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Jararhagin-derived RKKH Peptides Induce Structural Changes in α1I Domain of Human Integrin α1β1

2004; Elsevier BV; Volume: 279; Issue: 9 Linguagem: Inglês

10.1074/jbc.m312912200

1083-351X

Yvonne Nymalm, J. Santeri Puranen, Thomas K.M. Nyholm, Jarmo Käpylä, Heidi Kidron, Olli T. Pentikäinen, Tomi T. Airenne, Jyrki Heino, J. Peter Slotte, Mark S. Johnson, Tiina A. Salminen,

Biochemical and Structural Characterization

Integrin α1β1 is one of four collagen-binding integrins in humans. Collagens bind to the αI domain and in the case of α2I collagen binding is competitively inhibited by peptides containing the RKKH sequence and derived from the metalloproteinase jararhagin of snake venom from Bothrops jararaca. In α2I, these peptides bind near the metal ion-dependent adhesion site (MIDAS), where a collagen (I)-like peptide is known to bind; magnesium is required for binding. Published structures of the ligand-bound "open" conformation of α2I differs significantly from the "closed" conformation seen in the structure of apo-α2I near MIDAS. Here we show that two peptides, CTRKKHDC and CARKKHDC, derived from jararhagin also bind to α1I and competitively inhibit collagen I binding. Furthermore, calorimetric and fluorimetric measurements show that the structure of the complex of α1I with Mg2+ and CTRKKHDC differs from structure in the absence of peptide. A comparison of the x-ray structure of apo-α1I ("closed" conformation) and a model structure of the α1I ("open" conformation) based on the closely related structure of α2I reveals that the binding site is partially blocked to ligands by Glu255 and Tyr285 in the "closed" structure, whereas in the "open" structure helix C is unwound and these residues are shifted, and the "RKKH" peptides fit well when docked. The "open" conformation of α2I resulting from binding a collagen (I)-like peptide leads to exposure of hydrophobic surface, also seen in the model of α1I and shown experimentally for α1I using a fluorescent hydrophobic probe. Integrin α1β1 is one of four collagen-binding integrins in humans. Collagens bind to the αI domain and in the case of α2I collagen binding is competitively inhibited by peptides containing the RKKH sequence and derived from the metalloproteinase jararhagin of snake venom from Bothrops jararaca. In α2I, these peptides bind near the metal ion-dependent adhesion site (MIDAS), where a collagen (I)-like peptide is known to bind; magnesium is required for binding. Published structures of the ligand-bound "open" conformation of α2I differs significantly from the "closed" conformation seen in the structure of apo-α2I near MIDAS. Here we show that two peptides, CTRKKHDC and CARKKHDC, derived from jararhagin also bind to α1I and competitively inhibit collagen I binding. Furthermore, calorimetric and fluorimetric measurements show that the structure of the complex of α1I with Mg2+ and CTRKKHDC differs from structure in the absence of peptide. A comparison of the x-ray structure of apo-α1I ("closed" conformation) and a model structure of the α1I ("open" conformation) based on the closely related structure of α2I reveals that the binding site is partially blocked to ligands by Glu255 and Tyr285 in the "closed" structure, whereas in the "open" structure helix C is unwound and these residues are shifted, and the "RKKH" peptides fit well when docked. The "open" conformation of α2I resulting from binding a collagen (I)-like peptide leads to exposure of hydrophobic surface, also seen in the model of α1I and shown experimentally for α1I using a fluorescent hydrophobic probe. Integrins are a large family of cell surface glycoproteins that mediate cell-cell, cell-extracellular matrix, and matrix-matrix adhesion and transduce bidirectional signals between the cytoplasm and the extracellular matrix or other cells (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9014) Google Scholar). The adhesive function of integrins is important in various physiological processes such as platelet aggregation, inflammation, wound healing, tumor metastasis, cell migration during embryogenesis, viral infections, and other diseases (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9014) Google Scholar, 2Pigott R. Power C. The Adhesion Molecule Facts Book. Academic Press Ltd., London1993Google Scholar). Orthologues of human integrins have been identified in the genomes of birds, amphibians, and bony fish but so far not in the urochordates or in other invertebrates (3Johnson M.S. Tuckwell D. Gullberg D. I Domains in Integrins. Eurekah.com/Landes Bioscience, Georgetown, TX2003: 1-23Google Scholar). In humans, over 20 different integrin αβ heterodimers are formed from eight different β subunits (2Pigott R. Power C. The Adhesion Molecule Facts Book. Academic Press Ltd., London1993Google Scholar) and 18 different α subunits (4Velling T. Kusche-Gullberg M. Sejersen T. Gullberg D. J. Biol. Chem. 1999; 274: 25735-25742Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). The x-ray structure of integrin αVβ3 (5Xiong J.P. Stehle T. Diefenbach B. Zhang R. Dunker R. Scott D.L. Joachimiak A. Goodman S.L. Arnaout M.A. Science. 2001; 294: 339-345Crossref PubMed Scopus (1113) Google Scholar) defined much of the heterodimer common to all αβ integrins, where 12 domains assemble forming the "head" region where ligands bind plus two "tails" leading to the transmembrane helices that anchor integrins to the cell surface. The subunit interface between αV and β3 is mainly formed by a seven-bladed β-propeller domain (αV) and an I-like domain (β3). The structure of αVβ3 in complex with a cyclic pentapeptide with the arginine-glycine-aspartate (RGD) motif showed that the RGD sequence binds into a crevice between the β-propeller domain and the βI-like domain (6Xiong J.P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S.L. Arnaout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1403) Google Scholar). Upon binding the RGD pentapeptide, the tail regions of αVβ3 come closer to each other, and the β-propeller domain rotates slightly. Nine integrin α domains have an extra ∼200-residue inserted domain, the αI domain (for a review, see Ref. 7Gullberg D. Gullberg D. I Domains in Integrins: Molecular Biology Intelligence Unit. Eurekah.com/Landes Bioscience, Georgetown, TX2003Google Scholar). The αI domain is predicted to locate on the side and top of the β-propeller domain; currently, the only representative structure of an integrin heterodimer, αVβ3, lacks the αI domain (8Humphries M.J. Arthritis Res. 2002; 4: S69-78Crossref PubMed Scopus (31) Google Scholar). In the αI domain-containing integrins, the αI domain plays a major role in ligand binding, primarily to the metal ion-dependent adhesion site, MIDAS. 1The abbreviations used are: MIDAS, metal ion-dependent adhesion site; PBS, phosphate-buffered saline; bis-ANS, 1,1-bi(4-anilino)naphthalenesulforic acid. The collagen binding αI domains (α1, α2, α10, and α11) are distinguished from the αI domains of the immune system (αD, αE, αL, αM, αX) by having an additional α-helix, helix C, near MIDAS and can be divided into two groups according to differences in their collagen binding specificities (9Heino J. Gullberg D. I Domains in Integrins. Eurekah.com/Landes Bioscience, Georgetown, TX2003: 144-153Google Scholar). Integrin α2β1 and α11β1 bind best to fibrillar collagens I–III, whereas integrins α1β1 and α10β1 have higher affinity for network-forming collagen IV and for beaded filament-forming collagen VI (10Kern A. Eble J. Golbik R. Kuhn K. Eur. J. Biochem. 1993; 215: 151-159Crossref PubMed Scopus (181) Google Scholar, 11Tulla M. Pentikäinen O.T. Viitasalo T. Käpylä J. Impola U. Nykvist P. Nissinen L. Johnson M.S. Heino J. J. Biol. Chem. 2001; 276: 48206-48212Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 12Zhang W.M. Käpylä J. Puranen J.S. Knight C.G. Tiger C.F. Pentikäinen O.T. Johnson M.S. Farndale R.W. Heino J. Gullberg D. J. Biol. Chem. 2003; 278: 7270-7277Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Furthermore, α1β1 can attach to collagen XIII, whereas α2β1 cannot (13Nykvist P. Tu H. Ivaska J. Käpylä J. Pihlajaniemi T. Heino J. J. Biol. Chem. 2000; 275: 8255-8261Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). To date, only one complex structure with "collagen" is available, the α2I structure in complex with a collagen (I)-like peptide (14Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 101: 47-56Abstract Full Text Full Text PDF PubMed Scopus (843) Google Scholar). In comparison with the apo-form of α2I (15Emsley J. King S.L. Bergelson J.M. Liddington R.C. J. Biol. Chem. 1997; 272: 28512-28517Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar), a conformational change takes place on binding the collagen (I)-like peptide, which may reflect a general feature of ligand binding at MIDAS common to the collagen-binding I domains. Jararhagin is a snake venom metalloproteinase and a well known inhibitor of α2β1 integrin. Jararhagin and other class P-III snake venom metalloproteinases such as atrolysin A are composed of a metalloproteinase domain, a non-RGD-containing disintegrin domain and a cysteine-rich domain. The exact binding mechanism of snake venom metalloproteinases to α2β1 integrin is unknown. Previous studies have indicated that peptides derived from all three domains can inhibit α2β1 function (16Jia L.G. Wang X.M. Shannon J.D. Bjarnason J.B. Fox J.W. J. Biol. Chem. 1997; 272: 13094-13102Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 17Ivaska J. Käpylä J. Pentikäinen O. Hoffren A.M. Hermonen J. Huttunen P. Johnson M.S. Heino J. J. Biol. Chem. 1999; 274: 3513-3521Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 18Pentikäinen O. Hoffren A.M. Ivaska J. Käpylä J. Nyrönen T. Heino J. Johnson M.S. J. Biol. Chem. 1999; 274: 31493-31505Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 19Kamiguti A.S. Gallagher P. Marcinkiewicz C. Theakston R.D. Zuzel M. Fox J.W. FEBS Lett. 2003; 549: 129-134Crossref PubMed Scopus (68) Google Scholar), and therefore, snake venom metalloproteinases might contain several integrin recognition sites. We have shown that a cyclic RKKH peptide derived from metalloproteinase domain of jararhagin recognizes the α2I domain and prevents collagen binding to α2β1 integrin (17Ivaska J. Käpylä J. Pentikäinen O. Hoffren A.M. Hermonen J. Huttunen P. Johnson M.S. Heino J. J. Biol. Chem. 1999; 274: 3513-3521Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 18Pentikäinen O. Hoffren A.M. Ivaska J. Käpylä J. Nyrönen T. Heino J. Johnson M.S. J. Biol. Chem. 1999; 274: 31493-31505Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Already our first results suggested that RKKH might not be specific to α2I domain but that it may also bind to another integrin I domain, such as α1I (17Ivaska J. Käpylä J. Pentikäinen O. Hoffren A.M. Hermonen J. Huttunen P. Johnson M.S. Heino J. J. Biol. Chem. 1999; 274: 3513-3521Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Integrin binding to natural ligands, such as collagen, activates cellular signaling, and, based on current knowledge, ligand binding triggers a conformation change in the αI domain that leads to changes in the interaction between the α and β subunits, resulting in the separation of their intracellular domains (20Kim M. Carman C.V. Springer T.A. Science. 2003; 301: 1720-1725Crossref PubMed Scopus (642) Google Scholar). Importantly, jararhagin can trigger α2β1 integrin signaling in cells, and, therefore, jararhagin is a novel tool in the study of the integrin structure-function relationship (21Zigrino P. Kamiguti A.S. Eble J. Drescher C. Nischt R. Fox J.W. Mauch C. J. Biol. Chem. 2002; 277: 40528-40535Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Here, we have used RKKH peptides to study whether a small peptide can mimic natural ligands and induce the first step in integrin signaling, namely a conformational change in the integrin α1I domain. Peptides—Two different peptides cyclized via terminal cysteine residues were used in the binding studies. CTRKKHDC is directly based on the jararhagin metalloproteinase sequence (17Ivaska J. Käpylä J. Pentikäinen O. Hoffren A.M. Hermonen J. Huttunen P. Johnson M.S. Heino J. J. Biol. Chem. 1999; 274: 3513-3521Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar); in the CARKKHDC peptide, threonine of CTRKKHDC was replaced with alanine. All other experiments were made on the CTRKKHDC peptide only. Synthesis of the peptides was described in Ivaska et al. (17Ivaska J. Käpylä J. Pentikäinen O. Hoffren A.M. Hermonen J. Huttunen P. Johnson M.S. Heino J. J. Biol. Chem. 1999; 274: 3513-3521Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Binding Assay for Europium-labeled α1I—Human recombinant integrin α1I was expressed as reported earlier (13Nykvist P. Tu H. Ivaska J. Käpylä J. Pihlajaniemi T. Heino J. J. Biol. Chem. 2000; 275: 8255-8261Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). The integrin α1I binding assay to collagen I was performed as previously described (13Nykvist P. Tu H. Ivaska J. Käpylä J. Pihlajaniemi T. Heino J. J. Biol. Chem. 2000; 275: 8255-8261Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 17Ivaska J. Käpylä J. Pentikäinen O. Hoffren A.M. Hermonen J. Huttunen P. Johnson M.S. Heino J. J. Biol. Chem. 1999; 274: 3513-3521Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar); collagen I was precoated on microtiter plate wells, the wells were blocked with bovine serum albumin to prevent nonspecific binding, and α1I was allowed to bind to collagen in the absence or presence of inhibitory peptides. Detection of α1I binding to collagen was based on collagen-bound europium-labeled α1I and time-resolved fluorescence measured by fluorimetry (Delfia model 1232; Wallac/PerkinElmer Life Sciences). In order to measure the binding of α1I to the cyclic peptides, peptides were precoated on amine microtiter plate wells according to the manufacturer's instructions, and the integrin α1I binding assay was performed as described previously (17Ivaska J. Käpylä J. Pentikäinen O. Hoffren A.M. Hermonen J. Huttunen P. Johnson M.S. Heino J. J. Biol. Chem. 1999; 274: 3513-3521Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Wells were blocked with bovine serum albumin, and europium-labeled α1I was introduced. Bound α1I was detected using time-resolved fluorescence measured by fluorimetry, as above. Differential Scanning Calorimetry—In order to study the thermostability of α1I, differential scanning calorimetry (Nano II; Calorimetry Sciences Corp.) was used. For this purpose, 31.8 nmol of α1I in PBS with either 2 mm EDTA or 2 mm MgCl2 was prepared. In order to examine how the presence of peptide affected the stability of the α1I, 95.4 nmol of peptide was added to PBS containing 2 mm MgCl2. All samples were incubated for 7 h at room temperature (∼23 °C) without stirring and were degassed before being transferred to the cell for calorimetric measurements. The reference cell was filled with PBS. The heat capacity was recorded as a function of temperature, between 0 and 100 °C at a rate of 1 °C/min. The base line was subtracted, and the enthalpy (ΔH), the entropy (ΔS), and the transition midpoint temperature (Tm) were calculated using the software provided by Calorimetry Sciences Corp. The van't Hoff enthalpy (ΔHvH) was calculated in the usual way according to Equation 1, ΔHvH=4RTm2〈ΔCp〉tr,maxΔHcal(Eq. 1) where R is the gas constant, Tm is the temperature at which the base line-subtracted and concentration-normalized heat capacity function (〈ΔCp〉tr) has a maximum, and 〈ΔCp〉tr,max is the measured heat capacity at the peak maximum. The value of the ratio ΔHvH/ΔHcal is often used as a measure of cooperativity. In the case of small globular proteins, a folding/unfolding ratio of 1 is generally assumed to be evidence for a two-state transition (22Privalov P.L. Adv. Protein Chem. 1979; 33: 167-241Crossref PubMed Scopus (2203) Google Scholar, 23Sturtevant J.M. Annu. Rev. Phys. Chem. 1987; 33: 463-488Crossref Google Scholar), whereas larger values indicate the presence of additional intermediates along the unfolding pathway. Determination of the Free Mg2+Concentration—The fluorescent Mg2+ chelator Mag-Fura-2 (Molecular Probes, Inc., Eugene, OR) was used to monitor changes in the concentration of free Mg2+ in the sample buffer (24Murphy E. Freudenrich C.C. Levy L.A. London R.E. Lieberman M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2981-2984Crossref PubMed Scopus (92) Google Scholar). For this purpose, 100 nmol of α1I was incubated at room temperature in PBS containing 2 mm MgCl2 for 2 h, in order to saturate the protein with Mg2+. Excess Mg2+ was then removed, using a PD-10 column (Amersham Biosciences), after which the sample was transferred to a cuvette, and Mag-Fura-2 was added to a final concentration of 2.05 μm. The Mg2+ concentration was determined from the ratio of the intensities of emitted light following excitation at 340- and 398-nm wavelengths. The emission wavelength was set to 483 nm, and, as the excitation ratio was being monitored, 297 nmol of CTRKKHDC peptide was added. In order to calibrate the system, 100 nmol of MgCl2 was injected into the α1I solution before the peptide was injected. As a control, we added peptide and MgCl2 to PBS buffer pretreated in a similar manner as the α1I solution. Fluorescence Spectroscopy—In all of the fluorimetric experiments, we used a Quantamaster 1 steady-state spectrofluorimeter (Photon Technology International) with the slit width set to 4 nm unless stated otherwise. The experiments were conducted in a 10-mm quartz cuvette at room temperature. The solution was kept under constant stirring (300 rpm). The molar ratio of peptide to protein was 3:1 in all fluorimetric experiments. Tryptophan emission spectra were measured on solutions containing 10 μm α1I. In order to examine whether the emission spectra of α1I was affected by the presence of Mg2+, measurements were made in PBS with and without 2 mm MgCl2. The effect of peptide interactions with α1I was studied in the presence of 30 μm peptide and 2 mm MgCl2. The excitation wavelength was set to 295 nm. All spectra were recorded as the average of three scans. Quenching of tryptophan fluorescence by KI (Sigma) was measured with a solution containing 3.75 nmol of α1Iinthe presence and absence of MgCl2 and 10.5 nmol of peptide was added to the α1I solution with MgCl2. The excitation wavelength was set to 295 nm, and the emission due to tryptophan was measured at 328 nm, which is the emission maximum. KI was injected into the cuvette in 50-μmol increments from a 5 m stock solution. In order to prevent oxidation of iodide, 0.1 mm sodium thiosulfate (Na2S2O3; Merck) was included in the KI solution. Bis-ANS (Molecular Probes, Inc.) is a fluorescent probe that binds specifically to hydrophobic surfaces on proteins (25Rosen C.G. Weber G. Biochemistry. 1969; 8: 3915-3920Crossref PubMed Scopus (200) Google Scholar). Bis-ANS binding to 10 nmol of α1I was measured in a PBS solution in the presence and absence of 2 mm MgCl2 containing 1 nmol of bis-ANS. In order to examine how the presence of Mg2+ affected the fluorescence due to bis-ANS, we added 2 mm MgCl2 to the Mg2+-free buffer while monitoring the fluorescence intensity. Changes in hydrophobic surface due to α1I-peptide interactions was studied by the addition of 30 nmol of peptide to the 2 mm MgCl2-PBS while the fluorescence intensity was being recorded. The excitation wavelength was 370 nm, and the bis-ANS emission was measured at 470 nm. Light scattering was simultaneously measured on another detector (370 nm; a small slit width was used to reduce the amount of light reaching the detector) in order to detect aggregation. Crystallization and Data Collection—The protein was expressed, purified, and crystallized according to the previously described protocol (26Salminen T.A. Nymalm Y. Kankare J. Käpylä J. Heino J. Johnson M.S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1365-1367Crossref PubMed Scopus (13) Google Scholar). Briefly, the I domain (Swiss-Prot number P56199; residues 138–338) of the human integrin subunit α1 was expressed as a recombinant glutathione S-transferase fusion protein in Escherichia coli and purified by affinity chromatography. The protein was concentrated to 15–30 mg/ml, and initial crystallization conditions were screened with the sparse matrix screen (27Jancarik J. Kim S.-H. J. Appl. Crystallogr. 1991; 24: 409-411Crossref Scopus (2079) Google Scholar) (Hampton Research) using the vapor diffusion method. The initial crystallization conditions were systematically refined and shown to be suitable for x-ray analysis. The crystals were obtained using a drop containing 13 mg/ml protein and 0.25 mm ligand and the well containing 30% (w/v) polyethylene glycol 6000, 100 mm Tris, pH 8.5, 0.2 m sodium acetate, and 15% (v/v) glycerol. The x-ray data were collected from a single crystal at 100 K, flash-frozen using the Oxford cryosystem at the Turku Center for Biotechnology, using the X11 beamline at the EMBL-Hamburg Outstation and using a MAR CCD detector. The data were indexed, integrated, scaled, and reduced with the XDS software package (28Kabsch W. J. Appl. Crystallogr. 1993; 26: 795-800Crossref Scopus (3233) Google Scholar). The crystal has symmetry consistent with the space group P21 with cell dimensions a = 37.5 Å, b = 97.6 Å, c = 53.1 Å, α = γ = 90°, and β = 103.6°, and there are two α1I molecules in the asymmetric unit according to the calculated Matthews coefficient (29Matthews B.W. J. Mol. Biol. 1968; 33: 491-497Crossref PubMed Scopus (7922) Google Scholar) of 2.2 Å3/Da. Details from the data collection are presented in Table I.Table ICrystallographic data processing and refinement statistics for integrin α1IData processingValueSpace groupP21Unit cell parameters (Å2)a = 37.5, b = 97.6, c = 53.1Resolution limit (Å)25—1.87ObservationsaFirst and second values correspond to the completeness in the entire data range and in the highest resolution shell (1.87—2.0 Å)109,126/15,660Unique reflectionsaFirst and second values correspond to the completeness in the entire data range and in the highest resolution shell (1.87—2.0 Å)29,394/4909RedundancyaFirst and second values correspond to the completeness in the entire data range and in the highest resolution shell (1.87—2.0 Å)3.7/3.2CompletenessaFirst and second values correspond to the completeness in the entire data range and in the highest resolution shell (1.87—2.0 Å) (%)95.8/88.7RmergeaFirst and second values correspond to the completeness in the entire data range and in the highest resolution shell (1.87—2.0 Å),bRmerge = Σ|I(k) — 〈I〉|/ΣI(k), where I(k) and 〈I〉 are the kth individual and mean values of the intensity of a reflection, respectively (%)5.6/29.4I/σaFirst and second values correspond to the completeness in the entire data range and in the highest resolution shell (1.87—2.0 Å)15.6/4.5RefinementData range (Å)500—1.87No. of reflections27,922No. of waters279No. of glycerols2R-factorcR-factor = Σ|Fo — Fc|/Σ|Fo|, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively/RfreedPerformed on 10% of the reflections (%)19.1/24.0Average B-factor (Å2)20.1Root mean square deviation bond lengths (Å)0.01Root mean square deviation bond angles (degrees)1.3Residues with weak electron densityA moleculeArg218, Arg242, His257, Asn259, Lys263, Asn289, Ser291, Glu308B moleculeArg171, Lys177, Asn189, Lys210, Arg218, His257, Glu293, Lys294, Glu329a First and second values correspond to the completeness in the entire data range and in the highest resolution shell (1.87—2.0 Å)b Rmerge = Σ|I(k) — 〈I〉|/ΣI(k), where I(k) and 〈I〉 are the kth individual and mean values of the intensity of a reflection, respectivelyc R-factor = Σ|Fo — Fc|/Σ|Fo|, where Fo and Fc are the observed and calculated structure factor amplitudes, respectivelyd Performed on 10% of the reflections Open table in a new tab Structure Determination, Model Building, and Refinement—The α1I structure was solved by molecular replacement. CNS (30Brünger A.T. Adams P.D. Rice L.M. Structure. 1997; 5: 325-336Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar) was used in the molecular replacement work, whereas the CCP4 suite (31Collaborative Computational Project 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19770) Google Scholar) was used for refinement. The software O (32Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13011) Google Scholar) was used to display electron density maps and for model building. Before any structure solution was attempted, 5% of the reflections were randomly set aside and used only for calculations of the cross-validated error function Rfree (33Brünger A.T. Nature. 1992; 355: 472-475Crossref PubMed Scopus (3864) Google Scholar) but never in refinement or map calculations. Since we have already solved an α1I structure at 2.3 Å (26Salminen T.A. Nymalm Y. Kankare J. Käpylä J. Heino J. Johnson M.S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1365-1367Crossref PubMed Scopus (13) Google Scholar) (Protein Data Bank code 1QCY), we used that structure as the MR search model in order to solve the structure of α1I at 1.87-Å resolution. For refinement of the structure, the maximum likelihood method implemented in Refmac5 (34Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13870) Google Scholar) was used within the CCP4 suite. Water molecules, having reasonable hydrogen bonding geometry to the protein, were added to the models in places where the Fo - Fc electron density maps had a peak value 3 σ above the mean. Finally, the structure was checked with the Whatcheck program in the software package WHATIF (35Vriend G. J. Mol. Graph. 1990; 8: 52-56Crossref PubMed Scopus (3374) Google Scholar) and with Procheck (36Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-290Crossref Google Scholar). The coordinates have been submitted to the Protein Data Bank (code 1PT6). Peptide Modeling—A model structure of the TRKKHD peptide was created based on the theoretical model of the metalloproteinase jararhagin (18Pentikäinen O. Hoffren A.M. Ivaska J. Käpylä J. Nyrönen T. Heino J. Johnson M.S. J. Biol. Chem. 1999; 274: 31493-31505Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) (Protein Data Bank code 1C9G). A cysteine residue was added to both the amino terminus and the carboxyl terminus, and the peptide was cyclized via a disulfide bond built between them. For molecular dynamics, a simulation box containing the peptide was filled with solvent (water described by the simple point charge model) (37Berendsen H.J.C. Postma J.P.M. van Gunsteren W.F. Hermans J. Pullman B. Intermolecular Forces. Reidel, Dordrecht, The Netherlands1981: 331-342Google Scholar)). The δ-nitrogen of the histidine residue was protonated, whereas the ϵ-nitrogen was left unprotonated, enabling the formation of stabilizing intramolecular hydrogen bonds as seen in the metalloproteinase structures of adamalysin II from Crotalus adamanteus (Protein Data Bank code 1IAG (38Gomis-Ruth F.X. Kress L.F. Bode W. EMBO J. 1993; 12: 4151-4157Crossref PubMed Scopus (197) Google Scholar)) and acutolysin A from Agkistrodon acutus (Protein Data Bank code 1BUD (39Gong W. Zhu X. Liu S. Teng M. Niu L. J. Mol. Biol. 1998; 283: 657-668Crossref PubMed Scopus (107) Google Scholar)). The system was simulated for 1 ns at constant temperature (298 K; the temperature of the solvent and peptide were controlled separately) and pressure (1 bar), both controlled using the Berendsen weak coupling method (40Berendsen H.J.C. Postma J.P.M. Vangunsteren W.F. Dinola A. Haak J.R. J. Chem. Phys. 1984; 81: 3684-3690Crossref Scopus (23667) Google Scholar). Bond lengths were constrained using the LINCS algorithm (41Hess B. Bekker H. Berendsen H.J.C. Fraaije J.G.E.M. J. Comput. Chem. 1997; 18: 1463-1472Crossref Scopus (11739) Google Scholar). Short range electrostatic interactions were calculated with a cut-off of 1 nm, and long range electrostatic interactions were treated by the fast particle mesh Ewald method (42Darden T. York D. Pedersen L. J. Chem. Phys. 1993; 98: 10089-10092Crossref Scopus (21056) Google Scholar, 43Essmann U. Perera L. Berkowitz M.L. Darden T. Lee H. Pedersen L.G. J. Chem. Phys. 1995; 103: 8577-8593Crossref Scopus (15781) Google Scholar). The system was sampled with a time step of 1 fs using a leapfrog integrator (44Hockney R.W. Goel S.P. Eastwood J.W. J. Comput. Phys. 1974; 14: 148-158Crossref Scopus (823) Google Scholar). Periodic boundary conditions were used. Molecular dynamics was performed using GROMACS (45Berendsen H.J.C. van der Spoel D. van Drunen R. Comp. Phys. Comm. 1995; 91: 43-56Crossref Scopus (7261) Google Scholar, 46Lindahl E. Hess B. van der Spoel D. J. Mol. Mod. 2001; 7: 306-317Crossref Google Scholar) and the GROMACS force field. The CARKKHDC peptide was created by exchanging alanine for threonine of the simulated CTRKKHDC. Integrin α1I Modeling—The structure of the "open" conformation of the integrin α1I domain was modeled. The sequence of human α1I was aligned against the sequence of human α2I using the program MALIGN (47Johnson M.S. May A.C. Rodionov M.A. Overington J.P. Methods Enzymol. 1996; 266: 575-598Crossref PubMed Google Scholar, 48Johnson M.S. Overington J.P. J. Mol. Biol. 1993; 233: 716-738Crossref PubMed Scopus (266) Google Scholar) in the Bodil modeling environment. 2J. V. Lehtonen, V.-V. Rantanen, D.-J. Still, J. Ekholm, D. Björklund, Z. Iftikhar, M. Huhtala, A. Jussila, J. Jaakkola, O. Pentikäinen, T. Nyrönen, T. A. Salminen, M. Gyllenberg, and M. S. Johnson, manuscript in preparation. Since the structures of apo-α1I and apo-α2I are very similar to each other (when