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• W2013050330 abstract "Human kallikrein-related peptidase 2 (KLK2) is a tryptic serine protease predominantly expressed in prostatic tissue and secreted into prostatic fluid, a major component of seminal fluid. Most likely it activates and complements chymotryptic KLK3 (prostate-specific antigen) in cleaving seminal clotting proteins, resulting in sperm liquefaction. KLK2 belongs to the “classical” KLKs 1–3, which share an extended 99- or kallikrein loop near their non-primed substrate binding site. Here, we report the 1.9 Å crystal structures of two KLK2-small molecule inhibitor complexes. In both structures discontinuous electron density for the 99-loop indicates that this loop is largely disordered. We provide evidence that the 99-loop is responsible for two biochemical peculiarities of KLK2, i.e. reversible inhibition by micromolar Zn2+ concentrations and permanent inactivation by autocatalytic cleavage. Indeed, several 99-loop mutants of KLK2 displayed an altered susceptibility to Zn2+, which located the Zn2+ binding site at the 99-loop/active site interface. In addition, we identified an autolysis site between residues 95e and 95f in the 99-loop, whose elimination prevented the mature enzyme from limited autolysis and irreversible inactivation. An exhaustive comparison of KLK2 with related structures revealed that in the KLK family the 99-, 148-, and 220-loop exist in open and closed conformations, allowing or preventing substrate access, which extends the concept of conformational selection in trypsin-related proteases. Taken together, our novel biochemical and structural data on KLK2 identify its 99-loop as a key player in activity regulation. Human kallikrein-related peptidase 2 (KLK2) is a tryptic serine protease predominantly expressed in prostatic tissue and secreted into prostatic fluid, a major component of seminal fluid. Most likely it activates and complements chymotryptic KLK3 (prostate-specific antigen) in cleaving seminal clotting proteins, resulting in sperm liquefaction. KLK2 belongs to the “classical” KLKs 1–3, which share an extended 99- or kallikrein loop near their non-primed substrate binding site. Here, we report the 1.9 Å crystal structures of two KLK2-small molecule inhibitor complexes. In both structures discontinuous electron density for the 99-loop indicates that this loop is largely disordered. We provide evidence that the 99-loop is responsible for two biochemical peculiarities of KLK2, i.e. reversible inhibition by micromolar Zn2+ concentrations and permanent inactivation by autocatalytic cleavage. Indeed, several 99-loop mutants of KLK2 displayed an altered susceptibility to Zn2+, which located the Zn2+ binding site at the 99-loop/active site interface. In addition, we identified an autolysis site between residues 95e and 95f in the 99-loop, whose elimination prevented the mature enzyme from limited autolysis and irreversible inactivation. An exhaustive comparison of KLK2 with related structures revealed that in the KLK family the 99-, 148-, and 220-loop exist in open and closed conformations, allowing or preventing substrate access, which extends the concept of conformational selection in trypsin-related proteases. Taken together, our novel biochemical and structural data on KLK2 identify its 99-loop as a key player in activity regulation. Human kallikrein-related peptidases (KLKs) 2The abbreviations used are: KLKkallikrein-related peptidaseAMC7-amino-4-methylcoumarinBENACbenzamidine (BEN) affinity chromatographyEKenterokinaseIECion exchange chromatographyPCIproteinase C inhibitorpNApara-nitroanilineBzbenzoylPPACKH-D-Phe-Pro-Arg chloromethyl ketoner.m.s.d.root mean square deviationUPGMAunweighted pair group method with arithmetic mean. comprise 15 serine proteases that display the chymotrypsin fold (MEROPS clan PA, family S1). The first member of this family (KLK1) was described almost a century ago (1Frey E.K. Kraut H. Über einen von der Niere ausgeschiedenen, die Herztätigkeit anregenden Stoff.Hoppe-Seyler's Z. Physiol. Chem. 1926; 157: 32-61Crossref Scopus (25) Google Scholar) and subsequently named “kallikrein,” as it was detected in the pancreas, or καλλικρϵας (2Kraut H. Frey E.K. Werle E. Der Nachweis eines Kreislaufhormons in der Pankreasdrüse. (IV. Mitteilung über dieses Kreislaufhormon).Hoppe-Seyler's Z. Physiol. Chem. 1930; 189: 97-106Crossref Scopus (80) Google Scholar). Together with KLK3 (prostate-specific antigen), whose discovery dates back to the 1960s (3Rao A.R. Motiwala H.G. Karim O.M. The discovery of prostate-specific antigen.BJU Int. 2008; 101: 5-10Crossref PubMed Scopus (22) Google Scholar), and KLK2, whose corresponding gene was isolated in the 1980s (4Schedlich L.J. Bennetts B.H. Morris B.J. Primary structure of a human glandular kallikrein gene.DNA. 1987; 6: 429-437Crossref PubMed Scopus (278) Google Scholar), KLK1 belongs to the classical kallikrein subfamily. These proteases are more closely related to each other than to the new kallikreins 4–15 (called “new” because their gradual assignment to the KLK family started at the end of the 1990s (5Nelson P.S. Gan L. Ferguson C. Moss P. Gelinas R. Hood L. Wang K. Molecular cloning and characterization of prostase, an androgen-regulated serine protease with prostate-restricted expression.Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 3114-3119Crossref PubMed Scopus (205) Google Scholar)); KLK1–3 share a rather special surface loop that is 11 residues longer than the corresponding 99-loop of chymotrypsin (6Pavlopoulou A. Pampalakis G. Michalopoulos I. Sotiropoulou G. Evolutionary history of tissue kallikreins.PLoS ONE. 2010; 5: e13781Crossref PubMed Scopus (52) Google Scholar), and therefore, this loop is also designated the “kallikrein loop” (7Bode W. Chen Z. Bartels K. Kutzbach C. Schmidt-Kastner G. Bartunik H. Refined 2 Å x-ray crystal structure of porcine pancreatic kallikrein A, a specific trypsin-like serine proteinase. Crystallization, structure determination, crystallographic refinement, structure and its comparison with bovine trypsin.J. Mol. Biol. 1983; 164: 237-282Crossref PubMed Scopus (223) Google Scholar). kallikrein-related peptidase 7-amino-4-methylcoumarin benzamidine (BEN) affinity chromatography enterokinase ion exchange chromatography proteinase C inhibitor para-nitroaniline benzoyl H-D-Phe-Pro-Arg chloromethyl ketone root mean square deviation unweighted pair group method with arithmetic mean. KLK2, which was formerly called hK2 or human glandular kallikrein 1 (Uniprot identifier P20191, MEROPS entry S01.161), is relatively restricted to prostatic tissue and seminal fluid in healthy individuals (8Shaw J.L. Diamandis E.P. Distribution of 15 human kallikreins in tissues and biological fluids.Clin. Chem. 2007; 53: 1423-1432Crossref PubMed Scopus (311) Google Scholar). Current knowledge pinpoints the main physiological role of KLK2 to sperm liquefaction. On the one hand, KLK2 may activate the zymogen form of KLK3 (9Lövgren J. Rajakoski K. Karp M. Lundwall â. Lilja H. Activation of the zymogen form of prostate-specific antigen by human glandular kallikrein 2.Biochem. Biophys. Res. Commun. 1997; 238: 549-555Crossref PubMed Scopus (202) Google Scholar, 10Yoon H. Laxmikanthan G. Lee J. Blaber S.I. Rodriguez A. Kogot J.M. Scarisbrick I.A. Blaber M. Activation profiles and regulatory cascades of the human kallikrein-related peptidases.J. Biol. Chem. 2007; 282: 31852-31864Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), although there is contradictory evidence (11Denmeade S.R. Lövgren J. Khan S.R. Lilja H. Isaacs J.T. Activation of latent protease function of pro-hK2, but not pro-PSA, involves autoprocessing.Prostate. 2001; 48: 122-126Crossref PubMed Scopus (37) Google Scholar). KLK3 in turn dissolves the sperm coagulum via degradation of semenogelins 1 and 2 and fibronectin (12McGee R.S. Herr J.C. Human seminal vesicle-specific antigen is a substrate for prostate-specific antigen (or P-30).Biol. Reprod. 1988; 39: 499-510Crossref PubMed Scopus (73) Google Scholar). On the other hand, KLK2 itself is able to cleave the latter proteins at sites distinct from KLK3 (13Lövgren J. Airas K. Lilja H. Enzymatic action of human glandular kallikrein 2 (hK2). Substrate specificity and regulation by Zn2+ and extracellular protease inhibitors.Eur. J. Biochem. 1999; 262: 781-789Crossref PubMed Scopus (95) Google Scholar). Maximum KLK2 activity in sperm appears immediately after ejaculation, and it decreases within 10 min due to complex formation with PCI. Because the time course of semenogelin/fibronectin degradation and loss of KLK2 in vivo activity coincide, it is assumed that KLK2 complements KLK3 during sperm liquefaction (14Deperthes D. Frenette G. Brillard-Bourdet M. Bourgeois L. Gauthier F. Tremblay R.R. Dubé J.Y. Potential involvement of kallikrein hK2 in the hydrolysis of the human seminal vesicle proteins after ejaculation.J. Androl. 1996; 17: 659-665PubMed Google Scholar). However, KLK2 is aberrantly expressed in a range of human malignancies (15Kontos C.K. Scorilas A. Kallikrein-related peptidases (KLKs): a gene family of novel cancer biomarkers.Clin. Chem. Lab. Med. 2012; 50: 1877-1891Crossref PubMed Scopus (70) Google Scholar). Hence, elevated KLK2 levels in blood may constitute a valid marker for prostate cancer either alone or in combination with levels of various KLK3 isoforms (16Jansen F.H. Roobol M. Jenster G. Schröder F.H. Bangma C.H. Screening for prostate cancer in 2008 II: the importance of molecular subforms of prostate-specific antigen and tissue kallikreins.Eur. Urol. 2009; 55: 563-574Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Due to its narrow tissue distribution, KLK2 has been regarded as a potential drug target in prostate cancer (17Hekim C. Leinonen J. Närvänen A. Koistinen H. Zhu L. Koivunen E. Väisänen V. Stenman U.-H. Novel peptide inhibitors of human kallikrein 2.J. Biol. Chem. 2006; 281: 12555-12560Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) or as a prodrug activator in targeted chemotherapy (18Janssen S. Rosen D.M. Ricklis R.M. Dionne C.A. Lilja H. Christensen S.B. Isaacs J.T. Denmeade S.R. Pharmacokinetics, biodistribution, and antitumor efficacy of a human glandular kallikrein 2 (hK2)-activated thapsigargin prodrug.Prostate. 2006; 66: 358-368Crossref PubMed Scopus (47) Google Scholar). In prostate carcinoma, KLK2 may promote growth or metastasis of tumor cells by interacting with the urokinase-type plasminogen activator system. KLK2 is able to activate the zymogen form of urokinase-type plasminogen activator (19Frenette G. Tremblay R.R. Lazure C. Dube J.Y. Prostatic kallikrein hK2, but not prostate-specific antigen (hK3), activates single-chain urokinase-type plasminogen activator.Int. J. Cancer. 1997; 71: 897-899Crossref PubMed Scopus (113) Google Scholar), which may even initiate a positive feedback loop involving further activation of pro-KLK2 by urokinase-type plasminogen activator (11Denmeade S.R. Lövgren J. Khan S.R. Lilja H. Isaacs J.T. Activation of latent protease function of pro-hK2, but not pro-PSA, involves autoprocessing.Prostate. 2001; 48: 122-126Crossref PubMed Scopus (37) Google Scholar). Other cancer-related KLK2 targets include plasminogen activator inhibitor-1, an inhibitor of urokinase-type plasminogen activator (20Mikolajczyk S.D. Millar L.S. Kumar A. Saedi M.S. Prostatic human kallikrein 2 inactivates and complexes with plasminogen activator inhibitor-1.Int. J. Cancer. 1999; 81: 438-442Crossref PubMed Scopus (71) Google Scholar), insulin growth factor-binding proteins 2–5 (21Réhault S. Monget P. Mazerbourg S. Tremblay R. Gutman N. Gauthier F. Moreau T. Insulin-like growth factor binding proteins (IGFBPs) as potential physiological substrates for human kallikreins hK2 and hK3.Eur. J. Biochem. 2001; 268: 2960-2968Crossref PubMed Scopus (93) Google Scholar), and protease-activated receptor 2 (22Mize G.J. Wang W. Takayama T.K. Prostate-specific kallikreins-2 and -4 enhance the proliferation of DU-145 prostate cancer cells through protease-activated receptors-1 and -2.Mol. Cancer Res. 2008; 6: 1043-1051Crossref PubMed Scopus (76) Google Scholar). Although KLK1 (23Laxmikanthan G. Blaber S.I. Bernett M.J. Scarisbrick I.A. Juliano M.A. Blaber M. 1.70 Å x-ray structure of human apo kallikrein 1: structural changes upon peptide inhibitor/substrate binding.Proteins. 2005; 58: 802-814Crossref PubMed Scopus (58) Google Scholar), KLK3 (24Ménez R. Michel S. Muller B.H. Bossus M. Ducancel F. Jolivet-Reynaud C. Stura E.A. Crystal structure of a ternary complex between human prostate-specific antigen, its substrate acyl intermediate, and an activating antibody.J. Mol. Biol. 2008; 376: 1021-1033Crossref PubMed Scopus (70) Google Scholar, 25Stura E.A. Muller B.H. Bossus M. Michel S. Jolivet-Reynaud C. Ducancel F. Crystal structure of human prostate-specific antigen in a sandwich antibody complex.J. Mol. Biol. 2011; 414: 530-544Crossref PubMed Scopus (50) Google Scholar), and several new kallikreins (for review see Ref. 26Debela M. Beaufort N. Magdolen V. Schechter N.M. Craik C.S. Schmitt M. Bode W. Goettig P. Structures and specificity of the human kallikrein-related peptidases KLK 4, 5, 6, and 7.Biol. Chem. 2008; 389: 623-632Crossref PubMed Scopus (73) Google Scholar) are well characterized on the structural level, the structure of KLK2 has remained elusive. To close this knowledge gap within the classical kallikreins, we present here two crystal structures of KLK2 obtained from Escherichia coli expression and refolding. Furthermore, we characterized a series of KLK2 mutants to elucidate its Zn2+ inhibition and inactivation by proteolytic cleavage within the 99-loop. Kinetic properties of these mutants extend an in-depth comparison of KLK2 with related structures and investigate the diverse roles of the 99-loop in the regulation of KLK2 activity. First, KLK2 expression vectors were prepared from prostate adenoma cDNA by inserting the sequence of the mature protease (Ile-16 to Pro-245a) between the BamHI and HindIII sites of the pQE-30 plasmid (Qiagen, Hilden, Germany). Hence, the resulting plasmid pQE-30-pro(DDDDK)_KLK2 encoded an N-terminal artificial propeptide (MRGSHHHHHHGSDDDDK) with a hexahistidine tag preceding the canonical enterokinase (EK) recognition sequence (DDDDK). Second, round-the-horn site-directed mutagenesis 3S. Moore, unpublished data. was employed to generate two vectors with alternative cleavage sites in the propeptide: pQE-30-pro(SGDR)_KLK2 and pQE-30-pro(PSFR)_KLK2. Third, we generated six point mutants from these three pQE-30 derivatives by round-the-horn site-directed mutagenesis: H25A, H91A, K95eM, K95eQ, H95fA, H101A. DNA sequencing confirmed the correctness of all constructs. Enzymes for cloning were purchased from Thermo Scientific (Waltham, MA) or Stratagene (La Jolla, CA). KLK2 was expressed as inclusion bodies and folded in vitro essentially as described for the catalytic domain of EK (28Skala W. Goettig P. Brandstetter H. Do-it-yourself histidine-tagged bovine enterokinase: a handy member of the protein engineer's toolbox.J. Biotechnol. 2013; 168: 421-425Crossref PubMed Scopus (26) Google Scholar). In brief, E. coli M15[pREP4] cells (Qiagen) were transformed with the respective expression plasmid and grown in LB medium (supplemented with 100 μg/ml ampicillin and 30 μg/ml kanamycin) until the culture reached an A600 of 1.2. Protein expression was induced with 0.5 mm isopropyl β-d-1-thiogalactopyranoside for 4 h at 37 °C. Cells were disrupted by sonication, and insoluble matter was washed with Triton X-100- and EDTA-containing buffers. Washed inclusion bodies were solubilized 1:20 (w/v) in 7.5 m guanidine-HCl, pH 9, 50 mm Tris, 100 mm β-mercaptoethanol for 24 h, dialyzed against 5 mm citrate, pH 3.5–4.0, and resolubilized 1:10 (w/v) in 7.5 m guanidine-HCl, pH 4.0–4.5, 50 mm Tris for several hours. Dropwise dilution of this solution into the 100-fold volume of 500 mm arginine, 50 mm Tris, pH 8.3, 20 mm NaCl, 1 mm EDTA, 5 mm cysteine-HCl, and 0.5 mm cystine yielded 5–10% folded protein after 3 days at 16 °C. Wild type KLK2 and the mutants K95eM, K95eQ, and H101A were purified by (negative) ion exchange chromatography (IEC) and benzamidine (BEN) affinity chromatography (BENAC); purification of the mutants H25A, H91A, and H95fA comprised (positive) IEC, activation by EK, negative metal ion affinity chromatography, and BENAC (Fig. 1). Chromatography resins were obtained from GE Healthcare. After tangential flow concentration, the refolding solution was loaded onto a Q-Sepharose column equilibrated in IEC buffer (50 mm Tris-HCl, pH 8.0). The ratio of load to resin was about 50:1 (v/v) in this and all following affinity chromatography steps; all buffers contained 3 mm sodium azide. In negative IEC, the flow-through contained mature KLK2. In positive IEC, pro-KLK2 was eluted from the column with 3 resin volumes of IEC buffer supplemented with 150 mm NaCl. Zymogen forms of KLK2 from positive IEC were incubated with EK in a molar ratio of 1000:1 for 15 h at 20 °C. EK was produced in-house as previously described (28Skala W. Goettig P. Brandstetter H. Do-it-yourself histidine-tagged bovine enterokinase: a handy member of the protein engineer's toolbox.J. Biotechnol. 2013; 168: 421-425Crossref PubMed Scopus (26) Google Scholar). The digestion mixture was brought to 500 mm NaCl, 10 mm imidazole and loaded onto a Ni2+- or Co2+-Sepharose column equilibrated in 50 mm Tris-HCl, pH 8.0, 500 mm NaCl, 10 mm imidazole. The flow-through contained mature KLK2, whereas the resin-bound residual pro-KLK2 cleaved propeptide and EK. For BENAC, flow-through from negative IEC was brought to 500 mm NaCl and loaded onto a benzamidine-Sepharose column equilibrated in BENAC buffer (50 mm Tris-HCl, pH 8.0, 500 mm NaCl). Flow-through from metal ion affinity chromatography was directly loaded due to its proper sodium chloride concentration. After washing with 8 resin volumes of BENAC buffer, bound KLK2 was eluted with 3 × 2.5 resin volumes of BENAC buffer supplemented with 25, 50, and 100 mm benzamidine. As final polishing step, size exclusion chromatography was performed over a Superose 6 10/300 GL column connected to an ÄKTA FPLC system (GE Healthcare). To this end, the BENAC eluate was concentrated in Amicon Ultra-15 Centrifugal Filter Units, molecular weight cutoff 10 kDa (Millipore, Billerica, MA). Per run, 500 μl of concentrate were loaded onto the column at 4 °C (running buffer: 20 mm Tris-HCl, pH 8.0, 20 mm NaCl, 5 mm benzamidine). Fractions that corresponded to the monomeric KLK2 peak were combined and concentrated to 12 mg/ml. Chemicals of the highest purity available were either from AppliChem (Darmstadt, Germany), Carl Roth (Karlsruhe, Germany), Merck, or Sigma. Bz-PFR-pNA, H-GHR-AMC, H-PFR-AMC, H-Arg-AMC, and PPACK were obtained from Bachem (Weil am Rhein, Germany). Amidolytic activity was generally measured in 100 μl of assay buffer (50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 10% (v/v) DMSO, 0.1% (w/v) BSA) containing 400 ng (150 nm) of KLK2 and 250 μm chromogenic or fluorogenic substrate. pH values of the reaction mixtures were routinely checked to exclude any effects of pH changes. Time-dependent substrate cleavage corresponded to changes in absorbance at 405 nm (for pNA substrates) or fluorescence at 460 nm (for AMC substrates; excitation wavelength: 380 nm) and was recorded on an Infinite M200 microplate reader (Tecan, Männedorf, Switzerland). Protein concentrations were determined by absorbance at 280 nm using computed extinction coefficients and molecular weights. For calculating kcat values, we performed active site titration of the respective KLK2 variant with PPACK and corrected the data accordingly. The pH optimum was determined in 100 mm SPG buffer (12.5 mm succinate, 43.75 mm NaH2PO4, 43.75 mm glycine). Zn2+ inhibition curves were measured without BSA, as its metal binding sites sequestered Zn2+ ions from the reaction buffer. However, KLK2 adsorbed to the microplate walls in the absence of BSA, which interfered with the measurement of Michaelis-Menten kinetics at different Zn2+ concentrations. Thus, we saturated all Zn2+ binding sites in BSA by dialyzing BSA-containing assay buffer against the 200-fold volume of assay buffer with the desired Zn2+ concentration. Reactivity toward the burst reagent 4-nitrophenyl-4-guanidinobenzoate (NPGB) (29Chase Jr., T. Shaw E. Comparison of the esterase activities of trypsin, plasmin, and thrombin on guanidinobenzoate esters: titration of the enzymes.Biochemistry. 1969; 8: 2212-2224Crossref PubMed Scopus (380) Google Scholar) was determined by adding 75 μl of 100 μm KLK2 to 675 μl of 50 mm HEPES, pH 7.0, 150 mm NaCl, 50 μm NPGB and by detecting the concomitant change in absorbance at 405 nm. Substrate specificity was determined by positional scanning as previously described (30Choe Y. Leonetti F. Greenbaum D.C. Lecaille F. Bogyo M. Brömme D. Ellman J.A. Craik C.S. Substrate profiling of cysteine proteases using a combinatorial peptide library identifies functionally unique specificities.J. Biol. Chem. 2006; 281: 12824-12832Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar). Data were analyzed with nonlinear regression models as implemented in QtiPlot v0.9.8.8 (31Vasilief I. QtiPlot (Data Analysis and Scientific Visualization). Craiova, Romania2011Google Scholar). Crystals of active wild type KLK2 were grown at 20 °C by vapor diffusion in 500 nl of sitting drops of a 12 mg/ml protein solution in 20 mm Tris-HCl, pH 8.0, 20 mm NaCl, 10 mm benzamidine, 3 mm NaN3 that were mixed with 500 nl of precipitant. Crystals of benzamidine-inhibited KLK2 appeared within 10 days in 500 mm (NH4)2SO4, 1 m Li2SO4, 100 mm sodium citrate and were directly frozen in the nitrogen gas stream (100 K) without prior cryoprotection. Crystals of the KLK2-PPACK complex were obtained by soaking KLK2-benzamidine crystals for 1 h in mother liquor supplemented with 7 mm PPACK. Data sets were collected in-house (Bruker AXS microstar rotating anode, mar345 image plate) or at the beamline X12 (MarMosaic 225 charge-coupled device) at the DESY in Hamburg (see Table 1).TABLE 1Data collection and refinement statistics for KLK2StructureKLK2-BENKLK2-PPACKPDB ID4nfe4nffData collectionWavelength (Å)1.54180.97004Space groupP212121P212121Cell constants (Å)a = 59.65, b = 60.39, c = 67.68a = 60.10, b = 60.74, c = 66.80α = β = γ = 90°α = β = γ = 90°Resolution rangeaValues in parentheses are for the highest resolution shell. (Å)45.06-1.90 (2.00-1.90)30.37-1.90 (2.00-1.90)Number of observationsaValues in parentheses are for the highest resolution shell.127,875 (16,104)122,496 (10,451)Number of unique reflectionsaValues in parentheses are for the highest resolution shell.19,455 (2,731)19,421 (2,619)MultiplicityaValues in parentheses are for the highest resolution shell.6.6 (5.9)6.3 (4.0)CompletenessaValues in parentheses are for the highest resolution shell. (%)98.1 (96.5)97.9 (92.4)Mean 〈 I/σ(I)〉aValues in parentheses are for the highest resolution shell.13.0 (5.7)10.0 (2.3)RmergeaValues in parentheses are for the highest resolution shell.0.088 (0.224)0.150 (0.585)RrimaValues in parentheses are for the highest resolution shell.0.095 (0.247)0.163 (0.666)RpimaValues in parentheses are for the highest resolution shell.0.036 (0.099)0.062 (0.306)B factor from Wilson plot (Å2)16.514.2RefinementResolution rangeaValues in parentheses are for the highest resolution shell. (Å)29.83-1.90 (1.95-1.90)29.27-1.90 (1.95-1.90)CompletenessaValues in parentheses are for the highest resolution shell. (%)97.8 (95.6)97.7 (90.9)Reflections used in refinementaValues in parentheses are for the highest resolution shell..bCutoff criterion F > 0 σF.19,418 (1272)19,387 (1201)Reflections in working setaValues in parentheses are for the highest resolution shell.18,433 (1200)18,399 (1145)Reflections in test setaValues in parentheses are for the highest resolution shell.985 (72)988 (56)Rcryst (%)aValues in parentheses are for the highest resolution shell.17.7 (18.8)19.7 (25.2)Rfree (%)aValues in parentheses are for the highest resolution shell.21.3 (27.5)23.2 (31.4)Residues refined227229Non-hydrogen protein atomscAverage B values (Å2) in parentheses.1,754 (20.1)1,771 (14.0)Non-hydrogen ligand atomscAverage B values (Å2) in parentheses.33 (29.0)30 (14.6)Solvent water moleculescAverage B values (Å2) in parentheses.136 (26.8)100 (17.2)r.m.s.d. bond lengths (Å)0.0120.014r.m.s.d. bond angles (°)1.4731.770Ramachandran plotdRegions as defined by MolProbity (42).Favored regions97.3% (217/223)98.2% (221/225)Allowed regions2.7% (6/223)1.8% (4/225)Disallowed regions0% (0/223)0% (0/225)a Values in parentheses are for the highest resolution shell.b Cutoff criterion F > 0 σF.c Average B values (Å2) in parentheses.d Regions as defined by MolProbity (42Chen V.B. Arendall 3rd, W.B. Headd J.J. Keedy D.A. Immormino R.M. Kapral G.J. Murray L.W. Richardson J.S. Richardson D.C. MolProbity: all-atom structure validation for macromolecular crystallography.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 12-21Crossref PubMed Scopus (9858) Google Scholar). Open table in a new tab Diffraction data were integrated by iMosflm v1.0.5 (32Battye T.G. Kontogiannis L. Johnson O. Powell H.R. Leslie A.G. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM.Acta Crystallogr. D Biol. Crystallogr. 2011; 67: 271-281Crossref PubMed Scopus (2319) Google Scholar) and scaled with Scala v3.3.20 (33Evans P. Scaling and assessment of data quality.Acta Crystallogr. D Biol. Crystallogr. 2006; 62: 72-82Crossref PubMed Scopus (3754) Google Scholar) included in the CCP4 v6.2.0 program suite (34Winn M.D. Ballard C.C. Cowtan K.D. Dodson E.J. Emsley P. Evans P.R. Keegan R.M. Krissinel E.B. Leslie A.G. McCoy A. McNicholas S.J. Murshudov G.N. Pannu N.S. Potterton E.A. Powell H.R. Read R.J. Vagin A. Wilson K.S. Overview of the CCP4 suite and current developments.Acta Crystallogr. D Biol. Crystallogr. 2011; 67: 235-242Crossref PubMed Scopus (9246) Google Scholar). Initial phases were obtained for the KLK2-BEN data set by molecular replacement with Phaser v2.3.0 (35McCoy A.J. Grosse-Kunstleve R.W. Adams P.D. Winn M.D. Storoni L.C. Read R.J. Phaser crystallographic software.J. Appl. Crystallogr. 2007; 40: 658-674Crossref PubMed Scopus (14504) Google Scholar) using KLK3 (2zch/chain P) as the search model in the resolution range of 1.9–36.0 Å. The scores of the top solution were RFZ = 17.5, TFZ = 32.9, LLG = +1456, and R-factor = 45.8. Neither the rotational nor the translational searches yielded a second unrelated peak. Phases for the KLK2-PPACK data were obtained by molecular replacement using Phaser with the KLK2-BEN polypeptide model after refinement. Essentially, the parameters were similar to the initial search, resulting in an LLG = +2688 and an R-factor = 34.8. Both KLK2 structures contained one molecule in the asymmetric unit and had a Matthews coefficient of 2.33 and solvent content of 47.2%. Model building in Coot v0.6.2 (36Emsley P. Lohkamp B. Scott W.G. Cowtan K. Features and development of Coot.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 486-501Crossref PubMed Scopus (17187) Google Scholar) alternated with restrained maximum likelihood refinement in REFMAC v5.6.0117 (37Murshudov G.N. Skubák P. Lebedev A.A. Pannu N.S. Steiner R.A. Nicholls R.A. Winn M.D. Long F. Vagin A.A. REFMAC 5 for the refinement of macromolecular crystal structures.Acta Crystallogr. D Biol. Crystallogr. 2011; 67: 355-367Crossref PubMed Scopus (5982) Google Scholar) using standard target parameters (38Engh R.A. Huber R. Accurate bond and angle parameters for x-ray protein structure refinement.Acta Crystallogr. A Found. Crystallogr. 1991; 47: 392-400Crossref Scopus (2543) Google Scholar). Global quality indicators of the final models were in the typical range for the obtained resolution (see Table 1). Geometric restraints for the two covalent bonds between PPACK and KLK2 were determined in JLigand v1.0.36 (39Lebedev A.A. Young P. Isupov M.N. Moroz O.V. Vagin A.A. Murshudov G.N. JLigand: a graphical tool for the CCP4 template-restraint library.Acta Crystallogr. D Biol. Crystallogr. 2012; 68: 431-440Crossref PubMed Scopus (307) Google Scholar); two CIF files were generated, each describing one bond, and then manually merged (supplemental File S1). Correct Asn and Gln side chain rotamers were assigned by NQ-Flipper v2.7 (40Weichenberger C.X. Sippl M.J. NQ-Flipper: recognition and correction of erroneous asparagine and glutamine side-chain rotamers in protein structures.Nucleic Acids Res. 2007; 35: W403-W406Crossref PubMed Scopus (48) Google Scholar). Side chains of the following surface residues lacked interpretable electron density in both models unless otherwise indicated: Lys-24, Lys-60 (only KLK2-BEN), Lys-61, Pro-76, Arg-82 (KLK2-BEN), His-87, Glu-110, Lys-113, Asp-116 (KLK2-PPACK), Lys-119, Asn-128, Glu-129 (KLK2-BEN), Glu-148, Arg-153, Glu-174 (KLK2-BEN), Lys-175, Glu-178 (KLK2-BEN), Glu-218 (KLK2-BEN), Arg-235 (KLK2-BEN), Lys-236, Lys-239 and Pro-245a (KLK2-BEN). As recommended (41Rupp B. Detection and analysis of unusual features in the structural model and structure-factor data of a birch pollen allergen.Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2012; 68: 366-376Crossref PubMed Scopus (17) Google Scholar), these side chains were modeled in their most likely conformation with full occupancy, which resulted in high B values. Met-167-Sδ was modeled with two alternate conformations in the side chain beyond Cγ. Structures were validated by MolProbity v3.19 (42Chen V.B. Arendall 3rd, W.B. Headd J.J. Keedy D.A. Immormino R.M. Kapral G.J. Murray L.W. Richardson J.S. Richardson D.C. MolProbity: all-atom structure validation for macromolecular crystallography.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 1" @default.
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• W2013050330 date "2014-12-01" @default.
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• W2013050330 title "Structure-Function Analyses of Human Kallikrein-related Peptidase 2 Establish the 99-Loop as Master Regulator of Activity" @default.
• W2013050330 cites W1496675280 @default.
• W2013050330 cites W1512629615 @default.
• W2013050330 cites W1521494626 @default.
• W2013050330 cites W1538656685 @default.
• W2013050330 cites W1815177835 @default.
• W2013050330 cites W1823385938 @default.
• W2013050330 cites W1966110638 @default.
• W2013050330 cites W1966319203 @default.
• W2013050330 cites W1966581230 @default.
• W2013050330 cites W1967470075 @default.
• W2013050330 cites W1969561087 @default.
• W2013050330 cites W1970578328 @default.
• W2013050330 cites W1973011421 @default.
• W2013050330 cites W1973669051 @default.
• W2013050330 cites W1976339707 @default.
• W2013050330 cites W1985469117 @default.
• W2013050330 cites W1994568170 @default.
• W2013050330 cites W1996386189 @default.
• W2013050330 cites W1998568556 @default.
• W2013050330 cites W2000097682 @default.
• W2013050330 cites W2002228551 @default.
• W2013050330 cites W2002589261 @default.
• W2013050330 cites W2006910150 @default.
• W2013050330 cites W2008708467 @default.
• W2013050330 cites W2012845953 @default.
• W2013050330 cites W2013981530 @default.
• W2013050330 cites W2014998794 @default.
• W2013050330 cites W2016255772 @default.
• W2013050330 cites W2017569531 @default.
• W2013050330 cites W2017704986 @default.
• W2013050330 cites W2020412616 @default.
• W2013050330 cites W2024599256 @default.
• W2013050330 cites W2026968384 @default.
• W2013050330 cites W2028113711 @default.
• W2013050330 cites W2029582401 @default.
• W2013050330 cites W2030101006 @default.
• W2013050330 cites W2032092475 @default.
• W2013050330 cites W2034314059 @default.
• W2013050330 cites W2035503835 @default.
• W2013050330 cites W2037055211 @default.
• W2013050330 cites W2051524275 @default.
• W2013050330 cites W2056260465 @default.
• W2013050330 cites W2063308693 @default.
• W2013050330 cites W2065793660 @default.
• W2013050330 cites W2071485699 @default.
• W2013050330 cites W2074631079 @default.
• W2013050330 cites W2075393035 @default.
• W2013050330 cites W2075659901 @default.
• W2013050330 cites W2080418572 @default.
• W2013050330 cites W2087036480 @default.
• W2013050330 cites W2088956282 @default.
• W2013050330 cites W2091317550 @default.
• W2013050330 cites W2095249666 @default.
• W2013050330 cites W2095915834 @default.
• W2013050330 cites W2097908823 @default.
• W2013050330 cites W2098936754 @default.
• W2013050330 cites W2101341907 @default.
• W2013050330 cites W2102948567 @default.
• W2013050330 cites W2107132108 @default.
• W2013050330 cites W2107542943 @default.
• W2013050330 cites W2108921801 @default.
• W2013050330 cites W2109613748 @default.
• W2013050330 cites W2110539319 @default.
• W2013050330 cites W2113900104 @default.
• W2013050330 cites W2116275717 @default.
• W2013050330 cites W2117073162 @default.
• W2013050330 cites W2119548502 @default.
• W2013050330 cites W2121082629 @default.
• W2013050330 cites W2121897924 @default.
• W2013050330 cites W2124026197 @default.
• W2013050330 cites W2140179680 @default.
• W2013050330 cites W2140877018 @default.
• W2013050330 cites W2146087465 @default.
• W2013050330 cites W2147932547 @default.
• W2013050330 cites W2147949826 @default.
• W2013050330 cites W2152326664 @default.
• W2013050330 cites W2154714625 @default.
• W2013050330 cites W2158266834 @default.
• W2013050330 cites W2159211495 @default.
• W2013050330 cites W2160772317 @default.
• W2013050330 cites W2163341755 @default.
• W2013050330 cites W2163765109 @default.
• W2013050330 cites W2164548935 @default.
• W2013050330 cites W2165232049 @default.
• W2013050330 cites W2165594693 @default.

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