Identification of the Calcium Binding Site and a Novel Ytterbium Site in Blood Coagulation Factor XIII by X-ray Crystallography
1999; Elsevier BV; Volume: 274; Issue: 8 Linguagem: Inglês
10.1074/jbc.274.8.4917
ISSN1083-351X
AutoresBrian A. Fox, Vivien C. Yee, Lars C. Pedersen, Isolde Le Trong, Paul D. Bishop, Ronald E. Stenkamp, David C. Teller,
Tópico(s)Iron Metabolism and Disorders
ResumoThe presence or absence of calcium determines the activation, activity, oligomerization, and stability of blood coagulation factor XIII. To explore these observed effects, we have determined the x-ray crystal structure of recombinant factor XIII A2 in the presence of calcium, strontium, and ytterbium. The main calcium binding site within each monomer involves the main chain oxygen atom of Ala-457, and also the side chains from residues Asn-436, Asp-438, Glu-485, and Glu-490. Calcium and strontium bind in the same location, while ytterbium binds several angstroms removed. A novel ytterbium binding site is also found at the dimer two-fold axis, near residues Asp-270 and Glu-272, and this site may be related to the reported inhibition by lanthanide metals (Achyuthan, K. E., Mary, A., and Greenberg, C. S. (1989) Biochem. J. 257, 331–338). The overall structure of ion-bound factor XIII is very similar to the previously determined crystal structures of factor XIII zymogen, likely due to the constraints of this monoclinic crystal form. We have merged the three independent sets of water molecules in the structures to determine which water molecules are conserved and possibly structurally significant. The presence or absence of calcium determines the activation, activity, oligomerization, and stability of blood coagulation factor XIII. To explore these observed effects, we have determined the x-ray crystal structure of recombinant factor XIII A2 in the presence of calcium, strontium, and ytterbium. The main calcium binding site within each monomer involves the main chain oxygen atom of Ala-457, and also the side chains from residues Asn-436, Asp-438, Glu-485, and Glu-490. Calcium and strontium bind in the same location, while ytterbium binds several angstroms removed. A novel ytterbium binding site is also found at the dimer two-fold axis, near residues Asp-270 and Glu-272, and this site may be related to the reported inhibition by lanthanide metals (Achyuthan, K. E., Mary, A., and Greenberg, C. S. (1989) Biochem. J. 257, 331–338). The overall structure of ion-bound factor XIII is very similar to the previously determined crystal structures of factor XIII zymogen, likely due to the constraints of this monoclinic crystal form. We have merged the three independent sets of water molecules in the structures to determine which water molecules are conserved and possibly structurally significant. The biological importance of factor XIII (fXIII) 1The abbreviations fXIIIfactor XIIITGasetransglutaminaser.m.s.d.root mean square deviationMES4-morpholineethanesulfonic acid 1The abbreviations fXIIIfactor XIIITGasetransglutaminaser.m.s.d.root mean square deviationMES4-morpholineethanesulfonic acid(EC 2.3.2.13) lies in its ability to form new covalent bonds between protein chains. This activity was first recognized while studying blood coagulation; fXIII was required to form an insoluble clot (1Laki K. Lorand L. Science. 1948; 108: 280Crossref PubMed Scopus (173) Google Scholar). The activated form of fXIII covalently cross-links two fibrin molecules via an isopeptide bond between the side chains of a glutamine and a lysine located in the C-terminal region of the γ-chain. Over a longer time period in coagulation, it also forms cross-links between the α- and γ-chains of fibrin (2Lewis K.B. Teller D.C. Fry J. Lasser G.W. Bishop P.D. Biochemistry. 1997; 36: 995-1002Crossref PubMed Scopus (27) Google Scholar), and between α2-anti-plasmin and fibrin (3Ichinose A. Tamaki T. Aoki N. FEBS Lett. 1983; 153: 369-371Crossref PubMed Scopus (109) Google Scholar). fXIII has been shown to react with more than fibrin (4Muszbek L. Ádány R. Mikkola H. Crit. Rev. Clin. Lab. Sci. 1996; 33: 357-421Crossref PubMed Scopus (181) Google Scholar), and it has recently been found in brain tumors (5Bárdos H. Molnár P. Cdécsei G. Ádány R. Blood Coagul. Fibrinolysis. 1996; 7: 536-548Crossref PubMed Scopus (57) Google Scholar) and arthritic joints (6Carmassi F. De Negri F. Morale M. Song K.Y. Chung S.O. Semin. Thromb. Hemostasis. 1996; 22: 489-496Crossref PubMed Scopus (37) Google Scholar), and there are cases of fXIII deficiencies (7Kitchens C.S. Newcomb T.F. Medicine. 1979; 58: 413-428Crossref PubMed Scopus (76) Google Scholar, 8Board P.G. Losowsky M.S. Miloszewski K.J. Blood Rev. 1993; 7: 229-242Crossref PubMed Scopus (184) Google Scholar). Factor XIII is a member in the family of transglutaminases (TGases), which have a wide range of biological functions (9Greenberg C.S. Birckbichler P.J. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (926) Google Scholar). factor XIII transglutaminase root mean square deviation 4-morpholineethanesulfonic acid factor XIII transglutaminase root mean square deviation 4-morpholineethanesulfonic acid Like most coagulation factors, fXIII is synthesized as a zymogen and then cleaved by a protease to become an active enzyme. The structure of fXIII zymogen was determined several years ago (10Yee V.C. Pedersen L.C. Le Trong I. Bishop P.D. Stenkamp R.E. Teller D.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7296-7300Crossref PubMed Scopus (319) Google Scholar, 11Pederson L.C. Yee V.C. Bishop P.D. Le Trong I. Teller D.C. Stenkamp R.E. Protein Sci. 1994; 3: 1131-1135Crossref PubMed Scopus (137) Google Scholar). In this crystal form, the active site cysteine, Cys-314, is inaccessible to solvent and is not available for catalysis. Physiologically, calcium ions are required for fXIII activation and for TGase activity. In the blood, activation of circulating fXIII requires thrombin cleavage, calcium ions (1.5 mm) (12Curtis C.G. Brown K.L. Credo R.B. Domanik R.A. Gray A. Stenberg P. Lorand L. Biochemistry. 1974; 13: 3774-3780Crossref PubMed Scopus (131) Google Scholar, 13Cooke R.D. Holbrook J.J. Biochem. J. 1974; 141: 79-84Crossref PubMed Scopus (24) Google Scholar, 14Hornyak T.J. Shafer J.A. Biochemistry. 1991; 30: 6175-6182Crossref PubMed Scopus (56) Google Scholar), and fibrin(ogen) (15Credo R.B. Curtis C.G. Lorand L. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4234-4237Crossref PubMed Scopus (80) Google Scholar). High levels of calcium (>50 mm) can activate fXIII without the use of thrombin (15Credo R.B. Curtis C.G. Lorand L. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4234-4237Crossref PubMed Scopus (80) Google Scholar), and it has recently been shown that platelet fXIII can be activated nonproteolyticallyin vivo (16Muszbek L. Haramura G. Polgár J. Thromb. Haemostasis. 1995; 73: 702-705Crossref PubMed Scopus (42) Google Scholar). Based on the amino acid sequence, the calcium binding site was predicted to be in a region (residues 468–479) with high similarity to the EF-hand motif (17Takahashi N. Takahashi Y. Putnam F.W. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8019-8023Crossref PubMed Scopus (130) Google Scholar, 18Ichinose A. Davie E.W. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5829-5833Crossref PubMed Scopus (149) Google Scholar). The main calcium binding site, as seen in the preliminary crystallographic study (19Yee V.C. Le Trong I. Bishop P.D. Pedersen L.C. Stenkamp R.E. Teller D.C. Semin. Thromb. Hemostasis. 1996; 22: 377-384Crossref PubMed Scopus (48) Google Scholar), involves residues Asn-436, Asp-438, Ala-457, Glu-485, and Glu-490. C-terminal truncation experiments removing several residues from the calcium binding pocket show a total loss of enzymatic activity (20Lai T.S. Achyuthan K.E. Santiago M.A. Greenberg C.S. J. Biol. Chem. 1994; 269: 24596-24601Abstract Full Text PDF PubMed Google Scholar). Site-directed mutagenesis of the glutamate residues in the calcium binding region of guinea pig liver TGase decreases its sensitivity to calcium, while maintaining some enzymatic activity (21Ikura K. Yu C. Nagao M. Sasaki R. Furuyoshi S. Kawabata N. Arch. Biochem. Biophys. 1995; 318: 307-313Crossref PubMed Scopus (11) Google Scholar). There is further in vitro evidence that calcium and other divalent cations have effects on proteolytic susceptibility (22Mary A. Achyuthan K.E. Greenberg C.S. Arch. Biochem. Biophys. 1988; 261: 112-121Crossref PubMed Scopus (18) Google Scholar) and heat stability (23De Backer-Royer C. Meunier J.C. Int. J. Biochem. 1992; 24: 637-642Crossref PubMed Scopus (9) Google Scholar). Several lanthanide ions have been shown to replace calcium ions during fXIII activation (10–40 μm). Furthermore, these lanthanide ions can inhibit fXIII activity when the ion concentration is greater than 40 μm (24Achyuthan K.E. Mary A. Greenberg C.S. Biochem. J. 1989; 257: 331-338Crossref PubMed Scopus (10) Google Scholar). Most of these effects occur when the calcium ion is in the millimolar range, and with the lanthanide ion in the micromolar range. We have solved the structures of recombinant fXIII zymogen complexed with calcium, strontium, and ytterbium to determine the structural effects of these cations on fXIII. The goal in using the latter two cations was to positively identify the electron-rich ions in the electron density maps. All three ions bind to the same pocket. We have found an additional ytterbium binding site, hypothesized to be the lanthanide inhibition site. Crystals of recombinant human factor XIII A2 (166 kDa) (25Bishop P.D. Teller D.C. Smith R.A. Lasser G.W. Gilbert T. Seale R.L. Biochemistry. 1990; 29: 1861-1869Crossref PubMed Scopus (85) Google Scholar) zymogen were grown from 1, 2-propanediol (24%) and sodium potassium phosphate buffer (100 mm) at pH 6.2. The crystals show the symmetry of space group P21 with two monomers in the asymmetric unit. The ion-bound crystals were obtained by soaking zymogen crystals for 1–2 days in 90 mm CaCl2, 120 mmSrCl2, or 2 mm YbCl3 in an artificial mother liquor of 1,2-propanediol (24%) and MES buffer at pH 6.2. Data for the calcium soaked crystal were collected at the Stanford Synchrotron Radiation Laboratory on beamline 9–1 with a MAR image plate detector at −170 °C. For the strontium and ytterbium crystals, data were collected in-house at room temperature on an R-AXIS IIc image plate detector equipped with a Rigaku RU200 rotating anode generator. The calcium crystal yielded data to 2.1 Å, and the strontium and ytterbium data sets diffracted to 2.5 Å (Table I).Table IData collection and refinement statisticsCalciumStrontiumYtterbiumCation concentration (mm)901202Unit cell dimensionsa (Å)100.17101.90101.06b (Å)70.7672.3272.39c(Å)133.82135.03135.99β (°)106.1105.9106.1Temperature (°C)−1702525Data collectionSSRL 9–1R-AXIS IIcR-AXIS IIcCompleteness (%)Overall937876Last shell86 (2.15–2.10 Å)51 (2.54–2.50 Å)42 (2.54–2.50 Å)R sym (overall)4.3NA7.8I/ς (last shell)1.82.33.2Refinement resolution (Å)20.0–2.1aThe calcium structure includes anisotropic scaling of the data and the bulk solvent model. If the structure is refined with data from 8.0 to 2.1 Å and includes the anisotropic scaling and same bulk solvent model, the R and R free are reduced to 20.9% and 29.6%, respectively.10.0–2.510.0–2.5R factor (%)22.718.318.8R free (%)bR factor for 5% of the reflections which were omitted from all refinement steps.31.327.528.1No. of reflectionscThe total number of reflections in the refinement resolution range with F > 2ς.96,68250,77749,005No. of protein atoms11,29411,26011,296No. of water molecules1001230265Solvent B (Å2) (mean)47.239.040.8Overall B (Å2) (mean)dIncludes all atoms in the structure.38.134.036.1Geometry (r.m.s.d.)Bonds (Å)0.0170.0120.011Angles (°)2.01.71.7NA, not available; SSRL, Stanford Synchrotron Radiation Laboratory.a The calcium structure includes anisotropic scaling of the data and the bulk solvent model. If the structure is refined with data from 8.0 to 2.1 Å and includes the anisotropic scaling and same bulk solvent model, the R and R free are reduced to 20.9% and 29.6%, respectively.b R factor for 5% of the reflections which were omitted from all refinement steps.c The total number of reflections in the refinement resolution range with F > 2ς.d Includes all atoms in the structure. Open table in a new tab NA, not available; SSRL, Stanford Synchrotron Radiation Laboratory. Our previously refined P21fXIII zymogen structural model was used to generate initial phases for all three ion-bound structures. The initial R (Σ ‖‖F obs‖ − ‖F calc‖‖/Σ ‖F obs‖) was 48.2%, 36.8%, and 31.0% for the calcium, strontium, and ytterbium structures, respectively. The calcium refinement began with a series of rigid body minimization steps at 8–3.5 Å resolution using X-PLOR (26Brunger A.T. X-PLOR Version 3 (1): A System for X-ray Crystallography and NMR. Yale University Press, New Haven, CT1992Google Scholar). The first rigid body refinement used two monomers, while the next was composed of the eight domains; finally, a refinement with 193 secondary structure elements (helix, strand, loop) was performed. After those three steps, theR had dropped from 48.2% to 32.9%. This multistep rigid body procedure was used because of the cryogenic data collection and higher initial R factor. This rendered a 2.3% lowerR factor than if a simple two-monomer rigid body step was used. From here, the resolution was expanded to 2.2 Å in six steps by alternating rigid body (with 193 groups) and positional least square refinements. The R dropped to 27.9% with anR free of 38.6%. Simulated annealing with slow cooling from 2000 to 300 K, followed by a round of positional andB value least squares minimization gave a drop inR of 0.4% and R free of 1.6%. Refinement continued through several rounds of map fitting with the program XtalView (27McRee D.E. J. Mol. Graphics. 1992; 10: 44-46Crossref Google Scholar) and least squares and dynamics minimization. The program DDQ (28van den Akker F. Hol W.G.J. Acta Crystallogr. Sect. D. 1999; 55: 206-218Crossref PubMed Scopus (48) Google Scholar) was used to assess model and map quality during the refinement procedure. Waters were added at positions with difference electron density peaks greater than 3.0 ς, which also were within hydrogen bonding distance of the protein or other waters. In the end, the structure was refined with overall anisotropic scaling of the data along with a bulk solvent model and a resolution of 20.0–2.1 Å; this resulted in an R of 22.7% and R freeof 31.2%, with a total of 1001 waters (Table I). The strontium and ytterbium refinements were carried out over the resolution range of 10–2.5 Å for all steps. The X-PLOR refinement began with a positional and individual B value minimization of the starting model, which lowered the R to approximately 27%, and was followed by adding two ions per dimer at the highest peaks in the difference map (Table II). It then proceeded through more cycles of least squares minimization and manual map fitting in the program O (29Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (12999) Google Scholar). In the ytterbium refinement, three additional 10–12 ς difference peaks were observed near the non-crystallographic two-fold axis within the dimer, and three ions were added (sites 3, 4, and 5). Furthermore, one additional Yb3+ ion was added at a 10 ς difference peak (site 7), which is located at a crystallographic contact with another dimer. Following refinement of these four ions in the structure, one more difference peak appeared on the two-fold axis, so another ytterbium ion was added (site 6). After further refinement and map fitting, the R decreased to 19.8% (strontium) and 22.0% (ytterbium), and waters were placed at chemically reasonable 3 ς difference peaks. Additionally, a strong difference peak about 2 Å away from the main Yb3+ (site 2) in monomer B was identified, and a new Yb3+ ion (site 8) was added with an occupancy of 0.25, while the occupancy of the original Yb3+ion was reduced to 0.75. Several more rounds of least squares refinement, map fitting, and water addition led to a final Rof 18.3% and 18.8%, an R free of 27.5% and 28.1%, and 230 and 265 waters for the strontium and ytterbium complexes, respectively (Table I).Table IIStatistics for the cationsIonSite no.Initial peak height observed inF o - F c map (ς)Final peak height for F o - F c omitaThe omit map is constructed from the final refined structure which contains all atoms except the ion being measured.map (ς)Refined B (Å2) for ionOccupancy of ionB (Å2) of water at same locationCalcium13.89.1591.02324.611.1451.014Strontium17.821.6521.02bThe refinement program (X-PLOR) was configured such that 2.0 Å2 is the lower limit on B values.25.624.3461.02Ytterbium111.736.8551.02211.036.5360.752312.219.3170.252411.616.7230.252510.115.8230.25263.9cThis peak was only observed after other Yb3+ ions (1–5 and 7) were included in the refinement and map calculation.20.8100.252710.016.2640.50588.1dThis was the value observed with Yb3+ ions (1-7) already included in the structure and map calculation.14.1350.256a The omit map is constructed from the final refined structure which contains all atoms except the ion being measured.b The refinement program (X-PLOR) was configured such that 2.0 Å2 is the lower limit on B values.c This peak was only observed after other Yb3+ ions (1–5 and 7) were included in the refinement and map calculation.d This was the value observed with Yb3+ ions (1Laki K. Lorand L. Science. 1948; 108: 280Crossref PubMed Scopus (173) Google Scholar, 2Lewis K.B. Teller D.C. Fry J. Lasser G.W. Bishop P.D. Biochemistry. 1997; 36: 995-1002Crossref PubMed Scopus (27) Google Scholar, 3Ichinose A. Tamaki T. Aoki N. FEBS Lett. 1983; 153: 369-371Crossref PubMed Scopus (109) Google Scholar, 4Muszbek L. Ádány R. Mikkola H. Crit. Rev. Clin. Lab. Sci. 1996; 33: 357-421Crossref PubMed Scopus (181) Google Scholar, 5Bárdos H. Molnár P. Cdécsei G. Ádány R. Blood Coagul. Fibrinolysis. 1996; 7: 536-548Crossref PubMed Scopus (57) Google Scholar, 6Carmassi F. De Negri F. Morale M. Song K.Y. Chung S.O. Semin. Thromb. Hemostasis. 1996; 22: 489-496Crossref PubMed Scopus (37) Google Scholar, 7Kitchens C.S. Newcomb T.F. Medicine. 1979; 58: 413-428Crossref PubMed Scopus (76) Google Scholar) already included in the structure and map calculation. Open table in a new tab The Protein Data Bank codes for the calcium, strontium, and ytterbium structures are 1GGU, 1BL2, and 1GGY, respectively. Figures in this report were generated with Molscript (30Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), Raster3D (31Merritt E.A. Bacon D.J. Methods Enzymol. 1997; 277: 505-524Crossref PubMed Scopus (3866) Google Scholar), and XtalView (27McRee D.E. J. Mol. Graphics. 1992; 10: 44-46Crossref Google Scholar). Each monomer of fXIII in the dimer has 731 residues and consists of four domains: the β-sandwich, the catalytic core, and barrels 1 and 2 (Fig. 1). The active site residues Cys-314, His-373, and Asp-396 are not accessible to solvent, as in the zymogen structure. The calcium binding site is located in the core domain, near the surface of the protein. The root mean square deviation (r.m.s.d.) between all atoms from the strontium- and ytterbium-bound structures compared with the starting ion-free fXIII zymogen structure is less than 0.85 Å. The calcium structure, on the other hand, differs from the two cation structures and the starting structure with an r.m.s.d. of 1.1 Å. This higher atomic coordinate deviation is likely due to the cryogenic data collection for the calcium structure. Due to uninterpretable electron density, the monomers are missing various numbers of residues from the N terminus (residues 1–8), the linker between the activation peptide and the β-sandwich (residues 30–43), the region between the core and barrel 1 (residues 508–516), and the C terminus (residues 728–731). Most of the residues were well resolved, with 90% having average B values of less than 60 Å2. Less than 0.4% of the torsion angles are in the forbidden regions of the Ramachandran diagram, as reported by PROCHECK (32Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Three peptide bonds in each monomer are in thecis conformation (10Yee V.C. Pedersen L.C. Le Trong I. Bishop P.D. Stenkamp R.E. Teller D.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7296-7300Crossref PubMed Scopus (319) Google Scholar, 33Weiss M.S. Metzner H.J. Hilgenfeld R. FEBS Lett. 1998; 423: 291-296Crossref PubMed Scopus (134) Google Scholar). One involves a proline residue, 410–411, and the other two do not, 310–311 and 425–426. The structures are reasonable as judged by DDQ (28van den Akker F. Hol W.G.J. Acta Crystallogr. Sect. D. 1999; 55: 206-218Crossref PubMed Scopus (48) Google Scholar), Verify3D (34Eisenberg D. Luthy R. Bowie J.U. Methods Enzymol. 1997; 277: 396-404Crossref PubMed Scopus (1529) Google Scholar), ERRAT (35Colovos C. Yeates T.O. Protein Sci. 1993; 2: 1511-1519Crossref PubMed Scopus (2341) Google Scholar), and WHATIF (36Hooft R.W.W. Vriend G. Sander C. Abola E.E. Nature. 1996; 381: 272Crossref PubMed Scopus (1788) Google Scholar). The main ion binding site is near the interface between the catalytic core and barrel 1 (Fig. 1), and the ion binding helix (residues 485–501) is in contact with the other monomer. The calcium and strontium atoms superimpose within this site, while the main ytterbium ion is 2.7 Å from their location (Fig.2). The mean peak sizes in an early difference map were about 4, 7, and 11 ς for the calcium, strontium, and ytterbium ions, respectively (TableII). When water molecules replaced the calcium ions at these positions, their B values refined to about 14–23 Å2. However, for the strontium and ytterbium sites, the B values of the replaced water molecules refine down to 2 Å2, the minimum allowable value. This analysis implies that a water molecule in this location does not have enough electrons to adequately match the strontium and ytterbium x-ray diffraction data. The main cation site consists of a pocket with negatively charged side chains of Asn-436, Asp-438, Glu-485, and Glu-490 plus the main chain oxygen of Ala-457. All three ion-bound structures and the zymogen structure overlap quite closely in this region (Fig. 2). TableIII lists the oxygen atoms that are potentially involved in coordinating each cation. The main chain oxygen from Ala-457 is the main protein ligand for the Ca2+ and Sr2+ ions, as indicated by its low B value and its distance from the metals. The Yb3+ ion is farther from Ala-457 and closer to Glu-485 and Glu-490. The B values for the residues in the region from 484 to 490 are higher than average, and the electron density is weak.Table IIIThe distance and B value of all oxygen atoms within 4.0 Å of the specified ion of each siteResidueAtomMonomer AMonomer BDistance (Å)B (Å2)Distance (Å)B (Å2)CalciumAla-457O2.7742.62.5442.8Glu-485OE13.9475.5Wat-62LO2.2654.1Wat-402LO3.8189.2Wat-744LO2.8946.5Wat-745LO2.1438.9Wat-5LO2.6228.6Wat-23LO2.9069.9Wat-243LO2.8347.1Wat-300LO3.6663.8Wat-746LO2.5230.4StrontiumAsn-436O3.9421.9Asn-436OD13.4841.7Ala-457O2.5736.12.7029.0Glu-485OE13.6367.52.8671.2Glu-485OE23.9069.8Ytterbium(¾ Occ.)aThese refer to the strong and weak ytterbium ions, which are modeled as 0.75 and 0.25 occupancy, respectively. (¼ Occ.)aThese refer to the strong and weak ytterbium ions, which are modeled as 0.75 and 0.25 occupancy, respectively.Asn-436OD13.6833.12.68 2.5941.0Asp-438OD13.5429.7Ala-457O3.6229.9Glu-485OE12.2746.22.75 1.9866.7Glu-485OE22.1333.82.25 2.4963.4Glu-490OE13.4663.22.38 3.6832.4Glu-490OE23.6365.42.2928.0Wat-3006VO2.2136.9Wat-5034YO2.1420.7Wat-5063YO3.42a These refer to the strong and weak ytterbium ions, which are modeled as 0.75 and 0.25 occupancy, respectively. Open table in a new tab The ion site in monomer B of the calcium structure has six putative ligands arranged in a distorted bipyramidal arrangement (Fig.3). One protein atom (O–Ala-457) and up to five waters are within 4.0 Å of the ion. Residues Asp-438, Asn-436, and Glu-490 are hydrogen-bonded to several of these waters. Fewer waters were identified in the strontium and ytterbium structures due to the lower resolution data. The ytterbium ion has one monodentate (Asn-436) and two bidentate (Glu-485, Glu-490) ligands provided by the protein and nearby water molecules. The ion site in monomer B of the ytterbium structure has two alternate positions for the Yb3+ ion 1.8 Å from each other. The occupancies for each ion were chosen such that they refined to equal B values and summed to 1. It is unlikely that the weaker Yb3+ ion (site 8) is a negative chloride ion because it is very close to the position of the positive calcium ion from the calcium structure, which implies that its location is suitable for only positive ions. The ligand distances between the Yb3+ ion and the oxygen atoms are as low as 1.98 Å, but this is consistent with other Yb3+-oxygen distances, as observed in the Protein Data Bank (codes 1CNT (37McDonald N.Q. Panayotatos N. Hendrickson W.A. EMBO J. 1995; 14: 2689-2699Crossref PubMed Scopus (126) Google Scholar), 1NCG and 1NCH (38Shapiro L. Fannon A.M. Kwong P.D. Thompson A. Lehmann M.S. Grubel G. Legrand J.F. Als-Nielsen J. Colman D.R. Hendrickson W.A. Nature. 1995; 374: 327-337Crossref PubMed Scopus (967) Google Scholar), 1YTT (39Burling F.T. Weis W.I. Flaherty K.M. Brunger A.T. Science. 1996; 271: 72-77Crossref PubMed Scopus (218) Google Scholar), and 2BOP(40Hegde R.S. Grossman S.R. Laimins L.A. Sigler P.B. Nature. 1992; 359: 505-512Crossref PubMed Scopus (325) Google Scholar)). Four large electron density peaks near the non-crystallographic dimer two-fold axis form a nearly perfect tetrahedron with an edge length of approximately 3.6 ± 0.1 Å (Fig. 4 and Table II). Furthermore, a similar tetrahedron is observed in the ytterbium soak of the orthorhombic crystal form (10Yee V.C. Pedersen L.C. Le Trong I. Bishop P.D. Stenkamp R.E. Teller D.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7296-7300Crossref PubMed Scopus (319) Google Scholar) of fXIII (data not shown). After modeling four ytterbium ions at 0.25 occupancy into the electron density, the B values refined to 17, 23, 23, and 10 Å2. Placing waters at full occupancy in these locations gave B values that refined to 2 Å2. No ions or solvent molecules exist in the same location in the Ca2+- or Sr2+-bound crystal structures. Residues Asp-270, Asp-271, and Glu-272 of each monomer are in this region, but the electron density does not specify one distinct conformation for the side chains, and the B values are quite high. After the refinement of the three structures, waters were grouped into subsets based on their occurrence in each crystal. If waters from different structures were located within 2.4 Å from each other, they were considered equivalent, and given the same chain identification and residue number in the Protein Data Bank files. In some cases, the determination of equivalent waters was made on the basis of their B values and local protein differences between structures. A Venn diagram was constructed (Fig.5) showing the seven different subsets from the three independent groups. If a water from one structure had no equivalent waters from another structure, it was given a particular chain identifier (L for calcium, R for strontium, Y for ytterbium). If a water molecule was in only two different structures, it was given the same residue number and chain identification (U for calcium and strontium, V for calcium and ytterbium, W for strontium and ytterbium). Finally, water molecules that exist in all three structures were given the chain identification of S along with the same residue number. The average and standard deviation of B values and Cβ density for the water molecules are presented in TableIV. The Cβ density (41Sippl M.J. J. Comput. Aided Mol. Des. 1993; 7: 473-501Crossref PubMed Scopus (359) Google Scholar) estimates the amount of burial at each water site by counting the number of Cβ atoms within 10 Å of the water site. It has a numerical range of 2–30 atoms, which qualitatively ranges from highly exposed to deeply buried.Table IVWater subsets: average B values and Cβ densitiesWater subsetCa, Sr, and YbCa and SrCa and YbSr and YbCa onlySr onlyYb onlyPDB chain IDSUVWLRYNumber of H2O7968118137367055Ca2+ structure:〈B〉 Å236 (12Curtis C.G. Brown K.L. Credo R.B. Domanik R.A. Gray A. Stenberg P. Lorand L. Biochemistry. 1974; 13: 3774-3780Crossref PubMed Scopus (131) Google Scholar)a42 (15Credo R.B. Curtis C.G. Lorand L. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4234-4237Crossref PubMed Scopus (80) Google Scholar)39 (13Cooke R.D. Holbrook J.J. Biochem. J. 1974; 141: 79-84Crossref PubMed Scopus (24) Google Scholar)51 (18Ichinose A. Davie E.W. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5829-5833Crossref PubMed Scopus (149) Google Scholar)〈# Cβ〉19.3 (4.6)16.0 (4.8)18.4 (5.3)14.4 (5.5)Sr2+ structure:〈B〉 Å235 (14Hornyak T.J. Shafer J.A. Biochemistry. 1991; 30: 6175-6182Crossref PubMed Scopus (56) Google Scholar)42 (14Hornyak T.J. Shafer J.A. Biochemistry. 1991; 30: 6175-6182Crossref PubMed Scopus (56) Google Scholar)48 (21Ikura K. Yu C. Nagao M. Sasaki R. Furuyoshi S. Kawabata N. Arch. Biochem. Biophys. 1995; 318: 307-313Crossref PubMed Scopus (11) Google Scholar)44 (16Muszbek L. Haramura G. Polgár J. Thromb. Haemostasis. 1995; 73: 702-705Crossref PubMed Scopus (42) Google Scholar)〈# Cβ〉18.8 (4.3)15.4 (4.7)18.7 (5.2)11.4 (5.0)Yb3+ structure:〈B〉Å235 (14Hornyak T.J. Shafer J.A. Biochemistry. 1991; 30: 6175-6182Crossref PubMed Scopus (56) Google Scholar)48 (21Ikura K. Yu C. Nagao M. Sasaki R. Furuyoshi S. Kawabata N. Arch. Biochem. Biophys. 1995; 318: 307-313Crossref PubMed Scopus (11) Google Scholar)〈# Cβ〉18.7 (4.3)18.2 (5.1)19.5 (5.5)12.8 (6.6)Overall average:〈B〉 Å235423941504451〈# Cβ〉19161819141113Average Cβ density is the average of the number of Cβ atoms within 10 Å of each water in the subset. PDB, Protein Data Bank; ID, identification.a The root mean square deviation is shown in parentheses. Open table in a new tab Average Cβ density is the average of the number of Cβ atoms within 10 Å of each water in the subset. PDB, Protein Data Bank; ID, identification. a The root mean square deviation is shown in parentheses. Many of the water molecules are located in sites common to all three structures. Water molecules that are present in all three structures (subset S) have B values about 10 Å2 lower than unique waters. Likewise, these common waters are more buried, as shown by the 50% larger number of nearby Cβ atoms. Calcium and other cations have definite effects on fXIII behavior in solution. The ion soaking concentrations in the calcium and strontium crystals were above the level at which conformational changes, such as enzyme activation, are expected. No significant conformational differences between the ion complex and the zymogen structure are seen. Two complementary reasons for this lack of conformational change are: 1) the active conformation of the enzyme cannot pack in this crystal form, and 2) some other molecule, such as a substrate or inhibitor, is required to stabilize fXIII in its active form. Packing interactions in the monoclinic crystal form restrict any major conformational changes induced in fXIII. Crystallization of thrombin cleaved fXIII in the presence of 1.5 mm calcium has resulted in the same crystal form and the same conformation (42Yee V.C. Pedersen L.C. Bishop P.D. Stenkamp R.E. Teller D.C. Thromb. Res. 1996; 78: 389-397Abstract Full Text PDF Scopus (83) Google Scholar). Furthermore, co-crystallization of 45 mm CaCl2with fXIII has yielded this monoclinic crystal form with the same conformation (43.Pedersen, L. C., X-ray Structure Determination of Factor XIII.Ph.D. dissertation, 1994, University of Washington, Seattle, WA.Google Scholar). Structurally, one residue that must move to expose the active site Cys-314 is Tyr-560, located in barrel 1. In this space group, barrel 1 is trapped in place by crystal contacts. We therefore believe that this crystal form is incompatible with the active form of fXIII. The biochemical experiments used to demonstrate the effects of calcium and other cations on fXIII use either a substrate or inhibitor to measure whether the enzyme is active. We propose that one of these molecules is another requirement for the stabilization of the active form. In the presence of calcium, the inactive form of fXIII is in equilibrium with the active conformation. During crystallization, the inactive form is more likely to be crystallized; thus, mass action causes any active fXIII to change to its inactive conformation. The presence of a substrate or inhibitor might stabilize the active conformation further and allow crystallization in a new crystal form. This proposal is in line with the known effect of fibrin(ogen) in facilitating the activation of fXIII (14Hornyak T.J. Shafer J.A. Biochemistry. 1991; 30: 6175-6182Crossref PubMed Scopus (56) Google Scholar, 44Greenberg C.S. Achyuthan K.E. Fenton II, J.W. Blood. 1987; 69: 867-871Crossref PubMed Google Scholar). Some ideas about calcium activation can be gained from the location of the main binding site with respect to other features on the molecule. First, the calcium binding pocket is only 10 Å from barrel 1, which is responsible for partially blocking the active site via Tyr-560. The bound ion may induce some dynamical changes in nearby barrel 1, including exposure of the active site. Additionally, one end of the calcium binding helix forms close contacts with the other monomer. This may allow for allosteric communication between the calcium binding site of one monomer and the active site of the other, as described by Hornyak and Shafer (14Hornyak T.J. Shafer J.A. Biochemistry. 1991; 30: 6175-6182Crossref PubMed Scopus (56) Google Scholar). The residues involved in calcium binding are well conserved within the TGase family. The coordination geometry for the calcium and strontium is not ideal; many of the putative ligands are water molecules, and the metal-ligand distances are somewhat longer than observed for calcium complexes in other proteins (45McPhalen C.A. Strynadka N.C.J. James M.N.G. Adv. Protein Chem. 1991; 42: 77-144Crossref PubMed Scopus (257) Google Scholar) (Table III). The B values for these residues are not much different from those observed for the ion free structures. These observations are consistent with fXIII's millimolar binding affinity (15Credo R.B. Curtis C.G. Lorand L. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4234-4237Crossref PubMed Scopus (80) Google Scholar) for Ca2+ ions. The Yb3+ ion, which binds more tightly, is in a slightly different location and has more coordinating protein atoms with fewer water ligands. This ion has a smaller radius and has a higher charge than divalent cations, so the fact that it has a different binding location is not unreasonable. The four ytterbium ions on the dimer two-fold axis are modeled with ¼ occupancy each. The interatomic distances are too small to allow multiple positively charged ions to be present concurrently. They are time and space averaged throughout the crystal so that only one ion is in the site at any given time. This multiplicity of the ion has made it difficult to model the protein ligands. We postulate that this tetrahedron of partial occupancy ions is the lanthanide inhibition site. Achyuthan et al. (24Achyuthan K.E. Mary A. Greenberg C.S. Biochem. J. 1989; 257: 331-338Crossref PubMed Scopus (10) Google Scholar) have observed that the addition of lanthanide ions above 40 μmto thrombin-cleaved fXIII results in the non-competitive inhibition of fXIII. This inhibition cannot be reversed by a 200-fold molar excess of calcium ions. Furthermore, when the level of the lanthanide ion is between 10 and 40 μ m, fXIII can be activated by thrombin cleavage as in the presence of calcium. These results (24Achyuthan K.E. Mary A. Greenberg C.S. Biochem. J. 1989; 257: 331-338Crossref PubMed Scopus (10) Google Scholar) could be explained by multiple lanthanide binding sites. The main ion site, observed in all three ion-bound structures, is required for proper thrombin activation, and the novel Yb3+ site, which has a slightly weaker affinity, is responsible for the inhibition of fXIII. Given the soaking concentration of 2 mm in this data set, both ions sites are filled. Functionally, the location at the dimer interface could potentially destabilize the quaternary structure. Additionally, the residues that form the lanthanide inhibition site (Asp-270 to Glu-272) are only 5 residues away from Trp-279, which may play an important role in catalysis (11Pederson L.C. Yee V.C. Bishop P.D. Le Trong I. Teller D.C. Stenkamp R.E. Protein Sci. 1994; 3: 1131-1135Crossref PubMed Scopus (137) Google Scholar). Grouping the waters into subsets based on their presence in multiple structures has been helpful in identifying structurally significant waters. One example is that of water 6059S (Fig. 6). This water in the core domain binds to several residues that are fully conserved in the TGase family (Thr-466, Lys-467, Arg-333, and Tyr-204). The water is located at the separation fork of a pair of β strands. One half of each strand forms a paired sheet, and the other half of each strand diverges nearly at a right angle. Furthermore, these strands are linked with functionally important parts of the protein. One strand pairs with the strand containing the active site His-373; and the other strand contains a ligand (Ala-457) for the calcium binding site. Monomer B also contains a water molecule in this location (6009S). We believe this water is structurally important for maintaining the unique conformation of this pair of strands. Many other waters in this subset S are contacting conserved residues in this protein, and it is possible that some of these waters may help to explain natural fXIII deficiency mutations or other interesting structural and evolutionary features. In fact, of the 18 identified missense mutations (46Mikkola H. Palotie A. Semin. Thromb. Hemostasis. 1996; 22: 393-398Crossref PubMed Scopus (19) Google Scholar, 47Anwar R. Stewart A.D. Miloszewski K.J.A. Losowsky M.S. Markham A.F. Br. J. Haematol. 1995; 91: 728-735Crossref PubMed Scopus (67) Google Scholar, 48Aslam S. Yee V.C. Narayanan S. Duraisamy G. Standen G.R. Br. J. Haematol. 1997; 98: 346-352Crossref PubMed Scopus (19) Google Scholar, 49Takahashi N. Tsukamoto H. Umeyama H. Castaman G. Rodeghiero F. Ichinose A. Blood. 1998; 91: 2830-2838Crossref PubMed Google Scholar) 2H. Mikkola, personal communication. that cause a fXIII deficiency, half are contacting at least one conserved water from subset S. The probability of this happening by chance is 5%. There are no major conformational changes between the different ion-bound fXIII structures and the zymogen. This is likely due to crystal packing interactions and the lack of a substrate or inhibitor molecule to stabilize the active conformation. The location of the main ion binding site provides ideas about the mechanism of calcium activation, such as its proximity to barrel 1 and its contact with the other monomer. The novel ytterbium binding site, on the dimer two-fold axis, could be responsible for the biochemically observed lanthanide inhibition. The procedure of finding common waters between the structures is a new method for identifying particular waters and residues that may be structurally significant and important for the function of fXIII. Overall, these three structures positively identify the calcium binding site in factor XIII.
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