Biophysical Analysis, Crystallization and Preliminary X-ray Diffraction Characterization of the Pex4p:Pex22p Complex of

University of Groningen

Stapled peptides inhibitors Ali, Amina

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Chapter 5

Biophysical Analysis, Crystallization and Preliminary X-ray Diffraction Characterization of the Pex4p:Pex22p Complex of

Hansenula polymorpha

Ameena M. Ali, Jack Atmaj, Alaa Adawy, Sergey Lunev, Niels van Oosterwijk, Sun Rei Yan, Chris Williams and Matthew R. Groves

(Published in: Acta Crystal 2018, F74, 76-81)

Abstract

Peroxisomes are a major cellular compartment of eukaryotic cells. Peroxisomes are involved in a variety of metabolic functions and pathways according to species, cell types and environmental conditions. Their biogenesis relies on conserved genes known as PEX genes that encode Peroxin proteins. Peroxisomal membranes proteins and peroxisomal matrix proteins are generated in the cytosol and are subsequently imported into the peroxisome post-translationally. Matrix proteins containing a peroxisomal targeting signal type 1 (PTS1) are recognized by the cycling receptor Pex5p and transported to the peroxisomal lumen. Pex5p docking, releasing of the cargo into the lumen and recycling involve a number of Peroxins, but a key player is the Pex4p:Pex22p complex described in this manuscript. Pex4p from the yeast Saccharomyces cerevisiae is a ubiquitin- conjugating enzyme anchored on the cytosolic side of the peroxisomal membrane through its binding partner Pex22p, which acts as both a docking site and co-activator of Pex4p. As Pex5p undergoes recycling and release, the Pex4p:Pex22p complex is essential for monoubiquitination at the conserved Cysteine residue of Pex5p. Absence of the Pex4p:Pex22p inhibits Pex5p recycling and hence PTS1 protein import. In this paper we report the crystallization of Pex4p and the Pex4p:Pex22p complex from the yeast Hansenula polymorpha and data collection of the complex crystals protein structure at 2.0 and 2.85 Å resolution, respectively. The resulting structures are likely to provide important insights to understand the molecular mechanisms of the Pex4p:Pex22p complex and its role in peroxisome biogenesis.

1 Introduction

Peroxisomes are organelles involved in many metabolic functions and pathways, depending upon the species, cell type and environmental conditions. Such functions include the oxidation of fatty acids, the protection of cells from oxidative damage [96,97], the metabolism of specific carbon and/or nitrogen sources, e.g. methanol, D-alanine, primary amines or oleic acid in yeasts [98] and synthesis of plasmalogens, cholesterol and bile acids in mammals [99]. Their biogenesis relies on highly conserved genes known as PEX genes that encode peroxins, which mainly function in the formation of peroxisomes or the import of matrix and membrane proteins [96,100]. For example, Pex5p is a key cytosolic recycling-receptor for matrix proteins imported through the peroxisomal targeting signal type 1 (PTS1) pathway [101]. The Pex5p import cycle can be divided into the following steps: (1) recognition of PTS1-containg protein by Pex5p in the cytosol [102,103], (2) docking of Pex5p–cargo complex at the peroxisomal membrane, [104]

[105] (3) cargo translocation into the peroxisomal lumen[106,107] and (4) recycling of Pex5p for a new import cycle [108,109].

Although the recycling of the Pex5p receptor has been studied in detail, the mechanism of cargo release remains elusive [110]

[111].

The receptor recycling step involves Pex5p monoubiquitination at the conserved Cysteine residue

[112,113] and requires the action of a number of peroxins, including Pex1p, Pex4p, Pex6p, Pex15p

and Pex22p [108,114–116]. The ubiquitin conjugating (E2) enzyme Pex4p in the yeast

Saccharomyces cerevisiae (Sc) has been shown in several studies to be responsible for Pex5p

monoubiquitination [109,112,117]. Pex4p associates with the peroxisomal membrane via its

interaction with the membrane bound Pex22p [114]. However, Pex22p also acts as a co-activator of Pex4p, stimulating the E2 activity of Pex4p through an unknown mechanism [117–119].

In order to understand the molecular mechanisms guiding the assembly of the Pex4p:Pex22p complex, as well as the role of Pex22p as co-activator protein, further high-resolution structural models of the complex partners are required. Here we report the crystallization of Pex4p alone and in complex with the soluble domain of Pex22p (hereafter Pex22

) from the yeast Hansenula polymorpha. Moreover, we use MST to analyse the dissociation constant (K

) of the Pex4p:Pex22

complex in vitro.

2 Materials & Methods:

2.1 Cloning of PEX4 and PEX22

Escherichia coli plasmids for expression of His

-GST tagged wild type Pex4p and the soluble region of Pex22p (Pex22

; residues 26-160) were made as follows: PCR was performed on H.

polymorpha genomic DNA using the primer combinations HpP4 NcoI

(GCCATGGCTTCTACAGAAAAGCGG) and HpP4 HindIII

(GCGAAGCTTTATACATCATTAGATTCGTATGC) for Pex4p and HpP22 NcoI

(CCATGGCCTGGGCGTTGAAGACG) and HpP22 HindIII

(GCGAAGCTTTATATATAATCATTTATACGATCC) for Pex22

, the resulting fragments were digested with NcoI and HindIII and ligated into NcoI-HindIII digested pETM30 vector. For cloning details please refer to Table (1).

2.2 Expression and Purification of Pex4p and Pex22

BL21 [DE3] RIL E. coli competent cells were transformed with either pETM30-PEX4 or pETM30- PEX22

expression plasmids. These plasmids encode wild type Pex4p or soluble region of Pex22p, respectively, both fused to a N-terminal GST and a His

tag. The expression of both constructs was performed according to the following protocol. The transformed colonies were selected on LB agar plates supplemented with kanamycin (25 µg/ml) and chloramphenicol (35 µg/ml). A single colony was used to inoculate a 10 ml culture of LB supplemented with kanamycin (25 µg/ml) and chloramphenicol (35 µg/ml) and incubated in a shaking incubator at 310 K for 4-5 hours. The culture was then used to inoculate 1 L TB supplemented with kanamycin (25 µg/ml) and chloramphenicol (25 µg/ml) and allowed to grow at 310 K to an OD

of 0.8. Cells were then cooled to 294 K and induced with Isopropyl-β-D-thiogalactoside to a final concentration of 50 µM.

Both cultures were further incubated at 294 K for 18 hours. After harvesting the cell pellets were

resuspended in buffer 1 (50 mM Tris, 300 mM NaCl and 1 mM β-mercaptoethanol (BME) at pH

7.5) and lysed using a French press. The lysate was then clarified by centrifugation (18000rpm; SS-

34 (Sorvall)) before incubation with 5 ml of Glutathione S-Transferase (GST) resin (GE

Healthcare). The resin was further sequentially washed in Buffer1, Buffer2 (50 mM Tris, 1 M NaCl,

1 % Glycerol and 1 mM BME at pH 7.5) and Buffer3 (50 mM Tris, 150 mM NaCl, 1 % Glycerol

and 1 mM BME at pH 7.5). Target proteins were eluted with 20-30 ml Elution Buffer (Buffer3

supplemented with 20 mM reduced glutathione (GSH)). Subsequently, eluted Pex4p and Pex22

proteins were subjected to TEV cleavage (ratio 1 mg TEV to 25 mg fusion protein) overnight at 277

K without shaking. Both TEV and the N-terminal cleavage products containing a His

-tag were

removed by passing through Ni-NTA Agarose and the flow-through was collected. Pure Pex4p and Pex22

were present in the flow-through, which was concentrated using centrifugal concentrators (MWCO 10,000 Da (Vivaspin 20-Sartorius)). Proteins were further purified by size exclusion chromatography (SEC) using Superdex 75 16/60 column (GE Healthcare) equilibrated with GF buffer (25 mM Tris, 150 mM NaCl, 1 % glycerol, 1 mM BME at pH 7.4). The Pex4p:Pex22

complex was prepared by incubating a mixture of Pex4p and Pex22

(1:1.8 molar ratio as determined using UV spectroscopy and their tabulated extinction coefficients (www.expasy.org)) for 1 hour on ice. The sample was further concentrated prior to injection onto a SEC-column (Superdex 75 16/60; GE Healthcare) previously equilibrated in GF Buffer.

Table (1): Macromolecule production information (Pex22

and Pex4p)

Gene Pex22^s

Source organism Hansenula polymorpha

DNA source H. polymorpha genomic DNA

Forward primer (NcoI) 5’-CCATGGCCTGGGCGTTGAAGACG-3’

Reverse primer (HindIII) 5’-GCGAAGCTTTATATATAATCATTTATACGATCC-3’

Expression vector pETM30 Expression host E. coli Complete amino-acid sequence

of the construct produced^& GAMA26WALKTINPGLFEEPAKTSEASKSNGQSVSLVLTQKDLDFFSAAYLNEY PNLTVILHPSVDKSEFLSRFNVQRNSHQVIQVRTEESIFHVLKQLSSNINLITLGN LEMSANEVETFHLDKFLTNVHEVDRINDYI160

Gene Pex4p

Source organism Hansenula polymorpha

DNA source H. polymorpha genomic DNA

Forward primer (NcoI) 5’-GCCATGGCTTCTACAGAAAAGCGG-3’

Reverse primer (HindIII) 5’-GCGAAGCTTTATACATCATTAGATTCGTATGC-3’

Expression vector pETM30 Expression host E. coli

Complete amino-acid sequence of the construct produced^&

GAMAS2TEKRLLKEYRAVKKELTEKRSPIHDTGIVDLHPLEDGLFRWSAVIRGP DQSPFEDALWKLEIDIPTNYPLDPPKIKFVVFGEEKIRQLQRKTSSGARKVCYK MPHPNVNFKTGEICLDILQQKWSPAWTLQSALVAIVVLLANPEPLSPLNIDM ANLLKCDDTTAYKDLVHYYIAKYSAYESNDV188

& Post TEV-digestion

Cloning details for the truncated Pex22

and Pex4p from H. polymorpha. Restriction sites in the

primers for the wild type are shown in bold italic. Additional residues at the N-terminus are shown

in underlined. The beginning and ending numbers of the amino-acid sequences are shown as

subscripts.

2.3 Crystallization of Pex4p and the Pex4p:Pex22

complex

Purified Pex4p was concentrated using a Vivaspin 20 (Sartorius) concentrator to 12 mg ml

for crystallization trials. Pex4p crystallization conditions were established on the high-throughput crystallization platform at the EMBL (Hamburg) and initial crystals were identified in 0.1 M MES and 40 % (v/v) PEG-200 at pH 6.5. Further optimisations of the crystallization conditions resulted in high-quality crystals obtained in 0.1 M MES and 50 % (v/v) PEG-200 at pH 6.5 appropriate for X-ray diffraction analysis from plates that were incubated at 293 K. Similarly, Pex4p:Pex22

complex concentrated to 12 mg ml

and was used for crystallization trials using Mosquito high- throughput crystallization robot (TTP Labtech). The concentration was calculated based on an assumed 1:1 complex and the respective tabulated extinction coefficients (Expasy.org). Initial screening was carried out with two screening kits: PACT Premiere HT-96 and JCSG-plus (Molecular Dimensions, Ltd). The plates were incubated at 293 K for a week, and initial crystals were identified in 0.1 M BIS-Tris Propane, 0.2 M sodium sulphate, 20 % w/v PEG-3350 at pH 7.5.

After optimization of the crystallization buffer to 0.1 M BIS-Tris Propane, 0.2 M sodium sulphate, 22 % w/v PEG-3350 at pH 7.8, a single crystal suitable for X-ray diffraction analysis was obtained.

Table (2) summarizes the crystallization conditions. Several crystals for the complex were grown during optimization of conditions with fine needle-shapes, in a fan-like structure. These crystals were fragile and difficult to fish prior to diffraction studies. Seeding was not attempted.

Pex4p as well as Pex4p:Pex22

complex crystals were transferred to a cryo-buffer consisting of the reservoir buffer supplemented with 20 % (v/v) glycerol, and then flash-cooled in liquid nitrogen prior the data collection. The crystals were then shipped for diffraction data collection to the PETRA III synchrotron (Hamburg, Germany) using a dry-shipping container (Taylor–Wharton).

Table (2): Crystallization of Pex4p as well as the Pex4p:Pex22

complex, including final buffer composition and conditions in which crystals were grown.

Construct Pex4p Pex4p:Pex22^S complex

Method Hanging-drop vapour diffusion Sitting-drop vapour diffusion

Plate type 24-well XRL Plate (Molecular Dimensions cat#

MD11-00-100)

96-well Polystyrene MRC Crystallization Plate (Molecular

Dimensions; cat# MD3-11)

Temperature (K) 293 293

Protein concentration (mg ml^-1) 12 12

Buffer composition of protein solution

25 mM Tris, 150 mM NaCl, 1 % glycerol, 1 mM BME at pH 7.5

25 mM Tris, 150 mM NaCl, 1 % glycerol, 1 mM BME at pH 7.5 Buffer composition of reservoir

solution 0.1 M MES and 50 % (v/v) PEG-200 at pH 6.5

0.1 M BIS-TRIS Propane, 0.2 M sodium sulfate, 22 % w/v PEG-

3350 at pH 7.8

Volume and ratio of the drops 2 µl (1:1 ratio) 320 nl (1:1 ratio)

2.4 Microscale Thermophoresis (MST)

MST measurements were performed on a Nanotemper Monolith NT.115 instrument (Nanotemper Technologies, GmbH). Purified Pex4p was labelled with the Monolith Protein Labelling Kit RED according to the supplied protocol (Nanotemper Technologies, GmbH). Labelled protein was concentrated using a PES centrifugation filter (3 kDa cutoff, VWR), diluted with glycerol (final concentration 50 % v/v) and the aliquots were stored at 193 K. Measurements were performed in MST buffer (25 mM Tris pH 7.5, 125 mM NaCL, 1% Glycerol and 1 mM BME, 0.05 % Tween-20) in standard capillaries (K002, Nanotemper Technologies, GmbH). Labelled Pex4p was used at a final concentration of 10 nM. Pex22s was titrated in 1:1 dilutions starting at 2.82 µM. All binding reactions were incubated for 10 min at room temperature followed by centrifugation at 20,000 x g before loading into capillaries. All measurements were performed in triplicates at 20 % LED and 60

% MST power, Laser-On time was 30 sec, Laser Off time 5 sec.

3 Results:

3.1 Crystallization of Pex4p

Full-length Pex4p protein was purified from an E. coli-based expression system and the purified protein was crystallized at 293 K (Figure 1). The final crystals were grown in 0.1 M MES and 50 % (v/v) PEG-200 at pH 6.5. A single crystal was harvested in a mounted loop, directly flash-cooled in liquid nitrogen and transported to P11 beamline (PETRA III, Hamburg) for data collection and structural analysis.

Figure 1: Needle-shaped Pex4p crystals produced in 0.1 M MES and 50 % (v/v) PEG-200 at pH 6.5.

3.2 Crystallization of the Pex4p:Pex22

Complex

An initial crystal of Pex4p:Pex22

complex was grown in a solution of 0.1 M BIS-Tris Propane, 0.2 M sodium sulphate, 20 % w/v PEG-3350 at pH 7.5 from the PACT Premiere HT-96 screening kit (Molecular Dimensions, Ltd) as shown in Figure (2a). Figure (2b) illustrates the final crystal, which was grown in 0.1 M BIS-Tris Propane, 0.2 M sodium sulphate and 22 % (w/v) PEG-3350 at pH 7.8.

The crystal was directly harvested and flash-cooled for data collection and structural analysis as indicated above.

Figure 2: a) Initial crystals grown in 0.1 M Bis-Tris propane, 0.2 M ammonium sulphate and 20 % w/v PEG-3350 at pH 7.5 (PACT Premiere HT-96, Dimensions, Ltd). The drops were set-up by the sitting-drop method using a Mosquito robot (TTP Labtech) using drops consisting of 160 nl protein solution and 160 nl precipitant solution, and the plates were incubated at 293 K. b) The final crystals grown in 0.1 M Bis-Tris propane, 0.2 M ammonium sulphate and 22 % w/v PEG-3350 at pH 7.8. The drops were set-up by the sitting-drop method using a Mosquito robot (TTP Labtech) using drop sizes of 160nl:160nl (protein:precipitant) and plates incubated at 293 K.

3.3 Data Collection & Processing

The Pex4p crystal used for the experiment diffracted to a resolution of 2.0 Å and the X-ray data were collected using beamline P11 at the PETRA III synchrotron (DESY, Germany). The raw data were processed automatically using the software XDS [120] for integration and truncation of the data. The Pex4p crystal belonged to space group P4

2

2 with unit-cell parameters a = 46.35, b = 46.35, c = 206.41 (Å), α = β = γ = 90

. Similarly, diffraction data from the Pex4p:Pex22

complex crystal was also collected at beamline P11 of PETRA III synchrotron (DESY, Germany). The crystal diffracted to a maximal resolution of 2.85 Å. The crystal of the complex belonged to P1 space group with unit-cell parameters a = 44.7, b = 61.6, c = 78.4 (Å), α = 89.2

, β = 78.0

, γ = 84.1

The raw data were processed automatically using XDS [120]. Data-collection and processing statistics are summarized in Table (3). The Pex4p structure was solved with molecular replacement software MOLREP using the structure of S. cerevisae Pex4p as a search model (PDB: 2Y9M; 31/66

% identity similarity) with a solution with a Z-score of 10.0. The Pex4p:Pex22

complex

coordinates from S. cerevisae (PDB: 2Y9M)[118]; 31/66 % and 15/48 % identity/similarity, Pex4p

and Pex22

respectively) were also used as a search model to interpret the Pex4p:Pex22

data from

H. polymorpha, yielding a molecular replacement solution (Z-Score = 12.4) for two copies of each

partner in the complex protein (Pex4p: Pex22

).

Table (3): Data collection statistics

Pex4p Pex4p:Pex22^S complex

X-ray source PETRA III (Hamburg)

Beamline P11

Wavelength (Å) 1.03 0.98

Space group P41212 P1

Unit-cell parameters

a, b, c (Å) 46.35, 46.35, 206.41 44.7, 61.6, 78.4

α , β , γ (^°) 90, 90, 90 89.2, 78.0, 84.1

Resolution (Å) 45.22 – 2.0 (2.26 - 2.0) 76.7-2.85 (2.95-2.85)

Rmeas 0.147 (0.74) 0.123 (0.80)

Total No. of observations 59646 (1977) 71456 (9995)

Total No. of unique reflections 14409 (1206) 18181 (1822)

Mean I/σ (I) 6.21 (1.69) 6.03 (1.14)

Completeness (%) 92 (88) 95.02 (94.5)

Wilson B-factor 42.5 48.9

Multiplicity 4.1 (1.6) 3.9 (5.48)

CC1/2 0.995 (0.426) 0.996 (0.529)

Matthew’s Coefficient 2.63 2.92

Mosaicity 0.13 0.42

R-factor is defined as (

|𝐹

(ℎ𝑘𝑙) − 𝐹

(ℎ𝑘𝑙)| )/(

𝐹

(ℎ𝑘𝑙)), where F

and F

are observed and calculated structure factors of the reflection of hkl, respectively.

R

is defined as

|𝐼

ℎ𝑘𝑙 −< 𝐼(ℎ𝑘𝑙) > | /

𝐼

(ℎ𝑘𝑙) , where I

(hkl) is the i

intensity measurement of reflection hkl and <I(hkl)> is the average intensity from multiple observations.

Values in brackets represent data in the higher resolution shell.

3.4 Pex4p:Pex22

binding

Parameters of the Pex4p:Pex22

complex formation were assessed using Microscale

Thermophoresis (MST). MST relies on the motion of molecules in microscopic temperature

gradients to detect minute changes in charge, size and changes in the hydration shell of a molecule

[121,122]. In this experiment, fluorescently labelled Pex4p, previously purified to homogeneity

(See Materials and Methods) was titrated with Pex22

. Figure (3) shows an MST curve of Pex4p in

the presence of different concentrations of Pex22

. The dissociation constant for the Pex4p:Pex22

interaction was calculated to be 1.94 ± 0.39 nM. This is in good agreement with the reported

binding affinity between Pex4p and Pex22

from S. cerevisiae, as determined by isothermal titration calorimetry (ITC; 2.00 ± 0.08 nM) [118].

Figure 3: MST curve for the binding of Pex4p to Pex22^S, displaying 1:1 stoichiometry. The assay was performed using a fixed concentration of fluorescently labelled Pex4p (10 nM).

4 Discussion

The structure of H. polymorpha Pex4p was solved by molecular replacement using the Pex4p structure from S. cerevisiae as a search model (PDB entry 2y9m; 31% identity and 66% similarity to H. polymorpha Pex4p). The structure of the H. polymorpha Pex4p–Pex22

complex was solved using a model built from the structure of H. polymorpha Pex4p together with that of S. cerevisiae Pex22

from the S. cerevisiae Pex4p–Pex22

structure (PDB entry 2y9m; 15% identity and 48%

similarity to H. polymorpha Pex22

), yielding a clear molecular-replacement solution with two copies of H. polymorpha Pex4p– Pex22

. Our structural analysis of H. polymorpha Pex4p and the Pex4p–Pex22

protein complex will be reported elsewhere (Chapter 6).

Pex22p acts as a co-activator of Pex4p, stimulating the activity of the E2 enzyme through an as yet unknown mechanism (Williams et al., 2012, 2013; El Magraoui et al., 2014). Hence, we anticipate that the structures resulting from the data reported here will provide important insights into the molecular mechanism underlying the Pex22p-dependent co- activation of Pex4p and how this impacts on the role of Pex4p in peroxisome biogenesis.

The use of MST allowed more precise insight into complex formation. The low-nanomolar dissociation constant for complex formation in vitro (1.94 ± 0.39 nM) suggests that tight binding is required for the activation of Pex4p. A 1:1 stoichiometry for the binding is also supported by our MST data (Fig. 3), as at a 1:1 ratio (10 nM:10 nM) the curve is close to saturation. Our MST data are in agreement with previous ITC data provided for the Pex4p– Pex22

complex from S.

cerevisiae, in that the dissociation constant (K

) is equal to 2.0 ± 0.08 nM (Williams et al., 2012). It

should be borne in mind that these two affinity measurements of the Pex4p–Pex22

interaction,

while in good agreement, are from distinct species. As a result, a direct comparison of the methods

is difficult to defend, although the MST sample requirements are significantly lower.

5 Acknowledgements

The authors would like to thank the staff of the P11 beamline at the PETRA III synchrotron, DESY, Hamburg for beamline access. The authors would also like to thank Dr Christian Kleusch and Dr Katarzyna Walkiewicz from Nanotemper Technologies GmbH for technical support and advice.

6 Funding information

CW is supported by a VIDI Grant (723.013.004) from the Netherlands Organization for Scientific Research (NWO). AMA gratefully acknowledges the Qatar Research Leader- ship (QRLP)–Qatar Foundation for financial support.

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