Characterizing Protein Dynamics with Integrative Use of Bulk and

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Characterizing protein dynamics with integrative use of bulk and single-molecule techniques Zhu Liu, Zhou Gong, Yong Cao, Yuehe Ding, Meng-Qiu Dong, Yunbi Lu, Wei-ping Zhang, and Chun Tang Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00817 • Publication Date (Web): 25 Sep 2017 Downloaded from http://pubs.acs.org on September 26, 2017

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Biochemistry

Characterizing protein dynamics with integrative use of bulk and single-molecule techniques Zhu Liu1,2, Zhou Gong1*, Yong Cao3, Yue-He Ding3,4, Meng-Qiu Dong3, Yun-Bi Lu2, Wei-Ping Zhang2, Chun Tang1* 1

CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of

Magnetic Resonance and Atomic Molecular Physics, and National Center for Magnetic Resonance at Wuhan, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences, Wuhan, Hubei Province 430071, China 2

Department of Pharmacology, Institute of Neuroscience, Key Laboratory of Medical

Neurobiology of Ministry of Health of China, and Zhejiang Province Key Laboratory of Mental Disorder's Management, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province 310058, China 3

National Institute of Biological Sciences, Beijing, Beijing 102206, China

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RNA Therapeutics Institute, University of Massachusetts Medical School, 368 Plantation Street,

Worcester, MA 01605, USA *Correspondence should be addressed to [email protected] (C.T.) or [email protected] (Z.G.)

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ABSTRACT A protein dynamically samples multiple conformations, and the conformational dynamics enables the protein function. Most biophysical measurements are ensemble-based, with the observables averaged over all members of the ensemble. Though attainable, the decomposition of the observables to the constituting conformational states can be computationally expensive and ambiguous. Here we show that the incorporation of single-molecule fluorescence resonance energy transfer (smFRET) data resolves the ambiguity and affords protein ensemble structures of better precision and accuracy. Using K63-linked diubiquitin (K63-diUb), we characterize the dynamic domain arrangements of the model system, with the use of chemical cross-linking coupled with mass spectrometry (CXMS), small angle X-ray scattering (SAXS), and smFRET techniques. The CXMS allows the modeling of protein conformational states alternative to the crystal structure. The SAXS provides ensemble-averaged low-resolution shape information. Importantly, the smFRET affords state-specific populations, and the FRET distances validate the ensemble structures obtained by refining against CXMS and SAXS restraints. Together, the integrative use of bulk and single-molecule techniques affords better insight of protein dynamics, and shall find its wide implementation in structural biology.

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INTRODUCTION Proteins are dynamic. The dynamics is often manifested as alternative arrangements between the domains and subunits.1-3 Each arrangement is associated with a particular protein function. Thus characterization of the spatial arrangements of the multi-domain proteins can reveal the biophysical basis for protein interaction and signaling.4,5 Solution nuclear magnetic resonance (NMR) has been widely used to characterize protein dynamics.6,7 With a paramagnetic probe site-specifically introduced at one domain of a multidomain protein, paramagnetic relaxation enhancement (PRE) can be assessed for the other domain.8 The PRE measurement is proportional to the inverse sixth power of the distance between the paramagnetic probe and protein nuclei, and the PRE NMR technique has been successfully used to visualize low-populated conformations of multi-domain proteins and protein complexes.5,9-11 However, as the NMR data are averaged over tens of billions of molecules, decomposition of the experimental data by the constituting conformational states require exhaustive search and time-consuming computation.5,12 In addition, the NMR energy functions may not be steep enough to rank different solutions of ensemble structures, which may equally satisfying the experimental data. Small-angle X-ray scattering (SAXS) data provide low-resolution structural information of a protein in solution.13 For a protein predominately existing in a single conformation, the onedimensional SAXS profile can be used to reconstruct the three-dimensional model of the protein.14 On the other hand, for a dynamic protein, the SAXS profile corresponds to the population weighted average of all SAXS profiles for the constituting conformational states. A number of algorithms have been devised to resolve the conformational dynamics from the SAXS

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profile.15-17 As the SAXS data is low-resolution in nature, caution needs to be taken when decomposing the observable to each conformational state, especially when the SAXS is the sole experimental input. Chemical cross-linking coupled with mass spectrometry (CXMS) is increasingly used for modeling the structures of proteins and their complexes.18 Cross-linking reactions may only take place for the two reactive residues within the reach of the cross-linking agent.19 However, experimental cross-links often involve residues separated by a distance much larger than the maximum length of the cross-linker.20 In other words, the certain-length cross-linker cannot reach both residues when modeled onto the known structure of the protein. Recently, it has been shown that the “over-length” cross-links may proceed when the protein transiently switches to an alternative conformation. Therefore, the cross-links contain valuable information about protein dynamics.21-23 We have developed a program called DynaXL for modeling the ensemble structures of multi-domain proteins from the CXMS data.23 However, for mass spectrometry measurement, the population information for each conformational state cannot be obtained. Single-molecule fluorescence resonance energy transfer (smFRET) monitors the timedependent fluorescence fluctuations of donor and acceptor fluorophores conjugated to a single protein molecule.24 Unlike NMR and SAXS measurements, the smFRET is not ensembleaveraged, and therefore can discern the constituting conformational states.25,26 The smFRET data can be either collected with a wild-field detector in a total internal reflection fluorescence microscopy (TIRF) setup, or with time-correlated single photon counting (TCSPC) detectors in a confocal microscopy setup.27 Fluorescence contributions from incompletely labeled protein molecules can be eliminated with alternating or interleaving excitation in both setup schemes,2830

while the confocal setup may differentiate more rapidly interconverting conformational 4


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states.26 A recent tour de force study demonstrated that the ensemble structures of a multidomain protein, Hsp90, could be modeled from the smFRET data measured with more than 100 pairs of fluorophores strategically conjugated across the protein.31 For a single pair of conjugated fluorophores, the smFRET data do not provide a lot structural information per se. However, the smFRET data can be complementary to ensemble-based measurements. Here we show that on the basis of SAXS, CXMS and smFRET data, the conformational dynamics of a multi-domain protein can be visualized to high precision and accuracy, using K63diUb as the model system. By refining against extensive PRE NMR restraints, it has been previously shown that K63-linked diubiqitin (K63-diUb) adopts at least three conformational states.5 Each of the conformations, characterized by different arrangement between ubiquitin subunits, can recognize a specific partner protein through a conformational selection mechanism. The binding partners of K63-diUb include the tUIM domain from Rap80 involved in DNA repair,32 the Npl4 zinc-finger (NZF) domain from TAK1-binding proteins TAB2 or TAB3 involved in NF-κB activation,33 and the fourth zinc-finger (ZnF4) domain from A20 involved in NF-κB termination.34 The specific interaction in turn elicits downstream cell signal, whereas alterations in the relative populations of the preexisting conformational states can lead to changes in binding affinities and signaling strengths.5,35 In the present study, we use CXMS, SAXS and smFRET data conjointly, and the ensemble structures characterized for the ligand-free K63-diUb are similar to those determined previously using PRE NMR. Importantly, our approach provides a new tool of visualizing protein dynamics that integrates both bulk and single-molecule techniques and is not limited by the size of the protein system.

MATERIALS AND METHODS

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Protein sample preparation. Human ubiquitin was cloned to a pET-11a vector and expressed with BL21 star cells either in LB medium or in M9-minimum medium with

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N-

labeled NH4Cl as the sole nitrogen source. The cells were induced with 1 mM IPTG for 4 hours at 37 ºC. Harvested cells were lysed and the protein was purified over Sepharose SP and Sephacryl S100 columns used in tandem. To ensure a unique product, the K63-linked diubiquitin (K63-diUb) was prepared from distal Ub (K63R) and proximal Ub (77D with an aspartate appended at protein C-terminus). The ligation reaction was performed for 0.5 mM protein sample with the addition of 2.5 µM E1, 10 µM E2 (yeast Mms2/Ubc13 heterodimer), 2 mM ATP, and 5 mM MgCl2 for 5 hours at 30 ºC following the established protocol.36. The reaction was quenched with 5 mM DTT and 2 mM EDTA, and the mixture was transferred into Source-S column to further purify the product. For fluorophore conjugation, an N25C point mutant was introduced at the distal Ub, and a G76C mutation at the proximal Ub (without the additional aspartate). The NZF domain from TAB2 was cloned and prepared as previously described.5,33 Cross-linking reactions and identifications with mass spectrometry. The cross-linking reactions were performed on either a 30 µM K63-diUb protein with natural isotope abundance or a 30 µM K63-diUb protein with the distal Ub 15N-labeled. The protein was prepared in 20 mM pH 7.4 HEPES buffer containing 150 mM NaCl. The cross-linking reactions were performed at room temperature for 1 hour using the cross-linking reagents of 1 mM EGS, BS3, BS2G, or DST (Thermo Fisher). The reactions were quenched with the addition of 20 mM NH4HCO3. To exclude intermolecular cross-linking species, the cross-linked protein was resolved on SDSPAGE and the protein band corresponding to the diUb (~17 kDa) was excised for MS analysis. The gel pieces were trypsin digested, and the peptide products were extracted in acetonitrile, airdried, and resuspended in 20 µl 5% formic acid. The cross-linked peptides were analyzed on an

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Easy-nLC 1000 II HPLC (Thermo Fisher) coupled with a Q-Exactive HF mass spectrometer (Thermo Fisher). Peptides were loaded on a pre-column (75 µm ID, 6 cm long, packed with ODS-AQ 120 Å–10 µm beads from YMC Co., Ltd.) and were separated on an analytical column (75 µm ID, 12 cm long, packed with C18 1.8 µm 120 Å resin from Ultimate®) using an 0-28% acetonitrile gradient in 56 minutes at a flow rate of 200 nl/min. The top 15 most intense precursor ions from each full scan (resolution 60,000) were isolated for HCD MS2 (resolution 15,000; normalized collision energy 27) with a dynamic exclusion time of 30 s. The software pLink19 was used to identify 14Nα-14Nβ cross-linked peptides with precursor mass accuracy at 20 ppm, fragment ion mass accuracy at 20 ppm. The results were filtered with a cutoff of 5% false discovery rate and a cutoff of the best E-value of 1E-10. The intramolecular inter-subunit 15

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Nα-

Nβ or 15Nα-14Nβ cross-links were manually identified as described.21 Three technical repeats and

two biological repeats were performed. Single molecule FRET analysis. The cysteine mutant K63-diUb was conjugated with Alexa Fluor 488 C5 maleimide and Cy5 maleimide (Thermo Fisher). After conjugation, the protein was further purified using Source-Q ion-exchange column. The final product was prepared in 20 mM pH 7.4 HEPES buffer containing 150 mM NaCl. The smFRET experiments were performed at 25 ºC for 2 hours in a TCSPC confocal fluorescence microscopy setup with the protein concentration of ~150 pM. Data acquisition was performed as previously described.35 The smFRET profile was fitted with an in-house Matlab script, assuming Gaussian distribution for the inter-dye distance of each FRET species.37 For titration experiment, NZF protein was prepared in the same buffer, and was incubated with K63-diUb for 15 minutes before the smFRET data collection.

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SAXS data collection and analysis. The SAXS data were collected at room temperature for a 150 µM K63-diUb sample prepared in 20 mM pH 7.4 HEPES buffer containing 150mM NaCl. 20 consecutive frames of 1-second exposure were recorded and averaged, providing no obvious differences between the frames. The background scattering was recorded for the matching buffer and was subtracted from protein scattering data. The paired distance distribution probability function p(r) was obtained from the scattering intensity function I(q) using program GNOM. The theoretical scattering profiles for the members of the ensemble structures were calculated using program CRYSOL. GNOM and CRYSOL are components of software package ATSAS.38 Theoretical p(r) profiles for the different conformers of K63-diUb were calculated using CPPTRAJ from software package AMBER14 (University of California, San Francisco). Structural refinement using multiple experimental inputs. The intramolecular intersubunit cross-links identified for K63-diUb were converted to CXMS distance restraints with explicit representation of the cross-linkers and cross-linking reactions, as previously described.23 With the knowledge of the number of conformational states and their relative populations, the SAXS data were partitioned by giving different weight in the form of scaling factor, to each member of the ensemble; an N=3 ensemble was used, with the relative weight of 0.10, 0.23 and 0.67. The structure calculation was repeated 256 times using Xplor-NIH,39 each time with different random starting poses between the two subunits. The rigid portion of each subunit (residues 1-71) was grouped, while full torsional freedoms were given to the linkage between the to subunits (residues 72-76 of the distal Ub and the side chain of K63 of the proximal Ub). The structures with no violations against the CXMS restraints and with lowest overall energy were selected and analyzed. The structure images were rendered with PyMOL Molecular Graphics System, Version 1.8 (Schrödinger).

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RESULTS Intramolecular inter-subunit cross-links manifest K63-diUb dynamics. K63-diUb comprises two covalently linked ubiquitin subunits, in which the K63 side chain amine group of one ubiquitin is isopeptide-bonded to the C-terminal carboxylate group of another ubiquitin. The two covalently linked ubiquitin subunits are called proximal Ub and distal Ub, respectively. After the cross-linking reaction, we excised the band from protein gel that corresponds to the diubiquitin. With the mass spectrometry analysis performed for this protein band, only intramolecular cross-links are obtained. Since the two subunits of K63-diUb are identical in sequence, in order to assign which subunit the cross-linked peptide belongs to, we prepared a K63-diUb protein in which the distal Ub is 15N-labeled and the proximal Ub is 14N-labeled with natural isotope abundance. Thus a cross-link between heavy and light peptides has to be intersubunit,21,40 as illustrated in Figure 1.

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Figure 1. Identifications of intramolecular and inter-subunit cross-links for K63-diUb. (A) MS2 spectrum using BS3 for the cross-linked peptides involving residues K6 and K48. (B) Extracted ion chromatogram (XIC) for the K6-K48 cross-link. The αL and βL denote 14N-labeled peptide (from the proximal Ub), and αH and βH denote the 15N-labeled peptide (from the distal Ub). (C) A representative MS1 spectrum for K6-K48 inter-subunit cross-link. Both αL-βH and αH-βL cross-links are identified, indicating that the cross-linking reaction can go either way (c.f. Table 1). The mono-isotopic peak is marked with a star sign followed by additional isotopic peaks.

We used four different types of lysine-reactive cross-linking reagents of increasing spacer lengths, including disuccinimidyl tartrate (DST), bis(sulfosuccinimidyl) glutarate (BS2G), bis(sulfosuccinimidyl)suberate (BS3), and ethylene glycol bis(succinimidyl succinate) (EGS). For DST, BS2G, BS3, and EGS, the Nζ atoms of the cross-linked lysine residues should be separated by maximum distances of 6.4 Å, 7.7 Å, 11.4 Å, and 16.1 Å, respectively. We have thus identified a total of 10 intramolecular inter-subunits cross-links with high confidence, involving 8 unique pairs of lysine residues (Table 1). If a pair of lysine residues can be cross-linked by two types of cross-linking reagents, the shorter cross-link is used as the CXMS restraint. If a crosslink is identified only with relatively long cross-linking reagents, this means that the lysine residues are just too far away from each other to be cross-linked by the relatively short crosslinking reagents. In a few cases, the cross-links could only be identified with shorter crosslinking reagents but not with longer ones. This is likely because the longer cross-linking reagents have greater conformational flexibility and need to overcome larger entropic cost before reacting with the specific lysine residues.

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Table 1. Intramolecular inter-subunit cross-links identified for K63-diUb with high confidence. BS2G

DST

Cross-links

BS3

EGS

Num of spectra

Best Evalue

Num of spectra

Best Evalue

Num of spectra

Best Evalue

Num of spectra

Best Evalue

distal(K48)-proximal(K6)

28

6.20E-19

--

--

46*

4.11E-22

--

--


--

--

55

3.50E-16

46*

4.11E-22

--

--


--

--

--

--

129

2.01E-25

--

--


--

--

--

--

294

9.84E-21

--

--


--

--

54

7.90E-17

--

--

--

--


--

--

--

--

--

--

35#

2.40E-12


--

--

--

--

--

--

35#

2.40E-12


--

--

--

--

--

--

147

4.10E-11

* Both αL-βH and αH-βL cross-links are identified in XIC and MS1 (c.f. Figure 1), indicating that the cross-link can go both ways between distal Ub and proximal Ub. The number of spectra identified and the best E-value shown here are obtained from the analysis of MS2 and therefore are identical.

Using the intramolecular inter-subunit cross-links as the CXMS restraints, we modeled the ensemble structures of K63-diUb. The cross-links cannot be satisfied with a single conformation and can only be accounted for with a minimum of two conformers. In the N=2 ensemble, one of the conformers accounts for seven unique pairs of cross-links (Figure 2A), and the structure has a backbone root-mean-square deviation (RMSD) value of 2.22 ± 0.79 Å. The other conformer accounts for the DST cross-link between K48 of the proximal Ub and K6 of the distal Ub (Figure 2B), and has a larger backbone RMSD value of 3.22 ± 0.78 Å. Taken together, the intramolecular inter-subunit cross-links reveal that K63-diUb dynamically adopts at least two alternative conformational states. K63-diUb has been crystalized in fully open conformation.41 Nevertheless, if such a fully open conformation exists in solution, it would be not captured by cross-links owing to the large separation between the two subunits.

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Figure 2. The structural models for K63-diUb from the refinement against the CXMS restraints. The ensemble structures can account for (A) seven and (B) one “over-length” cross-links, respectively. With only the CXMS restraints as experimental input, the RMSD for backbone heavy atoms of the two structures are 2.26 ± 0.79 Å and 3.22 ± 0.78 Å, respectively.

Resolving the conformational dynamics of K63-diUb using smFRET. To determine whether K63-diUb also exists in fully open conformation in solution, and to assess the relative populations for the members of the ensemble, we resorted to smFRET measurement. Donor and acceptor fluorophores are conjugated at the N25C site of the distal Ub and at the G76C site of the proximal Ub (Figure 3A). Using the confocal microscopy setup with two TCSPC detectors, we collected the smFRET profile for a ~150 pM fluorophores-conjugated K63-diUb sample, which can be described as the sum of three FRET species (Figure 3B). Centering at FRET efficiencies of ~21%, ~44% and ~63%, the three species have respective populations of ~11%, ~22% and ~66%. The low-FRET species should correspond to the open state of K63-diUb, which has the largest separation between the two subunits. The two other species, both representing non-open compact states, should correspond to the two conformational states captured by inter-subunit cross-links. Importantly, the populations for the two compact states are populated at 1:3 ratio, which is consistent with the values previously determined from the PRE NMR refinement.5 We also doped the smFRET sample with 150 µM unlabeled K63-diUb protein, so that the experimental conditions are on par with the bulk measurements. The smFRET profile for the

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heavily doped sample can still be fitted to three overlapping FRET species with about the same efficiencies and populations (Figure 3C).

Figure 3. Single-molecule FRET analysis of K63-diUb. (A) The fluorophores are introduced at the G76C site of the proximal Ub and the N25C site of the distal Ub. (B) The smFRET profile of K63-diUb. The experimental profile is shown as 40 bins, and can be fitted as the sum of three overlapping FRET species, namely low-FRET, mediumFRET and high-FRET species. (C) The fluorophore-conjugated K63-diUb protein was doped with large quantity of unlabeled protein, thus to mimic the protein concentration commonly employed for bulk ensemble measurements.

Incorporation of SAXS restraint improves structural precision. We also collected smallangle X-ray scattering (SAXS) data. The SAXS profile of K63-diUb represents the populationweighted average of the SAXS profiles for the constituting conformational states (Figure 4). The

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knowledge of the relative populations of the ensemble members from the smFRET measurement allowed us to rapidly decompose the SAXS profile without going through extensive calculations.15,42

Figure 4. Small-angle X-ray scattering analysis of K63-diUb. The SAXS profile is shown as (A) the scattering intensity over scattering angle and as (B) paired distance probability. The experimental data are shown as black dots and black line in (A) and (B). The theoretical I(q) or p(r) profiles for high-FRET, medium-FRET and low-FRET structures are differently colored. A linear combination of the theoretical profiles of the three states can reproduce the experimental SAXS profile, with a χ value of 4.02 between the observed and calculated I(q) values.

Upon the incorporation of the SAXS restraint in the refinement, the structure for each conformational state becomes more converged, as compared to the refinement with the CXMS restraints alone. Given a 23% occupancy, the structure that accounts for a single DST cross-link between K48 of the proximal Ub and K6 of the distal Ub sees its backbone RMSD improved to

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2.01 ± 0.71 Å. Given 67% occupancy, the structure that account for the other seven cross-links sees its backbone RMSD improved to 1.07 ± 0.43 Å. The two compact states of K63-diUb bury solvent accessible area of 393 ± 101 Å2 and 767 ± 97 Å2, respectively, at the interface between two subunits. In addition, the fully open state of K63-diUb can be refined to an RMSD value of 3.95 ± 1.22 Å (Figure 5). Accordingly, the experimental SAXS profile can be reproduced from the linear combination of the theoretical profiles of all three states (Figure 4).

Figure 5. The ensemble structures of K63-diUb. Three representative conformers with the relative populations of (A) 10%, (B) 23%, and (C) 67%, which are obtained through joint refinement against SAXS and CXMS restraints. The three structures also displaying increasing compactness and are consistent with the FRET distances. The distal Ub is fixed in all three panels, and proximal Ub is show as transparent surface, with the atomic probability plotted at 15% threshold.

The ensemble structures of K63-diUb can be validated. The donor and acceptor

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fluorophores we used have a Förster distance of 52 Å.43 Thus for the three smFRET species centering at FRET efficiencies of ~21%, ~44% and ~63% (Figure 3), the distances between the aromatic centers of the fluorophores are about 64.8 Å, 54.1 Å and 47.5 Å, respectively. The FRET distance information was not used in the ensemble refinement. We patched the fluorophores to each structure of K63-diUb, optimized the dihedral angles between the protein backbone and the rigid fluorophore ring, and calculated the average inter-dye distance. The calculated FRET distances are 68.7 ± 7.4 Å, 55.3 ± 10.9 Å, and 50.7 ± 9.2 Å, for the three conformational states. Because the conjugated fluorophores are dynamic with respect to protein backbone, and because the FRET efficiency is related to the inverse sixth power of the distance between the fluorophores, the FRET efficiency is usually biased towards the shortest distance.43 Thus the calculated FRET distances are consistent with the experimental measurements.

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Figure 6. The smFRET titration of K63-diUb with the NZF domain of TAB2. (A) The smFRET profile of ligandfree K63-diUb showing the three FRET species. (B-D) Addition of increasing amount of NZF to K63-diUb. The titration enriches the medium-FRET species at the expense of the high-FRET species. Importantly, the centers of the FRET species remain unchanged during the titration. (E) The relative changes for the populations of the FRET species shown as histogram. Inset: the relative increase of the medium-FRET species over NZF concentration can be fitted to a single-site binding isotherm, affording the KD value.

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We also titrated the K63-diUb with the TAB2 NZF domain, and monitored the changes in smFRET profile. The medium-FRET species, i.e. the FRET species with a relative population of ~23% for ligand-free K63-diUb, is gradually enriched with the addition of the target protein of K63-diUb (Figure 6A-D). Importantly, the center of the FRET species shifts little during the titration, indicating that K63-diUb binds to NZF through a conformational selection mechanism. The relative increase for the medium-FRET species upon NZF titration can be fitted to a singlesite binding isotherm with the KD value of 17.0 ± 1.5 µM (Figure 6E), which is similar to the value previously determined with bulk techniques.5,33 Furthermore, the structure we have determined in this study for the ligand-free K63-diUb is similar to the structure previously determined using the PRE NMR technique, and is also similar to the crystal structure of K63diUb in its complex with NZF (Figure 7).

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Figure 7. Comparison between the structures of K63-diUb in different contexts and determined with different approaches. (A) The structure closest-to-the-mean for the medium-FRET species, as presented in this study. (B) The C2-state structure determined with PRE NMR (PDB code 2N2K). (C) The crystal structure of K63-diUb in complex with the NZF domain of TAB2 (PDB code 2WX0). The structure of the bound TAB2 NZF domain is not explicitly shown. The structures are shown with the same perspective of the distal Ub.

DISCUSSION We have shown here that by integrating different types of experimental inputs, one could determine the ensemble structures of a multi-domain protein to high precision and accuracy. In particular, single-molecule measurement does not suffer from ensemble averaging,44 and the smFRET can readily provide the relative populations of the constituting conformational states present for a dynamic protein system. Ensemble-based measurements with bulk techniques, in 19


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comparison, are averaged over the members of protein ensemble structures. Different types of observables are averaged differently. For example, the PRE NMR is ensemble averaged, and thus biased towards the conformer with the shortest distance between the paramagnetic label and observed protein nuclei.7 The SAXS measurement, on the other hand, is directly averaged.45 For the different types of averaging, there can be a unique solution of the ensemble structures satisfying all experimental observables. However, the process to identify the optimal combination of the conformational states can be extremely time-consuming, and the energy difference between the differently weighted ensemble structures can be hardly discernable. The optimization process is even more daunting when dealing with three or more conformational states. The ensemble structures of K63-diUb were constructed mainly based on intramolecular inter-subunit CXMS restraints. The cross-links were identified for a band excised from protein gel using high-resolution mass spectrometry. In comparison, for PRE NMR measurement, a paramagnetic probe has to be attached at a specific site of the protein, and the PRE contribution from transient intermolecular interactions has to be properly subtracted.5,46 Though the PRE NMR measurement can afford more details about protein ensemble structures,47,48 in many cases the structure information from the CXMS data concerning the relative orientations between the domains should suffice. Moreover, the CXMS measurement can be performed on proteins much larger than those handled by solution NMR. The cross-links identified for K63-diUb are incompatible with the crystal structure of the ligand-free protein captured in a fully open state.41 Therefore, these cross-links are considered “over-length”, if the open state were the sole conformation K63-diUb in solution. By refining against the intramolecular inter-subunit CXMS restraints, we obtained the structures for the two 20


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compact states of K63-diUb, with relative populations of ~23% and ~67%. In the past, “overlength” cross-links have been deemed artifacts, and the corresponding CXMS restraints were either discarded, or the distance restraints were generously relaxed. We have recently shown that the “over-length” cross-links contain valuable information about protein dynamics, and the cross-linking reactions may only proceed when the protein transiently takes up alternative conformations.23 Moreover, as shown in the present study, the over-length cross-links can be corroborated with other types of experimental data. In conclusion, we have shown that ensemble-based and single-molecule data provide complementary views about protein structure and dynamics. Future development in the integrative study of protein conformational dynamics with the use of various techniques shall afford better view of protein ensemble structures and better insight about the clockwork of protein function.

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Accession codes The small-angle X-ray scattering data described herein have been deposited at the SASDB (https://www.sasbdb.org) with the accession code of SASDCG7. The ensemble structures for the coordinates of the lowest-energy structures in the three preferred conformational states of ligand-free K63-diUb have been deposited in the integrative/hybrid structure database at https://pdb-dev.wwpdb.org. Acknowledgments We want to thank the staff at beamline BL19U2 of Shanghai Synchrotron Radiation Facility for assistance with SAXS data collection. Funding sources The work was made possible by the National Key R&D Program of China (2016YFA0501200 to C.T., and Z.G.), by the 973 program from the Chinese Ministry of Science and Technology (2013CB910200 to C.T. and W.-P.Z.), and by the National Natural Science Foundation of China (31400735 to Z.G., 31500595 Z.L., and 81573400 to W.-P.Z.). Z.L. also acknowledges China Postdoctoral Science Foundation for funding (2015M571860 and 2016T90537).

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For Table of Contents Use Only

Characterizing protein dynamics with integrative use of bulk and single-molecule techniques Zhu Liu1,2, Zhou Gong1*, Yong Cao3, Yue-He Ding3,4, Meng-Qiu Dong3, Yun-Bi Lu2, Wei-Ping Zhang2, Chun Tang1*

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Biochemistry

1 ~10% 2 3 4 5 6 7

CXMS

Page 28 of 28 Proximal Ub

smFRET

Distal Ub

SAXS

~67% ACS Paragon Plus Environment ~23%

Characterizing Protein Dynamics with Integrative Use of Bulk and

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