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Piecing Together the Allosteric Patterns of Chaperonin GroEL Jin Chen, Qian Zhang, Weitong Ren, and Wenfei Li J. Phys. Chem. B, Just Accepted Manuscript • Publication Date (Web): 21 Apr 2017 Downloaded from http://pubs.acs.org on April 24, 2017

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The Journal of Physical Chemistry

Article

Piecing Together the Allosteric Patterns of Chaperonin GroEL

Jin Chen*,†,‡,§,#, Qian Zhangǁ,#, Weitong Ren⊥, Wenfei Li⊥ †

Key Lab of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing 211166, China ‡ School of Public Health, Nanjing Medical University, Nanjing 211166, China § Okazaki Institute for Integrative Bioscience and Institute for Molecular Science, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan ǁ Florida State University, Department of Chemistry, Tallahassee, FL 32306, USA ⊥ National Laboratory of Solid State Microstructure, Department of Physics, and Collaborative Innovation Center of Advanced Microstructures, Nanjing University, Nanjing 210093, China ■ AUTHOR INFORMATION Corresponding Author *

E-mail: [email protected]; [email protected]

1

ACS Paragon Plus Environment

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ABSTRACT

Despite considerable effort, elucidating allostery of large macromolecular assemblies at a molecular level in solution remains technically challenging due to its structural complexity. Here we have employed an approach combining amide backbone hydrogen/deuterium exchange coupled with mass spectrometry, fluorescence spectroscopy and molecular simulations to characterize allosteric patterns of chaperonin GroEL, a ~800 kDa tetradecamer from E. coli. Using available crystal structures of GroEL, we quantitatively map out GroEL allosteric changes in solution by resolving exchange behaviors of 133 overlapping proteolytic peptides with more than 95% sequence coverage. This comprehensive analysis gives a refined resolution down to 5 residues to pilot the GroEL allosteric determinants, of which the localized dynamics is monitored by tryptophan-mutated GroEL. Furthermore, the GroEL conformational transition is evaluated by molecular dynamics simulations with an atomic-interaction-based coarse-grained model. Collectively, we provide a practical methodology to analyze GroEL allostery in solution.

2


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■ INTRODUCTION Allosteric regulation, which involves energetically coupled movements of distant sites of a protein, plays an important role in vast range of biological processes such as signal

transduction,

metabolism,

gene

regulation

and

apoptosis.1-4

The

allostery-driven system, chaperonin GroEL/GroES from E. coli, is indispensible for the cell to maintain cellular proteinstasis.5-7 The GroEL oligomer consists of 14 identical 57-kDa subunits arranged into two stacked homoheptameric rings. Each protomer is formed by three functional domains named as apical, intermediate, and equatorial.8-9 During its functional cycle, GroEL may undergo two distinctive allosteric transitions in structure (Fig. 1),5,10 which are ascribed to the intra-ring positive

cooperativity

and

inter-ring

negative

cooperativity

triggered

by

ATP-binding.11 Therefore, numerous studies on the allosteric regulation of GroEL by using ATP analogs have provided insightful evidences related to transient intermediate states of this molecular machinery along its functional cycles.10 Despite several techniques such as NMR spectroscopy and small-angle X-ray scattering used for the structural studies of GroEL,12-13 due to the increasing structural complexity, characterizing GroEL allostery at a molecular level in solution remains technically challenging. We therefore employed a hybrid approach to study GroEL allostery in solution using nucleotides. The amide backbone hydrogen/deuterium (H/D) exchange coupled with mass spectrometry (DXMS, see Methods) holds promising potential to improve structural knowledge of protein complexes that are oversized for NMR analysis or difficult to crystallize.14-17 Amide hydrogens, such as those involved in intra-molecular hydrogen bonds of α-helixes or β-stands in a protein structure, may display different deuteration changes. Through monitoring exchange behaviours of a protein by DXMS, we can 3



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obtain detailed information about its structural changes or dynamics. Therefore, in this study, we set out to combine DXMS, mutational methods and molecular simulation to study GroEL allostery in solution. Particularly, we focus on protein segments with different H/D exchange behaviours to dissect allosteric determinants of GroEL as specific sequence of protein may play crucial role in defining its motion.3,18 As Trp fluorescence is sensitive to its microenvironment which was used to probe the nucleotide-induced structural changes of GroEL,19-21 in a comparative analysis, we employ tryptophan fluorescence spectroscopy to monitor the localized allosteric kinetics of GroEL by using 4 ATPase-retained Trp mutants19-21 (R231W, Y360W in the apical domain and F44W, Y485W in the equatorial domain as shown in Fig. 2a). To gain more insights of GroEL allostery in terms of its large-amplitude conformational fluctuations, we conduct molecular simulations of conformational transitions

of

a

single

GroEL

ring

by

using

a

recently

developed

atomic-interaction-based coarse-grained model (AICG)18,22 together with the multiple-basin energy landscape model.23-24 Such coarse grained model has shown great success in modeling the ligand coupled large-amplitude conformational motions for proteins with large sizes.18,25-26

■ EXPERIMENTAL METHODS Protein and nucleotide. GroEL tryptophan-substituted mutants (Trp-GroEL: F44W, R231W, Y360W, Y485W) were constructed using site-directed mutagenesis. Cloning was performed using the Takara PrimeSTAR mutagenesis basal kit (TaKaRa). Both wide-type GroEL and mutants were expressed in E. coli BL21 (DE3) cells, and the proteins were purified to homogeneity as described previously.27-29 Protein quality was

verified

by

SDS

polyacrylamide

gel

electrophoresis

with

both 4


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Coomassie-brilliant blue and silver staining. The commercial nucleotides ADP, ATPγS (Sigma) were further purified to remove contaminants.30 Fluorescence spectroscopy. ATPγS- or ADP-AlFx-binding induced conformational changes of GroEL was measured by rapid mixing equal volumes of ligands (ATPγS or ADP-AlFx) and Trp-GroEL solutions using an Applied Photophysics SX20 stopped-flow spectrofluorimeter and by manual mixing using a Jasco FP-6500 spectrofluorometer, respectively. The assay temperature was kept constant at 5 oC using a water bath. The final concentration of protein was 0.5 µM. For the stopped-flow fluorescence measurement, the excitation wavelength was set to 295 nm, and the emission of the fluorescence was detected using a 320-nm cut-off filter. A 2-mm path length was used, and both the entrance and exit monochromator slit widths were set to 1 mm. The dead time of the mixing was about 2 ms. At least five transients were averaged for each data point. For the fluorescence measurement by manual mixing, 500 µl nucleotide solutions were pipetted into 500 µl pre-incubated Trp-GroEL solution and fluorescence signal changes were recorded. The excitation wavelength was set to 295 nm and fluorescence emission at 350 nm was measured. The excitation and emission slit width were 3 and 5 nm, respectively. The standard buffer used in the measurements contained 50 mM Tris-HCl, 10 mM MgCl2 and 10 mM KCl at pH 7.5. To prepare metal fluoride-ADP complex, 10 mM NaF and 300 µM AlF3 were included in the standard buffer. Hydrogen/deuterium Exchange. Apo GroEL tetradecamer with a monomer concentration at 140 µM was prepared in a standard buffer containing 20 µM HEPES, 10 mM MgCl2 and 10 mM KCl at pH 7.5. For GroEL-ADP complex, the 140 µM GroEL solution was prepared with the same standard buffer with additional 500 µM 5



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ADP. For GroEL-ADP-AlFx complex, the 140 µM GroEL solution was prepared with the standard buffer containing additional 500 µM ADP, 10 mM NaF and 300 µM AlF3. After the complexes were prepared, the samples were incubated on ice for 6 hours. Similar buffer conditions were applied for the preparation of D2O buffers, except that the nucleotides concentration was reduced to 50 µM. The DXMS experiments on apo GroEL, GroEL-ADP and GroEL-ADP-AlFx were performed

with

a

custom

modified

HTC

Pal

autosampler

(Eksigent

Technologies, Dublin, CA). To start the HDX reaction, 5 µL stock of apo or liganded GroEL was mixed with 45 µL corresponding buffer in D2O. For the blank control, the dilution was made in H2O buffer. The reaction was simultaneously quenched after different labeling duration. To maximize the protein digestion efficiency, 50 µL deuterated sample was rapid mixed with 25 µL of 200 mM TCEP, 8 M urea solution in 1.0% formic acid and 25 µL 5-fold dilution of saturated protease type XIII in 1.0% formic acid (final pH ~2.3).31 The in-solution protease digestion was performed for 2 min followed by injection for LC-MS analysis. Each exchange reaction and assay was performed in triplicate. To minimize the back-exchange level of peptides, all DXMS experiments were performed at ~1 ˚C using a water bath (Peter Huber, Offenburg, Germany). On-line LC ESI FT-ICR Mass. After proteolysis, peptides from GroEL were separated and desalted by a Jasco HPLC/SFC instrument (Jasco, Easton, MD). For LC, 45 µL of the protein digest was injected from a 50 µL loop to a Pro-Zap Expedite MS C18 column (Grace Davidson, Deerfield, IL), HR 1.5 µm particle size, 500 Å pore size, 2.1 × 10 mm2. A rapid gradient from 2% to 95% B in 1.5 min (A: acetonitrile/H2O/formic acid, 5/94.5/0.5; B: acetonitrile/H2O/formic acid, 95/4.5/0.5) was performed for eluting peptides at a flow rate of 0.3 mL/min.32 The LC elute was 6


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connected to an in-house ESI source with a post column splitter reducing the flow rate to ~1/1000. The ionized LC elute was directed to a custom-built hybrid LTQ 14.5 T FT-ICR mass spectrometer (ThermoFisher, San Jose, CA.33 Mass spectra were collected from 380 < m/z < 1300 at high mass resolving power (m/∆m50% = 100,000 at m/z 400). For each scan, 3 million LTQ-accumulated ions were transferred (~1 ms transfer period)34 through three octopole ion guides (2.2 MHz, 250 Vp–p) to a capacitively coupled35 closed cylindrical ICR cell (55 mm i.d.) for analysis. The ion accumulation period was typically less than 100 ms and the FTICR time-domain signal acquisition period was 767 ms (i.e., an overall duty cycle of ~1 Hz per acquisition). The total data acquisition period for each sample was 6.5 min. DXMS Data Analysis. Data was collected with Xcalibur software (Thermo-Fisher) and further processed by an in-house software package.36 The identified peaks for each deuterium-exchanged peptide were used to calculate the abundance weighted average and the deuterium uptake levels. To quantitatively characterize the allosteric state of GroEL upon nucleotide binding, we compared the deuteration level of identical peptides from two evaluated states by calculating their averaged relative D-uptake difference (ARDD, liganded minus apo, averaged over all HDX incubation periods, ti). A cut-off value of 5% was used in the study. For state L (liganded) and A (apo): N

ARDD (%) = (

∑ i =1

L(ti ) − A(ti ) ) / N × 100 L(ti )

(1)

where L(ti) and A(ti) is the D-uptake value at time ti. Molecular dynamics simulations. The molecular dynamics simulations were conducted by using the package CafeMol,37-38 which is a general-purpose coarse-grained

biomolecular

modeling

and

simulation

software.

The 7



atomic-interaction

based

coarse-grained

(AICG)

model

Page 8 of 28

was

used

in

the

simulations.18,22,25 The AICG model is developed based on the energy landscape theory of protein folding and all-atom force field by using a multiscale approach.18,22,25,39 In this model, each residue is represented by a bead located at Cα position. All beads are connected into a polymer chain by virtual bonds. The AICG energy function includes terms related to the virtual bonds, angles, dihedral angles, and non-bonded pairs of the beads. For a given reference structure, the AICG energy function builds up a funnel-like energy landscape with the bottom of the funnel corresponding to the reference structure. Due to the implement of the atomic interactions in the AICG model, the physicochemical features and chain flexibility of proteins can be well modeled with a coarse grained structure representation. The AICG model has been successfully used in modeling the folding of topologically complex proteins.22 In this work, the AICG2+ version of the AICG model was used. For simplicity, only one ring of the GroEL was included in the simulation system. The scheme developed in a previous work was used to construct the double-basin energy landscape for modeling the conformational transitions by interpolating two AICG energy functions corresponding to the R and T states, respectively.23-24 To construct the AICG energy functions, the crystal structure with PDB code of 1OEL8 was used as the reference structure of the T state. For the R state, the ring with ADP bound as shown in the crystal structure with PDB code of 1AON40 was used as the reference structure. Previously, a coarse grained self-organized polymer model has been successfully used in modeling the allosteric transitions of the GroEL by Hyeon and coworkers.41 To calculate the protection factors of H/D exchange from the simulations, we firstly reconstructed the all-atom details from 1000 coarse grained structures of the GroEL 8


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ring sampled in both the T and R states by using the softwares BBQ42 and SCWRL4.43 We then calculated the protection factor of H/D exchange for each residue in the T and R states separately by using the following formula.44

PI = ln Pf pred I

 PclI   1 = ln1 + I  = − ln λ (N m − N 0 )   Pop   e I 

  

(2)

I In this formula, Pf pred is the predicted protection factor of H/D exchange. PclI and

PopI represent the probability of protein in closed and open state, respectively.

is

the number of neighboring heavy atoms of the backbone nitrogen of residue I in a given structure.

represents the threshold number of neighboring heavy atoms of

nitrogen atom, and

is a scaling factor. Here, the threshold means the cut-off of the

packing density in defining the atomic contact number. When the number of the neighboring heavy atom of the NH group is less than a cut-off

, the local

conformation around the NH group was considered as fully open. When the number of the neighboring heavy atom of the NH group is greater than a cut-off

, it

contributes to the probability of the open conformation according to the term e (

1

λ N Im − N 0

) . So the probability of residue I in open conformations can be represented by

the average of

e (

1

λ N Im − N 0

= 0.3. When

) over all sampled snapshots. Here we chose

is smaller than

, we set

=

= 20 and

.

■ RESULTS AND DISCUSSION The

Overall

Conformational

Changes

of

GroEL

Induced

by

Nucleotide-binding. Nucleotide operates the conformational cycle of GroEL, which is initiated by the binding of ATP (Fig. 1). As metal fluoride-ADP (ADP-AlFx) 9



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promotes an allosteric transition of GroEL, we thus complexed and preincubated the apo GroEL (TT-state predominantly, referred as ‘T state’) with ADP-AlFx to mimic the allosteric states of GroEL locked at TR state (referred as ‘R state’).9,45 Although nucleotide-binding to GroEL was previously shown to be the preceding step of its functional cycling,

46-47

recent studies showed that substrate proteins may bind to

GroEL in the absence of nucleotide based on the symmetric GroEL-GroES folding chamber.48-49 Nevertheless, the ADP-AlFx-arrested state (R-state) may argue for a functionally important intermediate state. Despite the T state of GroEL previously reported,50 the crystal structure of the R-state of GroEL alone is not available until a GroELD83A/R197A-ADP complex resembling the R-state of GroEL that was recently solved by Lorimer and coworkers by removing two salt bridges of GroEL.51 As we mainly use wide-type GroEL in this study, we build our model of R-state GroEL corresponding to the folding-active GroEL/GroES/ADP-AlFx complex.44 Note that the structure of ATP-bound GroEL prior to the hydrolysis of ATP (TR’-state conceivably, referred as ‘R’ state’) resembles that of apo GroEL (T-state).52 Although the reason for such structural similarity between crystal structures of ATP-bound GroEL and apo GroEL is now recognized to be crystal packing forces, we simply acquired the DXMS data of apo GroEL and GroEL complexed with ADP-AlFx (allosteric state of T and R, respectively) to pilot its allosteric patterns as it is infeasible to monitor the deuteration of ATP-bound GroEL because of the nucleotide hydrolysis in solution. The overall DXMS data on GroEL show high sequence coverage. Altogether, about 500 proteolytic fragments were obtained, from which 133 overlapping peptides (≤ 30 residues) (Table S1 and Fig. S1) covering ~95% amino-acid sequence of GroEL were used for data analysis. The averaged relative difference (ARDD) of deuterium-uptake 10


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for identical peptides from T and R state is calculated to evaluate the allostery of GroEL (Fig. 2b). GroEL at different states displays difference on the deuterium-incorporation level (row i in Fig. 2b and Fig. 3a) for several regions in all of the three functional domains, indicating that ADP-AlFx-binding to GroEL brings pronounced yet segmental allosteric changes in the entire complex. Correspondently, ADP-AlFx binding-induced allosteric dynamics is also apparent for all tryptophan substituent (Fig. 3b-e) in the way that ADP-AlFx (red circles) induces an allosteric transition of GroEL. For comparison, we also calculated the conformational distributions and protection factors of H/D exchange for each residue at the T and R states based on the conformations sampled by molecular dynamics simulations43 (Fig. S2 and Fig. 4; In

Fig. 4a, the differences of the protection factors were plotted together with that from experimental measurements.). Notably, the results from our simulations accord with the experimental results to a great extent. The regions with larger solvent exposure, i.e., lower protection factor, revealed by the simulations (Fig. S2 and Fig. 4a right) well overlapped those identified in DXMS (Fig. 2b and Fig. 4a left). Moreover, in the simulations, the flexibility of GroEL increased significantly from the T to R state (Fig. 4b-e). Compared to the intra-subunit fluctuations, the inter-subunit fluctuations contribute to the high flexibility to a much larger extent as demonstrated by the wider conformational distributions of the complex of two adjacent protomers shown in Fig.

4c. We note that the protection factors for the two helices (located at the equatorial domain) labeled by the green circles are overestimated in the molecular simulations, possibly due to the inaccurate modeling of the inter-ring interactions in the simulations as discussed in the Method section.

11



The

Nucleotide-induced

bullet-shaped

Structural

GroEL/GroES/ADP

complex

Asymmetry was

found

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of

GroEL.

before.9,40,53

The Such

nucleotide-induced structural asymmetry of GroEL9,40,53 is also evident in DXMS spectra of proteolytic segments. In contrast to apo GroEL, the isotopic envelopes of several representative peptides (R state) show a bimodal pattern (Fig. 3f-i), in which the lower m/z part is similar with apo GroEL and the higher m/z part shows an abundance distribution generally at the same magnitude as judged from the intensity with marked deuterium uptake. Such bimodal distribution of identical fragments thus may reflect the population of two distinct conformations of protein or local structure in the solution.15 Considering the ring-ring composition of GroEL, we reason that the nucleotide preferentially binds and causes structural changes to one of its stacked rings, which demonstrates an asymmetric nucleotide-bound GroEL complex.40,53 Notably, such bimodal pattern was not observed for the control experiment of ADP-GroEL (Fig. S3).

The GroEL Apical Domain is the Prominent Allosteric Region. The prominent allosteric region is located in the apical domain. Altogether, more than 20% of residues comprising the apical domain display appreciable ARDD differences (>30%) between the T and R state (Fig. 2b and 3a). The result agrees well with previous structural findings that the apical domain undergoes dramatic rotation and lateral movement during the ATP-binding event.52,54 Particularly, peptide L221-V236 and the residues that compose the helix-H, the binding sites of substrate protein and GroES (Fig. 3a, top right), show noticeable differences in H/D exchange behaviours (Fig. 3a and j). The helix-H is shown to interact with GroES or substrate protein, which undergoes dramatic structural changes after the nucleotide binds to GroEL.41 12


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Accordingly, DXMS analysis indicates that relatively more amide hydrogens are exchanged at the R state (Fig. 3j) compared to the T state, reflecting a relatively enhanced solvent accessibility and/or structural flexibility formed in this region.10 The observed conformational change in helix H may account for its involvement in the binding interface for diverse substrate proteins. This observation is further corroborated by a relatively fast fluorescence-decreasing phase in the recorded kinetic progress curves of Y360W compared with that of R231W (Fig. 3b and c, red

circles). The simulation results also revealed that the apical domain undergoes remarkable changes during the conformational transition. We used the distance between residue E232 in one GroEL protomer and K245’ in the neighboring one to represent the distance of two proximal apical domains (Fig. 4d). It is evident that in T state, both E232 and K245’ were close in distance, which indicated that the two apical domains interacted with each other. At the R state, we found that these two residues were away from each other leading to the separation of the two apical domains, which suggested that residues like E232 or K245 belonging to the helix H may have more solvent accessible surface area in R state than in T state. Moreover, the wide distribution of the distance suggests the increased flexibility of R state (Fig. 4d).

The Breakdown of Salt-Bridge Involved in the GroEL Intermediate Domain. It was shown that the linking Helix F and M of the intermediate domain undergo a downward tilt with multiple salt-bridge reorientations41 during the ATP-binding event. Consequently, peptide V396-R406 that belongs to helix M contributes a pronounced difference in H/D exchange between T and R state of GroEL (Fig. 3a and Fig. S4) in which more deuteration is found for the R state. As several residues of the helix M including E386 and E409 are involved in a salt-bridge with 13



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the apical/equatorial domain to stabilize the T state of GroEL, this enhanced deuteration may indicate a more solvent accessibility and/or structural flexibility of the apical domain after the allosteric change. Furthermore, in the crystal structure of the complex (GroEL/GroES/ADP), helix M is found to form new contacts with residues in the equatorial domain after the binding of GroES.10 We reason that this flexible region may be further stabilized by GroES binding. Overall, the less-protected helix M from the R-state GroEL may highlight the role of this segment for transmitting allosteric signals triggered by nucleotide-binding. Similarly, segments of R-state GroEL which compose another connecting helix are also found to be less-protected (Helix G in Fig. 2b). The structural change of helix M during conformational transition was also revealed by the simulations. In the equatorial domain of each protomer, strand D is parallel with helix M at the T state while strand D tilts upward a bit making helix M more solvent accessible at the R state. We calculated the distribution of distance between E172 and T403 in each protomer and the results clearly showed that these two residues had a larger distance and wider distribution at the R state (Fig. 4e).

Nucleotide Binds to the GroEL Equatorial Domain Resulting in an Open Structure. Nucleotide binds to the equatorial domain of GroEL to trigger the allosteric signal transmitting, which was also demonstrated in our analyses. Kinetic traces obtained from F44W and Y485W upon nucleotide-binding exhibit distinguishable yet relatively fast decreasing phase (Fig. 3d and e, red circles) in comparison with that of Y360W (in the intermediate domain) and R231W (in the apical domain), which suggests dramatic allosteric conformational changes in the microenvironment around these two probes. As expected, both peptide D41-E63 and 14


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N467-D490 shows pronounced differences in H/D exchange behaviours between respective allosteric states (Fig. 3a, l and m). Since the local structural changes around peptide N467-D490, which consists of eight stabilizing amide hydrogens (two such H-bonds are labeled in Fig. 3a, bottom left), are facing exterior of the protein as judged from the crystal structure of the complex, this region is more solvent-exposed which is reflected by increased H/D exchange for the R-state of GroEL as compared to the T-state (Fig. 3m). Interestingly, peptide D41-E63 in strand 2 which forms interaction with strand 19 of the next subunit (Fig. 3a, bottom right) in the complex shows marked deuteration (Fig. 3l), suggesting a more solvent-accessible structure formed at R-state. Moreover, peptide N467-D490 locates in the inter-ring interface, where residue E461 and G459 of the adjoining subunit are in close contact with this region (Fig. 3a, bottom left). Therefore, our DXMS result may account for the role of E461 in the inter-ring cooperativity.55 This finding is further corroborated by previous cryo-electron microscopy studies, in which nucleotide-binding to GroEL results in differences in inter-ring contacts.54 Additionally, the N-terminal residues (helix A in

Fig. 2b and Fig. 3a, bottom right) were also found to be much more deuterated at R-state. Taken together, these less-protected fragments in the equatorial domain from R-state GroEL may reveal a more open structure in solution induced by nucleotide-binding.

Distinguishing the Minor Chemical Differences in Nucleotide by DXMS. As the γ-phosphate of ATP is crucial for the allostery or folding-active state of GroEL complex,45 we ask whether our measurement may distinguish the minor chemical differences in nucleotide. For this purpose, we performed a control experiment with the use of ADP as the addition of ADP-AlFx may induce a folding-active complex.45 15



Page 16 of 28

Interesting, the overall deuteration patterns of 81 assignable fragments show a high similarity between apo GroEL and the ADP-bound form (except N-terminal regions) (Fig. S5), indicating a discrimination of ADP-AlFx and ADP by GroEL. Such selectivity of GroEL towards the nucleotide is getting more evident around the ATP-binding site (such as peptide 467-490 in Fig. 2b). When an ATP binding-sensitive mutation (Y485W) is introduced nearby (encircled part in Fig. 2a), in contrast to ATPγS, a non-hydrolyzable ATP analog (Fig. S6), ADP-AlFx-binding to Y485W exhibits a more complicated kinetic behaviors (Fig. 2c) monitored by fluorescence spectroscopy. By the stopped-flow mixing, both nucleotide-binding to Y485W reflect a dominant bimolecular process27,47 (solid black lines). However, by manual mixing (circles), ADP-AlFx-binding to Y485W induces a significant fluorescence signal decrease (Fig. 2c) in comparison with ATPγS-binding (Fig. S6), demonstrating the contributing role of ADP-AlFx to induce GroEL allostery.

The Simulated Dynamics of Nucleotide-induced GroEL Conformational Transition. To further look at the dynamic process of GroEL allosteric conformational changes, we performed extensive molecular simulations on the transitions from T state to R state of the whole ring of GroEL. By averaging all the trajectories, we obtained the time evolution of a reaction coordinate Q(t) of intra-protomer and inter-protomer. Here, the Q(t) represents the fraction of formed native contacts of the R (or T) state at a conformation sampled at time t. As shown in

Fig. 4f and g, the kinetic traces of Q values were all fitted well with a double exponential function, (3) where kfast and kslow stand for the kinetic constants of the fast and slow phases, 16


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respectively. represents the average over different molecular dynamics trajectories. The fitting parameters were outlined in Table 1. The results suggest that the conformational changes that occur in each protomer or at the interface between adjoining subunits may be divided into two kinetically separated phases, of which the time scale of the slower one is two-order magnitude less than the fast one. Such simulated kinetic process meets well with the experimental results.

■ CONCLUSIONS In summary, our triple hybrid approaches integrating DXMS, fluorescence and molecular simulations reveal detailed information regarding the dynamics and conformation of GroEL allostery in solution. Importantly, our DXMS data provides detailed sequence composition encoding the allostery of GroEL at solution phase which was further corroborated by simulation results. The DXMS analysis identified several segments with multiple salt-bridge reorientations or rupture during T-R transition. Particularly, segments of the apical domain show a remarkable deuteration, which demonstrate an efficient long-range allosteric regulation in GroEL network. Using mutational studies, we have successfully monitored the localized dynamics around some hot regions, in which an apparent allosteric transition of GroEL induced by the binding of ADP-AlFx observed for all tryptophan substituent (Fig. 3i-l, red

circles). Intriguingly, our simulation also reveals a conformational change fitted well with two exponential functions. This finding together with equilibrium mutational results may suggest two structural transitions involved in the GroEL allostery though the underlying kinetic details remains to be clarified. It should be noted that many earlier studies on GroEL have relied on fluorescent labels or mutated proteins. By the 17



Page 18 of 28

use of DXMS, we evidently observe that a symmetric two-ring of wide-type GroEL becomes asymmetric at solution phase when adding the ATP analogue under the assay conditions while such asymmetric change does not occur when adding ADP. Taken together, by combining DXMS, mutational and simulation methods, we provide a practical method to study the allostery of large proteins in solution phase. In a long run, with emerging evidence of alternatively folding model of GroEL such as the formation of football-shaped GroEL in its complex along its functional cycling,48-49 the underlying conformational shift of GroEL induced by nucleotide-binding could be further refined considering the relatively low assay temperature used in our analysis.

■ ASSOCIATED CONTENT Supporting Information The DXMS data of proteolytic peptides as well as a table of fragments used for DXMS analysis. The difference of the protection parameter of H/D exchange at different allosteric states calculated from simulation.

■ AUTHOR INFORMATION Author contributions #

These authors contributed equally to this work.

Notes The authors declare no competing financial interest. Acknowledgements We thank professor Alan G. Marshall (Florida State University & National High Magnetic Field Laboratory, USA) for supports. We thank the financial support from a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), Jiangsu Shuangchuang Program, Open Funds of the State Key Laboratory for Chemo/Biosensing and Chemometrics (2016015), Jiangsu Specially-Appointed Professor project and the National Natural Science Foundations of China (11574132). This work was partially supported by NSF Division of Materials Research through DMR-06-54118 and the State of Florida, USA. 18


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Table 1. The fitting parameters of simulated conformational transition of GroEL.

value error value error value error value error value error residuals

-0.0047 5.290E-4 0.0603 1.241E-2 0.0557 1.732E-4 0.0025 2.882E-5 0.8932 2.127E-4 1.253E-6

0.0050 4.191E-4 0.0554 8.680E-3 -0.0468 1.438E-4 0.0025 2.885E-5 0.9271 1.702E-4 8.369E-7

-0.0108 3.128E-3 0.1114 5.469E-2 0.2456 7.706E-4 0.0024 2.679E-5 0.6358 9.554E-4 2.694E-5

0.0203 1.500E-3 0.0504 7.097E-3 -0.1703 5.470E-4 0.0025 3.026E-5 0.7544 6.315E-4 1.146E-5

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Figure legends Figure 1. Scheme for partitioned cooperativity of GroEL triggered by ATP binding. For simplicity, GroES and substrate proteins are not considered for the present nucleotide-induced GroEL allostery study. Other allosteric effectors such as salt and temperature are kept constant in the measurement. □ and ○ represent one ring of GroEL in substrate protein binding-competent and binding-incompetent states, respectively. Seven ATP molecules preferentially bind to seven subunits of one GroEL ring with a positive cooperativity resulting in an intermediate asymmetric complex (designated as T-R’, as R’ corresponding to the ATP-bound GroEL ring prior to the allosteric changes could not be captured by our technique, the R’ was shown with dash lines).11 Further allosteric conformational change of protein causes a T→R transition of GroEL followed by the hydrolysis of ATP. Conceivably, final state R’T is same as TR’ considering ring-ring symmetry. This process completes in which ADP is discharged when seven ATP molecules binds to the adjoining GroEL ring reflecting a negative cooperativity.11 The selectivity of GroEL to ATP and ADP operates its repeated functional cycle.

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Figure 2. a. Left, A GroEL protomer showing 4 tryptophan mutants distributing across its structural domains (apical is magenta, intermediate is green and equatorial is blue; mutated residue shown as red stick; PDB code: 1KP852). Right, The expanded region circle-shaded in b showing a portion of GroEL structure around nucleotide-binding site. b, The peptide coverage map of apo GroEL and GroEL-ADP-AlFx. For each peptide, the H/D exchange difference (ARDD) of GroEL-ADP-AlFx vs apo GroEL is colored according to the indicated heat map. The ARDD of 133 identifiable peptides are combined such that short fragments are mapped firstly then the longer ones are used to fill any gaps. GroEL secondary structural components are indicated by rectangles (α-helices) or arrows (β-strands) and extended strands with color coding corresponding to the representations in a. α-helices (A-R) and β-strands (1-19) are sorted as reported.9 c, Monitoring ADP-AlFx-binding induced allosteric conformational changes of GroEL using a tryptophan mutant (Y485 in a was substituted). The kinetic curves obtained both from stopped-flow (solid line) and manual mixing (open circles) are shown. The fast kinetic traces were fitted with exponential functions (light gray) and residual plots were shown in Fig. S7.

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Figure 3. a, Key allosteric residues including those in b-e and j-m and two peptides in the intermediate domain (M389-R404) and equatorial domain (K4-D25), respectively, are highlighted with dotted circles in cyan on a colored GroEL subunit. Coloring accords to the indicated heat map generated from H/D exchange difference (ARDD). b-e, Kinetic traces of GroEL allosteric conformational changes upon ADP-AlFx-binding (red), using dispersed tryptophan mutants. (stopped-flow mixing: solid line; manual mixing: open circles:). f-i, Mass-to-charge ratio spectra of four representative charged proteolytic peptides of GroEL at different states (T: apo GroEL; R: ADP-AlFx-bound). Peptide isotopic peak lists are extracted from the mass spectra. j-m, The number-average mass of four proteolytic peptides are plotted versus the H/D exchange time. (T: triangles; R: circles).

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Figure 4. a, The difference of the protection parameter of H/D exchange between T and R state calculated from simulation. b,c, The distribution of RMSD of one protomer (b) and two adjacent protomers (c).RMSDT and RMSDR represent the RMSD to native T and R state, respectively. d,e, The distribution of distance of two identified fragments (E232-K245' in d and E172-T403 in e) in T and R state, respectively. f,g, Time evolution of ( ) and ( ) in GroEL conformational transition of the one ring from T to R state. The curves are all fitted well with a double exponential function (light gray).

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