Structure and kinetics of phosphonopyruvate hydrolase from Variovorax sp. Pal2: new insight into the divergence of catalysis within the PEP mutase/isocitrate lyase superfamily

Phosphonopyruvate (P-pyr) hydrolase (PPH), a member of the phosphoenolpyruvate (PEP) mutase/isocitrate lyase (PEPM/ICL) superfamily, hydrolyzes P-pyr and shares the highest sequence identity and functional similarity with PEPM. Recombinant PPH from Variovorax sp. Pal2 was expressed in Escherichia coli and purified to homogeneity. Analytical gel filtration indicated that the protein exists in solution predominantly as a tetramer. The PPH pH rate profile indicates maximal activity over a broad pH range. The steady-state kinetic constants determined for a rapid equilibrium ordered kinetic mechanism with Mg2+ binding first (Kd = 140 +/- 40 microM), are kcat = 105 +/- 2 s(-1) and P-pyr Km = 5 +/- 1 microM. PEP (slow substrate kcat = 2 x 10(-4) s(-1)), oxalate, and sulfopyruvate are competitive inhibitors with Ki values of 2.0 +/- 0.1 mM, 17 +/- 1 microM, and 210 +/- 10 microM, respectively. Three PPH crystal structures have been determined, that of a ligand-free enzyme, the enzyme bound to Mg2+ and oxalate (inhibitor), and the enzyme bound to Mg2+ and P-pyr (substrate). The complex with the inhibitor was obtained by cocrystallization, whereas that with the substrate was obtained by briefly soaking crystals of the ligand-free enzyme with P-pyr prior to flash cooling. The PPH structure resembles that of the other members of the PEPM/ICL superfamily and is most similar to the functionally related enzyme, PEPM. Each monomer of the dimer of dimers exhibits an (alpha/beta)8 barrel fold with the eighth helix swapped between two molecules of the dimer. Both P-pyr and oxalate are anchored to the active site by Mg2+. The loop capping the active site is disordered in all three structures, in contrast to PEPM, where the equivalent loop adopts an open or disordered conformation in the unbound state but sequesters the inhibitor from solvent in the bound state. Crystal packing may have favored the open conformation of PPH even when the enzyme was cocrystallized with the oxalate inhibitor. Structure alignment of PPH with other superfamily members revealed two pairs of invariant or conservatively replaced residues that anchor the flexible gating loop. The proposed PPH catalytic mechanism is analogous to that of PEPM but includes activation of a water nucleophile with the loop Thr118 residue.     

Role of terminal nonhomologous domains in initiation of human red cell spectrin dimerization

Human erythrocyte spectrin is an antiparallel heterodimer comprised of a 280 kDa alpha subunit and a 246 kDa beta subunit which further associates into tetramers in the red cell membrane cytoskeleton. Lateral association of the flexible rodlike monomers involves a multiple-step process that is initiated by a high affinity association near the actin-binding end of the molecule (dimer nucleation site). In this study, recombinant alpha and beta proteins comprising two or four "spectrin type" motifs with and without adjacent, terminal nonhomologous domains were evaluated for their relative contributions to dimer initiation, and the thermodynamic properties of these heterodimer complexes were measured. Sedimentation equilibrium studies showed that in the absence of the heterologous subunit, individual recombinant proteins formed weak homodimers (K(d) > 0.3 mM). When 2-motif (alpha20-21 and beta1-2) and 4-motif (alpha18-21 and beta1-4) recombinants lacking the terminal nonhomologous domains were paired with the complementary protein, high affinity heterodimers were formed in sedimentation equilibrium analysis. Both the alpha20-21/beta1-2 complex and the alpha20-21EF/betaABD1-2 complex showed stoichiometric binding with similar binding affinities (K(d) approximately 10 nM) using isothermal titration calorimetry. The alpha20-21/beta1-2 complex showed an enthalpy of -10 kcal/mol, while the alpha20-21EF/betaABD1-2 complex showed an enthalpy of -13 kcal/mol. Pull-down assays using alpha spectrin GST fusion proteins showed strong associations between all heterodimer complexes in physiological buffer, but all heterodimer complexes were destabilized by the presence of Triton X-100 and other detergents. Complexes lacking the nonhomologous domains were destabilized to a greater extent than complexes that included the nonhomologous domains. The detergent effect appears to be responsible for the apparent essential role of the nonhomologous domains in prior reports. Taken together, our results indicate that the terminal nonhomologous domains do not contribute to dimer initiation nor are they required for formation of high affinity spectrin heterodimers in physiological buffers.     

Gonadotropin beta subunits determine the rate of assembly and the oligosaccharide processing of hormone dimer in transfected cells

The glycoprotein hormones lutropin (LH) and chorionic gonadotropin (CG) share a common structure consisting of an identical alpha subunit noncovalently linked to a hormone-specific beta subunit. While LH is produced in the anterior pituitary, CG is synthesized in placenta. To compare the assembly, processing, and secretion of human LH and CG in the same cell type, we have expressed their subunits, individually and together, in mouse C-127 mammary tumor cells. Analysis of transfected clones revealed an unexpected difference in the secretion of individually expressed subunits. Whereas alpha and CG beta subunits were rapidly and quantitatively secreted, only 10% of newly synthesized LH beta subunit reached the medium. The remaining subunit was found in an intracellular, endoglycosidase H (endo H)-sensitive pool that had a turnover rate of approximately 8 h. Coexpression with alpha subunit resulted in "rescue" of LH beta subunit by formation of LH dimer, which was efficiently secreted. However, combination of LH beta with alpha was slow, with an overall efficiency of only 50% despite the presence of excess alpha. In contrast, CG beta was rapidly assembled with the alpha subunit after synthesis. The two beta subunits also differed in their influence on the N-linked oligosaccharide processing of combined alpha. The oligosaccharides of LH dimer were endo H resistant, while those of CG dimer remained partially endo H sensitive. Thus, despite a high degree of homology between LH beta and CG beta, the two subunits differ in their secretion as free subunits, their rate of assembly with alpha subunit, and in their effect on the N-linked oligosaccharide processing of combined alpha.     

Evidence for two conformers of the beta subunit of tryptophan synthase in solution.

To explain our finding that the dimeric beta subunit of tryptophan synthase is only 50% inactivated by beta-chloro-L-alanine (Ahmed, S. A., Ruvinov, S. B., Kayastha, A. M., and Miles, E. W. (1991) J. Biol. Chem. 266, 21548-21557), we have extended our investigation using spectroscopic, steady-state kinetic, and electrophoretic methods. The spectroscopic properties of the half-active beta 2 dimer and the reactivation after alkali treatment show that the inactivation proceeds by an "enamine" mechanism. Although the fully active beta 2 dimer associates with the tryptophan synthase alpha subunit to form alpha 2 beta 2 complex, the inactive beta subunits in the half-active enzyme associate weakly or not at all with the alpha subunit. Our results provide evidence for two conformers of the beta subunit in solution: one is rapidly inactivated by beta-chloro-L-alanine and the other is not inactivated. Thermal inactivation studies and non-denaturing polyacrylamide gel electrophoresis of the half-active enzyme show that the beta 2 dimer exists in both homologous and heterologous combinations of these two forms. After removal of the reaction products and unreacted beta-chloro-L-alanine from the half-active beta 2 dimer by gel filtration, further incubation with beta-chloro-L-alanine results in the loss of 50% of the remaining activity. This result suggests that the subunits undergo rearrangement via an intermediate monomer form to regenerate the two conformers of the active beta subunit. This mechanism of rearrangement is supported by our finding that the extent of inactivation increases at lower concentrations of the beta 2 dimer.     

Mystery solved: discovery of a novel integrin ligand in the developing kidney

Mutant mice lacking the integrin alpha8 subunit exhibit variable defects in kidney development with most mutants missing both kidneys. Several lines of evidence indicate that the known extracellular matrix ligands for integrin alpha8beta1 are either dispensable for or not involved in alpha8beta1 signaling during kidney development. This suggests the presence of an unknown ligand. A novel alpha8beta1 ligand, nephronectin, has now been identified. Nephronectin is a new extracellular matrix protein associated with the Wolffian duct and the ureteric bud, epithelial structures with well-defined roles in kidney development.     

The beta subunit determines the ligand binding properties of synaptic glycine receptors

Inhibitory glycine receptors (GlyRs) regulate motor coordination and sensory signal processing in spinal cord and other brain regions. GlyRs are pentameric proteins composed of membrane-spanning alpha and beta subunits. Here, site-directed mutagenesis combined with homology modeling based on the crystal structure of the acetylcholine binding protein identified key ligand binding residues of recombinant homooligomeric alpha1 and heterooligomeric alpha1beta GlyRs. This disclosed two highly conserved, oppositely charged residues located on adjacent subunit interfaces as being crucial for agonist binding. In addition, the beta subunit was found to determine the ligand binding properties of heterooligomeric GlyRs. Expression of an alpha1beta tandem construct and affinity purification of metabolically labeled GlyRs confirmed a subunit stoichiometry of 2alpha3beta. Because the beta subunit anchors GlyRs at synaptic sites, our results have important implications for the biosynthesis, clustering, and pharmacology of synaptic GlyRs.     

Synthesis of multi-subunit domain gonadotropin complexes: a model for alpha/beta heterodimer formation

The human glycoprotein hormones chorionic gonadotropin (CG), thyrotropin (TSH), lutropin (LH), and follitropin (FSH) are heterodimers, composed of a common alpha subunit assembled to a hormone-specific beta subunit. The subunits combine noncovalently early in the secretory pathway and exist as heterodimers, but not as multimers. Little information is available regarding the steps associated with the assembly reaction. It is unclear if the initial alpha beta engagement results either in the formation of only mature heterodimer or if the nascent complex is reversible and can undergo an exchange of subunits or combine transiently with an additional subunit. This is relevant for the case of LH and FSH, because both are synthesized in the same cell (i.e., pituitary gonadotrophs) and several of the alpha subunit sequences required for association with either the LH beta or FSH beta subunits are different. Such features could favor the generation of short-lived, multi-subunit forms prior to completion of assembly. Previously, we showed that the CG beta or FSH beta subunit genes can be genetically fused to the alpha gene to produce biologically active single chains, CG beta alpha and F beta alpha, respectively. Studies using monoclonal antibodies sensitive to the conformation of the hCG subunits suggested that in contrast to the highly compact heterodimer, the interactions between the beta and alpha domains in the single chain are in a more relaxed configuration. That the tethered domains do not interact tightly predicts that they could combine with an additional subunit to form triple domain complexes. We tested this point by cotransfecting CHO cells with the genes encoding F beta alpha and the CG beta subunit or the CG beta alpha and FSH beta monomer. The CG beta subunit combined noncovalently with F beta alpha to form a F beta alpha/CG beta complex. Ternary complex formation was not restricted to a specific set of single chain/monomeric subunit, because a CG beta alpha/FSH beta complex was also detected implying that triple domain intermediates could be transiently generated along the secretory pathway. Monoclonal antibodies specific for the CG heterodimer recognized the F beta alpha/CG beta complex, which suggests that the epitopes unique for dimeric CG were established. In addition, media containing F beta alpha/CG beta displayed high-affinity binding to both CG and FSH receptors. The presence of CG activity is presumptive for the existence of a functional F beta alpha/CG beta complex, because neither F beta alpha nor the uncombined CG beta subunit binds to CG receptor. These data show that the alpha subunit of the tether, although covalently linked to the FSH beta domain, can functionally interact with a different beta subunit implying that the contacts in the nascent alpha beta dimer are reversible. The formation of a functional single chain/subunit complex was not restricted to the FSH single chain/CG beta subunit since CG single chain interacts with the monomeric FSH beta subunit and exhibits FSH activity. The presence of the triple domain configuration does not abolish bioactivity, suggesting that although the gonadotropins are heterodimers, the cognate receptor is capable of recognizing a larger ligand composed of three subunit domains.     

A Comparison of the Structures of the Alpha:Beta and Alpha:Gamma Dimers of Mouse Salivary Androgen-Binding Protein (ABP) and Their Differential Steroid Bindin

Mouse salivary androgen-binding protein (ABP) isa family of dimeric proteins that may play a pheromonalrole in Mus musculus. The protein dimer consists of acommon alpha subunit disulfide-bonded to avariable (beta or gamma) subunit. Here wereport N-terminal sequences of the beta and gammasubunits, showing that they are very similar to eachother while being quite different from the alphasubunit.We demonstrate differential androgen binding bythe two dimers. Both bind dihydrotestosterone to aboutthe same extent but the alpha:beta dimer bindssignificantly more testosterone than the alpha:gammadimer. We discuss the possible significance ofthis diversity of androgen binding with respect to thepossibility that androgen binding is related to aputative pheromonal role for the protein.     

Initiation of spectrin dimerization involves complementary electrostatic interactions between paired triple-helical bundles

The spectrin heterodimer is formed by the antiparallel lateral association of an alpha and a beta subunit, each of which comprises largely a series of homologous triple-helical motifs. Initiation of dimer assembly involves strong binding between complementary motifs near the actin-binding end of the dimer. In this study, the mechanism of lateral spectrin association at this dimer nucleation site was investigated using the analytical ultracentrifuge to analyze heterodimers formed from recombinant peptides containing two or four homologous motifs from each subunit (alpha20-21/beta1-2; alpha18-21/beta1-4). Both the two-motif and four-motif dimer associations were weakened substantially with increasing salt concentration, indicating that electrostatic interactions are important for the dimer initiation process. Modeling of the electrostatic potential on the surface of the alpha20 and beta2 motifs showed that the side of the motifs comprising the A and B helices is the most favorable for association, with an area of positive electrostatic potential on the AB face of the beta2 motif opposite negative potential on the AB face of the alpha20 motif and vise versa. Protease protection analysis of the alpha20-21/beta1-2 dimer showed that multiple trypsin and proteinase K sites in the A helices of the beta2 and alpha21 motifs become buried upon dimer formation. Together, these data support a model where complementary long range electrostatic interactions on the AB faces of the triple-helical motifs in the dimer nucleation site initiate the correct pairing of motifs, i.e. alpha21-beta1 and alpha20-beta2. After initial docking of these complementary triple-helical motifs, this association is probably stabilized by subsequent formation of stronger hydrophobic interactions in a complex involving the A helices of both subunits and possibly most of the AB faces. The beta subunit A helix in particular appears to be buried in the dimer interface.     

Human complement protein C8 gamma

Human C8 gamma is a 22 kDa subunit of complement component C8, which is one of five components (C5b, C6, C7, C8, C9) that interact to form the cytolytic membrane attack complex (MAC) of complement. C8 contains three nonidentical subunits (alpha, beta, gamma) that are products of different genes. These subunits are arranged asymmetrically to form a disulfide-linked C8 alpha-gamma dimer that is noncovalently associated with C8 beta. C8 alpha and C8 beta are homologous to C6, C7 and C9 and together these proteins comprise what is referred to as the 'MAC protein family'. By comparison, C8 gamma is distinct in that it belongs to the lipocalin family of small, secreted proteins which have the common ability to bind small hydrophobic ligands. While specific roles have been identified for C8 alpha and C8 beta in the formation and function of the MAC, a function for C8 gamma and the identity of its ligand are unknown. This review summarizes the current status of C8 gamma structure and function and the progress made from efforts to determine its role in the complement system.     

Amino acid sequence of the human fibronectin receptor

The amino acid sequence deduced from cDNA of the human placental fibronectin receptor is reported. The receptor is composed of two subunits: an alpha subunit of 1,008 amino acids which is processed into two polypeptides disulfide bonded to one another, and a beta subunit of 778 amino acids. Each subunit has near its COOH terminus a hydrophobic segment. This and other sequence features suggest a structure for the receptor in which the hydrophobic segments serve as transmembrane domains anchoring each subunit to the membrane and dividing each into a large ectodomain and a short cytoplasmic domain. The alpha subunit ectodomain has five sequence elements homologous to consensus Ca2+-binding sites of several calcium-binding proteins, and the beta subunit contains a fourfold repeat strikingly rich in cysteine. The alpha subunit sequence is 46% homologous to the alpha subunit of the vitronectin receptor. The beta subunit is 44% homologous to the human platelet adhesion receptor subunit IIIa and 47% homologous to a leukocyte adhesion receptor beta subunit. The high degree of homology (85%) of the beta subunit with one of the polypeptides of a chicken adhesion receptor complex referred to as integrin complex strongly suggests that the latter polypeptide is the chicken homologue of the fibronectin receptor beta subunit. These receptor subunit homologies define a superfamily of adhesion receptors. The availability of the entire protein sequence for the fibronectin receptor will facilitate studies on the functions of these receptors.     

Ions binding to S100 proteins. I. Calcium- and zinc-binding properties of bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) protein: Zn2+ regulates Ca2+ binding on S100b protein

Flow dialysis measurements of calcium binding to bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) proteins in 20 mM Tris-HCl buffer at pH 7.5 and 8.3 revealed that S100 proteins bind specifically 4 Ca2+ eq/mol of protein dimer. The specific calcium-binding sites had, therefore, been assigned to typical amino acid sequences on the alpha and beta subunit. The protein affinity for calcium is much lower in the presence of magnesium and potassium. Potassium strongly antagonizes calcium binding on two calcium-binding sites responsible for most of the Ca2+-induced conformational changes on S100 proteins (probably site II alpha and site II beta). Zinc-binding studies in the absence of divalent cations revealed eight zinc-binding sites/mol of S100b protein dimer that we assumed to correspond to 4 zinc-binding sites/beta subunit. Zinc binding to S100b studied with UV spectroscopy methods showed that the occupation of the four higher affinity sites and the four lower affinity sites on the protein dimer were responsible for different conformational changes in S100b structure. Zinc binding on the higher affinity sites regulates calcium binding to S100b by increasing the protein affinity for calcium and decreasing the antagonistic effect of potassium on calcium binding. Zinc-binding studies on S100a and S100 alpha alpha protein showed that the Trp-containing S100 proteins bind zinc more weakly than S100b protein. Calcium-binding studies on zinc-bound S100a proved that calcium- and zinc-binding sites were distinct although there was no increase in zinc-bound S100a affinity for calcium, as in S100b protein. Finally we provide evidence that discrepancies between previously published results on the optical properties of S100b protein probably result from oxidation of the sulfhydryl groups in the protein.     

Structural basis for ligand selectivity of heteromeric olfactory cyclic nucleotide-gated channels

In vertebrate olfactory receptors, cAMP produced by odorants opens cyclic nucleotide-gated (CNG) channels, which allow Ca(2+) entry and depolarization of the cell. These CNG channels are composed of alpha subunits and at least two types of beta subunits that are required for increased cAMP selectivity. We studied the molecular basis for the altered cAMP selectivity produced by one of the beta subunits (CNG5, CNCalpha4, OCNC2) using cloned rat olfactory CNG channels expressed in Xenopus oocytes. Compared with alpha subunit homomultimers (alpha channels), channels composed of alpha and beta subunits (alpha+beta channels) were half-activated (K(1/2)) by eightfold less cAMP and fivefold less cIMP, but similar concentrations of cGMP. The K(1/2) values for heteromultimers of the alpha subunit and a chimeric beta subunit with the alpha subunit cyclic nucleotide-binding region (CNBR) (alpha+beta-CNBRalpha channels) were restored to near the values for alpha channels. Furthermore, a single residue in the CNBR could account for the altered ligand selectivity. Mutation of the methionine residue at position 475 in the beta subunit to a glutamic acid as in the alpha subunit (beta-M475E) reverted the K(1/2,cAMP)/K(1/2,cGMP) and K(1/2, cIMP)/K(1/2,cGMP) ratios of alpha+beta-M475E channels to be very similar to those of alpha channels. In addition, comparison of alpha+beta-CNBRalpha channels with alpha+beta-M475E channels suggests that the CNBR of the beta subunit contains amino acid differences at positions other than 475 that produce an increase in the apparent affinity for each ligand. Like the wild-type beta subunit, the chimeric beta/alpha subunits conferred a shallow slope to the dose-response curves, increased voltage dependence, and caused desensitization. In addition, as for alpha+beta channels, block of alpha+betaCNBRalpha channels by internal Mg(2+) was not steeply voltage-dependent (zdelta approximately 1e(-)) as compared to block of alpha channels (zdelta 2.7e(-)). Thus, the ligand-independent effects localize outside of the CNBR. We propose a molecular model to explain how the beta subunit alters ligand selectivity of the heteromeric channels.     

Chimeric and truncated forms of human complement protein C8 alpha reveal binding sites for C8 beta and C8 gamma within the membrane attack complex/perforin region.

Human C8 is one of five components of the membrane attack complex of complement. It is an oligomeric protein composed of three subunits (C8 alpha, C8 beta, and C8 gamma) that are derived from different genes. C8 alpha and C8 beta are homologous and both contain a pair of tandemly arranged N-terminal modules [thrombospondin type 1 (TSP1) + low-density lipoprotein receptor class A (LDLRA)], an extended middle segment referred to as the membrane attack complex/perforin region (MACPF), and a pair of C-terminal modules [epidermal growth factor (EGF) + TSP1]. During biosynthetic processing, C8 alpha and C8 gamma associate to form a disulfide-linked dimer (C8 alpha-gamma) that binds to C8 beta through a site located on C8 alpha. In this study, the location of binding sites for C8 beta and C8 gamma and the importance of the modules in these interactions were investigated by use of chimeric and truncated forms of C8 alpha in which module pairs were either exchanged for those in C8 beta or deleted. Results show that exchange or deletion of one or both pairs of modules does not abrogate the ability of C8 alpha to form a disulfide-linked dimer when coexpressed with C8 gamma in COS cells. Furthermore, each chimeric and truncated form of C8 alpha-gamma retains the ability to bind C8 beta; however, only those containing the TSP1 + LDLRA modules from C8 alpha are hemolytically active. These results indicate that binding sites for C8 beta and C8 gamma reside within the MACPF region of C8 alpha and that interaction with either subunit is not dependent on the modules. They also suggest that the N-terminal modules in C8 alpha are important for C9 binding and/or expression of C8 activity.     

A method for assessing the reassembly of a multisubunit glycoprotein by western blotting

The Western blot procedure has been adapted to detect the reassembly of a two-subunit glycoprotein, urinary human chorionic gonadotropin (hCG), directly on the nitrocellulose. This glycoprotein is composed of two nonidentical subunits, alpha and beta. A simple procedure using immunoblotting has been developed to detect reassembly of the monomers to dimer. Three monoclonal antibodies were required for the development of this method: A109, which binds the alpha subunit or hCG; B105, which binds the beta subunit or hCG; and B107, specific for the intact hCG dimer. The alpha subunit and beta subunit of hCG were each electrophoresed and transferred to nitrocellulose, and the transfer was then incubated with the appropriate complementary subunit; reassembly of the dimer was determined by the binding of the monoclonal antibody B107. Evidence that the reassembly occurs directly on the nitrocellulose comes from the fact that B107 immunoreactivity is detected at the molecular weight position of the subunit and not at the dimer molecular weight. A genetically expressed recombinant form of the alpha subunit was also tested for its ability to recombine with the opposite subunit to produce the dimer. The recombinant alpha subunit was determined to have additional carbohydrate which interfered with the binding of the beta subunit. N-Glycanase digestion of the recombinant alpha subunit produced a form which, when incubated with the beta subunit, did recombine on the nitrocellulose and could be recognized by B107.     

The crystal structure of maleylacetate reductase from Rhizobium sp. strain MTP‐10005 provides insights into the reaction mechanism of enzymes in its original family

Maleylacetate reductase plays a crucial role in catabolism of resorcinol by catalyzing the NAD(P)H‐dependent reduction of maleylacetate, at a carbon–carbon double bond, to 3‐oxoadipate. The crystal structure of maleylacetate reductase from Rhizobium sp. strain MTP‐10005, GraC, has been elucidated by the X‐ray diffraction method at 1.5 Å resolution. GraC is a homodimer, and each subunit consists of two domains: an N‐terminal NADH‐binding domain adopting an α/β structure and a C‐terminal functional domain adopting an α‐helical structure. Such structural features show similarity to those of the two existing families of enzymes in dehydroquinate synthase‐like superfamily. However, GraC is distinct in dimer formation and activity expression mechanism from the families of enzymes. Two subunits in GraC have different structures from each other in the present crystal. One subunit has several ligands mimicking NADH and the substrate in the cleft and adopts a closed domain arrangement. In contrast, the other subunit does not contain any ligand causing structural changes and adopts an open domain arrangement. The structure of GraC reveals those of maleylacetate reductase both in the coenzyme, substrate‐binding state and in the ligand‐free state. The comparison of both subunit structures reveals a conformational change of the Tyr326 loop for interaction with His243 on ligand binding. Structures of related enzymes suggest that His243 is likely a catalytic residue of GraC. Mutational analyses of His243 and Tyr326 support the catalytic roles proposed from structural information. The crystal structure of GraC characterizes the maleylacetate reductase family as a third family in the dehydroquinate synthase‐like superfamily. Proteins 2016; 84:1029–1042. © 2016 Wiley Periodicals, Inc.     

Evidence that monoclonal antibodies directed against the integrin beta subunit plexin/semaphorin/integrin domain stimulate function by inducing receptor extension

The overall structure of integrins is that of a ligand-binding head connected to two long legs. The legs can exhibit a pronounced bend at the "knees," and it has been proposed that the legs undergo a dramatic straightening when integrins transit from a low affinity to a high affinity state. The knee region contains domains from both alpha and beta subunits, including the N-terminal plexin/semaphorin/integrin (PSI) domain of the beta subunit. The role played by the knee domains in the regulation of integrin-ligand binding is uncertain. Here we show that: (i) monoclonal antibodies (mAbs) N29 and 8E3 have epitopes in the beta(1) subunit PSI domain and stimulate ligand binding to alpha(5)beta(1); (ii) N29 and 8E3 cause long range conformational changes that alter the ligand binding activity of the head region; (iii) the stimulatory action of these mAbs is dependent on the calf-1 domain, which forms part of the alpha subunit knee; and (iv) the epitopes of 8E3 and N29 map close to the extreme N terminus of the PSI and are likely to lie on the side of this domain that faces the alpha subunit. Taken together, our data suggest that the binding of these mAbs results in a levering apart of the PSI and calf-1 domains, and thereby causes the alpha and beta subunit knees to separate. Several major inferences can be drawn from our findings. First, the PSI domain appears to form part of an interface with the alpha subunit that normally restrains the integrin in a bent state. Second, the PSI domain is important for the transduction of conformational changes from the knee to head. Third, unbending is likely to provide a general mechanism for control of integrin-ligand recognition.     

Chick integrin alpha V subunit molecular analysis reveals high conservation of structural domains and association with multiple beta subunits in embryo fibroblasts.

We have cloned and characterized a chick homologue of the human vitronectin receptor alpha subunit (alpha v) whose primary sequence is 83% identical with its human counterpart but less than 40% identical with any other known integrin alpha subunit. Comparison of the chick and human sequences reveals several highly conserved regions, including the cytoplasmic domain. The putative ligand binding domain contains alpha v-specific residues that may contribute to ligand binding specificity. These are concentrated in three regions that are located before and between the first three Ca2+ binding domains. Polyclonal antibodies raised against two peptides deduced from the putative cytoplasmic and extracellular domains of the chick alpha v sequence recognize specifically integrin heterodimers in chick embryo fibroblasts. At least three putative beta subunits coimmunoprecipitate with the chick alpha v subunit. In addition to a protein with the same molecular weight as beta 3 (94K), protein bands of Mr 84K and 110K are also coprecipitated. By successive immunodepletions, we demonstrate that this latter Mr 110K subunit is beta 1, which appears to be one of the alpha v-associated subunits in chick embryo fibroblasts.