A minimized human integrin alpha(5)beta(1) that retains ligand recognition

Two isolated recombinant fragments from human integrin alpha(5)beta(1) encompassing the FG-GAP repeats III to VII of alpha(5) and the insertion-type domain from beta(1), respectively, are structurally well defined in solution, based on CD evidence. Divalent cation binding induces a conformational adaptation that is achieved by Ca(2+) or Mg(2+) (or Mn(2+)) with alpha(5) and only by Mg(2+) (or Mn(2+)) with beta(1). Mn(2+) bound to beta(1) is highly hydrated ( approximately 3 water molecules), based on water NMR relaxation, in agreement with a metal ion-dependent adhesion site-type metal coordination. Each fragment saturated with Mg(2+) (or Mn(2+)) binds a recombinant fibronectin ligand in an RGD-dependent manner. A conformational rearrangement is induced on the fibronectin ligand upon binding to the alpha(5), but not to the beta(1) fragment, based on CD. Ligand binding results in metal ion displacement from beta(1). Both alpha(5) and beta(1) fragments form a stable heterodimer (alpha(5)beta(1) mini-integrin) that retains ligand recognition to form a 1:1:1 ternary complex, in the presence of Mg(2+), and induces a specific conformational adaptation of the fibronectin ligand. A two-site model for RGD binding to both alpha and beta integrin components is inferred from our data using low molecular weight RGD mimetics.     

Studies on the metal binding sites in the catalytic domain of beta1,4-galactosyltransferase

The catalytic domain of bovine beta1,4-galactosyltransferase (beta4Gal-T1) has been shown to have two metal binding sites, each with a distinct binding affinity. Site I binds Mn(2+) with high affinity and does not bind Ca(2+), whereas site II binds a variety of metal ions, including Ca(2+). The catalytic region of beta4Gal-T1 has DXD motifs, associated with metal binding in glycosyltransferases, in two separate sequences: D(242)YDYNCFVFSDVD(254) (region I) and W(312)GWGGEDDD(320) (region II). Recently, the crystal structure of beta4Gal-T1 bound with UDP, Mn(2+), and alpha-lactalbumin was determined in our laboratory. It shows that in the primary metal binding site of beta4Gal-T1, the Mn(2+) ion, is coordinated to five ligands, two supplied by the phosphates of the sugar nucleotide and the other three by Asp254, His347, and Met344. The residue Asp254 in the D(252)VD(254) sequence in region I is the only residue that is coordinated to the Mn(2+) ion. Region II forms a loop structure and contains the E(317)DDD(320) sequence in which residues Asp318 and Asp319 are directly involved in GlcNAc binding. This study, using site-directed mutagenesis, kinetic, and binding affinity analysis, shows that Asp254 and His347 are strong metal ligands, whereas Met344, which coordinates less strongly, can be substituted by alanine or glutamine. Specifically, substitution of Met344 to Gln has a less severe effect on the catalysis driven by Co(2+). Glu317 and Asp320 mutants, when partially activated by Mn(2+) binding to the primary site, can be further activated by Co(2+) or inhibited by Ca(2+), an effect that is the opposite of what is observed with the wild-type enzyme.     

The functional role of the binuclear metal center in D-aminoacylase: one-metal activation and second-metal attenuation

Our structural comparison of the TIM barrel metal-dependent hydrolase(-like) superfamily suggests a classification of their divergent active sites into four types: alphabeta-binuclear, alpha-mononuclear, beta-mononuclear, and metal-independent subsets. The d-aminoacylase from Alcaligenes faecalis DA1 belongs to the beta-mononuclear subset due to the fact that the catalytically essential Zn(2+) is tightly bound at the beta site with coordination by Cys(96), His(220), and His(250), even though it possesses a binuclear active site with a weak alpha binding site. Additional Zn(2+), Cd(2+), and Cu(2+), but not Ni(2+), Co(2+), Mg(2+), Mn(2+), and Ca(2+), can inhibit enzyme activity. Crystal structures of these metal derivatives show that Zn(2+) and Cd(2+) bind at the alpha(1) subsite ligated by His(67), His(69), and Asp(366), while Cu(2+) at the alpha(2) subsite is chelated by His(67), His(69) and Cys(96). Unexpectedly, the crystal structure of the inactive H220A mutant displays that the endogenous Zn(2+) shifts to the alpha(3) subsite coordinated by His(67), His(69), Cys(96), and Asp(366), revealing that elimination of the beta site changes the coordination geometry of the alpha ion with an enhanced affinity. Kinetic studies of the metal ligand mutants such as C96D indicate the uniqueness of the unusual bridging cysteine and its involvement in catalysis. Therefore, the two metal-binding sites in the d-aminoacylase are interactive with partially mutual exclusion, thus resulting in widely different affinities for the activation/attenuation mechanism, in which the enzyme is activated by the metal ion at the beta site, but inhibited by the subsequent binding of the second ion at the alpha site.     

Multiple activation states of integrin alpha4beta1 detected through their different affinities for a small molecule ligand

We have used the highly specific alpha4beta1 inhibitor 4-((N'-2-methylphenyl)ureido)-phenylacetyl-leucine-aspartic acid-valine-proline (BIO1211) as a model LDV-containing ligand to study alpha4beta1 integrin-ligand interactions on Jurkat cells under diverse conditions that affect the activation state of alpha4beta1. Observed KD values for BIO1211 binding ranged from a value of 20-40 nM in the non-activated state of the integrin that exists in 1 mM Mg2+, 1 mM Ca2+ to 100 pM in the activated state seen in 2 mM Mn2+ to 18 pM when binding was measured after co-activation by 2 mM Mn2+ plus 10 microgram/ml of the integrin-activating monoclonal antibody TS2/16. The large range in KD values was governed almost exclusively by differences in the dissociation rates of the integrin-BIO1211 complex, which ranged from 0.17 x 10(-4) s-1 to >140 x 10(-4) s-1. Association rate constants varied only slightly under the same conditions, all falling in the narrow range from 0.9 to 2.7 x 10(6) M-1 s-1. The further increase in affinity observed upon co-activation by divalent cations and TS2/16 compared with that observed at saturating concentrations of metal ions or TS2/16 alone indicates that the mechanism by which these factors bring about activation are distinct and identified a previously unrecognized high affinity state on alpha4beta1 that had not been detected by conventional assay methods. Similar changes in affinity were observed when the binding properties of vascular cell adhesion molecule-1 and CS1 to alpha4beta1 were studied, indicating that the different affinity states detected with BIO1211 are an inherent property of the integrin.     

Distinct roles of beta1 metal ion-dependent adhesion site (MIDAS), adjacent to MIDAS (ADMIDAS), and ligand-associated metal-binding site (LIMBS) cation-binding sites in ligand recognition by integrin alpha2beta1

Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding sites. In collagen-binding integrins, such as alpha2beta1, ligand recognition takes place exclusively at the alpha subunit I domain. However, activation of the alphaI domain depends on its interaction with a structurally similar domain in the beta subunit known as the I-like or betaI domain. The top face of the betaI domain contains three cation-binding sites: the metal-ion dependent adhesion site (MIDAS), the ADMIDAS (adjacent to MIDAS), and LIMBS (ligand-associated metal-binding site). The role of these sites in controlling ligand binding to the alphaI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to alpha2beta1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating monoclonal antibody TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between alphaI and betaI, whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of betaI. An activating mutation in the alpha2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca(2+), Mg(2+), and Mn(2+) on ligand binding to these mutants showed that the MIDAS is a ligand-competent site through which Mn(2+) stimulates ligand binding, whereas the LIMBS is a stimulatory Ca(2+)-binding site, occupancy of which increases the affinity of Mg(2+) for the MIDAS.     

Effect of divalent cations on the affinity and selectivity of alpha4 integrins towards the integrin ligands vascular cell adhesion molecule-1 and mucosal addressin cell adhesion molecule-1: Ca2+ activation of integrin alpha4beta1 confers a distinct ligand specificity

A microtiter plate assay measuring the binding of cells expressing integrins alpha4beta1 or alpha4beta7 to VCAM-1 and MAdCAM-1, expressed as Ig fusion proteins, was used to explore the interplay between the variables of integrin beta-chain, identity and density of ligand, and identity and concentration of activating cations. Both Mn2+ and Mg2+ supported binding of either integrin to either ligand. Ca2+ supported only the binding of alpha4beta1 to VCAM-Ig. Cation concentrations required for half-maximal binding (EC50) ranged from 0.8-280 microM for Mn2+ and 0.8-30 mM for Mg2+, being thus 2-3 logs lower for Mn2+ compared to Mg2+ independent of ligand. EC50 values for binding of alpha4beta1 to VCAM-Ig were 30-45-fold lower compared to MAdCAM-Ig, while alpha4beta7 showed an opposite 3-15-fold selectivity for MAdCAM-Ig over VCAM-Ig. The density of ligand required for adhesion via alpha4beta1 was markedly lower with Mn2+ versus Mg2+, and with VCAM-Ig versus MAdCAM-Ig. These results were interpreted in terms of a coupled equilibrium model, in which binding of activating metal ions and of integrin ligands each stabilizes activated integrin. We conclude that Mn2+ and Mg2+ bind to common regulatory sites with different affinities, producing similar activated states of the integrin. The resulting activated alpha4beta1 binds more strongly to VCAM-Ig versus MAdCAM-Ig by 30-45-fold, while similarly activated alpha4beta7 binds more strongly to MAdCAM-Ig versus VCAM-Ig by 3-15-fold. Inhibition studies showed that Ca2+ also binds to regulatory sites on both integrins. However, the Ca2+-activated state of alpha4beta1 is distinct from that achieved by Mn2+ and Mg2+, possessing increased selectivity for binding to VCAM-1 versus MAdCAM-1.     

Characterization of the metal ion-binding domains from rat alpha- and beta-parvalbumins

We have examined the metal ion-binding domains from rat alpha and beta parvalbumin. We find that the CD-EF fragments differ markedly in their tendency to self-associate. Whereas Ca(2+)-free alpha CD-EF is monomeric, the Ca(2+)-free beta peptide dimerizes weakly (K(2) = 2400 +/- 200 M(-1)). In buffer containing 1.0 mM Ca(2+), the apparent dimerization constant for beta CD-EF (191,000 +/- 29,000 M(-1)) is more than 50 times that of alpha (3400 +/- 200 M(-1)). Alpha CD-EF binds two Ca(2+) with positive cooperativity. Titration calorimetry data afford binding constants of 3.7(0.1) x 10(3) M(-1) and 8.6(0.2) x 10(4) M(-1). Beta CD-EF also binds two Ca(2+) cooperatively but with lower affinity. Equilibrium dialysis yields Adair constants of 4.2(0.1) x 10(3) and 6.1(0.2) x 10(3) M(-1). Significantly, the difference in Ca(2+) affinity is substantially smaller than that observed for the full-length proteins-suggesting that the AB domain can modulate divalent ion affinity. Analysis of beta calorimetry data requires explicit consideration of the self-association behavior. Data collected at low CD-EF concentration are consistent with preferential occupation of the EF site, dimerization of singly bound monomers, and cooperative filling of the CD sites. At higher concentrations, apo-protein dimerization can apparently precede cooperative occupation of the EF sites. In the presence of Ca(2+), alpha CD-EF exhibits higher thermal stability, consistent with its higher Ca(2+) affinity. However, the beta melting temperature shows greater concentration dependence, consistent with its greater tendency to dimerize. Neither fragment exhibits a sigmoidal melting curve in the Ca(2+)-free state, suggesting that the apo-peptides are disordered.     

The relative influence of metal ion binding sites in the I-like domain and the interface with the hybrid domain on rolling and firm adhesion by integrin alpha4beta7

We examined the effect of conformational change at the beta(7) I-like/hybrid domain interface on regulating the transition between rolling and firm adhesion by integrin alpha(4)beta(7). An N-glycosylation site was introduced into the I-like/hybrid domain interface to act as a wedge and to stabilize the open conformation of this interface and hence the open conformation of the alpha(4) beta(7) headpiece. Wild-type alpha(4)beta(7) mediates rolling adhesion in Ca(2+) and Ca(2+)/Mg(2+) but firm adhesion in Mg(2+) and Mn(2+). Stabilizing the open headpiece resulted in firm adhesion in all divalent cations. The interaction between metal binding sites in the I-like domain and the interface with the hybrid domain was examined in double mutants. Changes at these two sites can either counterbalance one another or be additive, emphasizing mutuality and the importance of multiple interfaces in integrin regulation. A double mutant with counterbalancing deactivating ligand-induced metal ion binding site (LIMBS) and activating wedge mutations could still be activated by Mn(2+), confirming the importance of the adjacent to metal ion-dependent adhesion site (ADMIDAS) in integrin activation by Mn(2+). Overall, the results demonstrate the importance of headpiece allostery in the conversion of rolling to firm adhesion.     

X-ray Structures of Magnesium and Manganese Complexes with the N-Terminal Domain of Calmodulin: Insights into the Mechanism and Specificity of Metal Ion Binding to an EF-Hand

Calmodulin (CaM), a member of the EF-hand superfamily, regulates many aspects of cell function by responding specifically to micromolar concentrations of Ca(2+) in the presence of an ～1000-fold higher concentration of cellular Mg(2+). To explain the structural basis of metal ion binding specificity, we have determined the X-ray structures of the N-terminal domain of calmodulin (N-CaM) in complexes with Mg(2+), Mn(2+), and Zn(2+). In contrast to Ca(2+), which induces domain opening in CaM, octahedrally coordinated Mg(2+) and Mn(2+) stabilize the closed-domain, apo-like conformation, while tetrahedrally coordinated Zn(2+) ions bind at the protein surface and do not compete with Ca(2+). The relative positions of bound Mg(2+) and Mn(2+) within the EF-hand loops are similar to those of Ca(2+); however, the Glu side chain at position 12 of the loop, whose bidentate interaction with Ca(2+) is critical for domain opening, does not bind directly to either Mn(2+) or Mg(2+), and the vacant ligand position is occupied by a water molecule. We conclude that this critical interaction is prevented by specific stereochemical constraints imposed on the ligands by the EF-hand β-scaffold. The structures suggest that Mg(2+) contributes to the switching off of calmodulin activity and possibly other EF-hand proteins at the resting levels of Ca(2+). The Mg(2+)-bound N-CaM structure also provides a unique view of a transiently bound hydrated metal ion and suggests a role for the hydration water in the metal-induced conformational change.     

Remarkable affinity and selectivity for Cs+ and uranyl (UO22+) binding to the manganese site of the apo-water oxidation complex of photosystem II.

The size and charge density requirements for metal ion binding to the high-affinity Mn2+ site of the apo-water oxidizing complex (WOC) of spinach photosystem II (PSII) were studied by comparing the relative binding affinities of alkali metal cations, divalent metals (Mg2+, Ca2+, Mn2+, Sr2+), and the oxo-cation UO22+. Cation binding to the apo-WOC-PSII protein was measured by: (1) inhibition of the rate and yield of photoactivation, the light-induced recovery of O2 evolution by assembly of the functional Mn4Ca1Clx, core from its constituent inorganic cofactors (Mn2+, Ca2+, and Cl-); and by (2) inhibition of the PSII-mediated light-induced electron transfer from Mn2+ to an electron acceptor (DCIP). Together, these methods enable discrimination between inhibition at the high- and low-affinity Mn2+ sites and the Ca2+ site of the apo-WOC-PSII. Unexpectedly strong binding of large alkali cations (Cs+ > Rb+ > K+ > Na+ > Li+) was found to smoothly correlate with decreasing cation charge density, exhibiting one of the largest Cs+/Li+ selectivities (>/=5000) for any known chelator. Both photoactivation and electron-transfer measurements at selected Mn2+ and Ca2+ concentrations reveal that Cs+ binds to the high-affinity Mn2+ site with a slightly greater affinity (2-3-fold at pH 6.0) than Mn2+, while binding about 10(4)-fold more weakly to the Ca2+-specific site required for reassembly of functional O2 evolving centers. In contrast to Cs+, divalent cations larger than Mn2+ bind considerably more weakly to the high-affinity Mn2+ site (Mn2+ > Ca2+ > Sr2+). Their affinities correlate with the hydrolysis constant for formation of the metal hydroxide by hydrolysis of water: Me2+aq --> [MeOH]+aq + H+aq. Along with the strong stimulation of the rate of photoactivation by alkaline pH, these metal cation trends support the interpretation that [MnOH]+ is the active species that forms upon binding of Mn2+aq to apo-WOC. Further support for this interpretation is found by the unusually strong inhibition of Mn2+ photooxidation by the linear uranyl cation (UO22+). The intrinsic binding constant for [MnOH]+ to apo-WOC was determined using a thermodynamic cycle to be K = 4.0 x 10(15) M-1 (at pH 6.0), consistent with a high-affinity, preorganized, multidentate coordination site. We propose that the selectivity for binding [MnOH]+, a linear low charge-density monocation, vs symmetrical Me2+ dications is functionally important for assembly of the WOC by enabling: (1) discrimination against higher charge density alkaline earth cations (Mg2+ and Ca2+) and smaller alkali metal cations (Na+ and K+) that are present in considerably greater abundance in vivo, and thus would suppress photoactivation; and (2) higher affinity binding of the one Ca2+ ion or the remaining three Mn2+ ions via coordination to form mu-hydroxo-bridged intermediates, apo-WOC-[Mn(mu-OH)2Mn]3+ or apo-WOC-[Mn(mu-OH)Ca]3+, during subsequent assembly steps of the native Mn4Ca1Clx core. In contrast to more acidic Me2+ divalent ion inhibitors of the high-affinity Mn2+ site, like Ca2+ and Sr2+, Cs+ does not accelerate the decay of the first light-induced intermediate, IM1, formed during photoactivation (attributed to apo-WOC-[Mn(OH)2]+). The inability of Cs+ to promote decay of IM1, despite having comparable affinity as Mn2+, is consistent with its considerably weaker Lewis acidity, resulting in the reprotonation of IM1 by water becoming the rate-limiting step for decay prior to displacement of Mn2+. All four different lines of evidence provide a self-consistent picture indicating that the initial step in assembly of the WOC involves high-affinity binding of [MnOH]+.     

Bistable regulation of integrin adhesiveness by a bipolar metal ion cluster

Integrin alpha(4)beta(7) mediates rolling adhesion in Ca(2+) and Ca(2+) + Mg(2+), and firm adhesion in Mg(2+) and Mn(2+), mimicking the two key steps in leukocyte accumulation in inflamed vasculature. We mutated an interlinked linear array of three divalent cation-binding sites present in integrin beta-subunit I-like domains. The middle, metal ion-dependent adhesion site (MIDAS) is required for both rolling and firm adhesion. One polar site, that adjacent to MIDAS (ADMIDAS), is required for rolling because its mutation results in firm adhesion. The other polar site, the ligand-induced metal binding site (LIMBS), is required for firm adhesion because its mutation results in rolling. The LIMBS mediates the positive regulatory effects of low Ca(2+) concentrations, whereas the ADMIDAS mediates the negative regulatory effects of higher Ca(2+) concentrations, which are competed by Mn(2+). The bipolar sites thus stabilize two alternative phases of adhesion.     

Different cation binding to the I domains of alpha1 and alpha2 integrins: implication of the binding site structure.

In the present work, we studied the interactions of recombinant alpha1 and alpha2 integrin I domains with cations Tb(3+), Mn(2+), Mg(2+) and Ca(2+). We observed that alpha1 and alpha2 I domains bind these cations with significantly different characteristics. The binding of Mg(2+) by the alpha1 I domain was accompanied by significant changes of tryptophan fluorescence which could be interpreted as a conformational change. Comparison of the alpha1 integrin I domain structure obtained by comparative modeling with a known structure of the alpha2 integrin I domain shows distinct differences in the metal ion binding sites which could explain the differences in cation binding.     

Fe binding properties of two soybean (Glycine max L.) LEA4 proteins associated with antioxidant activity

Late embryogenesis abundant (LEA) group 4 (LEA4) proteins play an important role in the water stress tolerance of plants. Although they have been hypothesized to stabilize macromolecules in stressed cells, the protective functions and mechanisms of LEA4 proteins are still not clear. In this study, the metal binding properties of two related soybean LEA4 proteins, GmPM1 and GmPM9, were tested using immobilized metal ion affinity chromatography (IMAC). The metal ions Fe(3+), Ni(2+), Cu(2+) and Zn(2+) were observed to bind these two proteins, while Ca(2+), Mg(2+) or Mn(2+) did not. Results from isothermal titration calorimetry (ITC) indicated that the binding affinity of GmPM1 for Fe(3+) was stronger than that of GmPM9. Hydroxyl radicals generated by the Fe(3+)/H(2)O(2) system were scavenged by both GmPM1 and GmPM9 in the absence or the presence of high ionic conditions (100 mM NaCl), although the scavenging activity of GmPM1 was significantly greater than that of GmPM9. These results suggest that GmPM1 and GmPM9 are metal-binding proteins which may function in reducing oxidative damage induced by abiotic stress in plants.     

Association of the AB and CD-EF domains from rat alpha- and beta-parvalbumin

Association of the parvalbumin AB and CD-EF domains was examined in Hepes-buffered saline, pH 7.4, employing fragments from rat alpha and beta. All of the interactions require Ca(2+). In saturating Ca(2+), the alpha AB/alpha CD-EF (alpha/alpha) complex displays an association constant of (7.6 +/- 0.4) x 10(7) M(-1). Ca(2+)-binding data for a mixture of the alpha fragments are compatible with an identical two-site model, yielding an average binding constant of (8.5 +/- 0.2) x 10(5) M(-1). The beta/beta interaction is significantly weaker, exhibiting an association constant of (3.0 +/- 0.6) x 10(6) M(-1). The Ca(2+)-binding constants for beta/beta are likewise diminished, at (1.0 +/- 0.1) x 10(5) and (2.3 +/- 0.2) x 10(4) M(-1). The magnitude of the apparent DeltaDeltaG(degree)' for Ca(2+) binding by alpha/alpha and beta/beta, at 3.4 kcal/mol, approaches that measured for the intact proteins (3.6 kcal/mol) and is substantially larger than the 1.5 kcal/mol value previously measured for the isolated CD-EF domains. This result suggests that the AB domain can modulate the Ca(2+) affinities of the CD and EF sites. Interestingly, the heterologous alpha/beta complex displays a larger association constant [(6.6 +/- 0.4) x 10(6) M(-1)] than the homologous beta/beta complex and heightened Ca(2+) affinity [binding constants of (1.3 +/- 0.1) x 10(6) and (8.8 +/- 0.2) x 10(4) M(-1)]. By contrast, beta/alpha associates more weakly than alpha/alpha and exhibits sharply reduced affinity for Ca(2+). Thus, the interaction between the beta AB domain and beta CD-EF domain may act to attenuate Ca(2+) affinity in the intact protein.     

Comparative assessment of the ligand and metal ion binding properties of integrins alpha9beta1 and alpha4beta1

Integrins alpha9beta1 and alpha4beta1 form a distinct structural class, but while alpha4beta1 has been subjected to extensive study, alpha9beta1 remains poorly characterized. We have used the small molecule N-(benzenesulfonyl)-(L)-prolyl-(L)-O-(1-pyrrolidinylcarbonyl)tyrosine (3) to investigate the biochemical properties of alpha9beta1 and directly compare these properties with those of alpha4beta1. Compound 3 has a high affinity for both integrins with K(D) values of < or =3 and 180 pM for alpha9beta1 in 1 mM Mn2+ (activating) and 1 mM Ca2+ and 1 mM Mg2+ (nonactivating) conditions and < or =5 and 730 pM for alpha4beta1 under the corresponding conditions. Ca2+ treatment promoted the binding of 3 to both integrins (EC50 = 30 microM Ca2+ in both cases). Compound 3 binding to both integrins was also stimulated by the addition of the activating monoclonal antibody TS2/16. These findings indicate that the mechanisms by which metal ions and TS2/16 regulate ligand binding to alpha9beta1 and alpha4beta1 are similar. The binding of 3 to both integrins induced the mAb 9EG7 LIBS epitope, a property consistent with occupancy of the receptor's ligand binding site by 3. But whereas EGTA treatment inhibited the binding of 9EG7 to alpha4beta1, it stimulated the binding of 9EG7 to alpha9beta1. The 9EG7 and TS2/16 effects point to contributions of the beta1-chains on binding. Cross-linking data revealed that the integrin alpha-chains are also involved in binding the small molecule, as stable linkages were observed on both the alpha9 chain of alpha9beta1 and the alpha4 chain of alpha4beta1. Extensive structure-activity analyses with natural and synthetic ligands indicate distinct features of the ligand binding pockets. Most notable was the estimated >1000-fold difference in the affinity of the integrins for VCAM-1, which binds alpha4beta1with an apparent K(D) of 10 nM and alpha9beta1 with an apparent K(D) of >10 microM. Differences were also seen in the binding of alpha9beta1 and alpha4beta1 to osteopontin. Compound 3 competed effectively for the binding of VCAM-1 and osteopontin to both integrins. While these studies show many similarities in the biochemical properties of alpha9beta1 and alpha4beta1, they identify important differences in their structure and function that can be exploited in the design of selective alpha9beta1 and alpha4beta1 inhibitors.     

Kinetics of conformational changes induced by the binding of various metal ions to bovine alpha-lactalbumin.

The kinetic effects of the binding of various metal ions (Ca(2+), Cd(2+), Co(2+), Mg(2+), Mn(2+), Sr(2+) and Zn(2+)) to apo bovine alpha-lactalbumin has been monitored by means of stopped-flow fluorescence spectroscopy. Our results show that the measured rate constant for the binding of metal ions to the Ca(2+)-site increases with increasing binding constant. This is, however, not the case for metal ions binding to the Zn(2+)-site. The binding experiments performed at different temperatures allowed us to calculate the activation energy for the transition from the metal-free to the metal-loaded state of the protein. These values do not depend on the nature of the metal ion but are correlated with the type of binding site. As a result, we were able to demonstrate that Mg(2+), a metal ion which was thought to bind to the Ca(2+)-site, shows the same binding characteristics as Co(2+) and Zn(2+) and therefore most likely interacts with the residues belonging to the Zn(2+)-binding site.     

Biosynthesis in vitro of SA-Lex and SA-diLex by alpha 1-3 fucosyltransferases from colon carcinoma cells and embryonic brain tissues.

The sialyl-fucosyl-lactosamine-epitope present in sialyl (SA)-Lex (NeuAc alpha 2-3Gal beta 1-4 [Fuc alpha 1-3]GlcNAc beta 1-3Gal beta 1-4Glc-Cer), a carcinoembryonic antigen, has been recognized recently as a ligand for the binding of leukocyte-endothelial cell adhesion molecule 1 (LECAM-1) to myeloid and tumour cell surfaces. We have recently detected the presence of an alpha 1-3 fucosyltransferase (FucT-3) activity in both embryonic chicken brain (ECB) and human colon carcinoma cells (Colo-205) which catalyses the biosynthesis in vitro of SA-Lex and SA-diLex. Fucosyltransferase activities from both sources are stimulated in the presence of divalent cations (Mn2+, Mg2+, Ca2+, Co2+ and Fe2+), although absolute metal requirement is not observed. Substrate specificity studies with this partially purified (ECB, 3000-fold; Colo-205, 100-fold) novel FucT-3 indicate the preference for terminally sialyl-substituted glycolipid acceptors, as observed by the lower Km values when sialyl-neolactotetraosyl ceramide, LM1, (Neu-Gc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4 Glc-Cer; Km = 0.048 mM) and sialyl-norhexaosylceramide, NeuGc-nLc6, (Neu-Gc alpha 2-3Gal beta 1-4 GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer; Km = 0.032 mM) were used as substrates. Fucosyltransferase from Colo-205 requires the presence of the acyl group of the ceramide moiety and an acetyl group on glucosamine in the acceptor glycolipid since lyso-LM1 was found to be completely inactive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cation charge and size selectivity of the C2 domain of cytosolic phospholipase A(2).

C2 domains regulate numerous eukaryotic signaling proteins by docking to target membranes upon binding Ca(2+). Effective activation of the C2 domain by intracellular Ca(2+) signals requires high Ca(2+) selectivity to exclude the prevalent physiological metal ions K(+), Na(+), and Mg(2+). The cooperative binding of two Ca(2+) ions to the C2 domain of cytosolic phospholipase A(2) (cPLA(2)-alpha) induces docking to phosphatidylcholine (PC) membranes. The ionic charge and size selectivities of this C2 domain were probed with representative mono-, di-, and trivalent spherical metal cations. Physiological concentrations of monovalent cations and Mg(2+) failed to bind to the domain and to induce docking to PC membranes. Superphysiological concentrations of Mg(2+) did bind but still failed to induce membrane docking. In contrast, Ca(2+), Sr(2+), and Ba(2+) bound to the domain in the low micromolar range, induced electrophoretic mobility shifts in native polyacrylamide gels, stabilized the domain against thermal denaturation, and induced docking to PC membranes. In the absence of membranes, the degree of apparent positive cooperativity in binding of Ca(2+), Sr(2+), and Ba(2+) decreased with increasing cation size, suggesting that the C2 domain binds two Ca(2+) or Sr(2+) ions, but only one Ba(2+) ion. These stoichiometries were correlated with the abilities of the ions to drive membrane docking, such that micromolar concentrations of Ca(2+) and Sr(2+) triggered docking while even millimolar concentrations of Ba(2+) yielded poor docking efficiency. The simplest explanation is that two bound divalent cations are required for stable membrane association. The physiological Ca(2+) ion triggered membrane docking at 20-fold lower concentrations than Sr(2+), due to both the higher Ca(2+) affinity of the free domain and the higher affinity of the Ca(2+)-loaded domain for membranes. Kinetic studies indicated that Ca(2+) ions bound to the free domain are retained at least 5-fold longer than Sr(2+) ions. Moreover, the Ca(2+)-loaded domain remained bound to membranes 2-fold longer than the Sr(2+)-loaded domain. For both Ca(2+) and Sr(2+), the two bound metal ions dissociate from the protein-membrane complex in two kinetically resolvable steps. Finally, representative trivalent lanthanide ions bound to the domain with high affinity and positive cooperativity, and induced docking to PC membranes. Overall, the results demonstrate that both cation charge and size constraints contribute to the high Ca(2+) selectivity of the C2 domain and suggest that formation of a cPLA(2)-alpha C2 domain-membrane complex requires two bound multivalent metal ions. These features are proposed to stem from the unique structural features of the metal ion-binding site in the C2 domain.     

Activation and inhibition of photoreceptor guanylyl cyclase by guanylyl cyclase activating protein 1 (GCAP-1): the functional role of Mg2+/Ca2+ exchange in EF-hand domains

Guanylyl cyclase activating protein 1 (GCAP-1), a Ca(2+)/Mg(2+) sensor protein that accelerates retinal guanylyl cyclase (RetGC) in the light and decelerates it in the dark, is inactive in cation-free form. Binding of Mg(2+) in EF-hands 2 and 3 was essential for RetGC activation in the conditions mimicking light adaptation. Mg(2+) binding in EF-hand 2 affected the conformation of a neighboring non-metal binding domain, EF-hand-1, and increased GCAP-1 affinity for RetGC nearly 40-fold compared with the metal-free EF-hand 2. Mg(2+) binding in EF-hand 3 increased GCAP-1 affinity for RetGC 5-fold and its maximal RetGC stimulation 2-fold. Mg(2+) binding in EF-hand 4 affected neither GCAP-1 affinity for RetGC, nor RetGC activation. Inactivation of Ca(2+) binding in EF-hand 4 was sufficient to render GCAP-1 a constitutive activator of RetGC, whereas the EF-hand 3 role in Ca(2+)-dependent deceleration of RetGC was likely to be through the neighboring EF-hand 4. Inactivation of Ca(2+) binding in EF-hand 2 affected cooperativity of RetGC inhibition by Ca(2+), but did not prevent the inhibition. We conclude that 1) Mg(2+) binding in EF-hands 2 and 3, but not EF-hand 4, is essential for the ability of GCAP-1 to activate RetGC in the light; 2) Mg(2+) or Ca(2+) binding in EF-hand 3 and especially in EF-hand 2 is required for high-affinity interaction with the cyclase and affects the conformation of the neighboring EF-hand 1, a domain required for targeting RetGC; and 3) RetGC inhibition is likely to be primarily caused by Ca(2+) binding in EF-hand 4.     

Metal ion binding to human hemopexin

Binding of divalent metal ions to human hemopexin (Hx) purified by a new protocol has been characterized by metal ion affinity chromatography and potentiometric titration in the presence and absence of bound protoheme IX. ApoHx was retained by variously charged metal affinity chelate resins in the following order: Ni(2+) > Cu(2+) > Co(2+) > Zn(2+) > Mn(2+). The Hx-heme complex exhibited similar behavior except the order of retention of the complex on Zn(2+)- and Co(2+)-charged columns was reversed. One-dimensional (1)H NMR of apoHx in the presence of Ni(2+) implicates at least two His residues and possibly an Asp, Glu, or Met residue in Ni(2+) binding. Potentiometric titrations establish that apoHx possesses more than two metal ion binding sites and that the capacity and/or affinity for metal ion binding is diminished when heme binds. For most metal ions that have been studied, potentiometric data did not fit to binding isotherms that assume one or two independent binding sites. For Mn(2+), however, these data were consistent with a high-affinity site [K(A) = (15 +/- 3) x 10(6) M(-)(1)] and a low-affinity site (K(A) 相似文献