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A Novel Adaptation of the Integrin PSI Domain Revealed from Its Crystal Structure

2004; Elsevier BV; Volume: 279; Issue: 39 Linguagem: Inglês

10.1074/jbc.c400362200

1083-351X

Jian-Ping Xiong, Thilo Stehle, Simon L. Goodman, M. Amin Arnaout,

Galectins and Cancer Biology

Integrin β-subunits contain an N-terminal PSI (for plexin-semaphorin-integrin) domain that contributes to integrin activation and harbors the PI(A) alloantigen associated with immune thrombocytopenias and susceptibility to sudden cardiac death. Here we report the crystal structure of PSI in the context of the crystallized αVβ3 ectodomain. The integrin PSI forms a two-stranded antiparallel β-sheet flanked by two short helices; its long interstrand loop houses Pl(A) and may face the EGF2 domain. The integrin PSI contains four cysteine pairs connected in a 1-4, 2-8, 3-6, 5-7 pattern. An unexpected feature of the structure is that the final, eighth cysteine is located C-terminal to the Ig-like hybrid domain and is thus separated by the hybrid domain from the other seven cysteines of PSI. This architecture may be relevant to the evolution of integrins and should help refine the current models of integrin activation. Integrin β-subunits contain an N-terminal PSI (for plexin-semaphorin-integrin) domain that contributes to integrin activation and harbors the PI(A) alloantigen associated with immune thrombocytopenias and susceptibility to sudden cardiac death. Here we report the crystal structure of PSI in the context of the crystallized αVβ3 ectodomain. The integrin PSI forms a two-stranded antiparallel β-sheet flanked by two short helices; its long interstrand loop houses Pl(A) and may face the EGF2 domain. The integrin PSI contains four cysteine pairs connected in a 1-4, 2-8, 3-6, 5-7 pattern. An unexpected feature of the structure is that the final, eighth cysteine is located C-terminal to the Ig-like hybrid domain and is thus separated by the hybrid domain from the other seven cysteines of PSI. This architecture may be relevant to the evolution of integrins and should help refine the current models of integrin activation. Integrins are heterodimeric (αβ) cell-matrix and cell-cell adhesion receptors, with each subunit containing a large extracellular domain, a single-pass transmembrane domain, and a short cytoplasmic tail (1Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6955) Google Scholar). Integrins are often expressed on the cell surface in an inactive state (unable to bind physiologic ligands) but can be rapidly activated by intracellular signals (inside-out activation) (2Xiong J.P. Stehle T. Goodman S.L. Arnaout M.A. Blood. 2003; 102: 1155-1159Crossref PubMed Scopus (159) Google Scholar). Once liganded, integrins cluster and initiate outside-in signals similar to classical receptors that modify cellular functions. The precise mechanism of integrin activation is incompletely understood. Insights into structure-activity relationships in integrins were greatly aided by our determination of the crystal structure of the ectodomain from integrin αVβ3 alone and in complex with the prototypical ligand RGD (3Xiong 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 (1118) Google Scholar, 4Xiong J.P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S.L. Arnaout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1410) Google Scholar). The structure has four domains in the αV subunit: an N-terminal seven-bladed propeller followed by an Ig-like "thigh" domain and two co-linear β-sandwich domains calf-1 and calf-2. The β-subunit ectodomain consists of eight domains. The N-terminal PSI domain (5Bork P. Doerks T. Springer T.A. Snel B. Trends Biochem. Sci. 1999; 24: 261-263Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) is followed by an Ig-like "hybrid" domain (with the ligand-binding vWFA domain (βA) emerging from the loop connecting its two β-sheets). The hybrid domain is then connected to four EGF 1The abbreviations used are: EGF, epidermal growth factor; MAD, multiple anomalous diffraction; SIRAS, single isomorphous replacement with anomalous scattering; PSI, plexin-semaphorin-integrin. -like domains and a novel β-tail domain (βTD). An unexpected feature of the crystal structure is that αVβ3 is genuflexed at the αV and β3 "knees" such that the head abuts the legs. Current models suggest that a straightening of the genu is required for physiologic ligand binding (in the switch-blade model of activation (6Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (943) Google Scholar)) or for ligand-induced outside-in signaling (in the deadbolt model (2Xiong J.P. Stehle T. Goodman S.L. Arnaout M.A. Blood. 2003; 102: 1155-1159Crossref PubMed Scopus (159) Google Scholar)). A better understanding of the basis of integrin activation requires structure determination of the activation-sensitive domains PSI, EGF1, and EGF2 (reviewed in Ref. 7Arnaout M.A. Immunol. Rev. 2002; 186: 125-140Crossref PubMed Scopus (94) Google Scholar), which are all located in the genuflexed β-subunit but were not resolved in the published structure. Although some features of the PSI domain, including two short helices, were visible in our electron density maps, our main chain tracing was inconsistent with published cysteine pairing (8Calvete J.J. Henschen A. Gonzalez-Rodriguez J. Biochem. J. 1991; 274: 63-71Crossref PubMed Scopus (158) Google Scholar) and predictions (5Bork P. Doerks T. Springer T.A. Snel B. Trends Biochem. Sci. 1999; 24: 261-263Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) especially between Cys5 and Cys435. Since several cysteines cluster close together, we were initially unable to build the domain with certainty. The density for EGF1 and EGF2 was even less well defined. The ∼50-amino acid PSI domain, first recognized based on primary sequence alignments, is present in one or more copies in more than 500 proteins (see smart.embl-heidelberg.de/smart/get_members.pl?WHAT=NRDB_COUNT&NAME=PSI). It is most commonly found in plexins, semaphorins, and integrins, glycoproteins that mediate cell growth, migration, and differentiation. Two recently determined structures of semaphorin 4D (SEMA4D) (9Love C.A. Harlos K. Mavaddat N. Davis S.J. Stuart D.I. Jones E.Y. Esnouf R.M. Nat. Struct. Biol. 2003; 10: 843-848Crossref PubMed Scopus (136) Google Scholar) and the plexin MET (10Stamos J. Lazarus R.A. Yao X. Kirchhofer D. Wiesmann C. EMBO J. 2004; 23: 2325-2335Crossref PubMed Scopus (199) Google Scholar), each containing a PSI domain, have now allowed us to build the integrin PSI domain into our αVβ3 maps without ambiguity. The salient and unexpected features of this structure as it relates to integrin architecture, activation, and disease are presented and discussed here. The original αVβ3 structure was determined using a combined phasing approach, with a lutetium derivative data set used for multiple anomalous diffraction (MAD) phasing and a platinum data set used for single isomorphous replacement with anomalous scattering (SIRAS) (3Xiong 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 (1118) Google Scholar). To solve the PSI structure, three maps were calculated: a MAD-phased map, a SIRAS-phased map based on the platinum derivative, and another SIRAS-phased map based on the lutetium data set collected at the peak wavelength. The three electron density maps were averaged using the RAVE package (11Kleywegt G.J. Bergfors T. Senn H. Le Motte P. Gsell B. Shudo K. Jones T.A. Structure (Lond.). 1994; 2: 1241-1258Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar) and displayed using program O (12Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). The averaged map showed clear density for a large portion of the PSI domain, including two helices and the central tryptophan residue (Trp25), allowing us to trace the PSI chain. The new model for αVβ3, including the PSI domain, was refined using the existing 3.1-Å data set (3Xiong 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 (1118) Google Scholar) and the program CNS (13Brunger A.T. Adams P.D. Acc. Chem. Res. 2002; 35: 404-412Crossref PubMed Scopus (55) Google Scholar). This resulted in R-factors of 29.0 (work set) and 35.6% (free set) and good model geometry. Structure Determination of the Integrin PSI—Our initial chain trace of the PSI did not agree with the published cysteine pairing, especially between Cys5 and Cys435. However, the recent structure determinations of two PSI domains (9Love C.A. Harlos K. Mavaddat N. Davis S.J. Stuart D.I. Jones E.Y. Esnouf R.M. Nat. Struct. Biol. 2003; 10: 843-848Crossref PubMed Scopus (136) Google Scholar, 10Stamos J. Lazarus R.A. Yao X. Kirchhofer D. Wiesmann C. EMBO J. 2004; 23: 2325-2335Crossref PubMed Scopus (199) Google Scholar), allowed us to establish the β3 PSI fold without ambiguity (Fig. 1A). We fitted the cysteine cores of SEMA4D and MET structures into our maps, and this allowed us to establish points of reference for the PSI domain fold. It became clear that Cys13, and not the published Cys5, pairs with Cys435 (Fig. 1B). It also became clear that the region around Cys435 substitutes for a portion of the PSI domain seen in the SEMA4D and MET structures, and thus Cys435 is integral to PSI. We were able to build residues 1–50 of the PSI domain by using the corrected disulfide bond pattern. The linker between PSI and hybrid domains (residues 51–53) is not well defined in our density maps and was not included. In our efforts to connect the PSI domain with the hybrid domain, we also noticed that the N-terminal strand of the hybrid domain was out of register by one residue. We have corrected this error, which has no bearing on previously published interpretations of αVβ3 structure and function. The location of the PSI domain in relation to the rest of the αVβ3 ectodomain is shown in Fig. 1C. Architecture of the Integrin PSI and Its Relationship to the Hybrid Domain—The integrin PSI domain forms a two-stranded antiparallel β-sheet, with two flanking short helices, connected by disulfides to the central sheet, and an N-terminal segment that may also be helical (Fig. 1B). The small hydrophobic core of the domain is formed by the highly conserved side chain of Trp25. The core integrin PSI structure can be largely superimposed onto those of SEMA4D and MET, including all three conserved cysteine pairs (Fig. 2, A and B). The fourth disulfide bridge is lacking in semaphorins and has a somewhat different conformation in plexins and integrins, perhaps a result of an additional C-terminal α-helix in the integrin PSI (Fig. 2, A and B). A key difference between the integrin PSI domain and the semaphorin and plexin PSI domains is a distinctively longer interstrand AB loop (Figs. 1B and 2B). While some of the side chains in this loop are solvent exposed, others may face the EGF2 domain, suggesting potential interactions. It is also apparent that the overall structures of the three PSI domains are quite different in the C-terminal half of the domain (Fig. 2, A and B). This suggests that this portion of PSI has a function defined by its specific structural context. Alignment of the PSI domains of SEMA4D, MET, and αVβ3 reveals that Cys435 of β3, located at the C terminus of the hybrid domain, is an integral part of the integrin PSI (Fig. 2A). Thus the hybrid domain is an insertion into the last loop of PSI. This unexpected architecture through which the PSI and hybrid domains are connected may explain the incorrect predictions of cysteine pairing of the PSI domain in integrins (5Bork P. Doerks T. Springer T.A. Snel B. Trends Biochem. Sci. 1999; 24: 261-263Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 8Calvete J.J. Henschen A. Gonzalez-Rodriguez J. Biochem. J. 1991; 274: 63-71Crossref PubMed Scopus (158) Google Scholar). The hybrid A′B loop contains an arginine (Arg93), conserved between integrin β-chains, that contributes to the interface with the PSI domain. Functional and Disease Implications—The integrin PSI domain contributes to integrin activation as evidenced by binding of activation-sensitive monoclonal antibodies such as AP5 (which binds the N-terminal six amino acids of PSI of β3 (14Honda S. Tomiyama Y. Pelletier A.J. Annis D. Honda Y. Orchekowski R. Ruggeri Z. Kunicki T.J. J. Biol. Chem. 1995; 270: 11947-11954Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar)) and activating amino acid substitutions (15Zang Q. Springer T.A. J. Biol. Chem. 2001; 276: 6922-6929Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 16Sun Q.H. Liu C.Y. Wang R. Paddock C. Newman P.J. Blood. 2002; 100: 2094-2101Crossref PubMed Scopus (57) Google Scholar) such as an alanine substitution of the Cys435, which is immediately N-terminal to EGF1 (Fig. 1B) (reviewed in Ref. 7Arnaout M.A. Immunol. Rev. 2002; 186: 125-140Crossref PubMed Scopus (94) Google Scholar). The PSI domain also harbors clinically important alloantigens that cause immune thrombocytopenias. A leucine-proline dimorphism at amino acid 33 (Leu33 → Pro) known as autoantigen Pl(A) is the one most frequently implicated in syndromes of immune-mediated platelet destruction, particularly neonatal alloimmune thrombocytopenia and post-transfusion purpura. Substitution of the common PlA1 alloantigen at Leu33 with Pro (PlA2 allele found in 15% of Caucasians) may also predispose to arterial thrombosis frequently manifested as acute coronary events that can lead to sudden death at a relatively young age (17Weiss E.J. Bray P.F. Tayback M. Schulman S.P. Kickler T.S. Becker L.C. Weiss J.L. Gerstenblith G. Goldschmidt-Clermont P.J. N. Engl. J. Med. 1996; 334: 1090-1094Crossref PubMed Scopus (702) Google Scholar). It is suspected that this predisposition is secondary to enhanced activation of platelet integrins (18Bussel J.B. Kunicki T.J. Michelson A.D. Hematology (Am. Soc. Hematol. Educ. Program). 2000; : 222-240Crossref PubMed Google Scholar), enhanced thrombin generation, and impaired antithrombotic action of aspirin at the site of microvascular injury (19Undas A. Brummel K. Musial J. Mann K.G. Szczeklik A. Circulation. 2001; 104: 2666-2672Crossref PubMed Scopus (158) Google Scholar). It has also been reported that the PlA2 allele is a risk factor for acute renal allograft rejection (20Salido E. Martin B. Barrios Y. Linares J.D. Hernandez D. Cobos M. Checa M.D. Hortal L. Fernandez A. Garcia J.J. Torres A. J. Am. Soc. Nephrol. 1999; 10: 2599-2605PubMed Google Scholar). The PSI domain also contributes to the binding of certain drugs that trigger drug-dependent antibodies to αIIbβ3, precipitating thrombocytopenia (21Peterson J.A. Nyree C.E. Newman P.J. Aster R.H. Blood. 2003; 101: 937-942Crossref PubMed Scopus (26) Google Scholar). The present crystallographic studies clarify a number of observations about structure-activity relationships in integrins. First, they establish the correct pairing of cysteines and domain boundaries for the integrin PSI. This will allow a reinterpretation of many functional studies and will be invaluable in devising new ones to evaluate the salient features of the structure. Second, the structure reveals that the N terminus of the activation-sensitive AP5 epitope is solvent-exposed (Fig. 1B), which may explain the ability of the AP5 monoclonal antibody to bind the ectodomain in solution (not shown). The Cys13-Cys435 disulfide bridge contributes to the PSI/hybrid interface and likely helps to restrict movement of the PSI with respect to the hybrid domain. The activating effect of the Cys435 to alanine substitution is expected to make this interface more flexible, and it may also allosterically alter putative contacts between PSI and EGF1/2, thus accounting for the activating nature of this mutation. Interruption of the adjacent Cys406-Cys433 disulfide bond in the hybrid domain may have a similar functional outcome. Third, Leu33 is located in a hydrophobic segment of the distinctively long AB loop (between strands A and B of the PSI domain, Fig. 1B). Its replacement with Pro, a conformationally restricted residue, may alter the structure of this loop leading to an autoantibody response and immune thrombocytopenia. Elucidation of the three-dimensional structure of the PlA1 alloantigen may be useful in averting the autoimmune consumption of platelets through drug design. Of note is that Leu33 is located in the AB loop that likely faces EGF2; its replacement with Pro may also alter this interface and promote integrin activation. Fourth, three quinine-dependent antibodies known to cause the precipitous destruction of platelets if a patient is exposed to the drug require a conformational epitope in the PSI, which includes Ala50 (Fig. 1B) and Arg62 and Asp66 in the first strand of the hybrid domain of β3. The present structure reveals that all three residues are located on the same side of the integrin. Finally, it is now apparent that two insertions have contributed to the current architecture of the integrin β-subunit, one in the last loop of PSI and the second in between the two sheets of the hybrid domain, setting the stage both for formation of the integrin heterodimer and its regulated ability to bind physiologic ligands.