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A Natively Monomeric Deubiquitinase UCH-L1 Forms Highly Dynamic but Defined Metastable Oligomeric Folding Intermediates Yun-Tzai Cloud Lee, and Shang-Te Danny Hsu J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.8b00815 • Publication Date (Web): 24 Apr 2018 Downloaded from http://pubs.acs.org on April 25, 2018

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The Journal of Physical Chemistry Letters

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A Natively Monomeric Deubiquitinase UCH-L1

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Forms Highly Dynamic but Defined Metastable

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Oligomeric Folding Intermediates

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Yun-Tzai Cloud Lee a,b and Shang-Te Danny Hsu a,b,* a

5 6

b

Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan

Institute of Biochemical Sciences, National Taiwan University, Taipei 10617, Taiwan

7

AUTHOR INFORMATION

8

Corresponding Author

9

Tel +886-2-27855696 ext5121; FAX: +886-2-27889759;

10

E-mail: [email protected]

11

ACS Paragon Plus Environment

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The Journal of Physical Chemistry Letters 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

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Abstract Oligomerization of misfolded protein species is implicated in many human disorders.

2

Here we showed by size-exclusion chromatography-coupled multi-angle light scattering (SEC-

3

MALS) and small angle X-ray scattering (SEC-SAXS) that urea-induced folding intermediate of

4

human ubiquitin C-terminal hydrolase, UCH-L1, can form well-defined dimer and tetramer

5

under denaturing conditions despite being highly disordered. Introduction of a Parkinson disease-

6

associated mutation, I93M, resulted in increased aggregation propensity and formation of

7

irreversible precipitants in the presence of moderate amount of urea. Since UCH-L1 exhibits

8

highly populated partially unfolded forms under native conditions that resemble urea-induced

9

folding intermediates, it is likely that these metastable dimer and tetramer can form under

10

physiological conditions. Our findings highlighted the unique strength of integrated SEC-

11

MALS/SAXS in quantitative analyses of the structure and dynamics of oligomeric folding

12

intermediates that enabled us to extract information that is inaccessible to conventional

13

biophysical techniques.

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TOC GRAPHICS

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KEYWORDS: aggregation, folding intermediate, Parkinson’s disease, SAXS, UCH-L1.


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Protein folding often involves the formation of folding intermediates that contain partially

2

unfolded forms (PUFs), which tend to self-associate leading to accumulation of aggregation-

3

prone toxic oligomers and eventually causing debilitating human disorders such as Alzheimer’s

4

disease and Parkinson’s disease (PD).1-2 While protein misfolding is a common feature shared

5

among many neurodegenerative diseases and non-neuropathic systemic amyloidosis, detailed

6

structural features of the early oligomers of the pathogenic proteins vary significantly.3

7

Continuous efforts have been devoted into gaining mechanistic insights of protein misfolding

8

and amyloid formation.4-5 In many cases, conformational rearrangements within the early

9

oligomers are found to be closely associated with their seeding effects.6-7 Extensive studies have

10

been reported to address the question of the exact number of monomers within different types of

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early oligomers and how they subsequently form proto-filaments and the eventual amyloid

12

fibrils.8-9

13

Human ubiquitin carboxyl-terminal hydrolase, UCH-L1 (also known as PGP9.5 or PARK5), is

14

a deubiquitinase that is highly expressed in neurons.10 It is a potential risk factor in PD and is

15

frequently found together with α-synuclein inclusions in Lewy bodies, the hallmark of PD. A

16

familial isoleucine to methionine mutation at residue position 93 in UCH-L1 (I93M) has been

17

identified in one German family with late onset autosomal dominant PD so that it is associated

18

with increased risk of PD.11 This mutation is located adjacent to the catalytic site, which could be

19

responsible for the reduced DUB activity,11 increased aggregation propensity,12 and aberrant

20

interactions with cellular factors such as molecular chaperones, e.g., Hsc70 and Hsp90, with the

21

potential to interfere with chaperone-mediated autophagy and accumulation of α-synuclein

22

aggregates.13

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aggregation propensity.14-17

Post-translational modifications by reactive metabolite can cause similar


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Using solution state NMR spectroscopy, we observed significant structural perturbations

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far beyond the I93M mutation sites evidenced by significant chemical shift perturbations18

3

despite the indistinguishable crystal structures between UCH-L1 wild type (WT) and the I93M

4

variant.19-20 We further demonstrated that the I93M mutation significantly reduces the folding

5

stability and accelerates the urea-induced unfolding kinetics of UCH-L1.18 Recently, we

6

identified two obligatory and distinct kinetic folding intermediates being populated along each of

7

the two parallel folding pathways connecting the native and denatured states of UCH-L1.21

8

Furthermore, native and pulsed-labeling NMR hydrogen-deuterium exchange (HDX) analyses

9

revealed the presence of highly populated PUFs of UCH-L1 that share the same structural

10

features with chemically induced folding intermediates.21 Although UCH-L1 is monomeric in

11

solution under the native condition, we observed significant protein concentration dependency in

12

the equilibrium intermediate population induced by urea (Figure S1) that was overlooked in

13

previous equilibrium analyses.18,

14

intermediate of UCH-L1 is oligomeric.

21

This result implies that the chemically induced folding

15

To further characterize the nature of these oligomeric folding intermediates, we carried

16

out analytical size-exclusion chromatography-coupled multi-angle static light scattering (SEC-

17

MALS) to determine the oligomeric states of UCH-L1 in the presence of varying amounts of

18

urea (Figure 1A).


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A

B 120 100 80 60 40 20 0

MW Calc /kDa

LS UV280 3.7M Urea

2.7M Urea

1

105 1.9M Urea

0.0M Urea

0

104 5 6 7 8 9 10 11 12 13

C Peak integration (normalized)

3.0M Urea

Tetramer Dimer

Monomer 0 25 50 75 100

MW Theo /kDa 1.0

1.0

0.5

0.5

0.0

Retention volume /mL 1

012345678

0.0

Fractional population

UV280& LS /A.U.

2

106

Molar mass /g mol-1

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


[Urea] /M

2

Figure 1. Urea-induced oligomerization of UCH-L1. (A) SEC-MALS profiles of UCH-L1 in the

3

presence of different urea concentrations as indicated. The calculated MWs are indicated at the

4

peak positions. The dotted line and solid line indicate light scattering (LS) and UV absorption at

5

280 nm (UV280). (B) Correlation between calculated and theoretical MWs (MWCalc and MWTheo)

6

associated with the individual elution peaks in (A). (C) Elution peak integrals corresponding to

7

monomer (open blue), dimer (open green) and tetramer (open orange) as a function of urea

8

concentration. Intrinsic fluorescence-derived native, intermediate and denatured state populations

9

(these populations were derived from an equilibrium three-state folding model at a protein

10

concentration of ca. 40 µM as shown in Figure S1) are shown in dash blue line, shaded gray area

11

and dashed red line, respectively.


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The results revealed the emergence of a well-defined dimeric UCH-L1 elution peak in the

2

presence of 1.9 M urea and subsequent emergence of tetrameric UCH-L1 elution peak on

3

increasing urea concentration, ranging from 2.7 to 3.7 M urea. The molecular weights (MWs) of

4

the dimer and tetramer elution peaks derived from SEC-MALS were in perfect match with the

5

theoretical values (Figure 1B and Table S1 in Supporting Information). Furthermore, the

6

populations of the different oligomeric states as a function of urea concentration agreed well with

7

the three-state population distributions derived from intrinsic fluorescence (Figure 1C and

8

Supporting Information). In bulk fluorescence measurements, the optical contributions of

9

different oligomeric states to the intermediate population could not be resolved. Therefore the

10

intermediate population (gray area in Figure 1c) corresponded to a lump sum of different

11

oligomeric forms. Time-dependent SEC-MALS analysis indicated that UCH-L1 fully converted

12

into dimer when mixed with 3.5 M urea within instrument dead time of 2.5 minutes; subsequent

13

emergence of tetrameric population was observed after 10 minute and beyond, indicating a

14

sequential oligomerization process for the chemically-induced folding intermediates (Figure S3).

15

Global fitting of the loss of dimeric population and the increase of tetrameric population of

16

UCH-L1 at 3.5 M urea yielded a slow apparent rate of the tetramer formation (kapp= 0.022 ±

17

0.005 h-1; Figure S3). Note that the kinetic analysis was carried out without considering the

18

protein concentration dependency. Due to instrument limitation, SEC-MALS analysis was

19

limited to urea concentration up to 4 M. To cover a wider range of urea concentrations, we

20

applied chemical cross-linking using (bis[sulfosuccinimidyl] suberate (BS3) in the presence of 0-

21

8 M urea, followed by SDS-PAGE imaging analyses to quantify the amount of dimer and

22

tetramer as a function of urea concentration. The results showed peak distributions of dimer and

23

tetramer populations that were general agreement with the intrinsic fluorescence-derived


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distributions, confirming that the intrinsic oligomerization propensity of urea-induced folding

2

intermediates of UCH-L1 (Figure S4).

3

SEC-MALS analysis of the PD-associated I93M variant of UCH-L1 showed a similar

4

trend of oligomerization of urea-induced folding intermediates with a much more pronounced

5

aggregation propensity (Figure 2A).

A

B

3.50 M

UV280& LS (A.U.)

1.0 UV280& LS (A.U.)

[Urea] 4.00 M

LS UV280

3.00 M 2.70 M

> MDa

1.89 M 106

0.8 0.6 55.9±6.2

0.4

105

28.0±2.0 kDa

104

0.2

Molar mass (g mol-1)

0.0 103 6 7 8 9 10 11 12 13 Retention volume (mL)

2.37 M 1.89 M

C

1.54 M

fN

OD550nm

2

1.00 M 0.00 M 5 6 7 8 9 10 11 12 13

1.0 fD fI

1

0.5

0

0.0 0

1

Retention volume (mL)

2 3 4 [Urea] (M)

Fractional population

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


5

6 7

Figure 2. Increased aggregation propensity of PD-associated I93M variant. (A) SEC-MALS

8

profiles of I93M in the presence of different urea concentrations as indicated. UV absorbance at

9

280 nm (UV280) and light scattering intensity (LS) are shown in solid and dashed lines,

10

respectively. (B) MW profiles of I93M variant in the presence of 1.89 M urea. The calculated


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Page 8 of 17

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MWs of monomer (blue), dimer (red) are indicated in kDa, and that of large aggregate is shown

2

in green down to the void volume (6 ml) (C) Sample turbidity (black bars) as a function of urea

3

concentration and was superimposed with the three-state fractional populations of I93M at a

4

protein concentration of ca. 40 µM.

5

In the presence of 1-2 M urea, higher oligomers were observed by SEC-MALS resulting

6

in a continuum of MW distribution to the void volume with an apparent MW beyond one

7

megadalton (Figure 2B). Prolonged overnight incubation under these conditions lead to visible

8

precipitates with significant turbidity that could be quantified by optical absorbance at 550 nm

9

(OD550nm; Figure 2C). The peak distribution of precipitating aggregates coincided with the

10

emergence of intermediate population, consistent with the previously reported increased

11

aggregation propensity caused by I93M mutation in vitro and in vivo.13-14

12

Collectively, our data suggested hierarchical formation of UCH-L1 oligomers upon urea-

13

induced unfolding starting from a partially unfolded monomeric folding intermediate followed

14

by the formation of dimer and tetramer (Figure S3). In the case of I93M, whose PUFs and

15

folding intermediates may be different from that of the WT, exhibited a much higher aggregation

16

propensity that could easily self-assemble into amorphous aggregates. To assess the structural

17

features of these oligomers under non-native conditions, we carried out size exclusion

18

chromatography-coupled small angle X-ray scattering analysis (SEC-SAXS),22-23 which has been

19

applied to study protein folding and self-association under a variety of experimental

20

conditions,24-26 to deconvolute the scattering contributions originated from the different

21

oligomeric states (Figure 3A).


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1 2

Figure 3. SAXS analyses for native and non-native forms of UCH-L1. (A) The SAXS profiles of

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the native state, monomeric and dimeric folding intermediates at 2.7 M urea were colored in

4

black, blue and orange, respectively. (B) The Kratky plots transformed from the SAXS profiles.

5

(C) The pair-wised distance distributions derive from the SAXS profiles using GNOM within the

6

ATSAS software suite. 27 (D) Rg values of UCH-L1 in their native and urea-denatured folding

7

intermediate states as a function of chain length. The red line and blue line are the empirical

8

Flory’s power law for chemically denatured proteins and for native proteins, respectively.17

9

SAXS data were collected from well-separated SEC elution peaks corresponding to the native

10

UCH-L1 without urea, and the monomeric and dimeric of UCH-L1 in the presence of 2.5, 2.7

11

and 3.0 M urea (grey shades in Figure S5A). The SEC-SAXS elution peak of the tetrameric


9


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folding intermediate of UCH-L1 was weak and overlapped with that of the dimeric intermediate

2

in the presence of high urea concentration, which prevented further quantitative analyses (Figure

3

S5A). For native, monomeric UCH-L1, the SAXS profile showed good agreement with the

4

back-calculated SAXS profile based on the reported crystal structure of UCH-L1; the scattering

5

contributions of the flexible hexahistidine tag at the C-terminus were included by using

6

AllosMod-FoXS (Figure S5A and S6A).28-29

7

For the monomeric folding intermediate of UCH-L1, a significant reduction in the peak height

8

of the Kratky plot was observed compared to that of the native profile (Figure 3B), indicating

9

that the monomeric folding intermediate is partially unfolded. This is corroborated by the pair-

10

wise distance distribution, P(r) (Figure 3C) and the increase radius of gyration (Rg) from 17.9 Å

11

to 29.3 Å, derived from Guinier approximation (Figures 3D and S6). In order to characterize this

12

partially folding intermediate, we performed molecular dynamic simulation to generate an

13

ensemble of structures that collectively match the experimental SAXS profile collected in the

14

presence of 3.3 M urea (Figure S6). Considering that a stable core structure encompassing the

15

central β-sheet and α-helix 2 of UCH-L1 was observed in our earlier pulsed-labeling NMR HDX

16

analysis,21 the structural protections were used as restrains during the ensemble modeling

17

(Supporting Information). A subset of conformations that closely resembles the monomeric

18

folding intermediate was selected and the results showed partially unfolded peripheral helices

19

and extended termini (inset in Figure 3D and Figure S6). Despite the partial unfolding, the

20

monomeric folding intermediate of UCH-L1 is relatively compact according to its Rg value in

21

comparison with other native proteins (Figure 3D).30-31 Due to the lack of independent local

22

structural information regarding the dimeric folding intermediate, we did not apply the same

23

modeling procedure to obtain a structural ensemble. Nonetheless, the corresponding Kratky plot,


10

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P(r) and Rg value (Figures 3) all indicated the presence of a highly disordered structure that

2

adopts an expanded random coil-like ensemble. How such a disordered ensemble manages to

3

form a well-defined dimer remains to be established. To the best of our knowledge, our findings

4

represent the first example defined sequential and reversible oligomerization events of

5

chemically induced folding intermediates starting from a monomeric native state.

6

In this work, we identified by SEC-MALS the formation of well-defined UCH-L1 dimer

7

and tetramer under non-native conditions (Figure 1). The oligomerization process of UCH-L1

8

WT was fully reversible upon removal of urea. Prolonged incubation of the PD-associated I93M

9

variant, however, lead to irreversible precipitation when incubated with moderate amounts of

10

urea, indicating that UCH-L1 is metastable and that the I93M mutation could tip the balance of

11

proteostasis towards aggregation, causing cellular stress.

12

restrained molecular modeling revealed a less compact monomeric folding intermediate

13

compared to the native monomer, and that partially unfolded UCH-L1 can assemble into specific

14

dimer and subsequently into tetramer. Our current findings highlighted the intrinsic shortcoming

15

of intrinsic fluorescence and far-UV circular dichroism that are insensitive to changes in the

16

oligomeric state of UCH-L1. The folding pathways of UCH-L1 therefore need to be revised in

17

light of our current findings.

Furthermore, SEC-SAXS and

18

We recently reported that UCH-L1 exhibits highly populated PUFs under native

19

conditions, which share common structural features with urea-induced folding intermediates.21 It

20

is conceivable that UCH-L1 may dimerize under native conditions despite being sparsely

21

populated. Indeed, dimerization of UCH-L1 under the native state has been reported to display

22

unusual ubiquitin ligase activity.32 Considering the geometry of ubiquitin binding site of UCH-

23

L1, it would require substantial conformational rearrangements for the binding crevice to


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simultaneously accommodate two ubiquitin molecules by dimeric UCH-L1 for isopeptide bond

2

formation. In summary, our findings echoed the notion of the fine balance between cellular

3

protein abundance and solubility in proteostasis as UCH-L1 is highly expressed in neuronal cells

4

with the ability to transiently populate oligomeric states with considerable conformational

5

disorder.21 Structural perturbation and destabilization by the PD-associated I93M mutation could

6

lead to irreversible precipitation when its folding intermediates are significantly populated and

7

perturbed, which may occur under physiological conditions with elevated oxidative stress or

8

aberrant proteostasis.14

9 10

ASSOCIATED CONTENT

11

Supporting Information.

12

Supporting Information Available: The Supporting Information detailing materials and methods

13

is available free of charge via the Internet at http://pubs.acs.org. It contains the thermodynamics

14

parameters derived from urea-induced equilibrium unfolding, chemical cross-linking, SEC-

15

MALS/SAXS and SAXS-based ensemble structural modeling results.

16 17

AUTHOR INFORMATION

18

Corresponding Author

19

Tel +886-2-27855696 ext5121; FAX: +886-2-27889759;

20

E-mail: [email protected]

21


12

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1 2

NOTE

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The authors declare no competing financial interests.

4

ACKNOWLEDGMENT

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This work is supported by the Ministry of Science and Technology (MOST104-2113-M-001-

6

016, MOST105-2113-M-001-005, and MOST 106-2113-M-001-004) and Academia Sinica,

7

Taiwan. S.-T.D.H. is a recipient of the Career Development Award (CDA-00025/2010-C) from

8

the International Human Frontier Science Program. We are grateful to the staff of BL23A1 of the

9

National Synchrotron Radiation Research Center (NSRRC, Taiwan) for their expert assistance in

10

SAXS data collection.

11

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A Natively Monomeric Deubiquitinase UCH-L1 ... - ACS Publications

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