Over-Production, Crystallization, and Preliminary X-Ray Crystallographic Analysis of a Coiled-Coil Region in Human Pericentrin

crystals

Communication Over-Production, Crystallization, and Preliminary X-ray Crystallographic Analysis of a Coiled-Coil Region in Human Pericentrin

Min Ye Kim, Jeong Kuk Park, Yeowon Sim, Doheum Kim, Jeong Yeon Sim and SangYoun Park *

School of Systems Biomedical Science, Soongsil University, Seoul 06978, Korea; [email protected] (M.Y.K.); [email protected] (J.K.P.); [email protected] (Y.S.); [email protected] (D.K.); [email protected] (J.Y.S.) * Correspondence: [email protected]; Tel.: +82-2-820-0456

Academic Editor: Jolanta Prywer Received: 12 September 2017; Accepted: 28 September 2017; Published: 2 October 2017

Abstract: The genes encoding three coiled-coil regions in human pericentrin were gene synthesized with Escherichia coli codon-optimization, and the proteins were successfully over-produced in large quantities using E. coli expression. After verifying that the puriﬁed proteins were mostly composed of α-helices, one of the proteins was crystallized using polyethylene glycol 8000 as crystallizing agent. X-ray diffraction data were collected to 3.8 Å resolution under cryo-condition using synchrotron X-ray. The crystal belonged to space group C2 with unit cell parameters a = 324.9 Å, b = 35.7 Å, c = 79.5 Å, and β = 101.6◦. According to Matthews’ coefﬁcient, the asymmetric unit may contain up to 3 −1 12 subunits of the monomeric protein, with a crystal volume per protein mass (VM) of 1.96 Å Da and a 37.3% solvent content.

Keywords: pericentrin; coiled-coil; centrosome; pericentriolar material (PCM)

1. Introduction The centrosome is the main microtubule organizing center (MTOC) in animal cells. In non-mitotic interphase cells, the centrosome is located near the nucleus to produce an assembly of microtubules that radiates towards the cell periphery, serving as tracks for motor protein-mediated transport of cellular compartments. During mitosis, microtubules reorganize from the centrosomes to form spindle poles that accurately segregate the duplicated chromosomes in two. The centrosome consists of a pair of orthogonally arranged centrioles surrounded by pericentriolar material (PCM). The PCM includes factors such as the γ-tubulin ring complex (γ-TuRC) that directly function to nucleate the microtubule arrays [1–4]. Pericentrin (PCNT) is a large ∼360 kDa protein that also exists within PCM [5] to act as a scaffold for anchoring multiple proteins of the PCM [6]. PCNT contains a series of predicted coiled-coil regions over most of their length [5], but a highly conserved PCM targeting motif called the PACT domain is found near the C-terminus [7] (Figure1). PCNT has been linked to many human disorders [6], and one of them is the loss-of-function mutations that cause microcephalic osteodysplastic primordial dwarﬁsm type II (MOPD II) [8]. In this study, three regions in the human PCNT which are predicted as coiled-coils were successfully over-produced in Escherichia coli using plasmids containing E. coli codon-optimized PCNT gene. The high α-helical contents for the three PCNT proteins were further conﬁrmed by circular dichroism analysis, and one of them was crystallized for preliminary X-ray crystallographic analysis.

Crystals 2017, 7, 296; doi:10.3390/cryst7100296 www.mdpi.com/journal/crystals Crystals 2017 7 Crystals 2017,, 7,, 296296 2 of 7

Figure 1. Regions of human pericentrin (PCNT) showing predicted coiled-coil (CC) and the PACTFigure domain. 1. Regions of human pericentrin (PCNT) showing predicted coiled-coil (CC) and the PACT domain. 2. Materials and Methods 2. Materials and Methods 2.1. Macromolecule Production 2.1. Macromolecule Production The DNA encoding three regions in human pericentrin (PCNT, full length of residues 1–3336) whichThe are DNA predicted encoding to encode three coiled-coilregions in (CC)human motifs pericentrin (CC1, 260–553; (PCNT, CC2,full length 676–831; of residues CC3, 1354–1684) 1–3336) werewhich synthesized are predicted with to encode an addition coiled-coil of nucleotides (CC) motifs encoding (CC1, 260–553; N-terminal CC2, His-tag 676–831; (Bioneer, CC3, 1354–1684) Daejeon, Korea)were synthesized for affinity with purification. an addition All genes of nucleotide were codon-optimizeds encoding N-terminal for expression His-tag in (Bioneer,E. coli and Daejeon, cloned intoKorea) pET28a for affinity vector purification. (Merck, Kenilworth, All genes NJ,were USA) codon-optimized using NdeI and for BamHexpressionI restriction in E. coli enzyme and cloned sites. Theinto generatedpET28a vector plasmids (Merck, were Kenilworth, all sequence NJ, verifiedUSA) using of the Nde insertI and region,BamHI restriction and were usedenzyme to transform sites. The thegeneratedE. coli plasmidsBL21 (DE3) were (Merck, all sequence Kenilworth, verified NJ, of the USA) insert cells region, using and heat were shock used at to 42 transform◦C (45 s). the The E. coli BL21 (DE3) (Merck, Kenilworth, NJ,◦ USA) cells using heat shock at 42 °C (45 s). The transformed transformed cells were grown at 37 C in 1 L of Luria-Bertani (LB) medium to an OD600 of ∼0.8 incells the were presence grown of at 25 37µ °g/mLC in 1 kanamycin.L of Luria-Bertani Expression (LB) medium of the recombinant to an OD600 of protein ∼0.8 in was the inducedpresence byof the25 μ additiong/mL kanamycin. of 0.5 mM Expressi isopropyl-D-thiogalactopyranosideon of the recombinant protein (IPTG) was induced at 22 ◦ C,by andthe addition cells were of allowed0.5 mM toisopropyl-D-thiogalactopyranoside grow for an extra 16 h. Cells were (IPTG) harvested at 22 °C, using and centrifugationcells were allowed at 4500 to grow× g for for 10 an min extra (4 16◦C). h. Cells were harvested using centrifugation at 4500× g for 10 min (4 °C). All three N-terminal His6- All three N-terminal His6-tagged proteins of PCNT were over-produced with soluble expression of the proteins.tagged proteins For protein of PCNT purification, were over-produced the bacterial cellwith pellets soluble were expression re-suspended of the in proteins. 50 mL ice-cold For protein lysis bufferpurification, (20 mM the Tris bacterial pH 7.5, cell 500 pellets mM NaCl, were andre-suspended 5 mM imidazole) in 50 mL and ice-cold lysed onlysis ice buffer by sonication. (20 mM TheTris homogenatespH 7.5, 500 mM were NaCl, centrifuged and 5 mM at 70,000imidazole)× g for and 30 lysed min (4on◦ C),ice by and sonication. supernatants The poured homogenates over a 5were mL Ni-nitrilotriaceticcentrifuged at 70,000 acid× agaroseg for 30 (Ni-NTA)min (4 °C), (Qiagen, and supernatants Hilden, Germany) poured over gravity a 5 column.mL Ni-nitrilotriacetic The columns wereacid agarose washed (Ni-NTA) with five (Qiagen, column volumes Hilden, Germany) of wash buffer gravity (20 column. mM Tris The pH columns 7.5, 20 mM were imidazole, washed with and 500five mMcolumn NaCl), volumes and the of proteins wash buffer were eluted(20 mM with Tris elution pH 7.5, buffer 20 mM (20 imidazole, mM Tris pH and 7.5, 500 200 mM mM NaCl), imidazole, and andthe proteins 500 mM NaCl).were eluted The elution with elution fractions buffer containing (20 mM the Tris PCNT pH proteins7.5, 200 weremM imidazole, checked using and Bradford500 mM assayNaCl). (BioRad, The elution Berkeley, fractions CA, USA),containing combined, the PCNT and added proteins with were 50 µL checked of 0.25U/ usingµL bovine Bradford thrombin assay (BioRad, Berkeley, CA, USA), combined, and added with 50 μL of 0.25 U/μL bovine thrombin◦ (Invitrogen, Carlsbad, CA, USA). After proteolysis of the His6-tag for 16 h incubation at 4 C, the protein(Invitrogen, samples Carlsbad, were further CA, USA). purified After using proteolysis a HiLoad of® the26/60 His Superdex6-tag for ®16200 h incubation size-exclusion at 4 column°C, the (SEC)protein pre-equilibrated samples were further with SEC purified buffer using (50 mMa HiLoad Tris pH® 26/60 7.5, 150Superdex mM NaCl,® 200 size-exclusion and 2 mM DTT). column The proteolysis(SEC) pre-equilibrated mixes were loadedwith SEC into buffer the column (50 mM connected Tris pH to7.5, an 150 ÄKTA mM FPLCNaCl, system and 2 (GEmM Healthcare,DTT). The Littleproteolysis Chalfont, mixes UK). were The loaded elution into profiles the column of all connected three proteins to an ÄKTA showed FPLC one system major peak(GE Healthcare, (Figure2), andLittle the Chalfont, fractions UK). were The concentrated elution profiles by Amiconof all three®ultracentrifugation proteins showed one filters major (Merck). peak (Figure Final protein2), and the fractions were concentrated by Amicon® ultracentrifugation filters (Merck). Final− 1protein−1 concentrations were estimated by A280 with a molar extinction coefficient (CC1, 15220 M cm ; −1 −1 CC2,concentrations 6970 M−1 werecm−1 estimated; CC3, 11380 by MA280−1 withcm− 1a) molar calculated extinction based coefficient upon the numbers (CC1, 15220 of tryptophan M cm ; CC2, and −1 −1 −1 −1 tyrosine6970 M residuescm ; CC3, [9 ].11380 Purity M and cm homogeneity) calculated based were assessedupon the usingnumb SDS-PAGEers of tryptophan analysis and (Figure tyrosine2). Theresidues concentrated [9]. Purity proteins and homogeneity in SEC buffer werewere flash-cooledassessed using and storedSDS-PAGE in liquid analysis nitrogen. (Figure 2). The concentrated proteins in SEC buffer were flash-cooled and stored in liquid nitrogen.

CrystalsCrystals2017 2017, 7,, 296 7, 296 3 of3 7 of 7

FigureFigure 2. Size-exclusion2. Size-exclusion chromatograms chromatograms of ofthree three PCNTPCNT coiled-coil proteins proteins (CC1, (CC1, CC2, CC2, and and CC3) CC3) and and SDS-PAGESDS-PAGE analysis analysis of of the the concentrated concentrated fractions fractions under under thethe elutionelution peaks (inset). (inset).

2.2.2.2. Circular Circular Dichroism Dichroism (CD) (CD) Studies Studies for for Secondary Secondary StructureStructure Estimation TheThe contents contents of theof secondarythe secondary structure structure elements elem inents the in expressed the expressed PCNT proteinsPCNT proteins were estimated were byestimated scanning ellipticityby scanning over ellipticity wavelength over (200–240wavelength nm) (200 using–240 a nm) JASCO using spectropolarimeter a JASCO spectropolarimeter (Model J-810, Tokyo,(Model Japan). J-810, TheTokyo, three Japan). PCNT The proteins three PCNT with pr theoteins final with concentration the final concentration of 0.3 µg/mL of 0.3 were μg/mL analyzed were usinganalyzed a 0.1 cm using path-length a 0.1 cm cuvettepath-length to obtain cuvette the to spectra. obtain the Secondary spectra. structureSecondary estimation structure programsestimation of K2D2programs [10] and of K2D3K2D2 [11[10]] were and appliedK2D3 [11] to thewere CD applied data for to approximation the CD data for of theapproximation secondary structureof the content.secondary Because structure the fitting content. of CD Because data forthe secondary fitting of CD structure data for contents secondary are highly structure dependent contents on are the exacthighly concentration dependent ofon the the protein exact concentration (which in our of case the wasprotein estimated (which fromin our calculated case was estimated molar extinction from coefficient),calculated up molar to two-fold extinction differences coefficient), in up the to protein two-fold concentrations differences in werethe protein allowed concentrations during the fits.were allowed during the fits. 2.3. Crystallization 2.3. Crystallization Although all three PCNT proteins were screened using commercial Wizard® solutions (Molecular ® Dimensions)Although for conditionsall three PCNT to obtain proteins crystals, we onlyre screened crystals using of PCNT commercial CC2 were Wizard obtained solutions (Table1 ). The(Molecular crystal drops Dimensions) were set byfor mixingconditions equal to obtain 1 µL volumes crystals, ofonly the crystals reservoir of solutionPCNT CC2 (10% were (w/v obtained) PEG 8K 100(Table mM potassium 1). The crystal phosphate drops were monobasic/sodium set by mixing equal phosphate 1 μL volumes dibasic pHof the 6.2, reservoir and 200 solution mM NaCl) (10% and (w/v) PEG 8K 100 mM potassium phosphate monobasic/sodium phosphate dibasic pH 6.2, and 200 the PCNT CC2 concentrate (60 mg/mL) in SEC buffer, and were equilibrated against 500 µL of the mM NaCl) and the PCNT CC2 concentrate (60 mg/mL) in SEC buffer, and were equilibrated against reservoir solution. Several crystals were harvested, gel-electrophoresed, and their tryptic digestion 500 μL of the reservoir solution. Several crystals were harvested, gel-electrophoresed, and their fragments were analyzed using LC-MS/MS (Table2). The crystals were transferred to a cryo-protectant tryptic digestion fragments were analyzed using LC-MS/MS (Table 2). The crystals were transferred solution of 30% glycerol supplemented to the reservoir solution, flash-cooled in liquid nitrogen for to a cryo-protectant solution of 30% glycerol supplemented to the reservoir solution, flash-cooled in storage,liquidand nitrogen transported for storage, to a and synchrotron transported facility to a synchrotron for diffraction facility experiment. for diffraction experiment.

Table 1. Macromolecule production information for PCNT CC2. Table 1. Macromolecule production information for PCNT CC2.

SourceSource Organism Homo Homo Sapiens Sapiens DNA source Synthesized DNA DNA source Synthesized DNA Cloning sites NdeI and BamHI Cloning sites NdeI and BamHI Cloning vector pET28a Cloning vector pET28a ExpressionExpression vector pET28a pET28a ExpressionExpression host E. coliE. BL21 coli BL21 (DE3) (DE3) 676 680 MGSSHHHHHHSSGLVPRGSHM-MGSSHHHHHHSSGLVPRGSHMEHKVQ-676EHKVQ680 681 710 LSLLQTELKEEIELLKIENRNLYGKLQHET681LSLLQTELKEEIELLKIENRNLYGKLQHET710 CompleteComplete amino amino acid acid sequence sequence of of the 711711RLKDDLEKVKHNLIEDHQKELNNAKQKTELRLKDDLEKVKHNLIEDHQKELNNAKQKTEL740 740 1 741741 770 770 theconstruct construct produced produced 1 MKQEFQRKETDWKVMKEELQREAEEKLTLMMKQEFQRKETDWKVMKEELQREAEEKLTLM 771771LLELREKAESEKQTIINKFELREAEMRQLQLLELREKAESEKQTIINKFELREAEMRQLQ800 800 801801DQQAAQILDLERSLTEQQGRLQQLEQDLTSDDQQAAQILDLERSLTEQQGRLQQLEQDLTSD831 831 1 1Non-native His6-tag and thrombin site are underlined. Non-native His6-tag and thrombin site are underlined.

Crystals 2017, 7, 296 4 of 7

Table 2. List of peptide fragments found from mass spectrometry analysis of the crystallized PCNT CC2.

Peptide Sequence 1 MH+ (Da) GSHM-676EHKVQLSLLQTELKEEIELLK696 2932.59 679VQLSLLQTELKEEIELLKIENR700 2638.52 678KVQLSLLQTELKEEIELLK696 2254.33 683LLQTELKEEIELLKIENR700 2211.26 676EHKVQLSLLQTELKEEIELLK696 2520.44 682SLLQTELKEEIELLKIENR700 2298.30 681LSLLQTELKEEIELLKIENR700 2411.38 793EAEMRQLQDQQAAQILDLER812 2385.19 684LQTELKEEIELLKIENR700 2098.18 679VQLSLLQTELKEEIELLK696 2126.23 682SLLQTELKEEIELLK696 1786.02 685QTELKEEIELLKIENR700 1985.09 681LSLLQTELKEEIELLK696 1899.11 683LLQTELKEEIELLK696 1698.99 721HNLIEDHQKELNNAK735 1802.92 686TELKEEIELLKIENR700 1857.03 685QTELKEEIELLK696 1472.82 719VKHNLIEDHQKELNNAK735 2030.08 684LQTELKEEIELLK696 1585.91 778AESEKQTIINKFELR792 1805.98 798QLQDQQAAQILDLER812 1768.92 687ELKEEIELLKIENR700 1755.99 767LTLMLLELREKAESEK782 1903.06 688LKEEIELLKIENR700 1626.95 798QLQDQQAAQILDLERSLTEQQGR820 2668.37 757EELQREAEEKLTLMLLELR775 2343.26 762EAEEKLTLMLLELR775 1687.93 762EAEEKLTLMLLELREK777 1945.07 1 Peptide hits are listed based on the sorted cross correlation score (XCorr) starting from 5.0 to 10.8 [XCorr is deﬁned by Proteome Discoverer 1.3 (Thermo Scientiﬁc, USA)].

2.4. Data Collection and Processing X-ray diffraction data were collected at 100 K using a CCD detector (ADSC Quantum 315r) at beamline 5C of the Pohang Light Source (PLS, Pohang, Korea). The crystal was rotated through a total of 180◦ with 1.0◦ oscillation range per frame. Data were processed in space group C2 using HKL2000 [12] (Table3).

Table 3. Data collection and processing.

Diffraction Source Pohang Light Source (PLS 5C) (Pohang, Korea) Wavelength (Å) 0.9795 Temperature (K) 100 Detector ADSC Quantum 315r Crystal–detector distance (mm) 450 Rotation range per image (◦) 1 Total rotation range (◦) 180 Exposure time per image (s) 1 Space group C2 a, b, c (Å) 324.9, 35.7, 79.5 α, β, γ (◦) 90.0, 101.6, 90.0 Mosaicity (◦) 1.1 Resolution range (Å) 50.0–3.80 (3.87–3.80)1 Total No. of reﬂections 16,221 No. of unique reﬂections 9074 Completeness (%) 97.3 (89.7) Redundancy 1.7 (1.6) hI/σ(I)i 18.1 (3.9) Rmerge 0.463 (0.120) Rp.i.m. 0.081 (0.303) CC1/2 (0.918) Overall B factor from Wilson plot (Å2) 78.7 1 Values for the outer shell are given in parentheses. Crystals 2017, 7, 296 5 of 7

Completeness (%) 97.3 (89.7) Redundancy 1.7 (1.6) 〈I/σ(I)〉 18.1 (3.9) Rmerge 0.463 (0.120) Rp.i.m. 0.081 (0.303) CC1/2 (0.918) Overall B factor from Wilson plot (Å2) 78.7 1 Crystals 2017, 7, 296 Values for the outer shell are given in parentheses. 5 of 7

3. Results and Discussion 3. Results and Discussion Various coiled-coil prediction algorithms such as COILS [13] suggest multiple occurrences of coiled-coilVarious regions coiled-coil in the predictionlarge-sized algorithms(3336 amino such acids) as human COILS PCNT [13] suggest (Figure multiple 1). Three occurrences human PCNT of coiled-coilconstructs regionsof three in thecoiled-coil large-sized regions (3336 were amino generated acids) human by PCNTgene synthesis (Figure1). with Three E. human coli codon- PCNT constructsoptimization of threefor bacterial coiled-coil recombinant regions were expression. generated The by geneproteins synthesis were withsuccessfullyE. coli codon-optimization over-produced in forE. coli bacterial as a soluble recombinant protein and expression. purified with The an proteins overall were yield successfully of >50 mg per over-produced 1 L of LB culture. in E. The coli SECas aelution soluble profiles protein of and the purified three coiled-coil with an overall regions yield on ofHiLoad >50 mg® 26/60 per 1 LSuperdex of LB culture.® 200 showed The SEC a elution single profilesmajor peak, of the and three SDS-PAGE coiled-coil regionsanalysis on of HiLoad the concentrated® 26/60 Superdex proteins® 200 under showed the a singlepeaks major indicated peak, andsuccessful SDS-PAGE expression analysis of ofthe the proteins concentrated (Figure proteins 2). The underestimated the peaksmolecular indicated masses successful of the proteins expression based of theon SDS-PAGE proteins (Figure standard2). The protein estimated markers molecular were ∼ masses35 kDa of (CC1), the proteins ∼17 kDa based (CC2), on SDS-PAGEand ∼40 kDa standard (CC3), proteinwhich were markers as expected were ∼35 from kDa the (CC1), calculated∼17 kDa mole (CC2),cular and masses∼40 of kDa the (CC3), protein which (CC1, were 35.5 as kDa; expected CC2, from19.2 kDa; the calculatedCC3, 38.7 molecularkDa). masses of the protein (CC1, 35.5 kDa; CC2, 19.2 kDa; CC3, 38.7 kDa). Predicted to form mostly coiled-coils, the secondarysecondary structure contents of PCNT were expected to be largelylargely αα-helical.-helical. Circular Circular dichroism dichroism (CD) (CD) analyses analyses of of the the purified purified PCNTs PCNTs confirmedconfirmed this prediction (Figure(Figure3a–c). 3a–c). When When the experimental the experimental ellipticity ellipticity values determined values determined at different wavelengthsat different (200–240wavelengths nm) (200 were–240 used nm) for were secondary used for structure secondary estimations, structure estimations, the results indicated the results that indicated large parts that of the proteins were mostly α-helical with <5% being β-strand (Figure3). For instance, PCNT CC1 large parts of the proteins were mostly α-helical with <5% being β-strand (Figure 3). For instance, was estimated with 69% (K2D2) or 81% (K2D3) α-helicity. Additionally, PCNT CC2 was estimated PCNT CC1 was estimated with 69% (K2D2) or 81% (K2D3) α-helicity. Additionally, PCNT CC2 was with 76% (K2D2) or 89% (K2D3) α-helicity, and PCNT CC3 with 63% (K2D2) or 67% (K2D3) α-helicity. estimated with 76% (K2D2) or 89% (K2D3) α-helicity, and PCNT CC3 with 63% (K2D2) or 67% (K2D3) Hence, the overall trend indicated that the three PCNT proteins were made up largely of α-helices as α-helicity. Hence, the overall trend indicated that the three PCNT proteins were made up largely of predicted from the amino acid sequences. α-helices as predicted from the amino acid sequences.

(a) (b) (c)

Figure 3. CircularCircular dichroism dichroism studies studies for for the the estimation estimation of ofsecondary secondary structure structure elements elements in PCNT in PCNT (a) (CC1,a) CC1, (b) (CC2,b) CC2, and and (c) (CC3.c) CC3.

Among the three purified PCNT proteins, crystals were obtained only from PCNT CC2. Among the three purified PCNT proteins, crystals were obtained only from PCNT CC2. Clustered Clustered crystals were grown against a reservoir solution of 10% (w/v) PEG 8K, 100 mM potassium crystals were grown against a reservoir solution of 10% (w/v) PEG 8K, 100 mM potassium phosphate phosphate monobasic/sodium phosphate dibasic pH 6.2, and 200 mM NaCl at 4 °C. Within two days, monobasic/sodium phosphate dibasic pH 6.2, and 200 mM NaCl at 4 ◦C. Within two days, the crystals the crystals grew to approximately 5 μm × 50 μm × 100 μm (Figure 4), which was sufficient in size for grew to approximately 5 µm × 50 µm × 100 µm (Figure4), which was sufficient in size for X-ray X-ray diffraction experiments. The mass spectrometry analysis on the tryptic digestion fragments of diffraction experiments. The mass spectrometry analysis on the tryptic digestion fragments of the the crystals confirmed the content of PCNT CC2 (Figure 4 and Table 2). The peptides found were crystals confirmed the content of PCNT CC2 (Figure4 and Table2). The peptides found were mapped mapped into the expressed PCNT CC2 protein sequence, resulting in 99.4% sequence coverage. Most into the expressed PCNT CC2 protein sequence, resulting in 99.4% sequence coverage. Most crystals crystals screened for X-ray diffraction using synchrotron radiation showed anisotropic diffraction. screened for X-ray diffraction using synchrotron radiation showed anisotropic diffraction. However, However, one out of ten crystals tested gave isotropic diffraction in all directions of oscillation to an one out of ten crystals tested gave isotropic diffraction in all directions of oscillation to an average resolution limit of 3.8 Å (Figure5). Alterations in PEG 8K concentrations, pH, as well as trials of different types of PEGs as the crystallization agent did not affect the overall diffraction quality. A total of 9074 unique reflections were measured and merged in the space group C2 (unit cell parameters of a = 324.9 Å, b = 35.7 Å, c = 79.5 Å, and β = 101.6◦). The merged dataset was overall 97.3% complete with Rmerge of 12% and Rp.i.m. of 8.1% (50–3.80 Å). The statistics for the collected data are summarized in Table3. According to the Matthews coefficient [ 14], the asymmetric unit may contain up to 12 Crystals 2017,, 7,, 296296 6 of 7 average resolution limit of 3.8 Å (Figure 5). Alterations in PEG 8K concentrations, pH, as well as trials of different types of PEGs as the crystallization agent did not affect the overall diffraction quality. A total of 9074 unique reflections were measured and merged in the space group C2 (unit cell parametersCrystals 2017, 7 of, 296 a = 324.9 Å, b = 35.7 Å, c = 79.5 Å, and β = 101.6°). The merged dataset was overall 97.3%6 of 7 complete with Rmerge of 12% and Rp.i.m. of 8.1% (50–3.80 Å). The statistics for the collected data are summarized in Table 3. According to the Matthews coefficient [14], the asymmetric unit may contain3 −1 subunits of the monomeric PCNT CC2 with a crystal volume per protein mass (VM) of 1.96 Å Da up to 12 subunits of the monomeric PCNT CC2 with a crystal volume per protein mass (VM) of 1.96 and 37.3% solvent content. An asymmetric unit containing eight subunits is also plausible with V Å3 Da−1 and 37.3% solvent content. An asymmetric unit containing eight subunits is also plausibleM of 2.93 Å3 Da−1 and 58.2% solvent content. A self-rotation function (kappa section = 180◦) revealed with VM of 2.93 Å3 Da−1 and 58.2% solvent content. A self-rotation function (kappa section = 180°) a peak corresponding to two-fold noncrystallographic symmetry, further suggesting that PCNT CC2 revealed a peak corresponding to two-fold noncrystallographic symmetry, further suggesting that exists as a dimer in the crystal. Because no model for PCNT exists in the Protein Databank (PDB), PCNT CC2 exists as a dimer in the crystal. Because no model for PCNT exists in the Protein Databank attempts for phasing via molecular replacement could not be made. (PDB), attempts for phasing via molecular replacement could not be made.

(a) (b)

Figure 4. ((a)) Clustered crystals of of PCNT CC2 and ( b) the SDS-PAGE analysisanalysis ofof harvestedharvested crystalscrystals used for MS analysis of tryptic digestion fragments.

Figure 5. Representative diffraction image of the PCNT CC2 crystal. Figure 5. RepresentativeRepresentative diffractiondiffraction imimage of the PCNT CC2 crystal.

In the future, future, we we plan plan to to determine determine the the high-res high-resolutionolution structure of the coiled-coil PCNT CC2 by improving thethe currentcurrent 3.8 3.8 Å Å resolution resolution crystal crystal and and also also by by direct direct phasing. phasing. Since Since no no information information on theon thestructure structure of PCNT of PCNT exists, exists, the structurethe structure would would give give insight insight towards towards understanding understanding the mechanismthe mechanism that thatgoverns governs thefunction the function of PCNT. of PCNT.

Acknowledgments: TheThe authors authors would would like like toto thank thank thethe staff staff at at PAL PAL 5C5C beamlinebeamline for their support and beam time. This This research research was supported by the Basic Science Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science,Science, ICTICT && FutureFuture PlanningPlanning (2016R1D1A1A09918187).(2016R1D1A1A09918187).

Author Contributions: Min Ye KimKim andand SangYoun ParkPark conceivedconceived andand designed the experiments; Min Ye Kim and Jeong Kuk Park performed the experiments; Jeong Kuk Park, Yeowon Sim, Doheum Kim and Jeong Yeon Sim andanalyzed Jeong the Kuk data; Park SangYoun performed Park the wrote experiments; the paper. Jeong Kuk Park, Yeowon Sim, Doheum Kim and Jeong Yeon Sim analyzed the data; SangYoun Park wrote the paper. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conflicts of Interest: TheThe authorsauthors declaredeclare nono conflictconflict ofof interest.interest. References

1. Sluder, G. Two-way trafﬁc: Centrosomes and the cell cycle. Nat. Rev. Mol. Cell Biol. 2005, 6, 743–748. [CrossRef][PubMed] Crystals 2017, 7, 296 7 of 7

2. Takahashi, M.; Yamagiwa, A.; Nishimura, T.; Mukai, H.; Ono, Y. Centrosomal proteins CG-NAP and kendrin provide microtubule nucleation sites by anchoring gamma-tubulin ring complex. Mol. Biol. Cell 2002, 13, 3235–3245. [CrossRef][PubMed] 3. Zimmerman, W.C.; Sillibourne, J.; Rosa, J.; Doxsey, S.J. Mitosis-specific anchoring of gamma tubulin complexes by pericentrin controls spindle organization and mitotic entry. Mol. Biol. Cell 2004, 15, 3642–3657. [CrossRef][PubMed] 4. Delaval, B.; Doxsey, S.J. Pericentrin in cellular function and disease. J. Cell Biol. 2010, 188, 181–190. [CrossRef] [PubMed] 5. Doxsey, S.; Zimmerman, W.; Mikule, K. Centrosome control of the cell cycle. Trends Cell Biol. 2005, 15, 303–311. [CrossRef][PubMed] 6. Doxsey, S.J.; Stein, P.; Evans, L.; Calarco, P.D.; Kirschner, M. Pericentrin, a highly conserved centrosome protein involved in microtubule organization. Cell 1994, 76, 639–650. [CrossRef] 7. Gillingham, A.K.; Munro, S. The PACT domain, a conserved centrosomal targeting motif in the coiled-coil proteins AKAP450 and pericentrin. EMBO Rep. 2000, 1, 524–529. [CrossRef][PubMed] 8. Rauch, A.; Thiel, C.T.; Schindler, D.; Wick, U.; Crow, Y.J.; Ekici, A.B.; van Essen, A.J.; Goecke, T.O.; Al-Gazali, L.; Chrzanowska, K.H.; et al. Mutations in the pericentrin (PCNT) gene cause primordial dwarfism. Science 2008, 319, 816–819. [CrossRef][PubMed] 9. Gill, S.C.; von Hippel, P.H. Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 1989, 182, 319–326. [CrossRef] 10. Perez-Iratxeta, C.; Andrade-Navarro, M.A. K2D2: Estimation of protein secondary structure from circular dichroism spectra. BMC Struct. Biol. 2008, 8, 1–5. [CrossRef][PubMed] 11. Louis-Jeune, C.; Andrade-Navarro, M.A.; Perez-Iratxeta, C. Prediction of protein secondary structure from circular dichroism using theoretically derived spectra. Proteins 2012, 80, 374–381. [CrossRef][PubMed] 12. Otwinowski, Z.; Minor, W. Processing of X-ray Diffraction Data Collected in Oscillation Mode. Methods Enzymol. 1997, 276, 307–326. [PubMed] 13. Lupas, A.; Van Dyke, M.; Stock, J. Predicting Coiled Coils from Protein Sequences. Science 1991, 252, 1162–1164. [CrossRef][PubMed] 14. Matthews, B.W. Solvent content of protein crystals. J. Mol. Biol. 1968, 33, 491–497. [CrossRef]

© 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).