Mutation of a Conserved Asparagine in the I-like Domain Promotes Constitutively Active Integrins αLβ2 and αIIbβ3*

The leukocyte β2 integrins are heterodimeric adhesion receptors required for a functional immune system. Many leukocyte adhesion deficiency-1 (LAD-1) mutations disrupt the expression and function of β2 integrins. Herein, we further characterized the LAD-1 mutation N329S in the β2 inserted (I)-like domain. This mutation converted αLβ2 from a resting into a high affinity conformer because αLβ2N329S transfectants adhered avidly to ligand intercellular adhesion molecule (ICAM)-3 in the absence of additional activating agent. An extended open conformation is adopted by αLβ2N329S because of its reactivity with the β2 activation reporter monoclonal antibodies MEM148 and KIM127. A corresponding mutation inβ3 generated constitutively activeαIIbβ3 that adhered to fibrinogen. This Asn is conserved in all human β subunits, and it resides before the last helix of the I-like domain, which is known to be important in activation signal propagation. By mutagenesis studies and review of existing integrin structures, we conjectured that this conserved Asn may have a primary role in shaping the I-like domain by stabilizing the conformation of theα7 helix and the β6-α7 loop in the I-like domain.

The integrins are type I membrane cell adhesion molecules formed non-covalently by two subunits (␣/␤). Despite having no intrinsic enzymatic properties, integrins are bidirectional signal transducers brought about by the recruitment of cytosolic proteins, many of which are signaling proteins, to their relatively short cytoplasmic tails (1). Structural data reveal a composite of distinct domains and folds found in an integrin molecule (2,3). Out of the 24 human integrins, nine contain in the ␣ subunit an additional inserted (I) domain that is the primary ligand-binding domain of these integrins (1). The I domain has a metal ion-dependent adhesion site (MIDAS) 3 that contains a divalent cation essential for ligand binding. Integrins that lack the I domain (henceforth referred to as I-less integrins) are found to bind ligand via the ␤ propeller of their ␣ subunit and the I-like domain of their ␤ subunit (2,3). The I-like domain is found in all integrin ␤ subunits, and it is structurally similar to that of the I domain. However, it contains a specificity-determining loop, which was reported to contribute toward ligand binding specificity and integrin ␣␤ subunit association (4), and it has two additional divalent cation-binding sites. The MIDAS of the I-like domain is flanked by the adjacent to MIDAS (ADMIDAS) and the ligand-induced metal-binding site (LIMBS), which serve as negative and positive regulatory sites, respectively (5)(6)(7). The conserved coordinating residues that are involved in the three cation-binding sites of the I-like domain are highlighted (Fig. 1A).
The leukocyte-restricted ␤ 2 integrins contain four members that differ in their ␣ subunits, the ␣ L ␤ 2 , ␣ M ␤ 2 , ␣ X ␤ 2 , and ␣ D ␤ 2 (1). These integrins maintain a functional immune system by their direct involvement in processes such as leukocyte adhesion and migration, phagocytosis, and antigen presentation. All four members contain the I domain that serves as the primary ligand-binding site. The structure of an I domain-containing integrin is lacking. Thus, the mechanism of I domain regulation by other domain(s) in an intact integrin is drawn largely from isolated I domain structures without or with ligands (8 -11), cogent mutagenesis studies that sculpted different I domain conformers reporting different ligand binding affinities (12,13), and extrapolation of possible I domain connectivity with other domain(s) from known structures of I-less integrins (14,15). Notably, I domain ligand binding was shown to be regulated allosterically by the I-like domain. An invariant glutamate residing in the last helix of the I domain serves as an intrinsic ligand for the I-like domain (16). Mutations in the I-like domain of the ␤ 2 integrins are known to generate receptors with impaired functions as exemplified in the inherited disorder leukocyte adhesion deficiency 1 (LAD-1) (17). The I-like domain mutations S116P and D209H generate ␤ 2 integrins that were expressed on the cell surface but dysfunctional (18,19). Ser 116 and Asp 209 are coordinating residues of the MIDAS/ADMI-DAS and LIMBS, respectively (Fig. 1A). Interestingly, the I-like domain mutation N329S, which was not inherited from either parent, was identified in a patient having another mutation causing aberrant splicing of the integrin ␤ 2 subunit (20). Although N329S mutation supported moderate integrin ␣ M ␤ 2 expression in a surrogate cell transfection system (20), it remains unclear as to the effect of this mutation on ␤ 2 integrin ligand binding function.
Previously, we reported the expression of a constitutively active ␣ L ␤ 2 N329S that adhered to intercellular adhesion molecule (ICAM)-1 (21). Accumulating evidence suggests that integrin ␣ L ␤ 2 may undergo conformational changes that generate the resting, intermediate, and high affinity states (22,23). Herein, we further characterize and report N329S as a mutation that promotes a high affinity ␣ L ␤ 2 . Similarly, the same mutation introduced into the I-less integrin ␣ IIb ␤ 3 produced an active receptor. Combinatorial analyses of N329S with S116P or D209H showed that the activating effect of N329S requires functional I-like domain MIDAS and LIMBS to allow propagation of the activating signal to the ␣L I domain. Of note, this Asn at position 329 in integrin ␤ 2 primary sequence is conserved in all integrin ␤ subunits. It may interact with neighboring residues to stabilize the ␤ I-like domain ␤6-␣7 loop that is required for the transmission of activation signal.
cDNAs, Expression Plasmids, and mAbs-The ␣ L , ␣ M , ␣ X and ␤ 2 pcDNA3 expression plasmids were described previously (32). The ␣ IIb and ␤ 3 pcDNA3 expression plasmids were kindly provided by P. J. Newman, Blood Center of Wisconsin and Medical College Wisconsin. Amino acid numbering of the integrins is based on Barclay et al. (33). All integrin mutants were generated using the QuikChange TM site-directed mutagenesis kit (Stratagene, La Jolla, CA) with the relevant primer pair. Integrins with more than one mutation (e.g. ␤ 2 N329S/D209H) were generated by sequential site-directed mutagenesis. The ␣ L c-c construct was generated by mutating I domain Lys 287 and Lys 294 into Cys to allow disulfide bridge formation (12). All constructs were verified by sequencing (Research Biolabs, Singapore).
Flow Cytometry Analyses-The preparation of samples for flow cytometry analyses was reported previously (35). Cell surface expression of ␤ 3 and ␤ 2 integrins was analyzed using the mAb 7E3 and the heterodimer-specific mAb MHM23, respectively, followed by flow cytometry analysis on a FACSCalibur using the software CellQuest (BD Biosciences). The expression level was represented by the expression index (EI) that was calculated by the percentage of cells gated positive ϫ geo-mean fluorescence intensity. An irrelevant mAb was used as background control in all preparations.
Cell Adhesion Assays-Adhesion of ␣ L ␤ 2 transfectants to ICAMs was performed as reported (35). Briefly, each Polysorb microtiter well (Nunc, Rosklide, Denmark) was coated with 0.5 g of goat anti-human IgG (Fc specific) (Sigma) in 100 l of 50 mM bicarbonate buffer (pH 9.2). Nonspecific binding sites were blocked with 0.5% (w/v) bovine serum albumin in PBS for 30 min at 37°C. Thereafter, 50 l of ICAM-Fc at 1 ng/l in PBS containing 0.1% (w/v) bovine serum albumin was added to each well and incubated for 2 h at room temperature. Wells were washed twice in RPMI wash buffer (RPMI medium containing 5% (v/v) heat-inactivated fetal bovine serum and 10 mM HEPES, pH 7.4) before assay. Cells labeled with 3.0 mM 2Ј,7Ј bis-(2carboxyethyl)-5-(and-6)-carboxyfluorescein fluorescent dye (Molecular Probes, Eugene, OR) were incubated in wash buffer containing Mg/EGTA (5 mM MgCl 2 and 1.5 mM EGTA) and/or activating mAb KIM185 (10 g/ml) to activate ␣ L ␤ 2 . ␣ L ␤ 2 -mediated adhesion specificity was demonstrated using MHM24 (10 g/ml). Fluorescence signal, which correlates with the number of cells adhering to the ligand-coated well, is measured using a FL600 fluorescent plate reader (Bio-Tek instruments, Winooski, VT). For ␣ IIb ␤ 3 transfectant adhesion to fibrinogen, fibrinogen at 1 g/ml in PBS was added into each microtiter well. The subsequent steps in the binding assay were the same as that of ␣ L ␤ 2 aforementioned. ␣ IIb ␤ 3 -mediated adhesion specificity was assessed using the function-blocking mAb 10E5 (10 g/ml). 1 mM MnCl 2 was used to activate ␣ IIb ␤ 3 .
Surface Labeling and Immunoprecipitation-Surface labeling of integrins with biotin was described previously (35). Cells were washed once in PBS and incubated in sulfo-NHS-biotin (Pierce) at 0.5 mg/ml in PBS for 20 min on ice. The reaction was terminated by washing surface-labeled cells once in PBS containing 10 mM Tris-HCl (pH 8.0). Thereafter, labeled cells were incubated in warm culture medium containing appropriate mAb MHM23, KIM127, or MEM148 (2 g each) in the absence or presence of Mg/EGTA for 30 min at 37°C. Cells were spun down and lysed in lysis buffer (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1% (v/v) Nonidet P-40) containing appropriate protease inhibitors (Roche Diagnostics, Basel, Switzerland) followed by immunoprecipitation with rabbit anti-mouse IgG coupled to Protein A-Sepharose beads (Amersham Biosciences, Buckinghamshire, UK). Bound proteins were resolved on a 7.5% SDS-PAGE gel under reducing conditions. Proteins were transferred onto Immobilon P membrane (Millipore, Bedford, MA), and biotinylated protein bands were detected with streptavidin-horseradish peroxidase followed by enhanced chemiluminescence detection using the ECL Plus kit (Amersham Biosciences).
Structural Images and Modeling-LSQKAB (Collaborative Computational Project, CCP4) (36) was used for molecular least-squares superposition of the three I-like domain conformers. Figs. 1B and 7 were created using PyMOL. The solventaccessible surface area of ␤ 3 Asn 339 of the three conformers was calculated using AREAIMOL (Collaborative Computational Project Number 4) with a probe of 1.7 Å radius: 46.1 Å 2 (conformer I); 16.5 Å 2 (conformer II); 68.8 Å 2 (conformer III). Structural models of wild-type ␤ 2 I-like domain or variants were generated using Modeler 9.

RESULTS
N329S Generates a High Affinity ␣ L ␤ 2 That Adheres to ICAM-1 and ICAM-3 Substrates Constitutively-The possibility of at least three affinity states of ␣ L ␤ 2 based on functional and structural studies prompted us to examine the affinity state of ␣ L ␤ 2 N329S (22,23). We showed previously that an intermediate affinity ␣ L ␤ 2 adhered to ICAM-1 but that a high affinity conformer was required for adhesion to ICAM-3 (22). Herein, ␣ L ␤ 2 N329S transfectant showed a high level of constitutive adhesion to ICAM-1 even in the absence of activating agent, whereas wild-type ␣ L ␤ 2 required activation with Mg/EGTA (Fig. 2). This is consistent with our first report on the constitutive activity of ␣ L ␤ 2 N329S with respect to ICAM-1 adhesion (21). Importantly, unlike wild-type ␣ L ␤ 2 transfectant, ␣ L ␤ 2 N329S transfectant also showed a high level of constitutive adhesion to ICAM-3 in the absence of activating agents Mg/EGTA and the integrin ␤ 2 -specific activating mAb KIM185 (Fig. 2). The expressions (represented as EI) of wild-type ␣ L ␤ 2 and ␣ L ␤ 2 N329S were comparable as assessed by flow cytometry using the ␤ 2 integrin heterodimer-specific mAb MHM23. The binding specificity was demonstrated using the ␣ L ␤ 2 -specific function-blocking mAb MHM24. Thus, the mutation N329S generates a high affinity ␣ L ␤ 2 with respect to ICAMs adhesions.
The Requirement of C␤ Instead of C␥ Amide Functional Group at Position 329 of the ␤ 2 I-like Domain-The Asn at position 329 of the ␤ 2 is conserved in all integrin ␤ subunits. Because the native structure of the ␤ 2 I-like domain has not been resolved and because of the fact that the corresponding residue in the ␤ 3 is Asn 339 , we made use of the resolved struc-ture of the ␤ 3 I-like domain of ␣ V ␤ 3 in complex with an Arg-Gly-Asp (RGD) ligand (37) to visualize the position of this conserved Asn and the three metal-binding sites. The ␤ 3 Asn 339 lies before the last helix of the I-like domain (Fig. 1B). Further, the position of LIMBS (gold), MIDAS (pink), and ADMIDAS (blue) cations and the location of Asp 217 (coordinating residue for LIMBS) and Ser 123 (coordinating residue for MIDAS) are illustrated.
In the liganded I domain, a significant downward displacement of its C-terminal helix was observed (8,38). Consistent with this observation, open conformation ␣ L and ␣ M I domains, which have high affinity ligand binding properties, were generated by introducing cystine that stabilized the last helix of the I domains (12,13). Similar to the I domain, the last helix of the ␤ 3 I-like domain was displaced in a ligand mimetic-bound ␣ IIb ␤ 3 (15). At present, it is unclear how ␤ 2 N329S confers ␣ L ␤ 2 constitutive propensity to adhere to ICAM-1 (21). The substitution of an amide to a hydroxyl side chain (Asn 3 Ser) hints at the possibility of functional group contribution toward the marked difference in the activity of ␣ L ␤ 2 with ␤ 2 Asn 329 or Ser 329 . Therefore, four other variants, ␣ L ␤ 2 N329T, ␣ L ␤ 2 N329Q, ␣ L ␤ 2 N329A, and ␣ L ␤ 2 N329D, were generated and tested for their constitutive capacities to adhere to the ICAMs (Fig. 3). All four transfectants constitutively adhered to ICAM-1 even in the absence of Mg/EGTA (Fig. 3A). Similarly, these transfectants adhered constitutively to ICAM-3 (Fig. 3B). The expression levels of ␣ L ␤ 2 N329T, ␣ L ␤ 2 N329Q, ␣ L ␤ 2 N329A, and ␣ L ␤ 2 N329D were determined by flow cytometry. The adhesion specificity was assessed by using the ␣ L ␤ 2 -specific functionblocking mAb MHM24. It is apparent that the introduction of a hydroxyl group as a result of N329S mutation does not have a primary role in generating a constitutively active ␣ L ␤ 2 because The reagents used were Mg/EGTA ((ME) 5 mM MgCl 2 and 1.5 mM EGTA), and KIM185 (10 g/ml) (␤ 2 integrin-activating mAb). Adhesion specificity was demonstrated using the ␣ L ␤ 2 -specific function-blocking mAb MHM24. The cell surface expressions of wild-type ␣ L ␤ 2 and ␣ L ␤ 2 N329S were assessed by flow cytometry analyses using mAb MHM23 (␤ 2 -specific heterodimer-recognizing mAb). The expression level was represented by the EI that was calculated by the percentage of cells gated positive ϫ geo-mean fluorescence intensity.
the substitutions N329Q, N329A, and N329D in ␤ 2 promoted comparable ICAM-adhesion activity. It is tempting to speculate that the altered binding property of ␣ L ␤ 2 N329S is attributed mainly to the loss of the side chain amide group at position 329 of the ␤ 2 . However, the ␤ 2 mutation N329Q, which had a similar activating effect on ␣ L ␤ 2 , suggests the requirement of C␤ instead of C␥ amide group at position 329 of the ␤ 2 I-like domain to maintain the functional integrity of ␣ L ␤ 2 .
The LIMBS and MIDAS of the I-like Domain Are Required for the Activating Effect of N329S in I Domain-containing ␣ L ␤ 2 -In I domain-containing integrins, the I-like domain allosterically regulates the ligand binding activity of the I domain (16). It is reasonable to suggest that structural changes at the locality of ␤ 2 N329S are propagated to the ligand-binding face of the I-like domain. This may trigger I-like domain binding of the invariant Glu in the last C-terminal helix of the ␣ L I domain, thus activating I domain ligand binding. Therefore, disrupting the ligand-binding sites of the I-like domain should abrogate the activating signal of N329S. Indeed, transfectants bearing ␣ L ␤ 2 having composite mutations N329S and S116P or D209H failed to adhere to ICAM-1 even in the presence of activating agents (Fig. 4). The adhesion specificity mediated by ␣ L ␤ 2 was demonstrated in all cases by complete abrogation of adhesion in the presence of mAb MHM24. The expression levels of these ␣ L ␤ 2 variants were comparable. Ser 116 is the third coordinating residue found in the signature motif DXSXS of the I-like domain MIDAS, and the function disrupting effect of S116P, identified in LAD-1 patient, has been reported (18). Asp 209 is a LIMBScoordinating residue. Conceivably, the LAD-1 D209H mutation abolished ␤ 2 integrin ligand binding capacity (19), which corroborates well with the role of LIMBS in stabilizing MIDASmediated firm adhesion (5). Collectively, these data suggest that the activating effect of N329S is propagated through the ligandbinding site(s) of the ␤ 2 I-like domain, which subsequently activates by allostery the ␣ L I domain.

Introduction of N339S in ␤ 3 , Which Corresponds to N329S in ␤ 2 , Generates a Constitutively Active I-less Integrin ␣ IIb ␤ 3 -The
Asn at position 329 of integrin ␤ 2 is conserved in all integrin ␤ subunits (Fig. 1A). To further demonstrate that the activating signal of Asn mutation is propagated to the ligand-binding site(s) of the I-like domain, we extended the investigation to the I-less integrin ␣ IIb ␤ 3 because the ␤ 3 I-like domain participates directly in extrinsic ligand binding (15,37). The corresponding Asn 339 in ␤ 3 (Fig. 1B) was substituted with Ser to generate ␣ IIb ␤ 3 N339S. Ser 116 and Asp 209 of the integrin ␤ 2 are also conserved in all integrin ␤ subunits. The corresponding residues in integrin ␤ 3 are Ser 123 and Asp 217 . Thus, the MIDAS variant ␣ IIb ␤ 3 S123P and the LIMBS variant ␣ IIb ␤ 3 D217H were constructed. In addition, the composite variants ␣ IIb ␤ 3 N339S/ S123P and ␣ IIb ␤ 3 N339S/D217H were generated (Fig. 5). Expression levels of ␣ IIb ␤ 3 and variants on transfectants were analyzed by flow cytometry using the ␤ 3 -specific mAb 7E3. Wild-type ␣ IIb ␤ 3 transfectants adhered avidly to its ligand fibrinogen only in the presence of activating manganese. By contrast, ␣ IIb ␤ 3 N339S showed constitutive adhesion to fibrinogen even in the absence of manganese. The specificity of ␣ IIb ␤ 3 -mediated adhesion was demonstrated using ␣ IIb -specific function-blocking mAb 10E5. Substitutions S123P and D217H in ␤ 3 abolished ␣ IIb ␤ 3 -mediated adhesion to fibrinogen. When S123P or D217H was introduced in combination with N339S, it attenuated the activating effect of N339S on ␣ IIb ␤ 3mediated adhesion to fibrinogen. Therefore, we conjectured that the mutations N329S in ␣ L ␤ 2 and N339S in ␣ IIb ␤ 3 affect the function of the respective I-like domains.
An Extended Conformation of ␣ L ␤ 2 N329S-The conversion from a severely bent to a highly extended conformation was FIGURE 4. Effect of N329S in combination with S116P or D209H on ␣ L ␤ 2mediated adhesion to ICAM-1. The mutations S116P and D209H abrogated the activating effect of Mg/EGTA (ME) or KIM185 on wild-type ␣ L ␤ 2 ICAM-1 adhesion. Similarly, these mutations abolished constitutive adhesion of ␣ L ␤ 2 N329S transfectants to ICAM-1. MHM23 was used for flow cytometry analyses and surface expression represented as EI.
FIGURE 5. Effect of N339S with S123P or D217H on I-less integrin ␣ IIb ␤ 3mediated adhesion to fibrinogen. Adhesion specificity was demonstrated using the ␣ IIb ␤ 3 -specific function-blocking mAb 10E5. 1 mM MnCl 2 (Mn) was used as the activating agent. The cell surface expressions of wild-type ␣ IIb ␤ 3 and variants were assessed by flow cytometry using mAb 7E3 (␤ 3 -specific mAb) and represented as EI.

An I-like Domain Mutation Promotes a High Affinity Integrin
shown to be an important event during integrin affinity state transition (15,23). We examined the conformation of ␣ L ␤ 2 N329S using two ␤ 2 integrin-specific reporter mAbs. The mAb KIM127 recognizes an epitope that is masked in the ␤ 2 integrin-epidermal growth factor-2 in a bent ␣ L ␤ 2 conformer. This epitope is, however, presented when ␣ L ␤ 2 adopts an extended conformation (39,40). The mAb MEM148 recognizes a masked epitope in the ␤ 2 hybrid domain, and it serves to report hybrid domain displacement (22). We chose immunoprecipitation over flow cytometry analyses in these experiments because in this case, we could detect a population of free ␤ 2 that is unassociated with ␣ L on the 293T transfectants. 4 This may complicate the analyses using the ␤ 2 reporter mAbs based on the method of flow cytometry because these mAbs will also stain the free ␤ 2 . The method of surface labeling and immunoprecipitation allows us to examine heterodimer reactivity with the ␤ 2 -specific reporter mAbs by detecting the level of ␣ L coprecipitated with the ␤ 2 . Cell surface ␣ L ␤ 2 and ␣ L ␤ 2 N329S on transfectants were labeled with biotin and immunoprecipitated with MHM23, KIM127, or MEM148 with or without Mg/EGTA. The ␣ L c-c was reported previously in which a disulfide bridge was engineered in the ␣ L I domain to "lock" the I domain in an active open conformation (12) (see "Experimental Procedures"). We determined that ␣ L c-c␤ 2 transfectants adhered constitutively to ICAM-1 but not ICAM-3, suggesting an intermediate affinity conformer (data not shown). Thus, ␣ L c-c␤ 2 was included for comparison with ␣ L ␤ 2 N329S on conformational changes. Wild-type ␣ L ␤ 2 was precipitated by MHM23 (a ␤ 2 integrin heterodimer-specific mAb that serves as a control in this experiment) (Fig. 6). However, wild-type ␣ L ␤ 2 was only precipitated by KIM127 or MEM148 in the presence of activating Mg/EGTA. A similar profile was detected for ␣ L c-c␤ 2 . By contrast, ␣ L ␤ 2 N329S was precipitated by KIM127 and MEM148 even in the absence of Mg/EGTA. These data suggest that ␣ L ␤ 2 N329S adopts an extended conformation with possible displacement of the hybrid domain. This conformation was proposed to depict a high affinity integrin (15), and it is in agreement with our binding data. The lack of ␣ L c-c␤ 2 precipitated by KIM127 in the absence of exogenous activation was also in par-allel with the previous observation that ␣ L c-c␤ 2 was stained weakly with KIM127 by flow cytometry (41).

DISCUSSION
Naturally occurring mutations identified in LAD-1 patients provide useful insights into the possible functions of residues and domains of the ␤ 2 integrins (17). Two such mutations S116P and D209H exemplify the importance of the MIDAS and LIMBS, respectively, of the ␤ 2 I-like domain in ␣ L ␤ 2 ligand binding function (18,19). Many LAD-1 mutations disrupt ␤ 2 integrin biosynthesis or heterodimer formation or generate dysfunctional cell surface-expressed ␤ 2 integrins (17,42). Previously, we reported two LAD-1 mutations, C568R and R571C, in the ␤ 2 that generated ␣ L ␤ 2 variants with precocious ligand binding activity (32). In addition, another ␤ 2 mutation N329S was found to generate constitutively active ␣ L ␤ 2 (21). This study further characterized the N329S mutation. The ␤ 2 Asn 329 is conserved in all human integrin ␤ subunits (Fig. 1A) and in the ␤ subunits of several other metazoans including coral and sponge (43). Our data suggest that a C␤ instead of C␥ amide group at position 329 of the ␤ 2 I-like domain is required to maintain ␣ L ␤ 2 functional integrity. Because of the availability of structural information of the ␤ 3 I-like domain, we first reviewed Asn 339 in ␤ 3 that corresponds to the Asn 329 in ␤ 2 . The ␤ 3 Asn 339 is located before the ␣7 helix (last helix) of its I-like domain (Fig.  7A). The orientations of the ␤6 strand and the ␣7 helix in the ␤ 3 I-like domain are different in the structures of a bent ␣ V ␤ 3 (red) (conformer I) (44), a bent-liganded ␣ V ␤ 3 (green) (conformer II) (37), and the fibrinogen mimetic-bound ␣ IIb ␤ 3 with open headpiece (blue) (conformer III) (15). The bent ␣ V ␤ 3 may represent a resting integrin conformer, whereas the fibrinogen mimeticbound ␣ IIb ␤ 3 with hybrid domain displaced may represent a high affinity conformer (2). The structure of the bent-liganded ␣ V ␤ 3 may require careful interpretation because the ␣ V ␤ 3 crystals were soaked in the RGD ligand instead of co-crystallization of an RGD and ␣ V ␤ 3 protein mixture, and no displacement of the ␣7 helix was observed in the structure (37). Thus, the repositioning of the Asn 339 herein was examined mainly between conformer I and conformer III. In conformer I, Asn 339 of the ␤ 3 I-like domain projects its amide side chain in an orientation that can allow hydrogen bonds to be established with the main chain carbonyl oxygen of Val 332 in the ␤6 strand and the side chain hydroxyl group of Ser 334 that resides in the loop connecting the ␤6 strand and the ␣7 helix. Interestingly, these are different in conformer III. Hydrogen bond can form between ␦ 1 O of Asn 339 with the main chain -NH of Val 332 but not between Asn 339 and Ser 334 . This observed difference between the two conformers is attributed to the downward movement of the ␣7 helix and the outward rotation of the Asn 339 in conformer III with respect to conformer I (Fig. 7B). The solvent-accessible surface area of Asn 339 of conformer I is 46.1 Å 2 , whereas that of conformer III is 68.8 Å 2 , which corroborates well with its outward reorientation in conformer III having a displaced ␣7 helix. It is, therefore, possible that the formation of polar contacts between Asn 339 with Val 332 and Ser 334 is required in part to maintain ␤ 3 I-like domain in a resting conformation. Disruption of these contacts as a result of a mutation such as N339S FIGURE 6. The N329S mutation transforms ␣ L ␤ 2 into an extended conformation. Cell surface-labeled wild-type ␣ L ␤ 2 , ␣ L c-c␤ 2 , and ␣ L ␤ 2 N329S were subjected to reporter mAbs MEM148 or KIM127 binding at 37°C with or without Mg/EGTA (ME) followed by precipitation using protein A-Sepharose beads (see "Experimental Procedures"). Immunoprecipitated integrins were resolved on a 7.5% SDS-PAGE gel under reducing conditions and detected by ECL. MHM23 was included as control mAb. may destabilize these interactions, thus generating an activated ␣ IIb ␤ 3 .
In ␤ 2 , similar polar contacts may be formed as depicted in a homology model of ␤ 2 I-like domain generated based on the bent ␣ V ␤ 3 structural coordinates (Fig. 7C). The Ser 324 side chain may hydrogen-bond with Asn 329 ␦ 1 O in the ␤ 2 I-like domain model. The residue at position 322 of ␤ 2 and 332 of ␤ 3 is different. This is, however, irrelevant because it is the backbone carbonyl of this residue that may hydrogen-bond with the conserved Asn. ␤ 2 I-like domain models incorporating Asn 329 substituted with Gln, Ala, Ser, Thr, or Asp (see "Experimental Procedures") showed potential disruptions in hydrogen-bond formation between these residues at position 329 with Ser 324 and main chain Glu 322 , which may account for the activating effect of these mutations on ␣ L ␤ 2 . Together, these suggest that the polar contacts between Asn 329 with Ser 324 and Glu 322 may serve to stabilize the ␤ 2 I-like domain in a fashion similar to that in ␤ 3 conformer I. It is noted that although ␤ 2 Ser 324 (␤ 3 Ser 334 ) can potentially hydrogen-bond with the conserved ␤ 2 Asn 329 (␤ 3 Asn 339 ), this should be extrapolated with caution to other integrins because ␤ 2 Ser 324 is conserved in ␤ 3 , ␤ 1 , and ␤ 7 but not in other ␤ subunits (Fig. 1A). In addition, the contribution of the conserved Asn toward the function of other ␤ subunits awaits further studies.
Integrin affinity states are governed by its varied conformations under different conditions (2,45). In a global setting, the bent integrin conformer depicts a resting low affinity state, and the extended integrin conformers are assigned as the active receptors (46). The extended integrin conformers are further divided into two major populations based on structural differences in their headpieces (15). Extended integrin conformers with or without hybrid domains displaced are assigned high or intermediate affinity states, respectively. In this study, the mutation N329S induced an extended ␣ L ␤ 2 conformation because in the absence of activating agent, ␣ L ␤ 2 N329S was immunoprecipitated by the reporter mAb KIM127 (40,47). Under the same conditions, ␣ L ␤ 2 N329S was precipitated by mAb MEM148, which reports hybrid domain displacement (22). These data suggest a high affinity ␣ L ␤ 2 generated by the ␤ 2 N329S mutation. Indeed, our functional data showing ␣ L ␤ 2 N329S binding constitutively and effectively to ICAM-3 supported the assignment of a high affinity receptor. It is unclear at present how N329S triggers such a dramatic global conformational change in ␣ L ␤ 2 . Not only does N329S incur activation of the I-like domain, as demonstrated in ␣ IIb ␤ 3 N339S, it also induces unbending of the entire integrin. However, this need not be unexpected because it was reported that one-turn deletion in the ␣7 helix of the ␤ 2 I-like domain promoted ␣ L ␤ 2 binding to ICAM-1 and exposed the epitopes of extension reporter mAb KIM127 and the activated I-like domain reporter mAb m24 (48).
In summary, we have further characterized the LAD-1 mutation N329S. This mutation generates a high affinity ␣ L ␤ 2 with an extended conformation. N329S activates the ␤ 2 I-like domain. The corresponding mutation in ␤ 3 N339S had similar activating effect on ␣ IIb ␤ 3 . The conservation of this Asn in all human integrin ␤ subunits through evolution suggests a primary role of this residue in the shaping of the I-like domain. Based on available structures of the ␤ I-like domains and structural predictions, this Asn may serve to stabilize the conformation of the ␣7 helix and its interaction with the preceding ␤6 strand. However, definitive revelation of its role in the shaping of the I-like domain will require structural studies of integrins incorporating the aforementioned mutation(s).