Tài liệu Báo cáo khoa học: Solution properties of full-length integrin aIIbb3 refined models suggest environment-dependent induction of alternative bent ⁄extended resting states doc

Solution properties of full-length integrin aIIbb3 refined models suggest environment-dependent induction of alternative bent ⁄extended resting states Camillo Rosano1 and Mattia Rocco2 Nanobiotecnologie, Istituto Nazionale per la Ricerca sul Cancro (IST), Genova, Italy Biopolimeri e Proteomica, IST, Genova, Italy Keywords blood coagulation; hydrodynamics; modeling; modular proteins; protein structure Correspondence M Rocco, Biopolimeri e Proteomica, IST c ⁄ o CBA, Largo R Benzi 10, I-16132 Genova, Italy Fax: +39-0105737-325 Tel: +39-0105737-310 E-mail: mattia.rocco@istge.it (Received 12 November 2009, revised May 2010, accepted 29 May 2010) doi:10.1111/j.1742-4658.2010.07724.x The recently published novel integrin aIIbb3 ectodomain crystallographic structure and NMR structures of its transmembrane ⁄ cytoplasmic segments were employed to refine previously developed molecular models Alternative complete aIIbb3 models were built and evaluated, and their shape was compared with EM maps and their computed hydrodynamic ⁄ conformational properties were compared with the available experimental data A partially extended ⁄ closed model, or a mixture of bent ⁄ closed and extended ⁄ closed conformations, are both compatible with the results of a recent small-angle neutron scattering study of Triton X-100-solubilized resting aIIbb3, while new electron microscopy evidence of nanodiscs-embedded aIIbb3 supports the bent ⁄ closed resting form However, only an extended ⁄ closed model matches well the hydrodynamics of either octyl-glucoside-solubilized or nanodiscs-embedded resting aIIbb3, suggesting that different solubilization strategies and substrate interactions might operate a conformational selection between alternative, stable states Furthermore, extended ⁄ open models are required to match the electron tomography map and the hydrodynamics following the priming-induced b3 hybrid domain swingout, but without immediate full tail separation Importantly, both extension and opening transitions can occur by pivoting at the recently identified b3 hinge point, which does not appear to be freely flexible The structure and mechanism of action of integrins thus seem to depend on discrete transitions and to be more tightly coupled to the local environment than previously thought Introduction Integrins are heterodimeric transmembrane (TM) cellular receptors involved in mechanical anchoring and two-way signaling [1] Each a and b subunit has a modular structure with a large extracellular portion, a single TM region and a cytoplasmic domain [1–3] The integrin activation mechanism is regulated by conformational changes, the details of which have not yet been fully elucidated [2,3] X-ray crystallography Abbreviations bc, bent ⁄ closed; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DMPG, 1,2-dimyristoyl-sn-glycero-3-phospho-(1¢-rac-glycerol); DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; ec, extended ⁄ closed; EGF, epidermal growth factor; EM, electron microscopy; eotc, extended ⁄ open ⁄ tails crossed; eots, extended ⁄ open ⁄ tail separated; ET, electron tomography; NMA, Normal Modes Analysis; OG, octylglucoside; pec, partially extended ⁄ closed; PSI, plexin ⁄ semaphorin ⁄ integrin; SANS, small-angle neutron scattering; TEM, transmission electron microscopy; TM, transmembrane 3190 FEBS Journal 277 (2010) 3190–3202 ª 2010 The Authors Journal compilation ª 2010 FEBS C Rosano and M Rocco has revealed similar bent shapes for resting and primed extracellular region constructs (‘ectodomains’) [4,5], while ligand binding-induced large structural re-arrangements in smaller constructs suggested extension, ‘opening’ and tails separation [3,6] Lower-resolution structural data, such as transmission electron microscopy (TEM) [7], cryo-electron microscopy (cryo-EM) [8] and electron tomography (ET) [9] have provided maps ranging from supporting the crystallographic models of the ectodomains, to being in accordance with the extended conformation after priming of full-length, detergent-solubilized samples Previously, we presented a multiresolution study of integrins avb3 (ectodomain) and aIIbb3 (full-length), in which crystallography ⁄ NMR-based models were fit in the EM ⁄ ET maps and their hydrodynamic parameters were then computed and compared with the experimental data [10] Our modeling work [10] suggested that full-length integrins might be already extended, with the transition to the open form (involving the swing-out of the b3 hybrid domain [6]) taking place without the requirement of full separation of the TM helices Since then, crystallographic structures of the aIIbb3, avb3 and axb2 ectodomains [11–13] and two NMR-based structures of the aIIbb3 TM helices – one embedded in a small bicelle [14] and the other solubilized in a mixed solvent and including the cytoplasmic domains [15] – have been published Furthermore, new low-resolution data of full-length aIIbb3 have very recently appeared: a small-angle neutron scattering (SANS) study after solubilization in Triton X-100 [16]; and an EM ⁄ analytical ultracentrifugation study after reconstitution in phospholipid nanodiscs [17] Importantly, the new aIIbb3 and avb3 ectodomain crystallographic structures reported the previously unresolved structure of the b3 I-epidermal growth factor (EGF)1-2 modules, revealing the potential hinge points for subunit extension [11,12] Because our previous aIIbb3 models [10] were developed by homology modeling of the aIIb subunit on the av template [4,5], we felt that the new data warranted a significant revision of the models This included inserting the recently published NMR-based structures of the TM helices ⁄ cytoplasmic domains for the resting integrin state, while for the primed state we employed the computer models [18,19] utilized in our previous work [10] Particular care was exerted when modeling the major, still-unresolved, loop in the calf-2 module of aIIb, and we resorted to using ab initio modeling procedures Extended ⁄ closed and extended ⁄ open models were derived from the fully bent ⁄ closed crystallographic model by fitting to the ET map, and two series of intermediate models were obtained by morphing between these initial and final Refined aIIbb3 models conformations The models were then assessed by comparing their hydrodynamic and conformational parameters (computed using the new UltraScan SOlution MOdeler (US-SOMO) bead-modeling implementation [20–22]) with experimental data [10,16,17] While the fully bent ⁄ closed crystallographic model was incompatible with all the available solution data for resting aIIbb3, differences remained between a partially extended ⁄ closed SANS-complying model and the extended ⁄ closed ET ⁄ hydrodynamics-based model However, the SANS data could also be interpreted as deriving from a mixture of bent and extended conformations Moreover, only an extended ⁄ open model without full tail separation was in accordance with both the ET and hydrodynamic data of primed aIIbb3 Our revised models further support an alternative view of the conformational states and mechanism of action of integrins, and suggest a more tightly coupled synergy than previously thought with the extracellular, TM and cytoplasmic environments Results Model building The first refined model, A2bB3-bent ⁄ closed (bc)), consisted of the new aIIbb3 ectodomain structure, 3FCS [11], to which the new TM ⁄ cytoplasmic domain NMR model, taken from the 2KNC structure [15], was attached The TM helices, which were nicely superimposable with those of the recent 2K9J NMR structure [14], were embedded in 50 octyl-glucoside (OG) molecules to mimic a solubilizing micelle [10] When reconnecting their N-terminal part to the C-terminal ends of the ectodomain, particular care was exerted to allow for reasonable mechanical coupling The intermediatelength loop in aIIb (E764-D774) was modeled using ModLoop [23], whereas the G75-S78 and D477-Q482 loops in b3 were taken from the new avb3 ectodomain structure, 3IJE [12] Given their probable flexible nature, no attempt was made to fully optimize the conformation of these segments As for the still-unresolved long loop at the end of the aIIb calf-2 module (G840Q873), alignment of all human integrin a subunits using ClustalW [24] showed that those with a proven or putative cleavage site in the calf-2 module have longer loops between conserved cysteine residues than uncleaved integrins (Supporting information Fig S1) Two structured stretches (mainly a-helical, some extended) were consistently predicted by ab initio modeling using Robetta [25] within this region, mostly preserved by the cleavage sites (see Fig S1; a gallery of predicted structures is shown in Fig S2) While a FEBS Journal 277 (2010) 3190–3202 ª 2010 The Authors Journal compilation ª 2010 FEBS 3191 Refined aIIbb3 models C Rosano and M Rocco single, robust conformation clearly cannot be obtained using these methods alone, we find interesting this prediction of a structured region in all integrins examined Therefore, and because a complete structure is required for reliable hydrodynamic computations, a best-fitting model generated, using Robetta, for the G840-Q873 segment was selected and inserted into the aIIb calf-2 module Finally, models of the carbohydrate chains were then attached at the previously defined N- and O-glycosylation points [10], with the only modifications that, on the basis of potential structural clashes and a new prediction of the O-glycosylation sites (see the Materials and methods), S218 and T615 were selected in place of T259 and T619, respectively, for two O-linked carbohydrates An overview of this model can be seen in Fig 1A,F A fully extended, closed conformation (A2bB3-ec; Fig 1C,H) was then modeled starting from our previous A2b-5 ⁄ ET model [10] and manually replacing by structural superimposition each module in aIIb with the corresponding ones from the A2bB3-bc model A slightly lower opening angle (134° versus 143°) between the thigh and calf-1 domains was employed to better accommodate the aIIb subunit in the ET map [9] The aIIb b-propeller and the b3 bA ⁄ hybrid ⁄ plexin ⁄ semaphorin ⁄ integrin (PSI) domains of the 3FCS structure [11] were inserted as a single block to preserve the aIIb ⁄ b3 interface The b3 I-EGF1-4 and bTD domains were then added in the bent conformation, superimposing the latter on its counterpart taken from the A2b-5 ⁄ ET original model While a span of $ 20° was very recently found for the interdomain angle between C A E B H F D I J G Fig An overview of the new, refined models Models A2bB3–bc (panels A and F), A2bB3-pec (panels B and G), A2bB3-ec (panels C and H), A2bB3-eotc (panels D and I) and A2bB3-eots (panels E and J), are shown as ribbons (protein only, panels A–E) and as surface (protein) and space-filling (carbohydrates and OG moieties) representations (panels F–J) The aIIbb3 modules are indicated in panel E, and color-coded in the same way in all models (in addition, carbohydrates are yellow, and OG molecules are orange) In panels H–J, a mesh representation (purple) of the ET map [9] is superimposed on the models 3192 FEBS Journal 277 (2010) 3190–3202 ª 2010 The Authors Journal compilation ª 2010 FEBS Refined aIIbb3 models C Rosano and M Rocco the I-EGF1-2 modules during the examination of 10 independent molecules in axb2 crystals [13], Normal Modes Analysis (NMA) [26] of this region did not reveal any freely extensible joint but only a sideways oscillation, as previously observed with the avb3derived models [10] Mutating in silico the hinge cysteine residues C473 and C503 to alanines did not substantially change the situation The extension of this segment was then performed by aligning the b3 C473C503 residues on the z-axis and manually performing an opening of $ 40° between the I-EGF1-2 modules along this hinge To reconnect this part of the chain with the bA ⁄ hybrid ⁄ PSI head domain, we first generated a pathway of 12 structures from the fully closed to the complete swung-out hybrid domain (taken from the 3FCU structure [6]), using the Yale morph server [27] Aligning all these structures on the bA domain in the A2bB3-extended ⁄ closed (ec) model framework revealed a conformation that could be linked to the extended I-EGF1-4 ⁄ bTD segment A swing-out, of $ 6°, of the hybrid domain was present in this conformation, consistent with that of $ 10° observed in the avb3 ectodomain fitted in the TEM map [7] Finally, after introduction of the mature aIIb cut at R856 [28], Robetta [25] was again employed to remodel the G847-R856 and D857-C879 stretches While an initial version of this work was undergoing evaluation, a SANS study of native, full-length aIIbb3 solubilized in Triton X-100 was published [16] In that ˚ study, low-resolution ($ 20 A) shape reconstruction from the I(q) versus q profiles using dammin [29] and related programs [16] produced dummy atom bead models that were judged to be compatible with the bent ⁄ closed conformation [16] A pairwise distance distribution function p(r) versus r curve was also derived from the experimental data (Fig 1b of ref [16]), allowing a direct comparison with the p(r) versus r that could be calculated from our models using the new US-SOMO implementation [22] Because the SANS data were collected at a content of 16% D2O, contrast-matching the Triton X-100 detergent, the OG moieties were removed from our models before the p(r) versus r computation was performed As can seen in Fig 2, neither the A2bB3-bc (blue line) nor the A2bB3-ec (magenta line) p(r) versus r curves matched the SANS-derived curve (black line-connected circles) A series of 30 intermediate conformations were then generated by morphing between the A2bB3-bc and the A2bB3-ec models, without the carbohydrates attached, using the Yale morph server [27], and their p(r) versus r curves were computed and compared with the SANS-derived curve The carbohydrates were then re-attached to a restricted number of models having Fig Comparison between the SANS- and the models-derived pairwise distance distribution function p(r) versus r The SANSderived p(r) versus r function, digitized from Fig 1b of ref [16], is shown as black circles connected with a black line Also shown are the calculated p(r) versus r functions for models A2bB3-bc (blue line), A2bB3-pec (orange line), A2bB3-ec (magenta line), and for a : mixture of models A2bB3-bc and A2bB3-ec (green line) OG moieties were removed from the models before computation of the p(r) versus r function (see the text for details) the closest fit, and a best-matching, partially extended ⁄ closed model, A2bB3-partially extended ⁄ closed (pec), was then individuated (Fig 2, orange line), having a angle of $ 80° between the thigh and calf-1 domains (Fig 1B,G) However, the less-than-optimal fit in the high r region suggested that the experimental data could also derive from a mixture of at least two welldefined conformations This was confirmed by the green curve in Fig 2, which was obtained by averaging, in a : ratio, the p(r) versus r of models A2bB3-bc and A2bB3-ec Although we did not attempt any further refinement, it is conceivable that a closer match could be obtained by mixing differently bent and extended models in appropriate ratios Finally, to allow a comparison to be made with the hydrodynamics of the other models, the OG moieties were re-attached to the A2bB3-pec model The transition to the extended-open forms was then achieved, starting from the A2bB3-ec model, by first restoring to 143° the angle between the thigh and calf1 domains, and then superimposing the aIIb b-propeller and the b3 bA ⁄ hybrid ⁄ PSI domains of the 3FCU structure [6] An initial extended ⁄ open ⁄ tail separated model [A2bB3-extended ⁄ open ⁄ tail separated (eots); Fig 1E,J] was then completed by allowing the b3 I-EGF1-4 and bTD domains to follow the swing-out, and repositioning the b3 TM helix on the same plane of its aIIb counterpart; each helix was surrounded by its own 50-molecule OG micelle We did not use a homology model of the b2 fully extended PSI ⁄ hybrid ⁄ I-EGF1-3 2P28 crystal structure [30] because it FEBS Journal 277 (2010) 3190–3202 ª 2010 The Authors Journal compilation ª 2010 FEBS 3193 Refined aIIbb3 models C Rosano and M Rocco would have pushed the b3 tail outside the hypothetical membrane plane [10] The other extended ⁄ open model, [A2bB3-extended ⁄ open ⁄ tails crossed (eotc) (Fig 1D,I)], was then made by first inserting the computer-based models of the TM helices [18,19] in the ‘open’ state, superimposing the two aIIb segments and re-attaching the NMR-based cytoplasmic domains The b3 subunit was then reconnected by first pivoting back the I-EGF3-4 and bTD domains by $ 29° along the C473C503 hinge, and then performing a slight rotation of the bTD module at the main chain of D606 to dock it against the calf-2 domain with its C-terminus in proximity of the N-terminus of the b3 ‘open’ TM helix Overall evaluation of the models The five new aIIbb3 models are shown in ribbon representation in Fig 1A–E, with their color-coded modules indicated in Fig 1E Surface and space-filling representations are then proposed in Fig 1F–J, where the ET map [9] is superimposed to the fully extended models Note how the aIIb extracellular modules, and the b3 bA module in the extended ⁄ closed conformation, fit extremely well in the map, with the b3 hybrid ⁄ PSI domains nicely occupying the remaining electron density blob in the extended ⁄ open conformations Furthermore, note how the left-side inferior lobe of the ET map is larger and could easily accommodate at least part of the b3 I-EFG4 ⁄ bTD domains in both the closed and open ⁄ tails-crossed models, again suggesting that in solubilized, primed aIIbb3, full tail separation is not the most populated state Close-up views of the b3 pivot region, highlighting the potential hinge movement, are shown in Fig 3A–D The C-terminal region of the models’ ectodomains, including the modeled E764-D774 and G840-Q873 loops, the attachment of the ectodomain to the TM helices, and the interfaces between the calf-2 and bTD modules, are also shown in Fig 3E–G Note the partial loss of the predicted P851-I861 helix within the G840-Q873 loop after introduction of the mature R856-D857 proteolytic cut In Table we present the computed sedimentation and diffusion coefficients for the five new models, compared with the experimental values (w and z ) for resting and primed aIIbb3 [10] As seen, the fully bent and partially extended models of the resting integrin are incompatible with the experimental data, while the extended ⁄ closed model is in excellent agreement, as previously noted for the less-refined A2b-5 ⁄ ET model [10] As for the extended ⁄ open structures, maintaining the TM helices in contact leads to hydrodynamic values in much better agreement (well 3194 within the experimental range) than those of the full swing-out model While this work was being prepared for resubmission, another work concerning the aIIbb3 structure appeared [17] In this study, purified full-length aIIbb3 was inserted into phospholipid nanodiscs [31], chromatographed and studied using EM and analytical ultracentrifugation In order to compare our models with this new evidence, we inserted them into atomic-scale models of the nanodiscs, which were prepared starting from a model containing a MSP1D1 membrane protein scaffold and · 80 1,2-dipalmitoyl-sn-glycero3-phosphocholine (DPPC) phospholipid molecules in the bilayer [31] (kindly provided by A Shih and S G Sligar, University of Illinois, IL, USA) The DPPC phospholipids were mutated in silico to 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) by removing the last two C atoms from the two hydro˚ phobic tails, followed by a translation, of $ A, of each layer in a direction perpendicular to the disc’s plane, thereby providing an approximate restoration of the bilayer interface, without any further optimization The total number of phospholipids was not changed, as MSP1 ⁄ DMPC nanodiscs were found to contain about the same number of units, 154 ± [32] Then, every second DMPC phosphocholine head was replaced with a phospho-(1¢-rac-glycerol) moiety to convert them into 1,2-dimyristoyl-sn-glycero-3-phospho-(1¢-rac-glycerol) (DMPG) phospholipids, assuming a : ratio of DMPC : DMPG in the nanodiscs used experimentally [17] The His-tag and TEV protease sequence (MGHHHHHHDYDIPTTENLYFQG), which was absent in the atomic model but present in the original MSP1D1 construct and not removed by Ye et al [17], was modeled by a combination of ab initio modeling using Robetta [25] and insertion from the 3GZH.pdb structure (YDIPTTENLYFQG) It was then grafted at the two N-termini of the MSP1D1-DMPC ⁄ DMPG nanodisc structure, again without any further optimization The computed molecular mass for this nanodisc model, 156 853 gỈmol)1, is in good agreement with the experimental value, of 181 500, measured by Ye et al [17], which was recalculated after correcting for the computed  of the nanodisc, 0.875 cm3Ỉg)1 (F Ye and v A Bobkov, personal communication) No further optimization of the DMPC : DMPG ratio was attempted, as it would have only slightly affected the computed hydrodynamic parameters About 18 DMPC : DMPG molecules were then removed to make space for the TM helices of the integrins, either from the center of the nanodisc or close to the protein belt, to take into account possible effects of the high gravitational field FEBS Journal 277 (2010) 3190–3202 ª 2010 The Authors Journal compilation ª 2010 FEBS Refined aIIbb3 models C Rosano and M Rocco A B C D E F G Fig Close-up views of selected regions in the new models Panels A-D: close-up views of the hinge region in four of the new models (A2bB3-bc, panel A; A2bB3-ec, panel B; A2bB3-eotc, panel C; A2Bb3-eots, panel D) The modules are aligned on the I-EGF3 module and color-coded as indicated in panel A, and the D477-Q482 loop (dark green) and the C473-C503 hinge disulfide (cyan) are clearly visible The blue sticks are the other disulfides present in the I-EGF1 ⁄ modules Panels E–F: close-up views of the bottom region of the ectodomain, connected to the TM helices, in models A2bB3-bc (panel E), A2bB3-ec (panel F) and A2bB3-eotc (panel G) The modules are color-coded as indicated in panel G The G840-Q873 modeled loop is clearly visible (orange-red), together with the post-translational cut at R856 and the new N-terminus at D857 (grey) The E764-D774 modeled loop is colored magenta present during ultracentrifugation In the lateral configuration, two different orientations of the integrins were considered (‘internal’ and ‘external’, see Fig 4), thus taking into account potential asymmetry effects These integrin ⁄ nanodisc models allowed computation of the sedimentation coefficients and their comparison with the published value [17], corrected to standard conditions Four representative models can be seen in Fig 4, and a comparison between experimental and computed s values is reported in Table Similarly to the OG-solubilized integrins, and in contrast with the EM images of Ye et al [17], the data in Table show FEBS Journal 277 (2010) 3190–3202 ª 2010 The Authors Journal compilation ª 2010 FEBS 3195 Refined aIIbb3 models C Rosano and M Rocco Table Comparison between experimental and computed hydrodynamic parameters for full-length, OG-solubilized aIIbb3 and the refined models Experimental ⁄ models w (S) Percentage ± SEM or percentage difference z (F) Percentage ± SEM or percentage difference aIIbb3 resting A2bB3-bc (bent ⁄ closed) A2bB3-pec (partially extended ⁄ closed) A2bB3-ec (extended ⁄ closed) aIIbb3 primed A2bB3-eots (extended ⁄ open ⁄ tails separated) A2bB3-eotc (extended ⁄ open ⁄ tails crossed) 8.18 ± 0.07a 9.08 8.99 8.20 7.65 Ä 7.97b 7.41 7.81 ± 0.9 + 11.0 + 9.9 + 0.2 )6.5 Ä )2.6c )9.4c )4.5c 3.07 ± 0.08a 3.31 3.27 2.98 2.64 Ä 2.99b 2.62 2.84 ± 2.6 + 7.8 + 6.5 )2.9 )14 Ä )2.5c )14.7c )7.5c a From a previous publication [10] experimental values b Range of experimental values as presented previously [10] that the fully bent integrin-conformation is at odds (+12 ) 14%) with the solution data, whereas the extended ⁄ closed conformation is in very good agreement with them (+1 ) 3%) Discussion The revised aIIbb3 models, which are now based on more complete structural evidence, allow a better evaluation of proposed alternative conformational states and the supposed transitions between them To begin with, the fully bent, crystallographic state observed for the avb3, aIIbb3 and axb2 ectodomains has now been found to be at odds with data on full-length, solubilized samples, either obtained by ‘bulk’ technologies, such as analytical ultracentrifugation [10,17], dynamic light scattering [10] and the recent SANS study [16], or by single particle averaging following ‘gentler’ EM methods, such as cryo-EM [8] and ET [9] Interestingly, from the hydrodynamic data, a strong consensus on the degree of extension between aIIbb3, solubilized either in OG [10] or in nanodiscs [17], is emerging Both data sets support a predominance of nearly fully extended structures in the resting state, fitting very well inside the ET map [9] (Tables and 2; Fig 1C,H; Fig 4B,D) However, the SANS data [16] are compatible with either an intermediate extension (Fig 1B,G), or, more likely, in our opinion, with an approximately equimolecular mixture of bent and extended structures (see Fig 2) Possible reasons for this discrepancy might derive from the different purification and solubilization procedures used in the SANS study, which involved a freeze-drying step and utilized Triton X-100 as the detergent [16] In particular, the micelles formed by Triton X-100 are much larger than those formed by OG (radii of 4.7 versus 2.3 nm, respectively [33,34]), and should clearly engulf also the cytoplasmic domains This could potentially deregulate any conformational control exerted at the inner membrane inter- 3196 c Percentage difference from the resting face, which should instead be preserved in the OG- and nanodiscs-solubilized samples The new aIIbb3 and axb2 ectodomain structures have been used to reinforce the scheme calling for bent resting integrins with a transition to fully extended, open, tails separated structures following activation [11,13] The recently published negative-staining EM images, derived either from ectodomain constructs [13] or from full-length, nanodisc-solubilized samples [17], also show a predominance of bent forms in the resting state Regarding the ectodomain studies, an important question can be raised of whether it is more ‘physiological’, for example, a truncated construct in which the TM and cytoplasmic regions are absent, immobilized either in a crystal lattice or on an EM grid, or is a full-length, native molecule isolated in a small micelles-forming mild detergent or in a confined lipid bilayer While good arguments could be made either way, is interesting to note that previous TEM work [7] has shown that integrin ectodomains can bind macromolecular ligands without relevant structural changes, suggesting that extension is not intrinsically needed for activation This was further supported by recent fluorescent lifetime imaging microscopy on full-length avb3 in live cells that did not detect any change in height following activation [12] However, it could instead be that the lack of the TM and cytoplasmic regions and ⁄ or the environmental conditions specifically deregulate fundamental conformational transitions in other integrins such as aIIbb3 The probable co-existence of bent and extended forms in Triton X-100, as suggested by our analysis of the recent SANS data [16] (see Fig 2), further reinforce the hypothesis that the cytoplasmic and TM regions are involved in this event However, it cannot be excluded that the purification procedures and the presence of detergent favor the extended conformation, although no signs of extra OG binding, besides presumably around the TM segments, were found in our solution studies [10], whereas the FEBS Journal 277 (2010) 3190–3202 ª 2010 The Authors Journal compilation ª 2010 FEBS Refined aIIbb3 models C Rosano and M Rocco A B C D Fig Nanodiscs-embedded aIIbb3 bent ⁄ closed and extended ⁄ closed models MSP1D1-DMPC ⁄ DMPG-nanodiscs centerembedded aIIbb3 models A2bB3-bc-ndc (panel A) and A2bB3-ec-ndc (panel B) and laterally embedded A2bB3-bc-ndle (panel C, ‘external’ orientation) and A2bB3-ec-ndli (panel D, ‘internal’ orientation) The aIIbb3 and MSP1D1 protein regions are shown in ribbon representation inside a semitransparent surface For aIIbb3, the color coding is as in Fig 1, while the two MSP1D1 identical subunits are orange and red, respectively The carbohydrates are shown as gold sticks, and the DMPC and DMPG lipids are shown in space-filling mode (light grey, DMPC; slate grey, DMPG) phospholipid moieties are confined within the scaffolds of the nanodiscs Furthermore, the extended ⁄ closed state appears to be a stable conformation in purified, solubilized full-length aIIbb3, which is fully able to make the transition to the extended ⁄ open state upon priming and to revert back to the extended ⁄ closed state when the priming agent is removed [35] In addition, the transition to the open state can take place on the cell membrane with changes in the integrin height that vary widely from system to system (e.g [36]; see also [37,38], and references therein) Instead, the recent studies with nanodiscs-embedded full-length aIIbb3 [17] provide apparently contradictory results: while the hydrodynamic data are fully consistent with an extended resting integrin, the EM images show a predominance of bent forms Importantly, the shape distribution appears to be rather bimodal, with the molecules assuming either a bent or an extended form, and an absence of well-defined intermediate conformations A similar bimodal distribution was also recently FEBS Journal 277 (2010) 3190–3202 ª 2010 The Authors Journal compilation ª 2010 FEBS 3197 Refined aIIbb3 models C Rosano and M Rocco Table Comparison between experimental and computed sedimentation coefficients for full-length aIIbb3 embedded in the MSP1D1-DMPC ⁄ DMPG nanodiscs and the new models Experimental ⁄ models w (S) Percentage difference aIIbb3 resting A2bB3-bc-ndc (bent ⁄ closed, nanodisc, centered) A2bB3-bc-ndli (bent ⁄ closed, nanodisc, lateral ⁄ internal orientation) A2bB3-bc-ndle (bent ⁄ closed, nanodisc, lateral ⁄ external orientation) A2bB3-pec-ndc (partially extended ⁄ closed, nanodisc, centered) A2bB3-ec-ndc (extended ⁄ closed, nanodisc, centered) A2bB3-ec-ndli (extended ⁄ closed, nanodisc, lateral ⁄ internal orientation) A2bB3-ec-ndle (extended ⁄ closed, nanodisc, lateral ⁄ external orientation) 9.02a 10.3 na + 14.2 10.3 + 14.2 10.1 + 12.0 10.1 + 12.0 9.31 + 3.2 9.26 + 2.7 9.13 + 1.2 a From a previous publication [17], after correction to standard conditions observed in the EM images of the axb2 ectodomain [13] Thus, true to the previously proposed ‘switchblade’ extension mechanism [39], the conformational change indeed appears to be snap-like, albeit not directly linked to priming or activation In this light, it could be hypothesized that interactions with the EM grid favor a snap-back to the bent conformation, thus offering an explanation for the apparent contradiction between the EM and the hydrodynamic data [17] Interestingly, the recent aIIbb3-nanodiscs EM study shows an increase of extended conformations upon binding of a talin head domain to the cytoplasmic tails [17], suggesting that this interaction indeed stabilizes the extended form, at least partially preventing a snapback to the bent form upon deposition on the EM grid As for the full tail separation, while it cannot be excluded that the constraints imposed by the solubilizing micelle could somewhat oppose it, it should be noted that in this system, after prolonged incubation with priming agents, the formation of dimers, trimers and oligomers seems not to be impaired [40], implying at least a re-arrangement at the TM level In addition, we would like to point out how the new EM images of talin head domain-bound full-length aIIbb3 integrin embedded in nanodiscs [17] are fully consistent with 3198 our previously developed model of extended ⁄ open integrins without tail separation [10], which has been further refined in the present study (Fig 1D,I; Table 1) The phospholipids environment and the relatively large radius of the nanodiscs should provide ample space to permit the integrin TM domains to fully separate on activation, and yet no clear-cut evidence of such an event emerges from the new EM images [17] Moreover, it is interesting to note that the pivot points probably utilized on extension [11] could as well act as a ‘universal’ joint, coupling the swingout to a simple conformational change in the TM helices In this respect, recent work has claimed that the b3 S527F mutation in aIIbb3 induces the high-affinity state by hindering the adoption of the bent conformation [41] However, the closest residue in the ‘head’ ˚ region, S401 of aIIb, is more than 20 A away in the bent conformation, while the S527F mutation perturbs a cluster of polar residues (R671 and N675 in aIIb, R498, D524, S527, R530, D546 and Y556 in b3) both in the bent and in the extended-closed conformations Furthermore, the S527-containing C523-C544 loop appears to be only slightly perturbed by the pivoting movement Therefore, it seems more likely that this mutation affects only the swing-out, thus favoring the high-affinity state, without any implication for the bent-to-extended transition Still in this respect, it has been proposed that integrins are inherently flexible, continuously exploring the conformations from bent to extended (e.g [3]), but NMA analysis does not support a fully flexible hinge at the I-EGF1-2 interface In addition, a Kratky plot of the SANS data has provided no evidence of intrinsic flexibility in Triton X-100-solubilized full-length aIIbb3 (see Fig S3 in [16]) While the extension could clearly take place around the b3 C473-C503 disulfide bond, this movement appears to require an ‘external’ driving force This could result from conformational changes at the cytoplasmic face, for instance following interactions with cytoskeleton components, or at the ectodomain ligand-binding region, such as in small moleculesinduced priming events However, given the complex disulfide pattern in this region (see Fig 3A–D), it could also be hypothesized that the extension is initially brought about under enzymatic control, as previously suggested (see [42], and references therein), perhaps following integrin insertion in the outer cell membrane Strong support for the bent-to-extended transition taking place on activation has instead been claimed from several antibody-binding studies (see [3], and references therein), recently revisited on the basis of the new axb2 structure [13] We have mapped the FEBS Journal 277 (2010) 3190–3202 ª 2010 The Authors Journal compilation ª 2010 FEBS C Rosano and M Rocco antibody-binding sites identified in aLb2 as markers of activation [43–45] on our refined models A2bB3-bc, A2bB3-ec and A2bB3-eotc (see Supporting information Fig S3) While the patches corresponding to the NKI-L16 and AO3 anti-aL antibody-binding sites seem to be more exposed in the extended ⁄ closed and extended ⁄ open conformations (Fig S3, panels B–C), they not appear to be buried in the bent ⁄ closed conformation (Fig S3, panel A) Noting that the majority of these epitope residues in the thigh module are close to the junction with the b-propeller module, it is possible that the differences in antibody binding stem from changes in the relative orientation of these two modules Indeed, a variation of $ 18° is observed between the thigh ⁄ b-propeller modules in 10 independent molecules in axb2 crystals [13], suggesting a degree of flexibility similar to that present at the I-EGF1 ⁄ interface These changes could be induced by binding events at the b-propeller ⁄ bI interface, or be a long-range propagation of the hybrid domain swing-out As for the CRB-LFA-1 ⁄ and KIM127 epitopes mapped on the b3 subunit, they are clearly buried in the bent conformation (Fig S3, panel A) However, antibody binding might be somewhat restricted also in the extended ⁄ closed conformation model (Fig S3, panel B), while all sites appear to be fully exposed in the extended ⁄ open conformation model (Fig S3, panel C) Finally, the activating mutations mapped to the E534 and M535 residues in b3 (steel blue spheres in Fig S3, panels B and C) are more difficult to reconcile with the extension being uncoupled from activation, as they are not in contact with other residues in the A2bB3-ec and A2bB3-eotc models We note, however, that in the extended ⁄ open model, the I-EGF3 module (where these residues reside) has undergone a clockwise rotation that would bring them facing, and possibly contacting, the aIIb subunit if this orientation was present also in the extended ⁄ closed state Our extended ⁄ closed model was conservatively developed, introducing the fewest possible changes not supported by experimental evidence However, it cannot be excluded that a rotation of the lower b3 leg already accompanies extension, bringing the activating residues into contact with other residues in the aIIb subunit In addition, some caution should be exerted in transferring the results of antibody-binding and mutational studies, carried out on different integrins, to the aIIbb3 integrin, which might be controlled by a different conformational regulatory system Given the complexity of the integrin activation ⁄ signaling network (e.g see [46] and references therein) further experimental work is clearly needed to fully resolve these issues Refined aIIbb3 models The putative structured segment in the calf-2 loop that was consistently generated by ab initio modeling in all integrin a subunits that undergo post-translational cleavage could be physiologically relevant, but caution should be exerted to avoid overinterpretations being made A more extensive study, for instance involving repeating the ab initio generation after scrambling the loop sequences, should be performed to strengthen the prediction, but this is outside the scope of the present study In any case, that this loop was not resolved in any ectodomain crystal structure indicates its inherent flexibility, which should be enhanced by the post-translational cleavage and is probably accompanied by loss of the putative secondary structure in aIIbb3, where the cut is located in the middle of the predicted helix Indeed, the two loop fragments are outside the ET map in the models shown in Fig Apart from studies demonstrating the necessity of this cleavage for the proper function of avb5 [47], little is known of its role in integrins lacking an a-I ⁄ A domain (see also [1]) With the above-mentioned caveats, it is tempting to speculate that the putative structure in this region is involved in interactions with other membrane proteins, perhaps helping integrins to assume or modulate their resting conformation NMR structural studies of this module, with and without proteolytic cleavage, could help to clarify this issue To conclude, our work further suggests that all evidence should be accounted for when proposing integrin activation mechanisms In particular, the bent-toextended transition has been repeatedly implied as a key step in integrin activation [3,11,13], but the solution evidence on full-length integrins seems to indicate otherwise Although the thermodynamic cost of such a huge conformational change could still be manageable, its physiological need is unclear, and alternative explanations for the role of the bent conformation, such as the ‘packaging for transport’ hypothesis that we have already proposed [10], should be investigated on a caseby-case basis The refined models presented here, which have been deposited in the public Protein Model DataBase (PMDB; http://mi.caspur.it/PMDB), should also aid the design of mutational studies aimed to fully clarify these important issues While we recognize that they clearly cannot provide definitive atomic details of unresolved, modeled segments, such as the calf-2 long loop and the relative orientation of the ectodomain ⁄ TM regions, or of the changes induced by extension not seen in crystal studies, we exerted great care and chose conservative assumptions during modeling Our refined models should also allow more realistic steered molecular dynamics simulations of the conformational FEBS Journal 277 (2010) 3190–3202 ª 2010 The Authors Journal compilation ª 2010 FEBS 3199 Refined aIIbb3 models C Rosano and M Rocco transitions of integrins (e.g [11]) to be performed, for instance by the use of nanodiscs-embedded models Materials and methods Molecular models were built and refined mostly as previously described [10] The Modeller ModLoop utility [23] (http://modbase.compbio.ucsf.edu/modloop) was used for intermediate-length loop modeling, while for the longer, non-resolved loop in aIIb, the Robetta ab initio approach [25] was employed on a dedicated webserver (http://robetta bakerlab.org/) with default parameters The hydrodynamic parameters were computed using the SOMO bead-modeling approach [20] with the US-SOMO implementation [21,22] (http://www.ultrascan.uthscsa.edu/) The US-SOMO set˚ tings were: accessible surface area cut-offs of 20 A and 50% for the atomic structures and for the beads in the models, respectively; synchronous overlap removal in all steps, with outward translation of the exposed side-chain beads; bead fusion thresholds of 70% between exposed beads; exclusion of the buried beads from the hydrodynamic computations and volume corrections; and stick boundary conditions and computation referred to the diffusion center The partial specific volume  of the singlev micelle models was 0.723 cm3Ỉg)1, while that of the twov micelles model was 0.731 cm3Ỉg)1 [10] The  values for DMPC and DMPG were 0.973 cm3Ỉg)1 (adjusted at 20 °C from the experimental value at 30 °C [48]) and 0.925 cm3Ỉg)1 (adjusted at 20 °C from the calculated [49] value at 25 °C), respectively The calculated  value at 20 °C for the v MSP1D1 nanodisc scaffold protein with the initial methionine, the His-tag and the TEV protease site, was 0.731 cm3Ỉ v g)1 For the nanodiscs-embedded integrins, the calculated  at 20 °C thus was 0.776 cm3Ỉg)1, assuming a : ratio of DMPC : DMPG and 142 total lipid molecules (160 ) 18 that were removed to make space for the TM regions of the integrins) The pairwise-distance distribution function utility of the new SAXS ⁄ SANS simulation module within US-SOMO [22] was used to compute the p(r) versus r for the models The experimental p(r) versus r data were digitized from the data in Fig 1b of ref [16] using the DigitizeIt 1.5 shareware program (http://www.digitizeit de/), and the p(r) was then normalized to 1.0 The YinOYang 1.2 [50] (http://www.cbs.dtu.dk/services/YinOYang/) was employed for O-glycosylation predictions NMA was performed utilizing the elNemo server [26] (http://www.igs cnrs-mrs.fr/elnemo/), as previously reported [10] Morphing between conformations was carried out with the Yale morph server [27] (http://molmovdb.org/morph/) using CNS (adiabatic mapping) interpolation Alignments were performed using ClustalW [24] on the UniProt server (http://www.uniprot.org/) with default settings With the exception of Fig S2, molecular graphics images were produced using the UCSF Chimera package [51] (alpha version 1.3, build 2577) from the Resource for Biocomput- 3200 ing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR01081; http://www.cgl.ucsf.edu/chimera/), which was also used for fitting models inside the ET map Paint Shop Pro (Jasc Sofware, Corel Inc., Mountain View, CA, USA; http://www.corel.com) was used to assemble all figures Acknowledgements We thank R.R Hantgan (Wake Forest University, NC, USA) for comments We are grateful to M.A Arnaout (Harvard Medical School, Charlestown, MA, USA) for very kindly providing us with the coordinates of the new avb3 ectodomain structure before public release; to F Ye, M Ginsberg (UCSD, CA, USA) and A Bobkov (The Burnham Institute, CA, USA) for providing important details of their experimental work and feedback on our calculations of the nanodiscs properties; and to A Shih and S.G Sligar (University of Illinois at Urbana-Champaign, IL, USA) for providing a nanodisc atomic model and related information We are indebted to E Brookes (UTHSCSA, San Antonio, TX, USA) for his constant and timely improvements to the US-SOMO program This work was partially supported by the Italy-USA project ‘Farmacogenomics oncology – Oncoproteomics’ (Grant 527B ⁄ 2A ⁄ 3) to MR The models accession codes in the PMDB database are PM0076386 (A2bB3-bc), PM0076372 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Nejjari M, Marvaldi J & Luis J (2004) The endoproteolytic processing of avb5 integrin is involved in cytoskeleton remodelling and cell migration FEBS Lett 557, 159–163 48 Greenwood AI, Tristram-Nagle S & Nagle JF (2006) Partial molecular volumes of lipids and cholesterol Chem Phys Lipids 143, 1–10 49 Durchschlag H & Zipper P (1994) Calculation of the partial volume of organic compounds and polymers Prog Colloid Polym Sci 94, 20–39 50 Gupta R & Brunak S (2002) Prediction of glycosylation across the human proteome and the correlation to protein function Pac Symp Biocomput 7, 310–322 51 Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC & Ferrin TE (2004) UCSF Chimera – a visualization system for exploratory research and analysis J Comput Chem 25, 1605–1612 Supporting information The following supplementary material is available: Fig S1 Alignment of the long loop region in the calf2 module of integrin’s a subunits Fig S2 A gallery of structures predicted by Robetta [20] for the long loop in the calf-2 region of integrins’ a subunits without the insertion domain Fig S3 Mapping the aL and b2 activation-sensitive antibodies binding sites on the aIIbb3 models A2bB3bc (panel A), A2bB3-ec (panel B) and A2bB3-eotc (panel C), shown in surface representation mode with the relevant residues highlighted in space-filling mode This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 277 (2010) 3190–3202 ª 2010 The Authors Journal compilation ª 2010 FEBS ... new models, compared with the experimental values (w and z ) for resting and primed aIIbb3 [10] As seen, the fully bent and partially extended models of the resting integrin. .. 2010 FEBS Refined aIIbb3 models C Rosano and M Rocco A B C D E F G Fig Close-up views of selected regions in the new models Panels A-D: close-up views of the hinge region in four of the new models. .. 2010 FEBS Refined aIIbb3 models C Rosano and M Rocco A B C D Fig Nanodiscs-embedded aIIbb3 bent ⁄ closed and extended ⁄ closed models MSP1D1-DMPC ⁄ DMPG-nanodiscs centerembedded aIIbb3 models A2bB3-bc-ndc