound-state structure have been reported for IDPs engaging in folding transitions coupled to partner binding50. In general, IDPs bind faster to their targets than ordered proteins; the kinetic advantage stems largely from the fewer total encounters required prior to formation of the final bound complex and from reduced binding free-energy barriers in evolving from the encounter complex to the final bound state51. In the proposed hypothetical mechanism for ICTL binding to LC8 (Figure 5), while the PPII structure may facilitate initial recognition and docking on the LC8 surface, the retained flexibility in this segment may allow it to adapt to the binding groove in LC8 and transition into its final β-strand structure in the process. The relatively open PPII conformation progresses easily to other conformational states52; only a relatively small φ-angle transition within Ramachandran space is required to reach βstrand structure. Other Intrinsically Disordered LC8 Partners Preferentially Sample Polyproline II.


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Given the similarity of Ramachandran population distributions for flexiblemeccano/ASTEROIDS ensemble selection based upon RDCs and CSs vs. selection from CSs alone for ICTL (Figure S6), flexible-meccano/ASTEROIDS ensembles were generated with CS selection for the other intrinsically disordered LC8 binding partners. For Chica:410–478, in addition to enhanced PPII content within the QT recognition motifs, we also observe a local maximum in PPII content within the α β linker between ‘QT3’ and ‘QT4’ (Figure 4A). Secondary (13C and 13C ) chemical shifts, coupled with

motional restriction inferred from NMR relaxation data, argued for transient structure within this linker segment9. Given the occurrence of multiple residue types capable of main-chain/side-chain hydrogenbond interactions within PPII structure (discussed above) in the sequence between Chica ‘QT3’ and ‘QT4’ motifs, the proposed transient structure is consistent with the significant PPII conformational sampling within this linker. For Nup159:1075–1178, patterns of peak disappearances during NMR titration with Dyn2 suggested a model in which Dyn2 “hops” along the Nup chain, reversibly binding to more than one QT recognition sequence8, almost as if in a dynamic equilibrium engagement scenario53,54. The enhanced PPII content in Nup159:1075–1178 (Figure 4B), coupled with the short linker length between successive QT recognition motifs, may facilitate this transient initial docking recognition between Dyn2 and multiple different sites in Nup159, as in our proposed hypothetical model for the role of significant PPII conformation in the initial docking interaction of ICTL with LC8 (Figure 5). For NMR titration of Pac11:1–87 with Dyn2(LC8), the peak disappearances are more discretely localized to the two QT recognition motifs for Dyn27; note that the flexible linker between these two QT recognition motifs is much longer (20 amino acid residues) than those in Nup159. Overall, the detailed ensemble calculations reported here for several intrinsically disordered LC8 binding partners reveal decreased sampling of β-strand conformations and increased PPII conformational sampling (relative to statistical coil) within or proximate to the LC8 binding regions. Contrary to


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expectations, the intrinsically disordered recognition motifs for LC8 (which assume β-strand structure in the bound state) do not preferentially sample β-strand structure in free state. Despite conflicting evidence as to the role of the PPII conformation in intrinsically disordered states of proteins 45,55–57, it is a perennially expressed assumption that the PPII conformation is common, widespread, or even dominant in disordered states of proteins. However, mounting evidence suggests that PPII is not the default or necessarily dominant backbone conformation but rather, is one of several different conformations in Ramachandran space (including the most favored regions: right-handed α-helix, β-strand, PPII helix, lefthanded α-helix, etc.) sampled by the dynamic ensemble of states populated by intrinsically disordered proteins 27,28,58. Aside from its architectural role in protein structural stability (as in the example of collagen), the major function of PPII structure is in the molecular recognition underlying protein–protein interactions49,52. The particular proteins examined in this study (dynein Intermediate Chain, Chica, and Nup159) happen to be intrinsically disordered scaffold proteins involved in macromolecular assembly; molecular recognition and binding to (diverse) partners (with very high binding density in some cases, such as for Nup159:1075–1178) figure prominently in the ‘job description’ of these particular proteins. Thus, it is not surprising that significant conformational sampling of PPII structure would occur along the examined segments of these scaffolding proteins. Predicted disorder and a TQT motif (or a ‘QT’ motif, where the first T is often replaced by a hydrophobic residue such as V or I) at the C-terminal of a 10–12 amino acid stretch have been used as strong predictors for LC8 binding. We propose a higher propensity of PPII over random coil (and decreased sampling of β-strand conformations) as an additional characteristic of LC8 recognition motifs. General Implication of Enhanced Polyproline II in Biology. The conformational transition of IDP regions from significant PPII conformation in the free state, to β-strand structure in an assembled state is an emerging mechanistic theme in the amyloidogenic transition of certain proteins59, including those involved in the major human neurodegenerative diseases


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Alzheimer’s and Parkinson’s. The intrinsically disordered human Tau (associated with Alzheimer’s disease) and α-synuclein (associated with Parkinson’s disease) proteins transition from soluble forms to large arrays of ordered β-structure (β-structured oligomeric aggregates) that compose neurofibrillary tangles and amyloid fibrils in the brain60,61. Using approaches similar to those reported here for the characterization of ICTL (e.g. generation of representative molecular ensembles from experimental data), recent detailed analyses of conformational preferences of Tau and α-synuclein have revealed enhanced PPII conformational sampling, most notably in aggregation-nucleation sites for each protein27,62 and in docking sites in the intrinsically disordered domain of MKK7 signaling protein that binds into an extended conformation upon interaction with the downstream kinase JNK63. We posit that the interaction of IC (and other IDPs such as Chica and Nup159) with LC8 represents another example (albeit one that is non-amyloidogenic and non-pathogenic) of an IDP segment that transitions from significant PPII structure in its free state, to β-strand structure in the bound state. It is widely accepted that LC8 binding to disordered partners promotes their assembly into dimeric bivalent complexes (reviewed in refs. 5 and 64). Preventing aggregation could be another universal role. Instead of the molecular assembly aggregation process in β-amyloid growth, LC8 appears to provide a β-sheet scaffold that binds the potentially aggregation-prone PPII structure in its intrinsically disordered binding partners and protects against aggregation by incorporating these segments into the LC8 fold. We also note that the binding of intrinsically disordered partners to LC8 constitutes an example of the ‘β-sheet augmentation’ class of protein–protein interactions, wherein the interaction is mediated by an unstructured segment in one protein contributing a β-strand to an exposed edge of a β-sheet in the binding partner65. The anti-parallel β-sheet domain in LC8(Dyn2) is augmented by anti-parallel addition of a β-strand from the flexible ligand segment, presumably via some as-yet-unknown ‘edge-docking’ mechanism. Whereas examples abound in the literature of IDPs that undergo binding-coupled folding transitions to α-helical structure, and studies have revealed mechanistic details of the interactions in such


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transitions50, less is known for IDPs that transition to β-strand structure upon partner binding. As more IDP complexes emerge and details of their intermolecular interactions are characterized, we predict that additional examples will also be realized wherein a disordered protein segment has enhanced or significant PPII conformation in its apo state and undergoes binding transition to β-strand structure— particularly in the case of IDPs that bind via ‘β-sheet augmentation’.


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ACKNOWLEDGMENTS We thank Jonathan Yih and Afua Nyarko for ICTL protein samples used in NMR experiments, and Gregory Benison for collecting the ICTL NMR relaxation data. We acknowledge the support of the nucleic acid and protein core and the mass spectrometry facilities and services core in the OSU Environmental Health Sciences Center (National Institutes of Health, NIEHS Grant 00210). We also acknowledge the support of the OSU NMR facility (HEI NIH1S10OD018518-01) and the TGIR-RMN-THC Fr3050 CNRS at the Institut de Biologie Structurale (Grenoble, France).

SUPPORTING INFORMATION Supplemental Figures 1–6. Figure S1. Comparison of ensemble-calculated RDCs with experimental values for ICTL. Figure S2. Comparison of ensemble-calculated secondary chemical shifts with experimental values for ICTL. Figure S3. Reduced spectral density mapping data for apo ICTL. Figure S4. Comparison of ensemble-calculated secondary chemical shifts with experimental values Figure S5. Conformational propensities in yeast Pac11:1-87. Figure S6. Ramachandran conformational sampling of ICTL for ensemble selection with or without RDC data.


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REFERENCES 1. Dyson, H. J., and Wright, P. E. (2005) Intrinsically unstructured proteins and their functions, Nat. Rev. Mol. Cell Biol. 6, 197–208. 2. Dunker, A. K., Cortese, M. S., Romero, P., Iakoucheva, L. M., and Uverksy, V. N. (2005) Flexible nets: the roles of intrinsic disorder in protein interaction networks, FEBS J. 272, 5129–5148. 3. Cortese, M. S., Uversky, V. N., and Dunker, A. K. (2008) Intrinsic disorder in scaffold proteins: getting more from less, Prog. Biophys. Mol. Bio. 98, 85–106. 4. Clark, S. A., Jespersen, N., Woodward, C., and Barbar, E. (2015) Multivalent IDP assemblies: unique properties of LC8-associated IDP duplex scaffolds, FEBS Lett. 589, 2543–2551. 5. Barbar, E. (2008) Dynein light chain LC8 is a dimerization hub essential in diverse protein networks, Biochemistry 47, 503–508. 6. Hall, J., Karplus, P. A., and Barbar, E. (2009) Multivalency in the assembly of intrinsically disordered dynein intermediate chain, J. Biol. Chem. 284, 33115–33121. 7. Jie, J., Lohr, F., and Barbar, E. (2015) Interactions of yeast dynein with dynein light chain and dynactin: general implications for intrinsically disordered duplex scaffolds in multiprotein assemblies, J. Biol. Chem. 290, 23863–23874. 8. Nyarko, A., Song, Y., Novácek, J., Zídek, L., and Barbar, E. (2013) Multiple recognition motifs in nucleoporin Nup159 provide a stable and rigid Nup159–Dyn2 assembly, J. Biol. Chem. 288, 2614–2622. 9. Clark, S., Nyarko, A., Löhr, F., Karplus, P. A., and Barbar, E. (2016) The anchored flexibility model in LC8 motif recognition: insights from the Chica complex, Biochemistry 55, 199–209. 10. Makokha, M., Hare, M., Li, M., Hays, T., and Barbar, E. (2002) Interactions of cytoplasmic dynein light chains Tctex-1 and LC8 with the intermediate chain IC74, Biochemistry 41, 4302–4311.


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11. Hall, J., Song, Y., Karplus, P. A., and Barbar, E. (2010) The crystal structure of dynein intermediate chain-light chain roadblock complex gives new insights into dynein assembly, J. Biol. Chem. 285, 22566– 22575. 12. Benison, G., Nyarko, A., and Barbar, E. (2006) Heteronuclear NMR identifies a nascent helix in intrinsically disordered dynein intermediate chain: implications for folding and dimerization, J. Mol. Biol. 362, 1082–1093. 13. Lee, S., Kim, D., Han, J., Cha, E., Lim, J., Cho, Y., Lee, C., and Han, K. (2012) Understanding prestructured motifs (PreSMos) in intrinsically unfolded proteins, Curr. Prot. Pept. Sci. 13, 34–54. 14. Fuxreiter, M., Simon, I., Friedrich, P., and Tompa, P. (2004) Preformed structural elements feature in partner recognition by intrinsically unstructured proteins, J. Mol. Biol. 338, 1015–1026. 15. Rückert, M., and Otting, G. (2000) Alignment of biological macromolecules in novel nonionic liquid crystalline media for NMR experiments, J. Am. Chem. Soc. 122, 7793–7797. 16. Rasia, R. M., Lescop, E., Palatnik, J. F., Boisbouvier, J., and Brutscher, B. (2011) Rapid measurement of residual dipolar couplings for fast fold elucidation of proteins, J. Biomol. NMR 51, 369–378. 17. Farrow, N. A., Muhandiram, R., Singer, A. U., Pascal, S. M., Kay, C. M., Gish, G., Shoelson, S. E., Pawson, T., Forman-Kay, J. D., and Kay, L. E. (1994) Backbone dynamics of a free and a phosphopeptide-complexed Src Homology 2 domain studied by 15N NMR relaxation, Biochemistry 33, 5984–6003. 18. Vuister, G., and Bax, A. (1993) Quantitative J correlation: a new approach for measuring homonuclear three-bond J(HNHα) coupling constants in 15N-enriched proteins, J. Am. Chem. Soc. 115, 7772–7777.


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Biochemistry

For Table of Contents Use Only The LC8 Recognition Motif Preferentially Samples Polyproline II Structure in its Free State

Jessica L. Morgan†, Malene Ringkjøbing Jensen‡, Valéry Ozenne‡X, Martin Blackledge‡, and Elisar Barbar*†


37

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. Residual secondary structure in ICTL.

A

Tctex1

LC8

B

C


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Page 39 of 42

p(αR)

p(βP)

p(βS)

Figure 2. Conformational sampling of the ICTL ensemble.

p(αL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry


Biochemistry

Figure 3. Dynamics of ICTL.

84

110

122 126

apo IC:84-143 Tctex1

T1 (seconds)

A

143

135

LC8

0.8 0.6 0.4 0.2 0

T2 (seconds)

B

84

90

100

84

90

100

84

90

100

110

120

130

140

0.4 0.3 0.2 0.1 0

C Isat / Iunsat

! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !

apo IC:84-143 110

120

130

140

110

120

130

140

0

-1

-2

! -3

! ! ! ! !

D J(ωN) / J(0.87ωH) (ns/rad)

!

12 20

9

16 12

6

8

3

"Bulkiness"

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

4

0

0

84

90

100

110 120 Residue Number Number Residue

130

140


Page 40 of 42

!

!

!

!

!

!

!

!

!

!

Biochemistry

Figure 4. Conformational propensities in human Chica and yeast nucleoporin Nup159.

‘QT2’

‘QT3’

1

p(β BetaS S)

0.8 0.6 0.4 0.2 0 410 1

420

430

440

450

460

420

430

440

450

460

420

430

440

450

460

p(β BetaP P)

0.8 0.6 0.4 0.2 0 410 1

p(αR) AlphaR

0.8 0.6 0.4 0.2 0 410 1

470

B 1 0.8 0.6 0.4 0.2 0 1 0.8 0.6 0.4 0.2 0 1 0.8 0.6 0.4 0.2 0

478

478

478

1080

1100

1120

1140

1160

1178

1080

1100

1120

1140

1160

1178

1080

1100

1120

1140

1160

1178

1080

1100

1120

1140

1160

1178

1

!

0.8

0.8

!

0.6 0.4

!

0.2 0 410

‘QT1’ ‘QT2’ ‘QT3’ ‘QT4’ ‘QT5’ ‘QT6’

p(β BetaS S)

‘QT1’

p(β BetaP P)

A

! ! ‘QT4’ ! ! ! ! ! ! ! 470 ! ! ! ! ! ! ! 470 ! ! ! ! !! !

p(αR) AlphaR

p(α AlphaL L)

!

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

p(α AlphaL L)

! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! !

Page 41 of 42

420

430

440

450

Residue Number

460

!

0.6 0.4 0.2

470

478

0

! ! ! ! ! ! !


Residue Number

Biochemistry

Figure 5. Hypothetical model for the role of PPII structure in IC docking with and binding to LC8.

180˚

β" ψ"

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 42

PPII

0˚

-180˚ -180˚ 110

123

0˚

φ"

180˚

122 126 135

(I)

136

(II)

(III)


(IV)

The LC8 Recognition Motif Preferentially Samples Polyproline II

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