How Natalizumab Binds and Antagonizes α4 Integrins*

Background: How does the multiple sclerosis therapeutic antibody natalizumab bind to α4 integrins? Results: Natalizumab binds the α4 β-propeller domain outside the ligand binding groove for domain 1 of vascular cell adhesion molecule (VCAM) and non-competitively antagonizes binding. Conclusion: Natalizumab may push domain 2 of VCAM into a non-preferred orientation upon integrin binding. Significance: Positioning of species-specific substitutions outside of ligand-binding sites leads to surprising antibody mechanisms of action. Natalizumab antibody to α4-integrins is used in therapy of multiple sclerosis and Crohn's disease. A crystal structure of the Fab bound to an α4 integrin β-propeller and thigh domain fragment shows that natalizumab recognizes human-mouse differences on the circumference of the β-propeller domain. The epitope is adjacent to but outside of a ligand-binding groove formed at the interface with the β-subunit βI domain and shows no difference in structure when bound to Fab. Competition between Fab and the ligand vascular cell adhesion molecule (VCAM) for binding to cell surface α4β1 shows noncompetitive antagonism. In agreement, VCAM docking models suggest that binding of domain 1 of VCAM to α4-integrins is unimpeded by the Fab, and that bound Fab requires a change in orientation between domains 1 and 2 of VCAM for binding to α4β1. Mapping of species-specific differences onto α4β1 and α4β7 shows that their ligand-binding sites are highly conserved. Skewing away from these conserved regions of the epitopes recognized by current therapeutic function-blocking antibodies has resulted in previously unanticipated mechanisms of action.

Structure Determination and Refinement-The structure was solved by molecular replacement using Phaser (17) first searching with the ␣ 4 ␤-propeller domain, then with different Fab, and then the ␣ 4 thigh domain (11). Models were iteratively improved by building with COOT (18), refinement with PHENIX (19), and validation with MOLPROBITY (20).
Various concentrations of 125 I-natalizumab Fab in 10 l of HBS were added to the cells to give final cation concentrations of 2 mM Mn 2ϩ and 0.2 mM Ca 2ϩ . After 2 h, bound ligand was quantified as described (21). Nonspecific binding was determined in the presence of 100 g/ml of cold natalizumab IgG for Fab concentrations of 10 nM or lower. At higher Fab concentrations, the IgG concentration was increased proportionally. Specific binding was calculated by subtracting nonspecific Fab binding from total binding.
Competition with increasing concentrations of non-labeled natalizumab Fab or IgG was performed similarly with 20 nM 125 I-natalizumab Fab. Protein concentrations were determined by measuring A 280 . Extinction coefficients for natalizumab Fab and IgG were calculated from amino acid sequences for the constant regions of human immunoglobulin ␥4 and chains (Uniprot) and the variable regions of natalizumab (22 To calculate ternary complex model constants, the same data set was fitted to the allosteric EC 50 shift model built into the Prism software (Equations 3 and 4) (24). Noncompetitive antagonism is described by the ternary complex model, in which the K D for a ternary complex equals K D /␣ for the binary complex (24). ␣, the ternary complex constant, describes how an allosteric modulator affects ligand binding, and K D,VCAM is the equilibrium dissociation constant for VCAM binding.

RESULTS
Natalizumab Fab co-crystallized with the integrin ␣ 4 -subunit from an ␣ 4 ␤ 7 headpiece preparation (Figs. 1A and 2) with one complex in the asymmetric unit. Resolution could be extended (16) to 2.84 Å ( Table 1). The ␤ 7 headpiece fragment dissociated during crystallization; only the Fab and ␣ 4 headpiece fragment are present in crystals (Figs. 1A and 2B). Dissociation was unrelated to natalizumab Fab binding, because natalizumab actually stabilizes association between ␣ 4 and ␤ 7 (Fig. 2, A and B), as previously demonstrated with vedolizumab (11). Furthermore, previous electron microscopy (EM) shows natalizumab binding to the ␣ 4 ␤ 7 headpiece with excellent density for the ␤ 7 subunit, and the same orientation between the ␣ 4 subunit and natalizumab Fab as seen in the crystal structure ( Fig. 1D) (11). The high pH of 9.0 and 5% organic solvent dioxane in crystallization may have contributed to ␣ 4 and ␤ 7 dissociation. The structure of the ␣ 4 ␤-propeller and thigh domains are essentially identical to those in the vedolizumab Fab complex, except for a difference in orientation of 8 to 24°at the flexible interface between the ␤-propeller and thigh domains (Fig. 1, A and B).
A previously undescribed (11) interaction between the thigh FGЈ loop and the ␤-propeller domain is conserved in all five examples of ␣ 4 ␤ 7 and ␣ 4 in crystal lattices. When superposition is based on the ␤-propeller domain, residues 563-569 in the highly extended thigh FGЈ loop vary little in position (Fig. 3A). In contrast, when superposition is based on the thigh domain, their position varies markedly (Fig. 3B). Stronger coupling of the thigh FGЈ loop to the ␤-propeller domain than to the thigh domain is consistent with extensive structural interactions.
Before describing this interaction, let us briefly review the integrin ␤-propeller structure (25). Each ␤-sheet, also termed a propeller blade or "W," has four anti-parallel ␤-strands arranged like legs of the letter W. The blades assemble around a 7-fold pseudosymmetry axis, with ␤-strand 1 lining a solventfilled central cavity and ␤-strand 4 forming the circumference of the propeller (Fig. 1C). The last three blades in ␣ 4 have Ca 2ϩbinding sites in their ␤1-␤2 loops (26). The sequence wraps around the propeller so blades 1 and 7 are adjacent; furthermore, in blade 7, ␤1-␤3 and ␤4 are from C-terminal and N-terminal portions of the ␤-propeller sequence, respectively (Fig.  1C).
The thigh FGЈ loop stabilizes the region of the ␤-propeller where its N and C termini are knit together in W7 (Figs. 1C and 3A). Thigh residue Phe-563 binds in a hydrophobic pocket formed by Leu-9 at the beginning of ␤-strand 4 and Leu-427 at the end of ␤-strand 3 in blade 7, and Ile-60 in the ␤3-␤4 loop of blade 6 ( Fig. 3A). Phe-563 also packs against the aliphatic portion of Asp-408 that coordinates Ca 2ϩ in the ␤1-␤2 loop of blade 7. Moreover, Arg-429 in the loop following the ␤3-strand of blade 7 forms a -cation bond to Phe-563, and hydrogen bonds to both Asp-408 and the backbone following Phe-563 (Fig. 3A). Leu-566 contributes a hydrophobic environment that stabilizes these polar interactions, and also interacts with Pro-432 and the aliphatic portion of Arg-431, which hydrogen bonds to the backbone of thigh residue 567. These interactions with the thigh domain may help the ␤-propeller domain to fold, and are consistent with findings for a large number of different integrins that heterodimer fragments containing ␣-subunits truncated after the thigh domain, but not after the ␤-propeller domain, are well expressed.
The Natalizumab Binding Site-Natalizumab binds to ␣ 4 ␤-propeller blades 2 to 4 ( Fig. 1C). The ligand binding groove in ␣ 4 ␤ 7 is formed at the interface between the ␣ 4 ␤-propeller and ␤ 7 ␤I domains ( Fig. 1B) (11). ␣ 4 forms one wall of this groove and part of its lower surface. Natalizumab binds beside this groove (Fig. 1A). Long loops in the ␤-propeller that help form this groove separate natalizumab from the groove (Fig. 1, A and  B). Among a large number of individual ␣ 4 ␤-propeller amino acid substitutions tested, only mutations of Tyr-187, Trp-188, and Gly-190 decreased binding to VCAM (27). Tyr-187 forms part of the ␣ 4 wall, Trp-188 forms part of the bottom of the groove (Fig. 1A) and each contacts a small molecule ␣ 4 antagonist (Fig. 1B). Notably, Tyr-187 and Trp-188 are distal from the natalizumab footprint (Fig. 1A). Gly-190 is in a highly conserved, buried position in the seven FG-GAP sequence repeats that form integrin ␤-propellers. The Ala side chain in the G190A mutation clashes with the backbone of Pro-183 and is expected to alter the conformation of the loop bearing Tyr-187 and Trp-188.
The circumference of integrin ␤-propellers is formed by the ␤4 strands; the ligand binding and ␤I domain-binding face is formed by the ␤4-␤1 and ␤2-␤3 loops, and the opposite face is formed by the ␤1-␤2 loops (some of which bind cations) and ␤3-␤4 loops. Natalizumab Fab binds to the circumference of the ␤-propeller, with its footprint extending all the way to the face bearing the ␤3-␤4 loops, but missing by 15 to 20 Å the face Pearson's correlation coefficient between average intensities of random half-datasets of the measurements for each unique reflection (16). c R work ϭ ⌺ hkl ʈF obs (hkl)͉ Ϫ ͉F calc (hkl)ʈ/⌺ hkl ͉F obs (hkl)͉. d R free is the R value obtained for a test of reflections. e Residues in favored, allowed, and outlier regions calculated with the MolProbity server (20).
of the ␤-propeller that binds the ␤I domain and ligand and bears Tyr-187 and Trp-188 ( Fig. 1A and Fig. 4). The epitope includes the ␤4-␤1 loop preceding W3, the ␤4-strand of W3, and the ␤3-␤4 loops of W3 and W4 (Fig. 4). The footprint extends to the tips of the ␤3-␤4 loops of W3 and W4 on the divalent cation binding ␤ face, but includes only tip-distal portions of the W3 ␤4-␤1 loop on the ligand binding face. Natalizumab induces no significant change in ␣ 4 conformation. The ␤-sheets and loops of the ␣ 4 ␤-propeller domain have the same conformations as when natalizumab is not bound and the ␤ 7 ␤I domain is bound (Fig. 4A). The side chains differ no more in rotamer than would be seen for the same protein in different crystal lattices.
Natalizumab buries a total solvent accessible surface area of 2060 Å 2 , with the heavy and light chains burying close to 75 and 25% of this area, respectively. The contact is dominated by the H chain CDR3 loop, which extends into a cleft on the cylindrical ␤-propeller surface formed between W2 and W3 (Fig. 4B). The central portion of the cleft is lined by invariant ␣ 4 hydrophobic residues that include Tyr-202 in ␤4 of W2, Pro-149 near the end of ␤4 of W2, and Leu-206 and Phe-162 of the ␤1-4 loops following W2 and W3, respectively (Fig. 4A). The long CDR H3 loop extends deep into this crevice, enabling its Tyr-101 and Tyr-108 residues both to bury hydrophobic residues and form hydrogen bonds to main chain atoms in ␤4 of W3 and in the segment following ␤4 of W2 (Fig. 4B). Contact even reaches deeper to Phe-193 in the ␤3-strand of W3 (Fig. 4A). ␤-Propeller loops that form and surround the hydrophobic pocket also contribute contacts, including the ␤4-␤1 loops following W1, W2, and W3 and the ␤3-␤4 loops of W3 and W4. Lys-201 in the W3 ␤4-strand and Lys-256 in the W4 ␤3-␤4 loop, each more peripheral than the central hydrophobic region yet still well within the antibody-binding interface, make important, charged hydrogen bonds to the antibody and insert in negatively charged antibody pockets. The Gln-152 side chain forms a hydrogen bond to the CDR H3 backbone (Fig. 4B).
Natalizumab recognizes an ␣ 4 region with significant sequence variation among human and mammals commonly used in disease models and toxicology (Fig. 5). By contrast, the long narrow binding groove for small molecule antagonists and the biological ligands MAdCAM and VCAM is highly conserved in ␣ 4 ␤ 7 (Fig. 5A) and invariant in ␣ 4 ␤ 1 (Fig. 5C). Domain 1 of VCAM can be docked in this groove with some precision (Fig.  5D) by placing the Asp side chain of its rigid, integrin-binding loop in the same ␤I domain MIDAS Mg 2ϩ -coordinating position as the carboxyl group of a co-crystallized antagonist (11) (Fig. 5A). This model is well supported by the excellent fit of D1 of VCAM into the ␣ 4 ␤ 1 or ␣ 4 ␤ 7 ligand-binding groove with the long axis of D1 aligned with the groove (11). Furthermore, this model places VCAM in contact with ␣ 4 residues Tyr-187 and Trp-188 (Figs. 1A and 5), shown mutationally to be important in binding VCAM (27). The orientation between D1 and D2 of VCAM-1 is variable among crystal structures (28,29) (Fig. 6). D1 of docked VCAM does not clash with natalizumab Fab; however, where D2 of VCAM emerges from the groove, it clashes in some orientations (Fig. 6, A and B). Our docking model thus predicts a noncompetitive mechanism of antagonism where natalizumab would lower affinity of ␣ 4 ␤ 1 by limiting the number of VCAM conformations accessible for binding (Fig. 6B).
Mechanism of Inhibition by Natalizumab-We set up an assay of competition between VCAM and 125 I-natalizumab Fab. Affinities were measured on Jurkat T lymphoblastoid cells in buffer containing 2 mM Mn 2ϩ and 0.2 mM Ca 2ϩ to activate integrin ␣ 4 ␤ 1 ; Jurkat cells express ␣ 4 ␤ 1 and lack ␣ 4 ␤ 7 . Binding was maximal after 2 h (Fig. 7A) and was saturable with 86,000 Ϯ 6,000 Fab bound per cell with a K D ϭ 19.6 Ϯ 4.6 nM (Fig. 7B). Given the importance of tyrosines in the CDR loops of natalizumab, we expected that their iodination might lower affinity. Non-labeled natalizumab Fab inhibited radiolabeled Fab binding with K I ϭ 6.4 nM (Fig. 7C). The difference in affinity between labeled and unlabeled Fab is significant and important for appreciating the affinity of the unlabeled Fab; however, it does not affect the measurements of competition with VCAM described in the following section. Inhibition of 125 I-natalizumab Fab by unlabeled natalizumab IgG occurs with an apparent K I ϭ 0.28 nM (Fig. 7C). The 20-fold higher avidity of the IgG than the affinity of the Fab demonstrates bivalent binding of natalizumab IgG to cell surface ␣ 4 ␤ 1 .
125 I-Natalizumab Fab binding was competed with a VCAM fragment containing domains 1 and 2 (D1D2) (Fig. 7D). The range of VCAM concentrations from 0.1 to 100 M spanned its K D for ␣ 4 ␤ 1 as shown below (Fig. 7D). Furthermore, the range of 125 I-Fab concentrations from 0.1 to 100 nM spanned the K D values of Fab for ␣ 4 ␤ 1 and extended to the concentration giving saturation binding in the absence of VCAM. All data were fit globally to an allosteric ternary complex model (24), which gave the lines shown in Fig. 7D and to a Gaddum-Schild equation, which gave the lines shown in Fig. 7E. The two methods yielded nearly identical estimates of the K D of VCAM (1.9 or 1.6 M) and the K D of 125 I-natalizumab Fab (24.7 or 22.9 nM). The Schild slope was S ϭ 0.595 Ϯ 0.084, with a 95% confidence interval of 0.429 to 0.760 (Fig. 7E). The data fit noncompetitive antagonism significantly better than competitive antagonism with S ϭ 1 (F-test, p ϭ 0.0003). The fit to the allosteric model yielded a ternary complex constant ␣ ϭ 0.083 (Fig. 7D). The ␣ value is significantly less than 1, showing that natalizumab is an allosteric inhibitor (24).
The allosteric coupling constant is the factor by which the affinity constant is modified by binding of the allosteric modulator (noncompetitive antagonist

DISCUSSION
Our study shows how a clinically important antibody binds to the ␤-propeller domain of ␣ 4 integrins, noncompetitively blocks binding of VCAM to ␣ 4 ␤ 1 , and illuminates a large number of previous studies on ␣ 4 integrin structure and function.
Integrin ␣ 4 antibodies were classified by effect on function and cross-competition as binding to epitopes A, B, or C (30). Antibodies to epitope B block binding to VCAM and fibronectin, whereas antibodies to epitopes A and C partially inhibit binding only to fibronectin or are non-inhibitory, respectively. The mouse anti-human precursor to natalizumab (31), TY21/6, binds the B epitope (32).
Among the ␣ 4 residues that natalizumab contacts, only four differ between mouse and human (Fig. 5, B and E). Previous chimera work showed that replacing the human ␣ 4 sequence FIGURE 5. Species-specific differences around the integrin ␣ 4 ␤ 7 and ␣ 4 ␤ 1 ligand-binding sites. Conservation on the solvent accessible surface is displayed from invariant (1, green or blue) to low (0.1, red) for A, C, and D using the species shown in panel E. In B, mouse-human sequence is shown as invariant (green or blue) or different (red). Invariant ␣ and ␤ subunit residues are shown in green and blue, respectively, to visualize the subunit interface. Additionally, invariant VCAM-binding ␣ 4 residues Tyr-187 and Trp-188 are in yellow and ␤ subunit MIDAS and ADMIDAS metal ions are shown as green spheres. Antibody footprints are outlined in yellow dashes (N for natalizumab) and key antigenic residues are labeled. A small molecule antagonist bound to ␣ 4 ␤ 7 is shown in stick with orange carbons (11). VCAM is shown in ribbon representation in the orientation found previously in docking to ␣ 4 ␤ 7 (11). A-D are in identical orientations. The ␣ 4 ␤ 1 model was made by superimposing the ␤ 1 ␤I domain from ␣ 5 ␤ 1 (44) onto ␣ 4 ␤ 7 (11). The location of the ligand binding groove is marked with dashed lines. Sequence conservation was calculated by AL2CO (51) with species equally weighted, using the sum of pairs measure with the BLOSUM62 matrix with normalization of the scoring matrix. There was no further normalization, so results for ␣ 4 , ␤ 1 , and ␤ 7 all use the same scale. E, sequence variation in the natalizumab epitope. Sequences are of species found to be positive (ϩ) or negative (Ϫ) for natalizumab reactivity in European Medicines Agency filings. Residues in the epitope are numbered. Residues most important for species reactivity are in bold. Residues that differ from human are red.
with mouse segments of 50 to 100 residues between residues 108 and 268, i.e. ␤-propeller blades 2 to 4, was sufficient to abolish or diminish reactivity of all tested B epitope antibodies (33,34). Because the smallest regions studied encompassed an entire ␤-propeller blade, whether antibodies mapped to the propeller circumference bearing the ␤4-strand, to the ␤I-proximal ␤4-␤1 or ␤2-␤3 loops, or to the cation-loop proximal ␤1-␤2 and ␤3-␤4 loops, could not be defined. Furthermore, because none of the studies demonstrated that a reciprocal swap would reconstitute the epitope, species-specific residues required for the epitope might have extended beyond the minimal mouse region required to eliminate binding, and indeed this was suggested by some swaps that did not completely eliminate reactivity. Therefore, little could be said previously about where B epitope antibodies bound, except that their epitopes included portions of ␤-propeller blades 2-4.
However, based on our structure, previous mapping data, and the finding that all B epitope antibodies cross-block one another, it now seems likely that all B epitope antibodies bind to a site that overlaps with natalizumab, and also do not overlap with the ligand-binding site. Thus, reactivity of HP1/2, HP2/1, HP2/4, L25, and P4C2 is abolished or decreased Ն80% by mouse residues 152-203 (34), which replace four human residues including two in the natalizumab epitope, Gln-152 and Lys-201 (Table 2). P4C2 and Z0E4 reactivity is abolished by mouse residues 108 -182 (33), which alter nine human residues including Gln-152 (Table 2). HP2/1 and SG/73 reactivity is abolished by mouse residues 195-268 (33), which replace four residues including Lys-201, Lys-208, and Lys-256 in the natalizumab epitope ( Table 2). In summary, there is substantial overlap between the species-specific residues recognized by natalizumab and the regions to which reactivity has been mapped for all previously characterized, function-blocking ␣ 4 integrin antibodies.
During development of natalizumab, it was tested on a range of species in disease model and toxicology studies. The species variation in reactivity with natalizumab (Fig. 5E) is in excellent agreement with our structural analysis in demonstrating the importance of residues Gln-152, Lys-201, and Lys-256. When any one of these differs from human, reactivity with natalizumab is lost (Fig. 5E). By contrast, Lys-208, on the edge of the epitope (Fig. 4A), is not required for reactivity (Fig. 5E).
The K D of natalizumab Fab for cell surface ␣ 4 ␤ 1 is 6.4 nM. The K D of its IgG is 0.28 nM, in agreement with previous measurements (22). The 20-fold higher affinity of the IgG strongly suggests that it binds bivalently to cell surface ␣ 4 integrins.
Although natalizumab starts out bivalent, after administration much of it becomes functionally monovalent. Like other IgG4 antibodies, its hinge region cysteines are partially reduced, and half-molecules containing a heavy and light chain pair exchange with other IgG4 molecules. Thus a substantial fraction of natalizumab becomes monovalent (35). Therefore in patients natalizumab is both bivalent and monovalent, and the monovalent IgG4 is expected to bind with a K D of 6.4 nM as measured here with Fab.
The VCAM D1D2 fragment has a K D of 2 M for Mn 2ϩactivated, cell surface integrin ␣ 4 ␤ 1 . The binding site centered in D1 includes a portion of D2, but there is no evidence it extends beyond D2 into D3 (36,37). MAdCAM, which contains only two IgSF domains, binds to integrin ␣ 4 ␤ 7 similarly to the docking model with VCAM, with much of D2 not in contact with the integrin; D3 if present would locate far from the integrin (11). VCAM is alternatively spliced, with IgSF domain 4 (D4) present in an isoform with 7 IgSF domains (7D) and absent in the isoform with 6 IgSF domains (6D) in the ectodomain. Domain 4 shows high sequence identity to domain 1, an identical integrin-binding motif, and binds to ␣ 4 ␤ 1 . The 7D isoform How Natalizumab Binds NOVEMBER 8, 2013 • VOLUME 288 • NUMBER 45 with D1 mutationally removed binds similarly to the native 6D isoform (38 -40).
The D1D2 fragment of VCAM used here ensures measurement of true, i.e. monovalent, affinity. Previous careful estimates of the affinity of the 7D fragment of VCAM for cell surface ␣ 4 ␤ 1 in Mn 2ϩ range from 9 to 33 nM (41). The much higher affinity estimates for 7D than D1D2 VCAM fragment strongly suggest that the 7D fragment binds bivalently to cell surface ␣ 4 ␤ 1 . In general, adhesion molecules have low affinity for their ligands compared with receptors for soluble ligands. A fulllength five IgSF ectodomain fragment of ICAM-1 binds to Mn 2ϩ -activated cell surface LFA-1 (integrin ␣ L ␤ 2 ) with a K D of 9 M (21), similar to the K D of 2 M measured here for binding of ␣ 4 ␤ 1 to VCAM D1D2.
The noncompetitive inhibition mechanism described here is a departure from previous concepts on how antibodies block function, particularly for integrins, which are known to undergo conformational change. Natalizumab clearly binds to a different site on ␣ 4 ␤ 1 than VCAM, making the noncompetitive mechanism conceptually easy to appreciate. A change in the conformational space accessible to D2 of VCAM that is imposed by natalizumab is the simplest explanation of the structural and ligand competition data. Crystal structures show multiple orientations between domains 1 and 2 of VCAM that differ by up to 35° (Fig. 6A). No doubt, greater variation in the D1-D2 orientation is possible than has yet been sampled by crystallography. D2 of VCAM has a role in binding integrin ␣ 4 ␤ 1 (37), and another interpretation of our results is that natalizumab would not only affect the D1-D2 orientation but also disrupt some of the interactions through D2 while leaving intact the more important interactions with D1. The net result is a 12-fold loss in affinity of ␣ 4 ␤ 1 for VCAM imposed by natalizumab. In contrast to these results, structural studies with several inhibitory antibodies to integrin ␤-subunits have directly demonstrated truly allosteric effects. These antibodies bind too far from the ligand-binding site to sterically hinder ligand binding, and stabilize the low-affinity, closed integrin headpiece relative to the high-affinity, open integrin headpiece (42)(43)(44).
Other B epitope antibodies inhibit VCAM binding to ␣ 4 ␤ 1 by a similar noncompetitive mechanism (32). The measurements were less quantitative, because they used bivalent IgG and ELISA with wash steps that disrupt equilibrium binding. Nonetheless, plots of single-reciprocal antibody binding were hyperbolic with increasing VCAM concentration ( Fig.  7E shows this type of plot), and suggested an allosteric or noncompetitive mechanism of action. These data were interpreted as an allosteric change induced by VCAM in the integrin ␣ 4 -subunit (32).
Formally, neither our structural or competition data can rule out this alternative mechanism of a VCAM-induced change in the conformation of the ␣ 4 ␤-propeller domain that lowers the affinity of natalizumab for its epitope. The epitope does not change conformation in ␣ 4 structures to date, i.e. when bound to natalizumab, when ␣ 4 is bound to the ␤ 7 subunit, which in turn is bound to vedolizumab, or when additionally a small molecule antagonist binds to ␣ 4 and ␤ 7 in the ligand-binding groove (11). As is the case for most integrin antagonists (45), this antagonist causes reshaping of the integrin ␤-subunit ␤I domain but not the ␣-subunit ␤-propeller domain (11). Whether VCAM induces a conformational change in ␣ 4 must await a co-crystal structure.
It is on structural grounds that we believe it is unlikely that VCAM binding would transmit conformational change through the ␤-propeller domain to the natalizumab epitope. ␤-Propellers are exceptionally large domains that are formed from unusually closely packed ␤-sheets (46). ␤-Sheets are rigid structural elements, and whereas most domains are two or three layers thick, ␤-propellers are eight layers thick across their diameter. Thus ␤-propellers are predicted to be unusually rigid. Indeed, their function in trimeric G proteins and integrins may be to serve as rigid platforms to stabilize their allosteric signaling ␤/␣ domain partners. Integrin ␤I domains, with their three layer ␤/␣ structures, transmit conformational change through motions of ␣-helices (45,47).
Our findings demonstrate that antibodies to integrins that inhibit by a noncompetitive mechanism can nonetheless be highly effective therapeutics. Strong blockade of adhesion in vitro is also not required. Consistent with noncompetitive inhibition, it is difficult to inhibit cell adhesion to VCAM with natalizumab in Mn 2ϩ , particularly at high VCAM density. 4 Because ligand-binding sites are well conserved across species (Fig. 4), the finding here that a function-blocking, speciesspecific antibody binds beside, rather than in, a ligand-binding site may be more common than not. Another species-specific therapeutic antibody, vedolizumab, also binds to the edge of the ligand-binding site, but on the opposite, ␤ 7 side (11) (Fig. 1B). Vedolizumab inhibits binding of MAdCAM, but not VCAM, to integrin ␣ 4 ␤ 7 (48). This unexpected result appears to be a consequence of binding close enough to the ligand-binding site to inhibit binding of MAdCAM, but not quite close enough to inhibit binding of VCAM. Similarly, efalizumab to LFA-1 binds to the ␣ L ␣I domain outside the binding site for D1 of ICAM-1, in a position where it would clash with or require bending away of D2 of ICAM-1 (49). The greater presence of species-specific differences outside of the ligand-binding site has thus skewed the current generation of therapeutic antibodies toward previously unanticipated mechanisms of action, which include noncompetitive antagonism and inhibition of binding of some and not other ligands. Currently, technology is maturing to generate synthetic antibody libraries that are devoid of tolerance to self (50). It will be interesting to see whether next generation antibodies recognize a broader range of epitopes including highly conserved ligand-binding epitopes, and reveal even further surprises.