Identity of the amino acid residues involved in C3bi binding to the I-domain supports a mosaic model to explain the broad ligand repertoire of integrin alpha M beta 2

Interactions between the complement degradation product C3bi and leukocyte integrin alpha(M)beta(2) are critical for host defense against foreign pathogens and in tumor cell surveillance. To gain insight into the mechanism by which the alpha(M)I-domain of the integrin interacts with C3bi, detailed mapping of the C3bi binding site was undertaken. Previous mutagenesis studies had implicated five small structural segments within the alpha(M)I-domain in recognition of this ligand. Sets of three amino acids within the five implicated segments were mutated to the corresponding alpha(L)I-domain residues. Then, within the affected mutants, single point mutations were introduced to precisely define the requisite residues. Ultimately, H148, F150, Q204, L205, R208, T211, T213, I256, P257 were identified as being critical for C3bi binding. A synthetic peptide approach confirmed the involvement of the specified residues with the complex midsegment, Q204-I215, in C3bi recognition. Furthermore, the alpha(D)I-domain, which has a low intrinsic affinity for C3bi, acquired high affinity for the ligand when the implicated residues were inserted. The residues necessary to engage C3bi reside on or adjacent to the cation binding MIDAS site of the alpha(M)I-domain. The amino acids involved in C3bi binding are distinct from those involved in interaction of previously mapped ligands with the alpha(M)I-domain. This divergence supports a mosaic model, in which different ligands engage different amino acids to bind to alpha(M)I-domain, accounting for the broad recognition capacity of integrin alpha(M)beta(2).     

Identification of critical residues for plasminogen binding by the alphaX I-domain of the beta2 integrin, alphaXbeta2

The beta2 integrins on leukocytes play important roles in cell adhesion, migration and phagocytosis. One of the beta2 integrins, alphaXbeta2 (CD11c/CD18), is known to bind ligands such as fibrinogen, Thy-1 and iC3b, but its function is not well characterized. To understand its biological roles, we attempted to identify novel ligands. The functional moiety of alphaXbeta2, the alphaX I-domain, was found to bind plasminogen, the zymogen of plasmin, with moderate affinity (1.92 X 10-(6) M) in the presence of Mg(2+) or Mn(2+). The betaD-alpha5 loop of the alphaX I-domain proved to be responsible for binding, and lysine residues (Lys(242), Lys(243)) in the loop were the most important for recognizing plasminogen. An excess amount of the lysine analog, 6-aminohexanoic acid, inhibited alphaX I-domain binding to plasminogen, indicating that binding is lysine-dependent. The results of this study indicate that leukocytes regulate plasminogen activation, and consequently plasmin activities, through an interaction with alphaXbeta2 integrin.     

Dual function for a unique site within the beta2I domain of integrin alphaMbeta2

Integrin activation has been postulated to occur in part via conformational changes in the I domain of the beta subunit (the betaI domain), especially near the F-alpha(7) loop, in response to "inside-out" signaling. However, direct evidence for a role of the F-alpha(7) loop in ligand binding and activity modulation is still lacking. Here, we report our finding that the F-alpha(7) loop (residues 344-358) within the beta(2)I domain has dual functions in ligand binding by alpha(M)beta(2). On the one hand, it supports intercellular adhesion molecule 1 (ICAM-1) binding to alpha(M)beta(2) directly as part of a recognition interface formed by five noncontiguous segments (Pro(192)-Glu(197), Asn(213)-Glu(220), Leu(225)-Leu(230), Ser(324)-Thr(329), and Glu(344)-Asp(348)) on the apex of the beta(2)I domain. On the other hand, it controls the open and closed conformation of the alpha(M)beta(2) receptor, thereby indirectly affecting alpha(M)beta(2) binding to other ligands. Switching the five constituent sequences of the ICAM-1-binding site within the beta(2)I domain to their beta(1) counterparts destroyed ICAM-1 binding but had no effect on the gross conformations of the receptor. Of the five ICAM-1 binding-defective mutants, four had normal or even stronger interaction with Fg and C3bi, as reported in our previous study. Synthetic peptides derived from the identified site inhibited alpha(M)beta(2)-ICAM-1 interaction and supported direct binding to ICAM-1. Most importantly, perturbation of the F-alpha(7) loop caused conformational changes within the beta(2)I domain, which was further propagated to other regions of alpha(M)beta(2). Altogether, our data demonstrate that inside-out signaling could modulate ligand binding directly by changing the ligand-binding pocket per se and/or indirectly by inducing multiple conformational changes within the receptor.     

Molecular basis for the interaction of low density lipoprotein receptor-related protein 1 (LRP1) with integrin alphaMbeta2: identification of binding sites within alphaMbeta2 for LRP1

The LDL receptor-related protein 1 (LRP1) is a large endocytic receptor that controls macrophage migration in part by interacting with β(2) integrin receptors. However, the molecular mechanism underlying LRP1 integrin recognition is poorly understood. Here, we report that LRP1 specifically recognizes α(M)β(2) but not its homologous receptor α(L)β(2). The interaction between these two cellular receptors in macrophages is significantly enhanced upon α(M)β(2) activation by LPS and is mediated by multiple regions in both LRP1 and α(M)β(2). Specifically, we find that both the heavy and light chains of LRP1 are involved in α(M)β(2) binding. Within the heavy chain, the binding is mediated primarily via the second and fourth ligand binding repeats. For α(M)β(2), we find that the α(M)-I domain represents a major LRP1 recognition site. Indeed, substitution of the I domain of the α(L)β(2) receptor with that of α(M) confers the α(L)β(2) receptor with the ability to interact with LRP1. Furthermore, we show that residues (160)EQLKKSKTL(170) within the α(M)-I domain represent a major LRP1 recognition site. Given that perturbation of this specific sequence leads to altered adhesive activity of α(M)β(2), our finding suggests that binding of LRP1 to α(M)β(2) could alter integrin function. Indeed, we further demonstrate that the soluble form of LRP1 (sLRP1) inhibits α(M)β(2)-mediated adhesion of cells to fibrinogen. These studies suggest that sLRP1 may attenuate inflammation by modulating integrin function.     

Identification of the key amino acids of glial cell line-derived neurotrophic factor family receptor alpha1 involved in its biological function

Glial cell line-derived neurotrophic factor (GDNF) plays a critical role in neurodevelopment and survival of midbrain dopaminergic and spinal motor neurons in vitro and in vivo. The biological actions of GDNF are mediated by a two-receptor complex consisting of a glycosylphosphatidylinositol-linked cell surface molecule, the GDNF family receptor alpha1 (GFRalpha1), and receptor protein tyrosine kinase Ret. Although structural analysis of GDNF has been extensively examined, less is known about the structural basis of GFRalpha1 function. In this study, based on evolutionary trace method and relative solvent accessibility prediction of residues, a set of trace residues that are solvent-accessible was selected for site-directed mutagenesis. A series of GFRalpha1 mutations was made, and PC12 cell lines stably expressing different GFRalpha1 mutants were generated. According to the survival and differentiation responses of these stable PC12 cells upon GDNF stimulation and the GDNF-GFRalpha1-Ret interaction assay, residues 152NN153, Arg259, and 316SNS318 in the GFRalpha1 central region were found to be critical for GFRalpha1 binding to GDNF and eliciting downstream signal transduction. The single mutation R259A in the GFRalpha1 molecule simultaneously lost its binding ability to GDNF and Ret. However N152A/N153A or S316A/N317A/S318A mutation in the GFRalpha1 molecule still retained the ability to bind with Ret. These findings suggest that distinct structural elements in GFRalpha1 may be involved in binding to GDNF and Ret.     

Identification of the binding site for fibrinogen recognition peptide gamma 383-395 within the alpha(M)I-domain of integrin alpha(M)beta2

The leukocyte integrin alpha(M)beta(2) (Mac-1, CD11b/CD18) is a cell surface adhesion receptor for fibrinogen. The interaction between fibrinogen and alpha(M)beta(2) mediates a range of adhesive reactions during the immune-inflammatory response. The sequence gamma(383)TMKIIPFNRLTIG(395), P2-C, within the gamma-module of the D-domain of fibrinogen, is a recognition site for alpha(M)beta(2) and alpha(X)beta(2). We have now identified the complementary sequences within the alpha(M)I-domain of the receptor responsible for recognition of P2-C. The strategy to localize the binding site for P2-C was based on distinct P2-C binding properties of the three structurally similar I-domains of alpha(M)beta(2), alpha(X)beta(2), and alpha(L)beta(2), i.e. the alpha(M)I- and alpha(X)I-domains bind P2-C, and the alpha(L)I-domain did not bind this ligand. The Lys(245)-Arg(261) sequence, which forms a loop betaD-alpha5 and an adjacent helix alpha5 in the three-dimensional structure of the alpha(M)I-domain, was identified as the binding site for P2-C. This conclusion is supported by the following data: 1) mutant cell lines in which the alpha(M)I-domain segments (245)KFG and Glu(253)-Arg(261) were switched to the homologous alpha(L)I-domain segments failed to support adhesion to P2-C; 2) synthetic peptides duplicating the Lys(245)-Tyr(252) and Glu(253)-Arg(261) sequences directly bound the D fragment and P2-C derivative, gamma384-402, and this interaction was blocked efficiently by the P2-C peptide; 3) mutation of three amino acid residues within the Lys(245)-Arg(261) segment, Phe(246), Asp(254), and Pro(257), resulted in the loss of the binding function of the recombinant alpha(M)I-domains; and 4) grafting the alpha(M)(Lys(245)-Arg(261)) segment into the alpha(L)I-domain converted it to a P2-C-binding protein. These results demonstrate that the alpha(M)(Lys(245)-Arg(261)) segment, a site of the major sequence and structure difference among alpha(M)I-, alpha(X)I-, and alpha(L)I-domains, is responsible for recognition of a small segment of fibrinogen, gammaThr(383)-Gly(395), by serving as ligand binding site.     

Concerted influence of key amino acids on the lipid binding properties of a single-spanning membrane protein: NMR and mutational analysis

Finding the combinations of key amino acids involved in the interaction network underlying the interfacial features of membrane proteins would contribute to a better understanding of their sequence-structure-function relationships and the role of anionic phospholipids. To further address these questions, we performed mutational analysis associated with NMR experiments on synthetic fragments of the single-spanning membrane protein PMP1 that exhibit binding specificity for phosphatidylserine (PS). The aromatic and glutamine residues of the helix part of the PMP1 cytoplasmic domain were mutated. (1)H NMR experiments were carried out using perdeuterated DPC micelles as a membrane-like environment, in the absence and presence of small amounts of either POPC or POPS lipids. From intermolecular NOEs and chemical shift data, specific and nonspecific aspects of peptide-phospholipid interactions were distinguished. The major finding of our study is to reveal the concerted influence of a tryptophan and a glutamine residue on the interfacial conformation and lipid binding specificity of the PMP1 cytoplasmic domain.     

The exponential model for a regulatory enzyme: Its extension to describe the binding of two ligands

The exponential model for a regulatory enzyme (Ainsworth, 1977a) is extended to deal explicitly with the presence in solution of a second ligand. This is achieved by introducing exponential interaction coefficients which respectively describe how the affinity of the free and bound forms of the protein for the ligand depend on its fractional saturation by the second ligand. The basic equations, so derived, are applied to binding experiments where the ligands bind independently or competitively and to rate experiments where the ligands represent two substrates or one substrate and a modifier which may be either competitive or non-competitive in type. The conditions required to display linkage between the binding of the two ligands are established and it is also shown that rate data may display a maximum as one ligand concentration is varied at a fixed concentration of the other. The equations that are derived are tested by application to experimental data and the conditions that have to be met to justify such an application are discussed.     

Deletion of the C-terminal 33 amino acids of cucumber mosaic virus movement protein enables a chimeric brome mosaic virus to move from cell to cell.

The movement protein (MP) gene of brome mosaic virus (BMV) was precisely replaced with that of cucumber mosaic virus (CMV). Infectivity tests of the chimeric BMV on Chenopodium quinoa, a permissive host for cell-to-cell movement of both BMV and CMV, showed that the chimeric BMV failed to move from cell to cell even though it replicated in protoplasts. A spontaneous mutant of the chimeric BMV that displayed cell-to-cell movement was subsequently obtained from a local lesion during one of the experiments. A cloned cDNA representing the genomic RNA encoding the MP of the chimeric BMV mutant was analyzed and found to contain a mutation in the CMV MP gene resulting in deletion of the C-terminal 33 amino acids of the MP. Directed mutagenesis of the CMV MP gene showed that the C-terminal deletion was responsible for the movement capability of the mutant. When the mutation was introduced into CMV, the CMV mutant moved from cell to cell in C. quinoa, though the movement was less efficient than that of the wild-type CMV. These results indicate that the CMV MP, except the C-terminal 33 amino acids, potentiates cell-to-cell movement of both BMV and CMV in C. quinoa. In addition, since C. quinoa is a common host for both BMV and CMV, these results suggest that the CMV MP has specificity for the viral genomes during cell-to-cell movement of the virus and that the C-terminal 33 amino acids of the CMV MP are involved in that specificity.     

Collagen binding is a key factor for the hemorrhagic activity of snake venom metalloproteinases

Snake venom metalloproteinases (SVMPs) are multifunctional enzymes involved in several symptoms following snakebite, such as severe local hemorrhage. Multidomain P-III SVMPs are strongly hemorrhagic, whereas single domain P-I SVMPs are not. This indicates that disintegrin-like and cysteine-rich domains allocate motifs that enable catalytic degradation of ECM components leading to disruption of capillary vessels. Interestingly, some P-III SVMPs are completely devoid of hemorrhagic activity despite their highly conserved disintegrin-like and cysteine-rich domains. This observation was approached in the present study by comparing the effects of jararhagin, a hemorrhagic P-III SVMP, and berythractivase, a pro-coagulant and non-hemorrhagic P-III SVMP. Both toxins inhibited collagen-induced platelet aggregation, but only jararhagin was able to bind to collagen I with high affinity. The monoclonal antibody MAJar 3, that neutralizes the hemorrhagic effect of Bothrops venoms and jararhagin binding to collagen, did not react with berythractivase. The three-dimensional structures of jararhagin and berythractivase were compared to explain the differential binding to collagen and MAJar 3. Thereby, we pinpointed a motif within the Da disintegrin subdomain located opposite to the catalytic domain. Jararhagin binds to both collagen I and IV in a triple helix-dependent manner and inhibited in vitro fibrillogenesis. The jararhagin-collagen complex retained the catalytic activity of the toxin as observed by hydrolysis of fibrin. Thus, we suggest that binding of hemorrhagic SVMPs to collagens I and IV occurs through a motif located in the Da subdomain. This allows accumulation of toxin molecules at the site of injection, close to capillary vessels, where their catalytic activity leads to a local hemorrhage. Toxins devoid of this motif would be more available for vascular internalization leading to systemic pro-coagulant effects. This reveals a novel function of the disintegrin domain in hemorrhage formation.     

Concurrent binding to multiple ligands: kinetic rates of CD16b for membrane-bound IgG1 and IgG2

CD16b (FcgammaRIIIb) is the most common receptor for the Fc domain of IgG on leukocytes. The binding of Fc receptors to immunoglobulin triggers a wide array of immune responses. In published assays measuring the reaction of CD16b with isotypes of soluble IgG, the affinity for IgG1 was low and that for IgG2 was undetectable. Here we report the first measurement of kinetic rates of CD16b binding to membrane-bound IgG isotypes-a physically distinct and physiologically more relevant presentation-using a recently developed micropipette method. In contrast to the soluble data, we found clearly measurable IgG2 binding, with a forward kinetic rate six-fold lower than that of IgG1 but with an equilibrium affinity only threefold lower. This suggests a nonnegligible role for IgG2 in Fc-mediated immune responses, particularly in longer duration contacts. The binding constants were measured from two sets of experiments. Single-isotype experiments were analyzed by an existing model (, Biophys. J. 75:1553-1572). The resulting kinetic rates were used as input to an extended model (, Biophys. J. 79:1850-1857.) to predict the results of mixed-isotype experiments. This design enabled rigorous validation of the concurrent binding model through a test of its predictive ability.     

Protein D2 channel of the Pseudomonas aeruginosa outer membrane has a binding site for basic amino acids and peptides

Protein D2 of Pseudomonas aeruginosa outer membrane is known to facilitate the specific permeation of imipenem (N-formimdoylthienamycin) across this membrane barrier. We have characterized the binding site in the protein D2 channel by studying the competitive inhibition, by various solutes, of imipenem diffusion into the periplasm. We found that basic amino acids, lysine, arginine, histidine, and ornithine, were effective inhibitors. L- and D-lysine were found to be competitive inhibitors with approximate Ki values of 0.6 and 0.3 mM, respectively. Peptides containing L-lysine at the carboxyl terminus, as well as dipeptides containing L-lysine at the amino terminus, were also able to inhibit the transport. Wild type cells transported tripeptide Thr-Ser-Lys into the periplasm three to four times as rapidly as the mutant cells lacking the D2 protein. These results suggest that protein D2 plays a physiologically significant role in the uptake of basic amino acids and peptides containing these amino acids across the outer membrane of P. aeruginosa.     

A mosaic multiple-binding model for the binding of caldesmon and myosin subfragment-1 to actin.

Binding of caldesmon to actin causes a decrease in the quantity of bound myosin and results in a reduction in the rate of actin-activated adenosine triphosphate hydrolysis. It is generally assumed that the binding of caldesmon and myosin to actin is a pure competitive interaction. However, recent binding studies of enzyme digested caldesmon subfragments directed at mapping the actin binding site of caldesmon have shown that a small 8-kD fragment around the COOH-terminal can compete directly with the myosin subfragment 1 (S-1) binding to actin; at least one other fragment that binds to actin does not inhibit the actin-activated adenosine triphosphate activity of myosin. That is, only a part of the caldesmon sequence may be responsible for directly blocking the binding of S-1 to actin. This prompts us to question the actual mode of binding of intact caldesmon and myosin S-1 to actin: whether the entire intact caldesmon molecule is competing with S-1 binding (pure competitive model) or just a small part of it (mosaic multiple-binding model). To answer this question, we measured the amount of myosin S-1 and caldesmon bound per actin monomer as a function of the total concentration of S-1 added to the system at constant concentrations of actin and caldesmon. A formalism for calculating the titration data based on the pure competitive model and a mosaic multiple-binding model was then developed. When compared with theoretical calculations, it is found that the binding of caldesmon and S-1 to actin cannot be pure competitive if no cooperativity exists between S-1 and caldesmon.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of three amino acids in the platelet-derived growth factor (PDGF) B-chain that are important for binding to the PDGF beta-receptor.

Platelet-derived growth factor (PDGF) is a disulfide-linked dimeric protein composed of two homologous polypeptide chains denoted A and B. Two types of PDGF receptors, alpha and beta, have been characterized. Whereas PDGF-AA binds only to PDGF alpha-receptors, PDGF-BB binds to both receptor types with high affinity. To map the regions of the PDGF B-chain that confer its ability to bind with high affinity to the PDGF beta-receptor, we expressed PDGF A/B-chain chimeras in COS cells and analyzed them with regard to PDGF alpha- and beta-receptor binding. A systematic analysis revealed that replacement of Asn-115, Arg-154, and Ile-158 of the PDGF B-chain with the corresponding A-chain amino acids led to a dramatic decrease in the affinity for the beta-receptor. Conversely, introduction of B-chain amino acids into the A-chain in the region spanning from Asn-115 to Ile-158 yielded a product with high affinity for the beta-receptor. These data thus indicate that Asn-115, Arg-154, and Ile-158 are likely to be part of the active site of the PDGF B-chain.     

The leech product saratin is a potent inhibitor of platelet integrin alpha2beta1 and von Willebrand factor binding to collagen

Subendothelial collagen plays an important role, via both direct and indirect mechanisms, in the initiation of thrombus formation at sites of vascular injury. Collagen binds plasma von Willebrand factor, which mediates platelet recruitment to collagen under high shear. Subsequently, the direct binding of the platelet receptors glycoprotein VI and alpha2beta1 to collagen is critical for platelet activation and stable adhesion. Leeches, have evolved a number of inhibitors directed towards platelet-collagen interactions so as to prevent hemostasis in the host during hematophagy. In this article, we describe the molecular mechanisms underlying the ability of the leech product saratin to inhibit platelet binding to collagen. In the presence of inhibitors of ADP and thromboxane A2, both saratin and 6F1, a blocking alpha2beta1 mAb, abrogated platelet adhesion to fibrillar and soluble collagen. Additionally, saratin eliminated alpha2beta1-dependent platelet adhesion to soluble collagen in the presence of an Src kinase inhibitor. Moreover, saratin prevented platelet-rich plasma adhesion to fibrillar collagen, a process dependent upon both alpha2beta1 and von Willebrand factor binding to collagen. Furthermore, saratin specifically inhibited the binding of the alpha2 integrin subunit I domain to collagen, and prevented platelet adhesion to collagen under flow to the same extent as observed in the presence of a combination of mAbs to glycoprotein Ib and alpha2beta1. These results demonstrate that saratin interferes with integrin alpha2beta1 binding to collagen in addition to inhibiting von Willebrand factor-collagen binding, presumably by binding to an overlapping epitope on collagen. This has significant implications for the use of saratin as a tool to inhibit platelet-collagen interactions.     

A cluster of aromatic amino acids in the i2 loop plays a key role for Gs coupling in prostaglandin EP2 and EP3 receptors

To assess the structural requirements for G(s) coupling by prostaglandin E receptors (EPs), the G(s)-coupled EP2 and G(i)-coupled EP3beta receptors were used to generate hybrid receptors. Interchanging of the whole i2 loop and its N-terminal half (i2N) had no effect on the binding of both receptors expressed in HEK293 cells. Agonist-induced cAMP formation was observed in wild type EP2 but not in the i2 loop- or i2N-substituted EP2. Wild type EP3beta left cAMP levels unaffected, whereas i2 loop- and i2N-substituted EP3 gained agonist-induced adenylyl cyclase stimulation. In EP2, the ability to stimulate cAMP formation was lost by mutation of Tyr(143) into Ala but retained by mutations into Phe, Trp, and Leu. Consistent with this observation, substitution of the equivalent His(140) enabled EP3beta to stimulate cAMP formation with the rank order of Phe > Tyr > Trp > Leu. The point mutation of His(140) into Phe was effective in another EP3 variant in which its C-terminal tail is different or lacking. Simultaneous mutation of the adjacent Trp(141) to Ala but not at the following Tyr(142) weakened the acquired ability to stimulate cAMP levels in the EP3 mutant. Mutation of EP2 at adjacent Phe(144) to Ala but not at Tyr(145) reduced the efficiency of agonist-induced cAMP formation. In Chinese hamster ovary cells stably expressing G(s)-acquired EP3 mutant, an agonist-dependent cAMP formation was observed, and pertussis toxin markedly augmented cAMP formation. These results suggest that a cluster of hydrophobic aromatic amino acids in the i2 loop plays a key role for G(s) coupling.