Purification, co-Crystallization and Structure Determination of

Plasmodium falciparum plasmepsins.

Master’s Thesis

Matthias BaroneÀ and Dr. Lars PradeÁ

ÀM.Sc Curriculum Nano-Sciences,

ÁActelion Pharmaceuticals Ltd, Gewerbestrasse 16, 4123 Allschwil, Switzerland

October 27, 2009

Supervised by Prof. Tilman Schirmer, Biozentrum, University of Basel, Switzerland

Abstract

In the erythrocytic stage of the life cycle, the malaria parasite Plasmodium falciparum degradeshemoglobin inside an acidic food vacuole. Degradation is initiated by a number of aspartic proteases,called plasmepsins. Three of these members were expressed in inclusion bodies of E. coli purified by aprotocol, which had been optimized for plasmepsin II during the master’s thesis.

Plasmepsin II and IV were co-crystallized with non-peptidometric inhibitors. Protocol optimizationled to crystals of three complexes which were solved in orthorhombic and triclinic crystal packings (spacegroups P212121 and P1) at a resolution limits of less than 2.7 and 1.95 A.

The complexes display a binding mode involving both hydrophobic and polar interactions. Therebythe inhibitors interact with the flap pocket and H-bonding to the catalytic dyad without need of acatalytic H2O.

Contents

1 Introduction 2

2 Materials and Methods 2

2.1 Chemicals . . . . . . . . . . . . . . 2

2.2 Main Protocol . . . . . . . . . . . . 3

2.2.1 Transformation . . . . . . . 4

2.2.2 Test Expression . . . . . . . 4

2.2.3 Expression . . . . . . . . . 4

2.2.4 Purification and Activation 5

2.2.5 Crystallization . . . . . . . 6

2.3 Data Acquisition and Processing . 6

3 Results and Discussion 6

3.1 Purification . . . . . . . . . . . . . 7

3.1.1 PMII . . . . . . . . . . . . 7

3.1.2 PMIV . . . . . . . . . . . . 123.1.3 PMI . . . . . . . . . . . . . 12

3.2 Crystallization . . . . . . . . . . . 143.2.1 Control of Protein Purity . 143.2.2 Crystal Growth . . . . . . . 14

3.3 Structure Determination . . . . . . 183.3.1 Compounds . . . . . . . . . 18

3.4 Structure Description . . . . . . . 213.4.1 Interfaces . . . . . . . . . . 21

3.5 Structure Comparison . . . . . . . 223.5.1 Diff. among Chains . . . . 223.5.2 Diff. ST47 and ST43 . . . . 233.5.3 Active Site & Flap . . . . . 24

4 Conclusion and Look-out 274.1 Purification . . . . . . . . . . . . . 274.2 Inhibitors . . . . . . . . . . . . . . 28

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2

1 Introduction

Tropical malaria constitutes one of the most im-poverishing infectious diseases, annually affecting200–500 million people worldwide and causing thedeath of 2.5 million people - alone one millionchildren in Africa[1, 2].

Malaria Pathogens and Their Life Cycle

The tropical disease malaria is caused by proto-zoan parasites. There are four pathogen specieswhich each cause a specific clinical picture: Plas-modium falciparum (Malaria tropica, the mostserious form), P. vivax (Malaria tertiana, thewidest spread form), P. ovale (Malaria tertiana)and P. malariae (Malaria quartana). The genusPlasmodium belongs to the phylum apicomplexa(old: sporozoa) and posses therefore a rather com-plex life cycle:

The most important mammalian reservoir is pro-vided by humans where the parasite spends itsasexual life cycle. The sexual life cycle is fin-ished in female mosquitoes of the genus Anophe-les. Mosquitos are infected by gametozytes. Af-ter maturation and fusion, the newly formed zy-gote resides within an oocyst on the basal side ofthe female Anopheline mosquito mid gut wall[3].The zygote produces in a later step infectingcells called sporozoites, which migrate into theanticoagulant-producing salivary gland[4].

During piercing a person’s skin the anticoagu-lant containing the infective form of Plasmod-ium is injected into the hepatic system of a newhost. Once in the blood stream, the sporo-zoites are removed by the liver and infect hep-atocytes. These cells serve as hosts in which thesporozoites multiply asexually and differentiateto merozoites. When the host cell is ruptured themature merozoites escape from the liver into theblood stream undetectable, thus beginning theerythrocytic stage of the life cycle[4].

The subsequent invasion of erythrocytes pro-duces all the characteristic symptoms known froma malaria infection: fever up to 40 ℃, followed byperiodic ague, may accompanied by vomitus andheadache. The continuous lysis of red blood cellsleads to anemia in a later phase.

During the erythrocytic stage, the protozoanparasite satisfies its requirements by degrad-ing hemoglobin within an acidic food vacuole.Therein plasmepsins I to IV (PMI to PMIV) ini-tiate degradation of hemoglobin. It is assumedthat all PMs degrade host cell hemoglobin. Thereare three isoforms of aspartic proteases (PMI, IIand IV) which all show in vitro degradation ofhemoglobin. PMIII is known as histo-asparticprotease (HAP).

The predicted coding sequences of PMI, II, IV,and HAP are 60–73% identical on amino acidlevel. High levels of sequence identity in bothpro- and mature sequence suggest that PMI, II,IV, and HAP have arisen through relatively re-cent gene duplication events[5].

Compared to PMII and I, HAP has several sub-stitutions including replacement of a catalytic as-partate with a histidine and changes in the con-served flap motif[6].

The aim of the work was to determine struc-tures of PM complexes with non-peptidometic in-hibitors synthesized by groups of Prof. Dr. Ger-hard Klebe (University of Marburg) and Prof.Dr. Francois Diederich (ETH Zurich).

2 Materials and Methods

2.1 Chemicals

Chemicals purchased from Fluka: polyethy-lene glycol 4000 (PEG 4K, ultra, 81268);glycerol 87% (92.10 g/mol+aq, 86 − 89%,49782); tris(hydroxymethyl)aminomethane (Tris,121.14 g/mol, ≥ 99%, 93352); sodium hy-droxide (NaOH, 40.00 g/mol, 71690); glycine

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(75.07 g/mol, ≥ 99%, 50049); ethylendiamine-tetraacetic acid disodium salt dihydrate (EDTA,372.24 g/mol, ≥ 99%, 03679); hydrochloricacid 32% (HCl, ≥ 32%, 84421); sodium ac-etate anhydrous (NaAc,82.04 g/mol, ≥ 99%,71180); acetic acid (60.05 g/mol, ≥ 99.8%,45731), sodium phosphate monobasic dihydrate(156.01 g/mol, ≥ 99%, 71500); tert.-butanol(74.12 g/mol, ≥ 99.7%, 19460), dimethylsulfox-ide (DMSO, 78.13 g/mol, ≥ 99.9%, 41640), imi-dazole (68.08 g/mol, ≥ 99.5%, 56750).

Chemicals delivered by Sigma: isopropyl-β-D-thiogalactoside (IPTG, 238.3 g/mol, 367-93-1), ampicillin sodium salt (371.39 g/mol,A9518-25G); (-)-glutathione, oxidized (GSSG,612.6 g/mol, 150568-5G); L-glutathione reduced(GSH, 307.32 g/mol, ≥ 99%, G4251-25G);2-mercaptoethanol (β-MeEtOH, 78.13 g/mol,1.115 g/l ≥ 98% M3148-100ML); 1-octanol 99%(111-87-5), LB Broth (L7275-500TAB); TerrificBroth, Modified EZMix Powder (T9179-1KG);urea (U4128-5KG); ethanolamine (E-6133);potassium phosphate dibasic (174.2 g/mol,P8281-500G)

Obtained from Invitrogen were: One ShotBL21(DE3) competent cells; S.O.C. medium(15544-034); MagicMedia E. coli expressionmedium (K6802); NuPAGE 4-12% Bis-Tris Gel(10 or 12 well, NP0322BOX); NuPAGE MOPSSDS running buffer (20×, NP0001); NuPAGELDS sample buffer (4×, NP0007); SeeBluePlus2 pre-stained standard (1× protein ladder,LC5925); pH 3-10 isoelectric focusing (IEF) gel(10 well, EC6655BOX); Novex IEF pH 3-10 cath-ode buffer (10×, LC5310); Novex IEF pH 3-10anode buffer (50×, LC5300).

Isopropyl-β-D-thiogalactoside (IPTG,238.3 g/mol, 10724815 001) was providedby Roche.

NaCl (58.44 g/mol; ≥ 99.5%; A1149,1000) waspurchased from AppliChem.

Except during expression SDS-PAGE samples

were stained as follows: 20µl sample, 5µl Nu-PAGE LDS sample buffer (4×), 2µl of 98% β-MeEtOH. Sample containing entire cells werestained with 80µl NuPAGE LDS sample bufferper OD600=1 and to every SDS sample, by de-fault, 5µl of 98% (β-MeEtOH). All SDS sam-ples were heated for 5min at 95 ℃ in heat block(Techne, DRI block DB·2D) and then centrifugedat 13000 rpm for 4min (Heraeus, Biofuge fresco).From protein ladder (MW marker) as well as sam-ples 10µl were loaded onto gel. 12 well gels wererun for 53min at 200V , 10 well gels for 44minin XCell surelock (Invitrogen Novex mini cell).

All substances were sufficiently pure and wereused without further purification. Ethanolamine,EDTA, 50% (w/v) PEG 4K, glycine, Tris, NaAc,MgCl2, and NaCl were dissolved in deionizedwater (milli-Q) and afterward filtered through0.45 µm Durapore membrane filters (Millipore).Stock solutions of Na/K phosphate (Na/K Pi,used for SEC running buffer) and sodium cit-rate (used for crystallization screens) were filteredwith 0.22 µm Steriflip vacuum filtration system(Millipore).

Crystals were grown at 293 K using the hanging-drop technique and vapor-diffusion method withCryschem plates HR3-275 (Hampton research).The crystallization solutions were first preparedmanually and 200 µl were used as reservoir. Thedroplet was formed by a ratio of 2:1 or 3:1 (v/v)protein to reservoir solution. For the whole pipet-ting procedure, a robot (Tecan, freedom evo) wasused.

2.2 Main Protocol

The following protocol defines the final versionachieved by continuous optimization of given pro-tocols (written by Andrew Jones for PMII pu-rification) and literature[7]. Main differences be-tween the final and the starting protocol are dis-cussed below. For the expression media following

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abbreviations were used: Terrific Broth (Tm), LBBroth (LB), and MagicMedia (Mm).

2.2.1 Transformation

Aliquots (50µl) of competent cells (Invitrogen,Escherichia coli One Shot BL21(DE3)) werethawed on ice. All tubes were held on ice un-til to each sample 1µl of plasmid (100ng/µl)was added. After incubating for 30min on ice,aliquots were heated for exactly 30 s at 42 ℃ in aheating block (Eppendorf, Thermo-mixer Com-fort) without shaking. After heat shock, tubeswere placed for 2min back on ice and afterward250µl of pre-warmed (37 ℃) S.O.C. medium wasadded (tubes were kept on ice until all had re-ceived S.O.C.). After incubating at 37 ℃ whileshaking at 300 rpm for 60min in a heating block,aliquots were spread onto 2 Petri dishes contain-ing 5 and 20µl of cell medium. To one aliquotof competent cells, 1µl of H2O instead of plas-mid was added as control. From this aliquot thewhole 301µl were spread onto one Petri dish. Af-ter drying for 5-10min, the plates were turnedupside-down and incubated at 37 ℃ over night(o/n).

2.2.2 Test Expression and Glycerol Stock

For test expression 5 clones were picked andincubated o/n at 37 ℃ in 3ml LB (Falcon14ml polystyrene round-bottom tubes) con-taining 50µg/ml ampicillin (Amp). Fromthe over-night cultures 0.5ml were used toinoculate 50ml (1:100 (v/v)) of prewarmedLB (50µg/ml Amp) and shaken (230 rpm) at37 ℃ until cultures reached an optical density at600nm (OD600) of 0.6. From every cell culture,a 3ml sample was placed on ice.

The first 2ml were used to form 10×200µl glyc-erol stock aliquots. Cell culture was mixed incryo tubes (Nunc, CryoTube vials 1.8ml) with1:1 (v/v) of 87% glycerol by finger flicking and

flash freezed in liquid nitrogen. Glycerol stock ofall 5 clones were stored at -80 ℃. The remaining1ml was used for SDS-PAGE (NuPAGE 4-12%Bis-Tris Gel).

The remaining 47ml of cell culture were inducedby addition of isopropyl-β-D-thiogalactoside(IPTG) to a final concentration of 1mM and in-cubated for 3h. Another SDS sample (stainedwith LDS buffer according to the new OD600)was then taken and an SDS-PAGE gel run tocompare the 5 clones.

2.2.3 Expression

According to SDS-PAGE one clone was pickedand an over-night culture with 50ml LB(50µg/ml Amp) was inoculated with 100µl of itsglycerol stock (using sterile tips) and stirred at160 rpm and 37 ℃. Together with the over-nightculture the expression medium may prewarmed(adding Amp before prewarming!).

According to preferred expression media, differ-ent procedures were needed for proper inoculationand induction.

LB Broth had to be induced manually byadding 1mM IPTG when the cell culturereached OD600 of 0.6. Therefore, it was im-portant to inoculate the medium as early aspossible in the morning. LB was inoculatedwith 1:100 (v/v) of o/n culture (10ml perliter LB).

Terrific Broth contained additionally to thepowder 0.8% (v/v) glycerol (9.2ml 87% glyc-erol per liter). Glycerol had been added be-fore autoclaving the medium. Tm was inocu-lated with 1:100 (v/v) of o/n culture, expres-sion had to be induced manually by adding1mM IPTG at an optical density of 0.6.

MagicMedia was designed as a two-componentmedium. Component A (SoluPouch) wasdissolved in 950ml H2O and autoclaved.

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Before inoculation, the medium was com-pleted by adding 50ml of component B and50µg/ml Amp. E. coli transformed PMIIcultures were grown o/n at 37 ℃, 220 rpmand harvested in stationary phase where theyreached an OD600 of about 5.

For a proper blanking of the OD meter inlater steps a 80µl sample of every uninoculatedmedium was stored at 4 ℃.

Before inducing LB and Tm, a 1ml sample hadbeen taken and prepared for SDS. If requiredone drop of 1-octanol acting as anti-foaming wasadded for proper aeration. After induction byIPTG, media were incubated for 4h before har-vested. Another 1ml sample was taken for induc-tion/expression control by SDS-PAGE .

Cells were harvested in 500ml centrifugationtubes (same number of tubes as liter of mediumused) at 7000 rpm, 10 ℃ for 7min (Sorvall, RC5C plus, SLA 3000-rotor, rotor code 30). Su-pernatant was discarded back into empty cultureflasks directly beside the ultracentrifuge. Pelletswere transferred by a spatula into 50ml-falcontubes, briefly centrifuged (Heraeus, Multifuge 1L-R) and frozen at -20 ℃.

2.2.4 Purification and Activation

Pellets stored at -20 ℃ were thawed on ice and re-suspended with approximately 40ml of buffer A(50mM Tris pH 8.0; 150mM NaCl) and stirredfor about 30min at 4 ℃. Before further process-ing, 20µl were transferred into an Eppendorftube and put on ice for SDS-PAGE.

Cells were disrupted by two rounds of frenchpress (1.33 kBar, 10 ℃). French press was rinsedand washed to a maximal final volume of 6×35ml(the maximal centrifuge tube volume), 210ml ofbuffer A.

Cell lysate was transferred into 4 to 6 35ml-centrifuge tubes and centrifuged at 14000 rpm for

15min at 15 ℃ (Sorvall, RC 5C plus, SS34-rotor,rotor code 5). While supernatant was kept onice, tubes were refilled with buffer B1 (100mMglycine pH 11.0; 50mM β-MeEtOH). Homoge-neously colored pellets were not resuspended.Tubes were vortexed for a few seconds and af-terward centrifuged at 14000 rpm for 15min at15 ℃.

After the supernatant of buffer B was alsostored on ice, pellets were diluted with 500ml ofpre-cooled buffer C1 (100mM Tris pH 8.0; 6Murea; 1mM glycine; 1mM EDTA; 50mM β-MeEtOH). After 2h of dissolving at 4 ℃, an SDS-PAGE had been run containing the stored pellet,french press samples, supernatants A and B, plusthe diluted protein in urea-buffer C.

Complete dilution in buffer C took place o/nat 4 ℃. Aliquots (50ml) were stored at -20 ℃ asan oxidation during the storage led to significantlower yield. Samples of 50ml were diluted withP960 (AKTApurifier 10) with 0.1ml/min into 1 lof buffer D (10mM Tris pH 8.5, sterile filteredand pre-cooled to 4 ℃). The most rapid dilutionwas achieved if the tubing was placed in the mid-dle of a vigorously stirring renaturation buffer.The final dilution of 1:20 (v/v) (final 300mMurea) was enough for rapid refolding. The yieldof properly folded material decreased if renatu-rated protein in buffer D was stored longer than24h.

The first liter of diluted protein solution wastherefore directly processed further by loadingonto a HiTrap Q HP ion exchange column (GEHealthcare, 1ml or 5ml bed volume) with max-imum flow (depending on back pressure of thesystem). After washing the column with 20 −30×C.V. running buffer (0.1M Tris/HCl pH 8.5)with 1− 2ml/min (starting conductivity around3mS/cm) protein was eluted by a gradient over

1As buffer B and C contained temperature-sensitivematerial as β-MeEtOH or EDTA they were mixed directlybefore application.

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35 × C.V., 1.5ml/min to 100% elution buffer(0.1M Tris/HCl pH 8.5; 1M NaCl, conductivityof around 100mS/cm).

Depending on the elution profile, two sampleswere pooled: 4ml containing the peak-top anda second sample with the adjacent fractions in-cluding the populations behind the main peak.Pools were stored at 4 ℃. Although 1 month oldadjacent peaks were able to activate themselves,the samples contained significant populations ofmisfolded and aggregated protein.

For activation, the sample was brought to roomtemperature (RT), mixed with 1:4 (v/v) of activa-tion buffer (1M sodium acetate (NaAc) pH 4.5)and gently shaken for 1.5 − 2h. Activation wasdetectable after 15min on SDS gels. The ac-tivated main-peak sample (4.8ml) was directlyloaded onto a HiLoad 16/60 Superdex 200 pg sizeexclusion column (SEC) at 4 ℃.

The HiTrap pool containing the adjacent peaksusually was bigger than 5ml (recommended sam-ple volume for a 16/60 SEC). Because the crystal-lization conditions lay around pH 3-5 and there-fore the protein seemed to have a significantlylower solubility in acidic environment, the acti-vated sample (pH 4.5) was brought to pH 8.37 be-fore centrifugation by addition of 10% (v/v) 1MNaOH of the HiTrap pool volume (or 50% (v/v)of activation buffer used). Samples for SEC loadwere concentrated at 4 ℃ (Amicron ultra cen-trifugal filter, 4.5ml max. volume, 10 kD cut-off,2850 rpm, cycles of maximally 4min).

SEC had been run at 4 ℃, 1ml/min with fol-lowing running buffer: 5mM Na/K Pi pH 6.5;100mM NaCl. Correctly folded PMII passed the16/60 SEC after 86ml. Pooled protein was puton ice and concentrated with 4.5ml-Amicron fil-ter up to 10mg/ml at 4 ℃, 2850 rpm, 4 − 2minper run (merging the whole solution after eachrun).

2.2.5 Crystallization

Inhibitors were dissolved to a concentration of30mM in DMSO or tert.-butanol and stored at20 ℃. Purified protein was acidified in SEC run-ning buffer with 1:400 (v/v) of 1M HCl to a fi-nal pH 4.2 and afterward mixed by finger-flickingwith an equimolar, 1:20, or 1:10 (v/v) ratio withinhibitors and incubated o/n at 20 ℃. Beforepipetting the sample was centrifuged at RT at13000 rpm for 4min (Heraeus, Biofuge fresco).

2.3 Data Acquisition and Processing

Crystals were measured using synchrotron radia-tion light source at the PXIII (X06DA) beamlineof the Swiss Light Source (Paul Scherrer Insti-tute, Villigen AG, Switzerland). Datasets wereindexed using go.com[8], imosflm[9, 10], or XDS[11].

Molecular replacement was done withMOLREP[12] using a pruned PMII as a searchmodel. This model contained only the conservedcore of PMII without three flexible loops. Thestructures were refined using REFMAC5[13] andrebuilt by Coot[14] or Moloc[15]. Assessment ofthe models against their native structure factorswere done using Sfcheck[12].

Protein models (Cα-trace or cartoon) were ren-dered using PyMOL[16] and finished in Photoshop(highlighting, labeling, erase irrelevant chains oradding artificial objects).

3 Results and Discussion

As an overview of the crucial steps, the follow-ing itemization lists the modified and improvedsteps and conditions. Each point is discussed sep-arately:

• The main differences to the starting proto-col (written by Andrew Jones) lie in crucialsteps before and after rapid dilution. The

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old protocol used, after activation of proP-MII a concentration step, a H4848 affinitycolumn followed by another concentration bycentrifugation for SEC load. After the SEC,a third concentration step was necessary forcrystallization.

Thereby the H4848 showed no significantgain in purification. After checking the lit-erature, the first two lossy concentrationsand the H4848 column were replaced by amuch more efficient loading of proPMII (noactivation) onto a HiTrap column (analogueoriginal literature). Using HiTrap columnswe reached more than 250×-concentration(1− 4 l-load pooled as 4ml fractions).

• During renaturation proPMII is susceptibleto unfolding if processed or stored at RT.Refolding, purification as well as concentra-tion yielded significantly higher amounts ofcorrectly folded populations whenever run at4 ℃.

• The activation of proPMII in acidic environ-ment was no longer run for 2h at 37 ℃. AnSDS-PAGE-tracing during the activation atRT showed complete activation after 2h. In-stead of 10% we used 20% (v/v) of 1M NaActo reach the final pH of 4.5.

• If the activated sample was too voluminous,the concentration step for SEC load was runat high pH (8.5) to prevent precipitation.For the same reason, ultracentrifugation wasrun at 4 ℃ and 2850 rpm.

• Crystal growth took place in a solution con-taining two different buffers. PMII was in-cubated in SEC running buffer (5mM Na/KPi pH 6.5, acified by HCl to pH 4.2), whereas0.1M sodium citrate was used to obtain thepH of the crystallization condition (bufferedpH 2.5 to 7.0). There is evidence that dur-ing pipetting and crystal growth the pH of

crystallization droplets did not shift to thedesired value - especially at pH 2.5 to 4.0.For this purpose the initial concentration of20mM Na/K Pi SEC running buffer was re-duced to 5mM Na/K Pi keeping the pH at6.5.

• We observed that after refolding by rapiddilution, proper oxidation of PMII’s cysteinresidues does not require addition of oxidiz-ing and reducing agents like GSSG and GSH.The crucial factor is the flow by which the de-naturated protein is injected into the renat-uration buffer. To control it we optimizedthe renaturation using the P960 membranepump at 4 ℃ with a flow of 0.1ml/min. Nei-ther the time-consuming drop-by-drop injec-tion by hand (as in the old protocol) nor us-ing Bernoulli’s principle could reach the sam-ple quality as a slow, flow-controlled injec-tion by P960 did.

3.1 Purification

3.1.1 PMII

Test expression and glycerol stock forming wererun according to the main protocol.

Pellet Purification

proPMII was expressed in E. coli in inclusionbodies, which facilitated the pellet purification bycentrifugation after cell disruption. Fig. 2 showsthat dissolved impurities were removed mainly bycentrifugation of buffer A. The bright white colorof the pellets allowed us to conclude that inclu-sion bodies do not get disrupted by two roundsof french press. According to an SDS the subse-quent centrifugation in 100mM glycine pH 11.0of buffer B did not improve the purity. As thisadditional centrifugation step lasted only 12min,it was kept in the protocol.

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Figure 1: SDS-PAGE controlling the test expres-sion of proPMII in three clones. Bands: MWmarker, À clone A before, Á after induction, Â

clone B before, Ã after induction, Ä clone C be-fore, Å after induction. Clone C was used for ex-pression as this clone grew faster than the other(data not shown).

All subsequent steps after pellet purification wereperformed at 4 ℃ or tubes were kept on ice un-til further processed (e.g. during concentration).Pellets were dissolved in urea with a pre-cooledbuffer C while tubes were vigorously stirred overnight at 4 ℃.

Figure 2: SDS-PAGE to check the pellet purifi-cation of proPMII. Bands from left to right: MWmarker, À pellet stored at -20 ℃, Á after tworounds of french press, Â supernatant buffer Aafter centrifugation, Ã supernatant buffer B af-ter centrifugation, Ä proPMII diluted in bufferC.

Controlling the Refolding by HiTrap Elu-

tion Profiles

The implementation of a HiTrap Q ion exchangerhad two main advantages: a) proPMII diluted inrather large volumes could be bound to a HiTrapand concentrated by a NaCl-gradient elution, andb) correctly folded and misfolded proPMII gotseparated by the gradient as shown by isoelectricfocusing (IEF, Fig. 3).

Using HiTrap columns with 1ml bed volume anda long NaCl-gradient led to the convenient sideeffect of a main-peak pool with a volume around4ml. This was the maximum volume which couldbe loaded after activation directly on a SEC with-out concentrating the sample. The rest of theelution profile was also pooled. It was concen-trated while the main-peak sample was passedthrough SEC. However, since all subsequent SECflow-through profiles showed, this off-peak mate-rial was of less quality (SEC peak was therebyoften flanked by shoulders).

By comparing the elution profiles of a HiTrap Qwe were able to optimize the refolding step whichwas not possible with the old protocol. Thereby,we found several parameters which all influencethe efficiency of the refolding. A sample of goodquality should have a low ratio between misfoldedand correctly folded protein. This ratio can beeasily estimated by the peak areas of a HiTrapelution profile.

There are mainly four criteria which influencethe efficiency of the refolding by rapid dilution:

There is evidence to suggest that the expressionmedium influences the quality of the protein ininclusion bodies. Compared to LB and especiallyTm, the elution profiles of proPMII expressedfrom E. coli in Mm showed throughout betterquality.

The time in which proPMII stays in renaturationbuffer is crucial and should be kept as short aspossible because the subsequent step - the load-

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Figure 3: Elution profile of the first batch proPMII expressed in LB and processed according to theold protocol. The IEF bands À to È correspond to fractions A12 to B5 of the elution profile. Behindthe main peak (A13 and A14) a second population (which is thought to be misfolded proPMI) with adifferent isoelectric point (pI) is detectable. According to the IEF-marker the population in A13 has apI around pH 4.8, whereas the population behind the main peak reveals a pI at pH 6.3. IEF

ing on a HiTrap with 1ml/min still lasts 17.5hper 1 l. For this purpose only small aliquots(50ml) were diluted in 1 l of renaturation buffer.Such small samples can be diluted with a smallflow rate which increases the quality significantlywithout loosing much time in which already re-naturated protein stays in solution.

It seems that the ratio between folded and mis-folded proPMII cannot be influenced by oxi-dizing/reducing agents. Even if used at non-equimolar amounts, the ratio could not be shiftedtoward correctly folded protein. To highlight thisconclusion, Fig. 4 shows an overlay of elutionprofiles run at 4 ℃. One sample (dotted line) wasrenaturated in buffer D according to the mainprotocol. For comparison, the elution profile of asample renaturated in buffer D containing 1mMGSSG and 0.5mM GSH is printed in straightlines.

The most relevant factor is the tempera-ture. Samples which were renaturated, loaded,and eluted at 20 ℃ showed significantly higheramounts of misfolded populations compared tosamples at 4 ℃. This fact holds for both nor-mal buffer D (Fig. 5) and buffer D containing

GSSG/GSH (Fig. 6). In both Figures the sam-ple renaturated and eluted at 20 ℃ are plottedwith dotted lines. These curves show beside themain peak a dominant second population whichis thought to be misfolded proPMII, as an IEFshowed (Fig. 3).

Figures 5 and 6 show that proPMII detachedfrom HiTrap Q resin at around 175 (buffer D plusGSSG/GSH) to 195mM (normal buffer D) NaCl.The second population got eluted at around 350(buffer D plus GSSG/GSH) to 380mM NaCl(normal buffer D). The longer the gradient waschosen the better the two populations got sepa-rated.

Activation and Concentration

proPMII activates itself at low pH (4.5) by cleav-age of the N-terminal pro-sequence. Following tothe old protocol, the incubation time for activa-tion was 2h at 37 ℃. After activation, PMII de-grades in a later step the hydrolysed pro-sequenceand other impurities, mainly misfolded proteinpopulations. This is impressively visible on theright SDS gel in Fig. 7. Within 2h, nearly all

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Figure 4: Comparison of HiTrap elution profiles run at 4 ℃. Straight lines: renaturation buffer containing1mM GSSG and 0.5mM GSH, dotted line: normal renaturation buffer. Absorbance at 280nm blue,absorbance at 254nm red. The black broken line represents the actual percentage of elution buffer(called %B in right scale). The difference of peak-top absorbtion had arisen of loading different amountsof material (normal renaturation buffer contained 35% less material). There is no qualitative differencebetween the two renaturation buffers.

impurities as well as the pro-sequence fade away.After 15min (band Á in the right SDS gel), thecut pro-sequence is easily visible.

Complete activation and degradation of impuri-ties were finished after about 2h at RT. Longeractivation - up to one month at 4 ℃ - showed noloss of material. Thus, the activation of PMIIper se can be used as purification of the samplewhen run long enough. Note that the band Á

of the left SDS gel in Fig. 7 did not attenuateduring 4.5h. Compared to this main-peak sam-ple, bands  and à in Fig. 9 show an activationof an adjacent peak pool. Therein only a smallamount of proPMIV could be activated while therest got degraded during activation.

After activation, the small degraded fragmentshave to be separated from the active protein.This task might be fulfilled by either runningan affinity column (H4848) or a SEC (Superdex16/60). The H4848 would have the advantage ofeluting the protein in 20mM Tris pH 8. This pHfirst improves the solubility of PMII during a laterconcentration step and inactivates the protease,thereby possibly slowing auto-degradation.

Figure 7: SDS-PAGE monitoring the activa-tion of a HiTrap main-peak sample expressedin Mm(2/4), renaturated in buffer D containing1mM GSSG and 0.5mM GSH. Bands from leftto right: MW marker, À before activation, Á af-ter 0.5h, Â 1h, Ã 1.5h, Ä 2h, Å 2.5h, Æ 3h,Ç 3.5h, and È 5h. In the right SDS the twopopulations of proPMII, active form and the cutpro-sequence are visible after 15min of activationat RT (Á). After 2h (Â) the activation appearsto be complete.

11

Figure 5: Comparison of HiTrap elution profiles run at 4 and 20 ℃ loaded from normal renaturationbuffer. Straight lines: 4 ℃, dotted line: 20 ℃. Absorbance at 280nm blue, absorbance at 254nm red.The black broken line represents the actual percentage of elution buffer (called %B in right scale) Thedifference of peak-top absorbtion had arisen of loading different amounts of material (the sample at20 ℃ contained 38% less material). At 20 ℃ the elution profile shows clearly a second population elutedlater then the main peak. Especially at 254nm the second (misfolded) proPMII population is visible.

Figure 6: Comparison of HiTrap elution profiles run at 4 and 20 ℃ eluted proPMII in renaturation buffercontaining 1mM GSSG and 0.5mM GSH. Straight lines: 4 ℃, dotted line: 20 ℃. Absorbance at 280nmblue, absorbance at 254nm red. The black broken line represents the actual percentage of elution buffer(called %B in right scale) The difference of peak-top absorbtion had arisen of loading different amountsof material (the sample at 20 ℃ contained 20% less material). Renaturation buffer at 20 ℃ shows at280nm a dominant peak shoulder.

12

We decided to skip the H4848 because of sev-eral practical reasons. First of all H4848 showedno better purification of the sample compared toSEC. Additionally, the H-4848 inhibitor is ex-pensive and the column has to be prepared andpacked manually. In contrast to the H4848, sizeexclusion columns have two advantages: In theelution profile aggregates as well as pepide frag-ments are easily detectable and SECs have amore convenient handling: the sample is directlyloaded via super-loop and flowed through within2h without time consuming elution and regener-ation steps.

3.1.2 PMIV

Test expression and glycerol stock forming wererun according to the main protocol.

Figure 8: SDS-PAGE controlling the test expres-sion of proPMIV in four clones. Bands: beforeinduction À clone A, MW marker, Á clone B, Â

clone C (two bands), after induction à clone A,Ä clone B, Å clone C, Æ clone D. Clone A wasused for expression as this clone used most of theenergy to express proPMIV.

SDS-PAGE in Figure 8 showed significantlyslower bacterial growth compared to PMII. In-terestingly, clone A grew much faster if expressedafterward in Tm or Mm.

Purification

Pellet purification of proPMIV expressed in Tmwas run according to an ‘intermediate’ protocol.

This means that the protocol was already opti-mized in certain steps whereas some values werenot yet modified. The differences to the final pro-tocol in section 2.2 were added in parentheses.

From PMIV only one batch (a pellet from 1 lof Tm) was purified. This procedure is brieflysummarized in the following section:

Around 50ml of buffer A was used to resuspendthe pellet at RT under stirring. French press wasrun two times and rinsed with buffer A to a finalvolume of 110ml. After centrifugation of bufferA in 3 tubes, the pellet showed less impuritiesand supernatant was a little muddier comparedto PMII Tm samples. SDS-PAGE of pellet pu-rification and activation is shown in Fig. 9.

After dilution of proPMIV in 250ml buffer C(containing 8M urea), the sample was dilutedwith P960 at 4 ℃, 0.2ml/min into 10 l bufferD and loaded afterwards on a 5ml-HiTrap with2ml/min. This lasted 3.5 days.

The elution profile with a gradient over 30×C.V.(150ml, 2ml/min) is shown in Fig. 10. Mainpeak sample was activated and directly loadedon SEC, where PMIV passed the SEC after thesame volume as PMII (86ml).

3.1.3 PMI

Compared to the other two PMs, PMI was clonedwith a (His)10-tag. From PMI only one batch (apellet from 1 l of Tm) was purified. This proce-dure is briefly summarized in the following sec-tion:

Around 30ml of buffer A was used to dilute thepellet thawed on ice. As the pellet did not resus-pend properly and stayed slimy, it was vortexedand afterward stirred at 4 ℃ for 10min. Frenchpress was run two times and rinsed with buffer Ato final volume of around 130ml.

After centrifugation of buffer A, subsequent re-filling and centrifugation with buffer B, pelletswere dissolved in 500ml of buffer C.

13

Figure 10: HiTrap elution profile of proPMIV expressed in Tm. Absorbance at 280nm blue, absorbanceat 254nm red. The green line represents the actual percentage of elution buffer (called %B in rightscale), the conductivity is drawn in brown.

Figure 9: Left: Pellet purification of proPMIVexpressed in Tm. Bands from left to right: MWmarker, À -20 ℃ stored pellet, Á after two roundsof french press,  supernatant buffer A after cen-trifugation, à supernatant buffer B after centrifu-gation, Ä diluted in buffer C. Right: Activationof HiTrap main peak (before À and after 90minÁ) and adjacent peak (before  and after activa-tion Ã). During activation of adjacent peak pool,the misfolded protein running at the same heightis degraded by active PMIV.

Figure 11: SDS-PAGE controlling the test ex-pression of PMI in four clones. Bands: MWmarker, before induction À clone A, Á clone B, clone C, à clone D, after induction Ä clone A,Å clone B, Æ clone C, Ç clone D. Clone C wasused for expression as this clone grew faster thanthe other (data not shown).

14

Figure 12: SDS-PAGE controlling the pellet pu-rification of proPMI. Bands: MW marker, À -20 ℃ stored pellet, Á first and  second runfrench press, à supernatant buffer A after cen-trifugation, Ä supernatant buffer B after centrifu-gation, Å proPMI dissolved in buffer C

SDS-PAGE showed material running at the sameheight as proPMI in supernatant A. 130ml su-pernatant of buffer A were directly loaded on aHisTrap HP (GE Healthcare, 1ml bed volume)with 1ml/min. HisTrap binding buffer contained20mM Tris/HCl pH 8.0; 500mM NaCl; 20mMimidazole (buffer was adjusted to pH 8.07 withHCl, final conductivity around 45mS). Super-natant buffer A after centrifugation was loadedon HisTrap o/n.

HisTrap was washed with 20×C.V. binding bufferand proPMI eluted with a gradient over 25 ×C.V., 1ml/min to 100% elution buffer (20mMTris/HCl pH 8.0; 500mM NaCl; 500mM imida-zole (adjusted with HCl to pH 8.04) conductivityof 48mS). The elution profile shown in Fig. 13displayed no PMI in supernatant A. Therefore itwas assumed that proPMI was also expressed ininclusion bodies.

A small sample (20ml) of proPMI dissolvedin buffer C was renaturated with 0.1ml/mininto 500ml buffer D. Afterward, the sample wasloaded on a 1ml-HiTrap (as the backpressurerose constantly, HiTrap was eluted after loading200ml buffer D).

Elution HiTrap elution profile visible in Fig. 14showed a massive amount of misfolded protein.There was no time to optimize the refolding ofPMI.

3.2 Crystallization

3.2.1 Control of Protein Purity

The quality of purified PMII according to the op-timized protocol was tested by co-crystallizationwith a known in-house inhibitor (ACT-061133,deposited PDB code: 2BJU). PMII (8.96mg/ml;5mM Na/K Pi pH 6.5; 100mM NaCl) was mixedin a 1:20 ratio with ACT-061133 (30mM in tert.-butanol, stored at -20 ℃ for 5 years) and pipettedwith a drop ratio of 3:1 (v/v) protein to reservoirsolution. Crystals grew in pH 6.5 and 18% PEGafter 3-4 weeks and showed the known P21212crystal form with an according resolution around1.7 A. This corresponds to previous quality.

3.2.2 pH Shift During Crystal Growth

Native crystals of the first batches in which theSEC running buffer contained 20mM Na/K Pigrew within 3 to 4 weeks. All of them showedstrong diffraction up to 2.1 A. Because of hugemosaicity and twinned diffraction pattern crys-tals were not suitable for processing. Fig. 15shows the diffraction image of a crystal from thefirst batch.

An experiment showed that the pH - using a dropratio of 2:1 (v/v) protein (20mM Na/K Pi pH6.5) to reservoir solution (0.1M sodium citrate)- lay around 0.37 to 0.41 above the desired pHof the reservoir solution (measured pH values 3.0;3.25; 3.75). This problem was detected as crystalsshowed less mosaicity if soaked with cryo bufferin which the pH was shifted +0.25 (relative to thepH of the reservoir solution).

Despite shifting the pH of the cryo buffers by+0.25 or +0.5 units, crystals from the first

15

Figure 13: HisTrap elution PMI expressed in Tm. Left image: Elution profile, absorbance at 280nmblue, absorbance at 254nm red. Green line represents actual percentage of elution buffer (called %B inright scale), the conductivity is drawn in brown. Rigth image: SDS-PAGE controlling the loading andelution of HisTrap. Bands: MW marker, À HisTrap load, Á HisTrap flowthrough during load, Â 1.ml ofwash (visible in elution profile), Ã 20× C.V. wash, Ä pool of peak fractions A4-A9.

Figure 14: HiTrap elution PMI expressed in Tm. Left image: Elution profile, absorbance at 280nmblue, absorbance at 254nm red. Green line represents actual percentage of elution buffer (called %B inright scale), the conductivity is drawn in brown. Rigth image: SDS-PAGE controlling the loading andelution of HiTrap. Bands: MW marker, À fraction A3, Á A5, Â A6, Ã A7, Ä A9, Å A10, Æ A11, Ç

A12, È A13

16

batches still showed huge mosaicity problems. Toensure that the pH stayed constant during thegrowth of a crystal, the concentration of the SECrunning buffer was reduced to 5mM Na/K PipH 6.5. Crystals with co-crystallized inhibitorST47 grew thereby within 24h, other crystalswithin two to three weeks. Crystals which greweven slower showed huge mosaicity problems andcould not be processed. Soaking experiments us-ing native ST43 and ST47 crystals with moreaffine inhibitors were not successful.

Acidification for Incubation

Inhibitors were added in a ratio of 10:1 or 20:1(v/v) or equimolar amount directly to activeprotein in SEC running buffer (5mM Na/K PipH 6.5; 100mM NaCl). To ensure a completeprotonation of the inhibitors, the pH was shiftedbefore incubation with 1:400 (v/v) of 1M HClto a final pH 4.2. This shift led to much smallerpellets in the subsequent centrifugation step (be-fore pipetting the samples were centrifugated toremove unbound inhibitors and suspended solid)which may have been caused by a higher occu-pancy of the active site. Fig. 16 shows a diffrac-tion image of a crystal grown with acidified solu-tion. Compared to the first batches (Fig. 15) theproblem of mosacity had been solved successfully.

With the acidification during incubation a thirdinhibitor (mzd 1) crystallized. We assume thatfurther investigations in acidification provideeven more stable complexes and might lead tomore crystals.

Screening

Initial screening covered nearly the whole pHrange between 3.0 and 5.75 (0.25 increments)and 10-30% PEG 4K (2% increments). In thebeginning each screen was expanded by adding30µl 4M NaCl as additive to reservoir solu-tion. This led to a reservoir concentration of

Figure 15: Diffraction image of PMII-ST47 com-plex. This crystal belonged to the fist batch. SECrunning buffer contained 20mM Na/K Pi, crys-tal grew in pH 3.0 and 20% PEG 4K. Cryo buffer0.1M sodium citrate pH 3.0; 35% PEG 4K. De-tector distance 220mm, λ = 1.0001 A

Figure 16: Diffraction image of PMII-mzd 1 com-plex. PMII was incubated in 5mM Na/K Piand acidified with HCl. Inhibitor was added toequimolar amount, crystal grew in pH 4.0 and20% PEG 4K. Cryo buffer 0.1M sodium citratepH 3.75; 25% PEG 4K; 20% glycerol. Detectordistance 280mm, λ = 1.0097 A

17

522mM NaCl (200µl reservoir plus 30µl 4MNaCl). By a drop ratio of 2:1, the final NaClconcentration in the droplet reached theoreti-cally 722mM in equilibrium with the reservoir(protein buffer contained in all screens 100mMNaCl). Droplets (2:1) without additive reachedtheoretically 200mM NaCl as final concentra-tion.

Each screen was pipetted in parallel with andwithout NaCl as additive. From the beginning weeasily could observe that the final NaCl concen-tration was too high: Plates with additive showedprecipitation in all conditions whereas withoutadditive the same conditions stayed clear. There-fore in later batches the additive volume was re-duced to 20µl (364mM NaCl in reservoir) lead-ing to a final theoretical droplet concentration of564mM (2:1) and 664mM (3:1).

The influence of 20µl NaCl additive by a dropratio of 2:1 was impressive mainly with two in-hibitors (screens with 8.66mg/ml LBM-09-2 and9.85mg/ml PMII-mzd 1 complex). In total 8screens (mainly covering pH 2.75 to 3.75) the ad-ditive shifted the phase boundary to around 6-10% less PEG 4K (e.g mzd 1 from 22% PEG 4Kto 14% at pH 3.25) indicating that NaCl pushesthe precipitation.

Especially in screens of high pH and high PEG4K the supplemental NaCl led to heavy precipi-tation in all conditions. Therefore, the additionof any NaCl was stopped to save material. In re-turn, the ratio of 2:1 was changed to 3:1 (v/v)protein to reservoir to increase slightly the con-centration of NaCl stemming from SEC buffer.This new ratio was successful with inhibitor ST47but not with ST43 (which crystallized in 2:1 butprecipitated in 3:1 screens).

None of the screens above pH 5 led to diffract-ing crystals. We assume that above a criticalpH the inhibitors get deprotonated and there-fore the complex is not stable anymore. Espe-cially the compounds synthesized by Diederich et

al. (ETH) contained an electron withdrawing sul-fonic acid beside the protonated secondary amide.From these compounds only one complex could bemeasured and processed (complex with inhibitormzd 1 scattered to 2.7 A).

Interestingly the most affine inhibitors did notcrystallize. Two of them have values of Ki of 30and a third inhibitor 100nM . In the first batchesthese complexes stayed completely clear. Afterremoving glycerol from the SEC buffer (not men-tioned in the main protocol or in the discussion)and reducing the SEC running buffer to 5mMNa/K Pi pH 6.5; 100mM NaCl phase boundarieswere noted between pH 3-3.75; 14-24% PEG 4K.An addition of 20µl 4M NaCl to reservoir ledin nearly all conditions to precipitate, except forthose at pH 3 and low PEG 4K. After designing anew screen with even lower pH and less PEG 4K(pH 2.5-3.25 and 10-20% PEG 4K) phase bound-ary was furthermore observable but at the bound-ary no crystals grew.

Figure 17: Observation during one month of aPMII-ST43 complex twinned crystal growing inpH 4.5; 24% PEG 4K.

Crystals of the PMII-ST43 complex grew within2 weeks in pH 4.75; 24% PEG 4K. PMII(10.07mg/ml; 5mM Na/K Pi; 100mM NaCl)was mixed by 20:1 (v/v) ratio with ST43 (30mMin DMSO). Droplets were pipetted with a dropratio of 2:1 (v/v) reservoir to protein solution.Crystals grew as huge polarizing plates, most ofthem twinned. During growth of crystals pre-cipitate dissolved reversibly. Figure 17 shows atwinned crystal growing at pH 4.5; 24% PEG 4K.During the first days, crystals grew while the pre-

18

cipitate dissolved. In a later phase, continuousforming of microcrystals in the droplet led to aslower growth of the crystal.

Crystals of the PMII-ST47 complex grewwithin 24h in pH 3.0; 20% PEG 4K. PMII(10.07mg/ml; 5mM Na/K Pi; 100mM NaCl)was mixed with ST47 (30mM in DMSO) at a20:1 ratio and pipetted with a drop ratio of 2:1(v/v) reservoir to protein. Crystals grew as hugepolarizing plates shown in Fig. 18.

Figure 18: Image of PMII co-crystallized ST47.These crystal grew in pH 3.0; 20% PEG 4K andwere used for solving the structure.

3.3 Structure Determination

Only PMII complexes could be processed. Allthree inhibitors are non-peptidomimetic chiralcompounds synthesized by the group of Prof. Dr.Gerhard Klebe.

3.3.1 Compounds

Data collection and structure refinement statis-tics are listed in Table 2.

Inhibitor ST43

Assignment to space group P1 (No. 1) withthe cell constants a = 63.380 b = 74.220 c =74.840 A, α = 113.98 β = 106.08 γ = 104.89yielded an Rmerge of 4.1 (26.4 for the highest

Name Chemical Structure Ki

ST43 1µM

ST47 100nM

mzd 1 680nM

Table 1: Listing of the chemical structures of in-hibitors ST43, ST47 and mzd 1 and the corre-sponding affinities to PMII.

resolution shell between 2.30 and 1.90 A). Ac-cording to the Matthews coefficient calculation,3 monomers are in the asymmetric unit (VM =2.53 A3/Da, 36.9 kDa).

The phase problem was solved by molecular re-placement (MR) using CCP4 suite and a prunedPMII (source PDB code: 2BJU) as search model.Molecular replacement ended with a Rfact of29.51% and Rfree=30.38%. Initial NCS-based re-finement was done without inhibitors. After im-proving the electron density by adding H2O en-ergy minimized inhibitors were fit in.

Inhibitor ST47

Assignment to space group P1 (No. 1) withthe cell constants a = 64.000 b = 74.510 c =74.590 A, α = 113.47 β = 106.89 γ = 104.66yielded an Rmerge of 4.8 (31.5 for the highestresolution shell between 2.30 and 1.90 A). Ac-cording to the Matthews coefficient calculation,3 monomers are in the asymmetric unit (VM =

19

2.56 A3/Da, 36.9 kDa).

Data reduction of ST47 complex displayed thesame crystal form as ST43. ST47 was solvedwithout MR, but was directly refined with thestructure of ST43 (first refinement cycles withoutinhibitor). After improving the electron densityby adding H2O energy minimized inhibitors werefit in.

Inhibitor mzd 1

The structure of a third complex with inhibitormzd 1 synthesized by the group of Prof. Dr.Francois Diederich was solved right before finish-ing this report. Because of lack of time duringfinishing the report, this complex (its packing andcrystal contacts) will not be mentioned in the dis-cussion about structure determination and will beused only in reference to non-peptidomimetic in-hibitors in section 3.5.3.

Assignment to space group P212121 with the cellconstants a = 94.99 b = 110.63 c = 134.13, α =β = γ = 90 yielded an Rmerge of 6.3 (58 for thehighest resolution shell between 2.9 and 2.70 A).

Data reduction of mzd 1 complex displayed 4 en-zymes in the asymmetric unit. The phase prob-lem was solved by molecular replacement (MR)using CCP4 suite and a pruned PMII (sourcePDB code: 2BJU) as search model.

20

Inhi

bito

rN

ame

ST43

ST47

mzd

1C

ryst

alSy

stem

Tri

clin

icT

ricl

inic

Ort

horh

ombi

cSp

ace

grou

pP

1P

1P

2 12 1

2 1C

ell

dim

ensi

ons

[A,°]

a=

63.3

80b

=74.2

20c

=74.8

40a

=64.0

00b

=74.5

10c

=74.5

90a

=94.9

9b

=11

0.63

c=

134.

13α

=11

3.98

β=

106.

08γ

=10

4.89

α=

113.

47β

=10

6.89

γ=

104.

66α

=β

=γ

=90

Wav

elen

gth

[A]

1.00

01.

000

1.00

0R

esol

utio

n[A

]35.9

5−

1.98

(2.3

0−

1.90

)35.6

8−

1.98

(2.3

0−

1.90

)38.9

3(2.9−

2.7)

Uni

que

Refl

ecti

ons

6844

5(2

3340

)66

612

(222

06)

3836

8(6

761)

Red

unda

ncy

1.95

(1.9

0)1.

95(1

.89)

7.37

(7.4

1)C

ompl

eten

ess

[%]

79.6

(62.

3)76

.7(5

8.7)

97.1

(90.

1)I/σ

(I)

14.6

1(3

.08)

12.7

6(2

.89)

26.0

8(4

.05)

Rmrgd

[%]

4.1

(26.

4)4.

8(3

1.5)

6.3

(58)

Val

ues

inpa

rent

hese

sre

fer

toda

tain

the

high

est

reso

luti

onsh

ell.

Res

olut

ion

[A]

35.9

5−

1.98

35.6

8−

1.98

38.9

3−

2.71

Refl

ecti

onus

ed65

022

6328

036

449

Rfact

[%]

20.6

923

.26

22.4

4Rfree

[%]

26.0

728

.78

32.9

5A

vera

geB

-fac

tor

[A2]

44.5

441

.49

63.9

3rm

sdbond

leng

th[A

]0.

013

0.01

90.

010

rmsdbond

angl

e[°]

1.55

61.

858

1.59

0

Tab

le2:

Dat

aco

llect

ion

and

stru

ctur

alre

finem

ent

stat

isti

csof

co-c

ryst

alliz

edP

MII

wit

hin

hibi

tor

ST43

,ST

47an

dm

zd1.

21

3.4 Structure Description

PMII shows the typical fold of eukaryotic asparticproteases like mammalian (pepsin, renin, gastric-sin and cathepsin D) and fungal (endothiapepsinand rhizopuspepsin) enzymes[17] (see Fig. 19 foroverview).

The active enzyme is formed by a single chainconsisting of 329 amino acids. It is foldedinto two topologically similar N and C-terminaldomains[18], each of which contributes a catalyticaspartate residue to the active site. The two do-mains contact each other along an intersection inwhich the binding cleft containing the catalyticdyad, Asp34 and Asp214, is buried. Behind theactive site, a six-stranded interdomain β-sheetanchors the two domains. This structure is com-posed of 5 β-strands which were stabilized at oneend by an N-terminal sheet.

The N-terminal domain consists mainly of 7 β-sheets which form a rib cage. At the top end ofthe ”costal arch” a distinctive single β-hairpin,known as flap, forms directly above the bindingcleft a finger-like structure and interacts with abound substrate.

3.4.1 Interfaces of the Crystal Packing

Both complexes PMII-ST47 and -ST43 revealthree enzymes in the asymmetric unit (calledchain A to C, see Fig. 20). In the asymmetricunit two different crystal contacts were found:

The first contact between chain A and B consistsof several H-bonds between the two C-domains.Thereby 3 amino acids in a row form a short an-tiparallel β-sheet structure with the neighboringPMII: Lys163A-Thr165A and Tyr184B-Leu182Bwith totally 3 H-bonds. Directly beside this con-tact, a fourth H-bond between the amide groupof the side chain Asp328A and the backbone ofGlu262B completes this polar crystal contact. Itscontact area between chain A and B in the unitcell is computed to 560.6 A2 (ST43, 3.7% of total

Figure 20: Crystal packing of PMII-ST43 com-plex. The cartoon shows the unit cell containingchains A (yellow), B (magenta), and C (cyan)with the respective inhibitors in green. Both crys-tal contacts are visible: hydrophobic contact be-tween chains B and C in the proximity of thebinding cleft of chain B, and the polar interac-tion between chains A and B.

surface) and 549.8 A2 (ST47, 3.6% of total sur-face).

PISA[19] found several supplemental interactions.The local density at the crucial amino acidsis not accurate enough to determine the exactposition: There might be weak H-bonds be-tween Ser262A/Asn250A and Arg64B and a sec-ond weak interaction between Asn145A/Asp316Aand Lys72B. All four interactions are more than3.2 A long and therefore are mentioned here onlyfor the sake of completeness.

The contact interface between chain A and Bcontributes only little to the entire solvation freeenergy (∆G) loss upon formation of the inter-faces. This is because the contact interface be-tween chain A and B involves no hydrophobicinteractions. The major driving force of crystalgrowth arose upon formation of the second con-tact interface (between chains B and C in unitcell). PISA calculated 757.1 A2 (ST43, 5.0% oftotal surface) and 733.8 A2 (ST47, 4.5% of totalsurface) for the hydrophobic interface between Band C in the unit cell.

22

Figure 19: Ribbon diagram of the complex of PMII with ST47 showing disulfide bridges (magenta),catalytic dyad with inhibitor (green), flap (blue), interdomain β-sheet (brown), and proline-rich loop inyellow. Right image: 180° turned and slabbed ribbon diagram, same coloring.

The interaction between chain B and C was al-ready found in other protease structures (PDBcodes: 2BJU, 2IGX and 2IGY). The mainly hy-drophobic character explains the high contribu-tion to ∆G (PISA calculated more than 50% ofthe total loss of solvation Gibbs energy due toformation of this hydrophobic interface). It hasarisen from a special conformation of hydrophobicloop interacting in the proximity of the neighbor-ing binding cleft and the bound inhibitor:

The space at the far side of the pyrrolidine ringis filled by a loop of the coterminous chain Cinteracting directly with the inhibitor bound inthe active site of chain B. The interacting loopis natively positioned at the surface but revealswithin its 10 amino acids an extremely atypi-cal hydrophobic pattern: V236IKVPFLPFYV246.Due to the hydrophobic properties, this loopis thought to interact with the heterodimerichemoglobin by partially disrupting the inter-molecular interactions, thereby making the quar-ternary structure susceptible for degradation byPMs.

PISA found 3 residues of this loop staying in hy-drophobic contact with the inhibitor: Pro240Cand Phe241C at the very end of the loop and thealiphatic chain of Lys238C.

Already two amino acids away from Lys238C

- around 16 A from the hydrophobic interactionwith the inhibitor - the same loop forms a hy-drophobic pocket in which the proline-rich loopof chain B fits in. Thereby mainly Phe294B,Pro295B, and Val296B of the proline-rich loopare involved in interactions with the pocket-forming Leu227C, Leu231C, Val236C, Tyr245C,and Val246C.

Due to the hydrophobic interactions at the farside of the inhibitor, the loop of chain C getssandwiched between the inhibitor and the flapof chain B, consequentially also interacting withthe flap. As a result, the hydrophobic loopforms a 2.92 A long H-bond from the backbone ofPhe241C to the backbone of Val78B (tip of theflap). This H-bond fixes the flap in a well definedposition and completes this crystal contact. It isshown in Fig. 21. Beside these interactions PISAfound one additional H-bond between Gly134Band Val280C (3.1 A).

3.5 Structure Comparison

3.5.1 Differences among Chains in Crys-

tal

Although both datasets of ST43 and ST47 couldbe indexed with rhombohedral lattice (spacegroup R3, No. 146), processing yielded a Rmerge

23

Figure 21: Stick model of the H-bond between thebackbone of Phe241C to the backbone of Val78B.The drawn 2.92 A long H-bond presumably fixedthe flap in a highly conserved arrangement foundthroughout in all chains of ST47 and ST43.

value of 50%. Processing was only possiblewith triclinic crystal system (space group P1).Slightly differing arrangements at distinct posi-tions among chain A to C may broke the symme-try down to P1.

In the ST47 structure mainly three irregularitieswere found (all shown in Fig. 22):

1. The N-domain-near naphtyl ring in chain Cis turned by 180° still lying in the same planeas the other two naphtyl rings in front of theflap pocket.

2. The ester bond of the C-domain-nearnaphtyl ring bound to chain B is turnedaway and oriented toward a H2O moleculewhich stays in H-bonding contact (2.7 A) toThr217B.

3. An overlay shows striking irregularities onlyin a flexible loop in the C-domain. This loopcontains amino acids His276 to Leu284.

3.5.2 Differences between ST47 and ST43

Both structures show very similar packing andnearly the same cell constants in P1. An overlay

Figure 22: Stick model of superimposed chainsA (yellow), B (magenta), and C (cyan) of ST47showing in the upper image the three inhibitorsand the catalytic dyad. The chain C naphtylring near the flap pocket is coplanarly flipped andstays in the same region as the other two. Theester bond of the inhibitor bound to chain B isturned away and oriented towards a H2O (notdrawn). The C-domain near naphtyl ring in thebackground of the image is tightly bound to S1’pocket. The lower image shows the not congru-ent flexible loop in C-domain (His276 to Leu284)mentioned in the text.

24

of the two inhibitors ST43 and ST47 displayed theprotonated nitrogen of the pyrrolidine ring nearlyat same position, indicating a strongly bound in-hibitor to the catalytic dyad (not shown). Inan overlay, the backbone showed no significantstructural differences. Congruent chains displaya mean-square displacement of 0.454 A (withoutH2O; all atoms compared). Greater displace-ments up to 1.3 A were found in the α-helixlike structure directly beside the flap of chain C(Phe111C-Phe120C).

The almost identical 3D structures of PMII com-plexes indicate that ST43 and ST47 bind to PMIIin a very similar manner. Both occupy with onenaphtyl ring to the entrance of the flap pocket(N-domain-near). The second aromatic ring in-teracts with the hydrophobic pocket S1’ of theC-domain. This groove serves as an anchor forthe first C-terminal residue of the substrate be-ing hydrolyzed by PMII.

The structural analysis showed no significant dif-ferences in the binding geometry. According tothe binding mode, there is no obvious reason whyST47 binds 10 times better than ST43. We as-sume that ST47 forms a more relaxed structure insolution and therefore binds with a higher prob-ability to the active site. Another explanationmight be the naphtyl ring interacting with theS1’ pocket. If α-connected (ST47), this ring ex-tends deeper into the hydrophobic groove, servingthereby as better anchor for the inhibitor.

3.5.3 Active Site and Flap Rearrange-

ment

Both discussed inhibitors are chiral non-peptidomimetic hydrophobic compounds. Thesecompounds interact directly with the catalyticdyad without need of a conserved catalytic H2Olocated between the two aspartic acids. TheH-bonding partner of the catalytic dyad is 3,4-(s,s)-dihydroxy-pyrrolidine, a heterocyclic amine

ring. Unsubstituated pyrrolidine has a pKa of11.27[20]. The two hydrophobic naphtyl rings areconnected to the pyrrolidine by an acetic ester.The naphtyl ring is connected at α (ST47) or βposition (ST43) to the acetic acid.

Peptidomimetic Inhibitors

During the first part of the discussion about theactive site we will reference to a well knownPMII-pepstatin complex published by Binkert etal.[21](PDB code: 1XDH), shown in Fig. 23. Pep-statin is a classical peptidomimetic compoundwhich binds to PMs with not less than 10 H-bonds (not all are visible in Figures 23 and 24).Beside amino acids located at the surface of thebinding cleft the tip of the flap provides inter-actions at the far side of the catalytic dyad. Inall complexes with peptidomimetic inhibitors theflap (N-domain) is therefore lowered down, ori-enting and stabilizing the substrate during cleav-age.

Due to the lowered orientation of the flap thespace underneath it is completely filled by hy-drophobic residues, whereby the bottom and oneside of the pocket are occupied by Trp41 andPhe111. The entrance to this pocket is blockedby an inward-turned Tyr77 at the tip of the flap(see Figure 23).

As the two inhibitors ST47 and ST43 provide noH-bonding partners at the far side of the pyrro-lidine ring, the flap does not interact with themand stays therefore in a rather open conforma-tion. Due to missing interactions in the prox-imity of the binding cleft, the tip of the flap(Val78) moves relative to the pepstatin structure7-8 A away from the active site. This open flaparrangement entails a new binding mode for non-peptidomimetic inhibitors.

The flap opening has consequences for mainlythree amino acids: Trp41, Tyr77, and Phe111.Because of the lost steric inhibition, Trp41 is

25

able to turn outside the flap pocket along χ1 byaround -120° (looking from Cα to Cβ). Due to theflap drift, Tyr77 is getting repressed from its loca-tion in front of the pocket by one naphtyl ring. Atthis position the naphtyl ring near the N-domainside of the inhibitor blocks the entrance of the flappocket. This allows Phe111 from the backsideto slip into the enlarged pocket. The movementof Phe111 inward the pocket draws the backboneabout 2 A relative to the pepstatin structure. Fig.24 shows an overlay of the pepstatin and ST47structure.

Figure 23: Stick model of PMII-pepstatin com-plex. The flap coming from behind in the upperhalf of the image is lowered down and interactswith two hydrogen bonds with pepstatin (green).At the tip of the flap Tyr77 is inward-turned andblocks together with Phe111 at the right side theentrance of the flap pocket. In the backgroundTrp41 lying on the bottom of the pocket is alsovisible. (PDB code: 1XDH)

For the above-mentioned reasons ST43 and ST47interact in a different way with PMII comparedto peptidomimetic inhibitors, whose compoundclass is associated with relatively low bioavail-ability and high production costs[22]. Especiallythe latter reason makes non-peptidomimetic com-pounds more interesting in drug design, asmalaria stays a main problem in developing coun-tries.

Figure 24: Overlay of stick models of PMII-pepstatin (yellow with pale yellow pepstatin) andPMII-ST47 (orange with green ST47) complex.Due to missing H-bonds with the inhibitor the tipof the flap moves by 7-8 A upwards away from theactive site. Thereby Trp41 turns by 120° aroundχ1 outside the pocket (under half of the upperimage). The position of Tyr77 at the tip of theflap gets thereby occupied by the N-domain-nearnaphtyl ring of ST47. This position closes theflap pocket which allows the Phe111 in the back-ground of the image to slip inside this pocketand thereby draws the backbone of Phe111 about2 A toward the flap (rearrangement of Phe111 isshown in lower 180° turned image). (PDB codefor pepstatin: 1XDH)

26

Figure 25: Overlay of stick models of 2IGX.pdb(yellow with pale yellow inhibitor) and PMII-ST47 (orange with green ST47) complex. Upperimage shows the flap pocket which gets occupiedby the n-pentyl chain of the 2IGX-inhibitor, re-pressing thereby Phe111 from the pocket (back-ground of the image). As ST47 only covers thepocket, Phe111 is able to slip in to the flap-pocket (orange). The lower image shows the flappocket from the right side and the catalytic dyad(side chains of ST47 model). The C-domain-nearnaphtyl ring is bound to the same pocket (S1’) asthe 2-methylene-imidazole unit.

Non-peptidomimetic Inhibitors

There are several non-peptidomimetic structuresreported[22, 23] and deposited (PDB codes: 2BJU,2IGX and 2IGY). This class is characterized byabsent interactions with the tip of the flap. Thecomplete absence of hydrogen bonds between thetightly bound inhibitor and the flap, as well as di-minished attractive hydrophobic interactions leadto a opened flap conformation[22].

The movement of the tip of the flap awayfrom the catalytic dyad opens a cylinder-shapedlipophilic pocket, which is not filled but coveredby the naphtyl rings of ST43/47. According tothe electron density we assume that this naphtylring is not exactly positioned and may reveal twodifferent orientations. D. Bur, L. Prade et al.(as well as the third solved PMII-mzd 1 complex)have shown that the flap pocket can be openedcompletely by repressing Phe111.

Fig. 25 shows one of these (achiral)non-peptidomimetic inhibitors (PDB code:2IGX) in overlay with ST47. By comparing thebinding of the two inhibitors one can see that

1. the 2IGX-inhibitor binds tightly with an n-pentyl chain into the pocket, whereas thenaphtyl ring of ST47 only covers the entranceof the pocket,

2. the C-domain-near naphtyl ring is bound tothe same pocket (S1’) as the 2-methylene-imidazole moiety of the 2IGX-inhibitor. Thisoutcome was rather unexpected because thisnaphtyl ring could reach parts of the S1and S3 pockets, in which the 4’-substitutedbiphenyl-methylene unit binds (this unit isvisible at the right side in the lower image ofFig. 25),

3. ST43/47 repressed a very strong hydro-gen bonded catalytic water molecule (notdrawn). This water molecule was not ex-

27

pelled from its position between the two car-boxylates of the catalytic aspartates[22].

The third solved complex with the inhibitormzd 1 binds to the flap pocket in a similar man-ner as the above mentioned inhibitor 2IGX does(see Fig. 26). The structure of mzd 1 is shownin Table 1 and reveals also an n-pentyl moietywhich interacts with the lipophilic flap pocket.Compared to 2IGX, this hydrophobic interactionis weaker because the pentyl chain fills not thewhole pocket (visible in an overlay of 2IGX andmzd 1 inhibitor in lower image of Fig. 26). Thesecond moiety (naphtyl ring) does neither inter-act with S1’ (as ST47 and ST43) nor with parts ofthe S1 and S3 pockets (as the biphenyl-methyleneunit of 2IGX) but remains completely unboundand was therefore not visible in the electron den-sity.

The electron density of the tip of the flap (espe-cially the above mentioned Tyr77 at the entranceof the pocket) is missing (not shown). In contrastto ST43 and ST47, mzd 1 revealed a completelydifferent packing in which no H-bond between thetip of the flap and the hydrophobic loop was ob-servable. This missing H-bond as well as a weakinteraction between the n-pentyl unit with Tyr77may explain the lack of electron density at the tipof the flap.

4 Conclusion and Look-out

4.1 Purification

The protocol was optimized for PMII purifica-tion. The modifications targeted on maximalyield in rapid refolding and concentration stepsas well as most efficient binding during incuba-tion. As the elution profile in Fig. 10 of a PMIVsample shows, the protocol has to be optimizedfor PMIV but it works for this protease as well.

The purification protocol had been optimizedduring the whole master’s thesis and therefore

Figure 26: Stick model of PMII-mzd 1 complex.Compared to ST43 and ST47, mzd 1 (green) con-tains an n-pentyl moiety which interacts with theflap pocket, thereby repressing Phe111 from thepocket. Its binding mode to the flap pocket iscomparable to 2IGX-inhibitor (shown in lowerimage: PMII yellow with pale-yellow inhibitor).The electron density of the naphtly ring is notvisible, suggesting that this ring does not inter-act with parts of the S1 and S3 pocket and staysunbound in the vicinity of the binding cleft.

28

some points still remain unanswered. Duringwriting the report we found several issues thatwould have been of interest.

We never checked whether the PMs are prop-erly folded within the inclusion bodies. It wouldbe interesting to dilute a sample in renaturationbuffer D after french press. Sadly I always forgotto branch off a small sample after centrifugationof buffer B.

For denaturation in buffer C it has to be takeninto account that the yield of PMII expressedfrom E. coli grown in different media can differby factors (yield of Tm is around five times higherthan in LB). The diametrical differences betweenthe expression media may caused by an insuffi-ciently denaturated proPMII or due to too highfinal urea concentrations in buffer D. Measuringthe circular dichroism of denaturated proPMII inbuffer C should clarify whether the protein gotcompletely linearized.

As the binding of the inhibitor involves the for-mation of only two H-bonds this process seems tobe mainly entropically driven. Incubation of theinhibitors may be even more efficient when runat higher temperatures.

It would be also worth trying to reduce the NaClin SEC buffer and increasing the PEG 4K concen-tration during crystallization instead. This maylead to complexes which grow in other packingthan P1. By measuring only 180°, P1 led dur-ing data reduction to a rather low redundancywhich created noisy electron density maps. Dur-ing NCS-based refinement the packing caused anda lot of work because of 3 enzymes in the unitcell. It would be much easier for processing ifthe PMII-complexes grew in other conditions andhopefully different crystal packings.

4.2 Inhibitors

The pryrrolidine ring of ST43 and ST47 bindstightly to the catalytic dyad. The structure re-

veals within the unit cell different conformationsof the ester bonds of ST47.

Thereby the six ester bonds (3 PMII per unitcell, two different inhibitors) of the C-domain-near naphtyl rings (S1’-pocket) display two dif-ferent conformations. Four of them show a cis-conformation along the methylene-carbonyl bond(carbonyl double bond is oriented in nearly 90° toThr217-alcohol), two show gauche conformations(carbonyl oriented toward Phe241, an amino acidof the hydrophobic loop of the neighboring chain).To provide a better binding the ester group mightbe exchanged by an amide bond, forcing the es-ter to stay in gauche conformation. The protonof the amide bond will thereby be oriented di-rectly to Asp214 of the catalytic dyad in about3 A distance.

Most of the inhibitors contained electron with-drawing groups (sulfuric acid). If these groupswere exchanged, the secondary amine serving asbinding partner of catalytic dyad might be evenbetter protonated at incubation pH. An exchangeof these groups may lead to crystals growing inhigher pH and different packing.

Acknowledgment

Many thanks go to Dr. Lars Prade for the sci-entific steering of the master’s thesis, as well asto Daniel Bur for helpful discussions. I grate-fully acknowledge the excellent technical assis-tance of Alain Chambovey and Antoinette Am-rein and Silvia Rogers for critically reading themanuscript. Finally I would like to thank Prof.Tilman Schirmer for supervising my master’s the-sis.

29

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