Involvement of alpha-cysteine-62 and beta-tryptophan-135 in human pyruvate dehydrogenase catalysis.

Pyruvate dehydrogenase (E1), a heterotetramer (alpha(2)beta(2)), is the first catalytic component of the mammalian pyruvate dehydrogenase complex (PDC). To investigate the roles of cysteine-62 of E1alpha (alphaC62) and tryptophan-135 of E1beta (betaW135) (identified previously as active site residues using chemical modifications) in E1 catalysis, two recombinant human E1 mutants were generated using site-directed mutagenesis: alphaC62A and betaW135L. Compared to wild-type, k(cat) values for alphaC62A and betaW135L measured by PDC assay were markedly reduced to 7.2 and 11. 6%, respectively. Apparent K(m) values for thiamin pyrophosphate (TPP) were increased approximately sixfold for both mutants, resulting in catalytic efficiency for TPP of only 1-2% of the wild-type E1. K(m) values for pyruvate increased only moderately (twofold). The alphaC62A and betaW135L mutants were less thermostable than wild-type E1. The conformations of the mutant apo-E1s determined by spectral analysis were different from that of the wild-type apo-E1. CD spectral analysis indicated that TPP binding was affected for both the alphaC62A and betaW135L mutant E1s. The substrate analogs, fluoropyruvate and bromopyruvate, were shown to be active site-directed inhibitors of human E1; in the absence of TPP, bromopyruvate (but not fluoropyruvate) inhibited human E1 due to SH-group modification. Pyruvate induced inactivation of human E1 could be restored by thiol reagents. Cysteine-62 (and maybe another group) is proposed to be involved in E1 inhibition by the substrate and substrate analogs. Taken together these results indicate that alphaC62 and betaW135 facilitate coenzyme binding, and alphaC62 could be near the substrate-binding site.     

Characterization of point mutations in patients with pyruvate dehydrogenase deficiency: role of methionine-181, proline-188, and arginine-349 in the alpha subunit.

Human pyruvate dehydrogenase (E1), a heterotetramer (alpha2beta2), is the first component of the pyruvate dehydrogenase complex (PDC). E1 catalyzes the thiamin pyrophosphate (TPP)-dependent decarboxylation of pyruvate and the reductive acetylation of the dihydrolipoamide acetyltransferase component. Site-directed mutagenesis was employed to recreate three point mutations in the alpha subunit identified in E1-deficient patients, M181V, R349H, and P188L (P188A mutant E1 was used because of the very low level of expression of P188L), to investigate the functional roles of these three amino acid residues. P188A mutant E1 was much less thermostable than the wild-type E1. The kcats of M181V and P188A mutant E1s determined in the PDC reaction were 38 and 24% of that of the wild-type enzyme, respectively. The apparent Km for TPP for M181V increased significantly (approx 250-fold when determined in the PDC assay), while the apparent Km for pyruvate increased by only about 3-fold. In contrast, P188A had similar Kms for the coenzyme and the substrate as the wild-type. Km values for R349H were not determined due to the extremely low activity of this mutant (1.2% of the wild-type E1-specific activity measured in the PDC assay). Wild-type E1 displayed a lag phase in the progress curve of the PDC reaction measured in the presence of low TPP concentrations (below 1 microM) only. All mutants had a lag phase that was not eliminated even at very high TPP concentrations, suggesting modifications in the conformation of the active site. Kinetic analysis indicated thiamin 2-thiothiazolone pyrophosphate (ThTTPP) to be an intermediate analog for wild-type human E1. M181V required a higher concentration of ThTTPP for inactivation than the wild-type and P188A E1s. The results of circular dichroism spectropolarimetry in the far UV region indicated that there were no major changes in the secondary structure of M181V, P188A, and R349H E1s. These mutant enzymes exhibited negative dichroic spectra at about 330 nm only in the presence of high TPP concentrations. This study suggests that arginine-349 is critical for E1's activity, methionine-181 is involved in the binding of TPP, and proline-188 is necessary for structural integrity of E1.     

Critical amino acid residues for ligand binding are clustered in a predicted beta-turn of the third N-terminal repeat in the integrin alpha 4 and alpha 5 subunits.

Integrin alpha 4 beta 1 is a receptor for vascular cell adhesion molecule (VCAM)-1 and fibronectin (CS-1). The alpha 4 beta 1-ligand interaction is involved in the pathogenesis of diseases and is, therefore, a therapeutic target. Here, we identified critical residues of alpha 4 for ligand binding using alanine-scanning mutagenesis of the previously localized putative ligand binding sites (residues 108-268). Among 43 mutations tested, mutations of Tyr187, Trp188 and Gly190 significantly inhibited cell adhesion to both VCAM-1 and CS-1. This inhibition was not due to any gross structural changes of alpha 4 beta 1. These critical residues are clustered in a predicted beta-turn structure (residues 181-190) of the third N-terminal repeat in alpha 4. The repeat does not contain divalent cation binding motifs. Notably, the mutations within the corresponding region of alpha 5 significantly reduced fibronectin-alpha 5 beta 1 interaction. These findings suggest that the predicted beta-turn structure could be ubiquitously involved in ligand binding of non-I domain integrins.     

Structural requirements for interaction of sodium channel beta 1 subunits with ankyrin

Sodium channel beta subunits modulate channel kinetic properties and cell surface expression levels and function as cell adhesion molecules. beta 1 and beta 2 participate in homophilic cell adhesion resulting in ankyrin recruitment to cell contact sites. We hypothesized that a tyrosine residue in the cytoplasmic domain of beta 1 may be important for ankyrin recruitment and tested our hypothesis using beta 1 mutants replacing Tyr(181) with alanine (beta 1Y181A), phenylalanine (beta 1Y181F), or glutamate (beta 1Y181E), or a truncated construct deleting all residues beyond Tyr(181) (beta 1L182(STOP)). Ankyrin recruitment was observed in beta 1L182(STOP), showing that residues Ile(166)-Tyr(181) contain the major ankyrin recruiting activity of beta 1. Ankyrin recruitment was abolished in beta 1Y181E, suggesting that tyrosine phosphorylation of beta 1 may inhibit beta 1-ankyrin interactions. Ankyrin(G) and beta 1 associate in rat brain membranes and in transfected cells expressing beta 1 and ankyrin(G) in the absence of sodium channel alpha subunits. beta 1 subunits are recognized by anti-phosphotyrosine antibodies following treatment of these cell lines with fibroblast growth factor. beta 1 and ankryin(G) association is not detectable in cells following treatment with fibroblast growth factor. Ankyrin(G) and beta 1Y181E do not associate even in the absence of fibroblast growth factor treatment. beta 1 subunit-mediated cell adhesion and ankyrin recruitment may contribute to sodium channel placement at nodes of Ranvier. The phosphorylation state of beta 1Y181 may be a critical regulatory step in these developmental processes.     

Role of pairwise interactions between M1 and M2 domains of the nicotinic receptor in channel gating

The adult form of the nicotinic acetylcholine receptor (AChR) consists of five subunits (alpha(2)betaepsilondelta), each having four transmembrane domains (M1-M4). The atomic model of the nicotinic acetylcholine receptor shows that the pore-lining M2 domains make no extensive contacts with the rest of the transmembrane domains. However, there are several sites where close appositions between segments occur. It has been suggested that the pair alphaM1-F15' and alphaM2-L11' is one of the potential interactions between segments. To determine experimentally if these residues are interacting and to explore if this interhelical interaction is essential for channel gating, we combined mutagenesis with single-channel kinetic analysis. Mutations in alphaM1-F15' lead to profound changes in the opening rate and slighter changes in the closing rate. Channel gating is impaired as the volume of the residue increases. Rate-equilibrium linear free-energy relationship analysis reveals an approximately 70% open-state-like environment for alphaM1-F15' at the transition state of the gating reaction, suggesting that it moves early during the gating process. Replacing the residue at alphaM1-15' by that at alphaM2-11' and vice versa profoundly alters gating, but the combination of the two mutations restores gating to near normal, indicating that alphaM1-F15' and alphaM2-L11' are interchangeable. Double-mutant cycle analysis shows that these residues are energetically coupled. Thus, the interaction between M1 and M2 plays a key role in channel gating.     

Utilizing temperature-sensitive association of Pluronic F-127 with lipid bilayers to control liposome-cell adhesion

Genetic defects in pyruvate dehydrogenase complex (PDC) cause lactic acidosis, neurological deficits, and often early death. Most mutations of PDC are localized in the alpha subunit of the pyruvate dehydrogenase (E1) component. We have kinetically characterized a patient's missense mutation alphaH44R in E1alpha by creating and purifying three recombinant human E1s (alphaH44R, alphaH44Q, and alphaH44A). Substitutions at histidine-15 resulted in decreased V(max) values (6% alphaH44R; 30% alphaH44Q; 90% alphaH44A) while increasing K(m) values for thiamine pyrophosphate (TPP) compared to wild-type (alphaH44R, 3-fold; alphaH44Q, 7-fold; alphaH44A, 10-fold). This suggests that the volume of the residue at site 15 is important for TPP binding and substitution by a residue with a longer side chain disrupts the active site more than the TPP binding site. The rates of phosphorylation and dephosphorylation of alphaH44R E1 by E1-kinase and phospho-E1 phosphatase, respectively, were similar to that of the wild-type E1 protein. These results provide a biochemical basis for altered E1 function in the alphaH44R E1 patient.     

Inter- and intrasubunit interactions during the formation of RNA polymerase assembly intermediate

We used yeast two-hybrid and in vitro co-immobilization assays to study the interaction between the Escherichia coli RNA polymerase (RNAP) alpha and beta subunits during the formation of alpha(2)beta, a physiological RNAP assembly intermediate. We show that a 430-amino acid-long fragment containing beta conserved segments F, G, H, and a short part of segment I forms a minimal domain capable of specific interaction with alpha. The alpha-interacting domain is held together by protein-protein interactions between beta segments F and I. Residues in catalytically important beta segments H and I directly participate in alpha binding; substitutions of strictly conserved segment H Asp(1084) and segment I Gly(1215) abolish alpha(2)beta formation in vitro and are lethal in vivo. The importance of these beta amino acids in alpha binding is fully supported by the structural model of the Thermus aquaticus RNAP core enzyme. We also demonstrate that determinants of RNAP assembly are conserved, and that a homologue of beta Asp(1084) in A135, the beta-like subunit of yeast RNAP I, is responsible for interaction with AC40, the largest alpha-like subunit. However, the A135-AC40 interaction is weak compared with the E. coli alpha-beta interaction, and A135 mutation that abolishes the interaction is phenotypically silent. The results suggest that in eukaryotes additional RNAP subunits orchestrate the enzyme assembly by stabilizing weak, but specific interactions of core subunits.     

The solution structure of the guanine nucleotide exchange domain of human elongation factor 1beta reveals a striking resemblance to that of EF-Ts from Escherichia coli.

BACKGROUND: In eukaryotic protein synthesis, the multi-subunit elongation factor 1 (EF-1) plays an important role in ensuring the fidelity and regulating the rate of translation. EF-1alpha, which transports the aminoacyl tRNA to the ribosome, is a member of the G-protein superfamily. EF-1beta regulates the activity of EF-1alpha by catalyzing the exchange of GDP for GTP and thereby regenerating the active form of EF-1alpha. The structure of the bacterial analog of EF-1alpha, EF-Tu has been solved in complex with its GDP exchange factor, EF-Ts. These structures indicate a mechanism for GDP-GTP exchange in prokaryotes. Although there is good sequence conservation between EF-1alpha and EF-Tu, there is essentially no sequence similarity between EF-1beta and EF-Ts. We wished to explore whether the prokaryotic exchange mechanism could shed any light on the mechanism of eukaryotic translation elongation. RESULTS: Here, we report the structure of the guanine-nucleotide exchange factor (GEF) domain of human EF-1beta (hEF-1beta, residues 135-224); hEF-1beta[135-224], determined by nuclear magnetic resonance spectroscopy. Sequence conservation analysis of the GEF domains of EF-1 subunits beta and delta from widely divergent organisms indicates that the most highly conserved residues are in two loop regions. Intriguingly, hEF-1beta[135-224] shares structural homology with the GEF domain of EF-Ts despite their different primary sequences. CONCLUSIONS: On the basis of both the structural homology between EF-Ts and hEF-1beta[135-224] and the sequence conservation analysis, we propose that the mechanism of guanine-nucleotide exchange in protein synthesis has been conserved in prokaryotes and eukaryotes. In particular, Tyr181 of hEF-1beta[135-224] appears to be analogous to Phe81 of Escherichia coli EF-Ts.     

The molecular pathway for the allosteric regulation of tryptophan synthase

The pyridoxal 5'-phosphate (PLP)-dependent tryptophan synthase is a alpha(2)beta(2) complex. The alpha-beta subunit interaction plays a critical role both in the reciprocal activation of the individual subunits and in the allosteric regulation. We have investigated whether mutations of alpha loop6 Gly(181) and beta helix6 Ser(178) affect intersubunit communication. The loss of the hydrogen bond between these residues, achieved by proline substitution, does not significantly influence the intersubunit catalytic activation, but completely abolishes ligand-induced intersubunit signaling. The comparison of the crystal structure of the wild type and beta Ser(178)Pro mutant, in the absence and presence of alpha-subunit ligands, indicates that the removal of the interaction between beta Ser(178) and alpha Gly(181) strongly affects the equilibrium between active (closed) and inactive (open) conformations of the alpha-active site, the latter being stabilized in both mutants.     

Critical residues of alphaX I-domain recognizing fibrinogen central domain

Integrin alphaXbeta2 (CD11c/CD18), which binds several ligands such as fibrinogen and iC3b, has important roles in leukocyte functions including phagocytosis and migration. Establishment of structure and functional relationship in alphaX I-domain, which is a ligand-binding moiety, is important in understanding leukocyte biology and integrin function. Previously we showed that two loops (alpha3-alpha4, betaD-alpha5) around a ligand-binding face of alphaX I-domain are important for the binding of the fibrinogen molecule. In this study, we took the further step of identifying critical residues in these loops and in a supportive loop (betaF-alpha7) for fibrinogen fragment E, the central domain of fibrinogen. The residues S(199) and Q(202) in the alpha3-alpha4 loop and K(243), Y(250) in the betaD-alpha5 loop are critical for the ligand. The residues K(242), D(249), K(251), and D(252) are important but less critical for fibrinogen fragment E. The involvement of the residues in the 3-dimensional model of the I-domain suggests that several amino acid sequences in fibrinogen fragment E are responsible for alphaX I-domain. Sequence comparisons with alphaM I-domain reveal that most of the critical residues shown in alphaX I-domain are also conserved in alphaM and may have important roles in fibrinogen central domain recognition in alphaM I-domain as well.     

Tight Binding of the Phosphorylated α Subunit of Initiation Factor 2 (eIF2α) to the Regulatory Subunits of Guanine Nucleotide Exchange Factor eIF2B Is Required for Inhibition of Translation Initiation

Translation initiation factor 2 (eIF2) is a heterotrimeric protein that transfers methionyl-initiator tRNA(Met) to the small ribosomal subunit in a ternary complex with GTP. The eIF2 phosphorylated on serine 51 of its alpha subunit [eIF2(alphaP)] acts as competitive inhibitor of its guanine nucleotide exchange factor, eIF2B, impairing formation of the ternary complex and thereby inhibiting translation initiation. eIF2B is comprised of catalytic and regulatory subcomplexes harboring independent eIF2 binding sites; however, it was unknown whether the alpha subunit of eIF2 directly contacts any eIF2B subunits or whether this interaction is modulated by phosphorylation. We found that recombinant eIF2alpha (glutathione S-transferase [GST]-SUI2) bound to the eIF2B regulatory subcomplex in vitro, in a manner stimulated by Ser-51 phosphorylation. Genetic data suggest that this direct interaction also occurred in vivo, allowing overexpressed SUI2 to compete with eIF2(alphaP) holoprotein for binding to the eIF2B regulatory subcomplex. Mutations in SUI2 and in the eIF2B regulatory subunit GCD7 that eliminated inhibition of eIF2B by eIF2(alphaP) also impaired binding of phosphorylated GST-SUI2 to the eIF2B regulatory subunits. These findings provide strong evidence that tight binding of phosphorylated SUI2 to the eIF2B regulatory subcomplex is crucial for the inhibition of eIF2B and attendant downregulation of protein synthesis exerted by eIF2(alphaP). We propose that this regulatory interaction prevents association of the eIF2B catalytic subcomplex with the beta and gamma subunits of eIF2 in the manner required for GDP-GTP exchange.     

Identification of the gamma-subunit interaction sites in the retinal cyclic-GMP phosphodiesterase beta-subunit.

Using synthetic peptides, the identification of the retinal cyclic-GMP phosphodiesterase (cGMP PDE) interaction sites for the inhibitory gamma-subunit in the catalytic alpha-subunit were recently localized to residues #16-30 and 78-90 in the alpha-subunit (1). In this study, a binding radioimmunoassay (RIA) showed a weak interaction between PDE gamma and PDE beta subunits in PDE beta residues #15-34, and stronger interaction sites were found in residues #91-110 and 211-230. Sequence comparison between PDE alpha and PDE beta illustrate some differences in these regions, particularly in PDE alpha 16-30 and PDE beta 15-34 regions. Differences in interaction sites in PDE alpha and PDE beta for PDE gamma may account for the differences in affinities observed between PDE gamma and the catalytic subunits.     

Alpha 3 beta 1 adhesion to laminin-5 and invasin: critical and differential role of integrin residues clustered at the boundary between alpha 3 N-terminal repeats 2 and 3

Integrin/ligand interaction is a therapeutic target for many diseases. We previously reported that residues critical for ligand binding are clustered in N-terminal repeat 3 (in the predicted 2-3 loop) of alpha 4, alpha 5 and alpha IIb. Here we have localized residues critical for ligand binding in the alpha 3 subunit of integrin alpha 3 beta 1 with distinct ligand specificity (laminin-5). We identified an alpha 3 epitope common to several function-blocking anti-alpha 3 antibodies at the boundary between repeats 1 and 2 (residues 75-80). We found that swapping the predicted 4-1 loop (residues 153-165) at the boundary between repeats 2 and 3 with the corresponding alpha 4 sequence and mutating Thr-162 and Gly-163 residues in this predicted loop block laminin-5 binding. Thr-162 and Gly-163 and the antibody epitope are separated in the primary structure; however, they are close to each other in the proposed beta-propeller model. Mutating residues recently reported to block (Tyr-186 and Trp-188) or enhance (Asp-122) laminin-5 binding to alpha 3 beta 1 [Krukonis, E. S., Dersch, P., Eble, J. A., and Isberg, R. R.(1998) J. Biol. Chem. 273, 31837-31843] did not affect laminin-5 binding under the assay conditions used. Thr-162 and Gly-163 are not critical for adhesion to invasin, indicating that laminin-5 and invasin may use different recognition mechanisms, and that mutation of Thr-162 and Gly-163 does not drastically affect the integrity of alpha 3 beta 1. These results suggest that residues critical for ligand binding may be similarly (but not identically) located in repeat 3 of the alpha subunit regardless of ligand specificity.     

Hydrogen bonds between the alpha and beta subunits of the F1-ATPase allow communication between the catalytic site and the interface of the beta catch loop and the gamma subunit

F(1)-ATPase mutations in Escherichia coli that changed the strength of hydrogen bonds between the alpha and beta subunits in a location that links the catalytic site to the interface between the beta catch loop and the gamma subunit were examined. Loss of the ability to form the hydrogen bonds involving alphaS337, betaD301, and alphaD335 lowered the k(cat) of ATPase and decreased its susceptibility to Mg(2+)-ADP-AlF(n) inhibition, while mutations that maintain or strengthen these bonds increased the susceptibility to Mg(2+)-ADP-AlF(n) inhibition and lowered the k(cat) of ATPase. These data suggest that hydrogen bonds connecting alphaS337 to betaD301 and betaR323 and connecting alphaD335 to alphaS337 are important to transition state stabilization and catalytic function that may result from the proper alignment of catalytic site residues betaR182 and alphaR376 through the VISIT sequence (alpha344-348). Mutations betaD301E, betaR323K, and alphaR282Q changed the rate-limiting step of the reaction as determined by an isokinetic plot. Hydrophobic mutations of betaR323 decreased the susceptibility to Mg(2+)-ADP-AlF(n)() inhibition and lowered the number of interactions required in the rate-limiting step yet did not affect the k(cat) of ATPase, suggesting that betaR323 is important to transition state formation. The decreased rate of ATP synthase-dependent growth and decreased level of lactate-dependent quenching observed with alphaD335, betaD301, and alphaE283 mutations suggest that these residues may be important to the formation of an alternative set of hydrogen bonds at the interface of the alpha and beta subunits that permits the release of intersubunit bonds upon the binding of ATP, allowing gamma rotation in the escapement mechanism.     

N-terminal amino acid sequence analyses of the alpha and beta polypeptides from four distinct subsets of sheep major histocompatibility complex class II molecules

Four monoclonal antibodies, SBU.II 28-1, 37-68, 38-27, and 42-20, each recognizing a distinct, non-overlapping subset of sheep class II molecules, were used to purify class II molecules from a single sheep. Four class II alpha subunits designated 28-1 alpha, 37-68 alpha, 42-20 alpha, and 38-27 alpha and five class II beta subunits designated 28-1 beta, 37-68 beta 1, 37-68 beta 2, 42-20 beta, and 38-27 beta were compared by N-terminal sequence analyses. Two distinct alpha subunits were identified; the 28-1 alpha, 37-68 alpha, and 42-20 alpha subunits all had identical N-terminal amino acids sequences, which exhibited about 75% homology with HLA-DR alpha and mouse E alpha polypeptides. In contrast, the 38-27 alpha sequence exhibited about 80% sequence homology with HLA-DQ alpha and mouse A alpha polypeptides. In general, sheep beta subunits displayed insufficient sequence homology to enable correlation with human beta-chain sequences; however, the 38-27 beta-chain sequence showed homology with the HLA-DQ beta sequence. The conserved sequence surrounding the site for N-linked glycosylation within human/mouse beta polypeptides (residues 19 to 21) was not present in sheep beta sequences and in contrast with the beta-chains of mouse and man, sheep beta polypeptides contained between 1 and 3 positionally variable cysteine residues (residues 13 to 15 inclusive). Individual sheep beta subunits exhibited extensive sequence heterogeneity and each consisted of a unique population of beta polypeptide species. At least 16 different beta polypeptide sequences were identified from a single sheep and the existence of no fewer than nine non-allelic beta genes was inferred from the sequence data. We have previously provided evidence suggesting that the sheep has multiple major histocompatibility complex class II alpha and beta genes related to those of all three HLA-D subregions. The present results suggest that a number of these genes encode HLA-DQ-like heterodimers and that a sheep DR-like alpha gene product is shared with the products of a large and heterogeneous sheep beta gene family.     

Gating at the mouth of the acetylcholine receptor channel: energetic consequences of mutations in the alphaM2-cap

Gating of nicotinic acetylcholine receptors from a C(losed) to an O(pen) conformation is the initial event in the postsynaptic signaling cascade at the vertebrate nerve-muscle junction. Studies of receptor structure and function show that many residues in this large, five-subunit membrane protein contribute to the energy difference between C and O. Of special interest are amino acids located at the two transmitter binding sites and in the narrow region of the channel, where C<-->O gating motions generate a low<-->high change in the affinity for agonists and in the ionic conductance, respectively. We have measured the energy changes and relative timing of gating movements for residues that lie between these two locations, in the C-terminus of the pore-lining M2 helix of the alpha subunit ('alphaM2-cap'). This region contains a binding site for non-competitive inhibitors and a charged ring that influences the conductance of the open pore. alphaM2-cap mutations have large effects on gating but much smaller effects on agonist binding, channel conductance, channel block and desensitization. Three alphaM2-cap residues (alphaI260, alphaP265 and alphaS268) appear to move at the outset of channel-opening, about at the same time as those at the transmitter binding site. The results suggest that the alphaM2-cap changes its secondary structure to link gating motions in the extracellular domain with those in the channel that regulate ionic conductance.     

Axolotl hemoglobin: cDNA-derived amino acid sequences of two alpha globins and a beta globin from an adult Ambystoma mexicanum

Erythrocytes of the adult axolotl, Ambystoma mexicanum, have multiple hemoglobins. We separated and purified two kinds of hemoglobin, termed major hemoglobin (Hb M) and minor hemoglobin (Hb m), from a five-year-old male by hydrophobic interaction column chromatography on Alkyl Superose. The hemoglobins have two distinct alpha type globin polypeptides (alphaM and alpham) and a common beta globin polypeptide, all of which were purified in FPLC on a reversed-phase column after S-pyridylethylation. The complete amino acid sequences of the three globin chains were determined separately using nucleotide sequencing with the assistance of protein sequencing. The mature globin molecules were composed of 141 amino acid residues for alphaM globin, 143 for alpham globin and 146 for beta globin. Comparing primary structures of the five kinds of axolotl globins, including two previously established alpha type globins from the same species, with other known globins of amphibians and representatives of other vertebrates, we constructed phylogenetic trees for amphibian hemoglobins and tetrapod hemoglobins. The molecular trees indicated that alphaM, alpham, beta and the previously known alpha major globin were adult types of globins and the other known alpha globin was a larval type. The existence of two to four more globins in the axolotl erythrocyte is predicted.     

Functional classification of ADAMs based on a conserved motif for binding to integrin alpha 9beta 1: implications for sperm-egg binding and other cell interactions

ADAMs (a disintegrin and metalloproteases) are members of the metzincin superfamily of metalloproteases. Among integrins binding to disintegrin domains of ADAMs are alpha(9)beta(1) and alpha(v)beta(3), and they bind in an RGD-independent and an RGD-dependent manner, respectively. Human ADAM15 is the only ADAM with the RGD motif in the disintegrin domain. Thus, both integrin alpha(9)beta(1) and alpha(v)beta(3) recognize the ADAM15 disintegrin domain. We determined how these integrins recognize the ADAM15 disintegrin domain by mutational analysis. We found that the Arg(481) and the Asp-Leu-Pro-Glu-Phe residues (residues 488-492) were critical for alpha(9)beta(1) binding, but the RGD motif (residues 484-486) was not. In contrast, the RGD motif was critical for alpha(v)beta(3) binding, but the other residues flanking the RGD motif were not. As the RX(6)DLPEF alpha(9)beta(1) recognition motif (residues 481-492) is conserved among ADAMs, except for ADAM10 and 17, we hypothesized that alpha(9)beta(1) may recognize disintegrin domains in all ADAMs except ADAM10 and 17. Indeed we found that alpha(9)beta(1) bound avidly to the disintegrin domains of ADAM1, 2, 3, and 9 but not to the disintegrin domains of ADAM10 and 17. As several ADAMs have been implicated in sperm-oocyte interaction, we tested whether the functional classification of ADAMs, based on specificity for integrin alpha(9)beta(1), applies to sperm-egg binding. We found that the ADAM2 and 15 disintegrin domains bound to oocytes, but the ADAM17 disintegrin domain did not. Furthermore, the ADAM2 and 15 disintegrin domains effectively blocked binding of sperm to oocytes, but the ADAM17 disintegrin domain did not. These results suggest that oocytes and alpha(9)beta(1) have similar binding specificities for ADAMs and that alpha(9)beta(1), or a receptor with similar specificity, may be involved in sperm-egg interaction during fertilization. As alpha(9)beta(1) is a receptor for many ADAM disintegrins and alpha(9)beta(1) and ADAMs are widely expressed, alpha(9)beta(1)-ADAM interaction may be of a broad biological importance.     

Amino acid sequence of the alpha and beta subunits of Methanosarcina barkeri ATPase deduced from cloned genes. Similarity to subunits of eukaryotic vacuolar and F0F1-ATPases

The atpA and atpB genes coding for the alpha and beta subunits, respectively, of membrane ATPase were cloned from a methanogen Methanosarcina barkeri, and the amino acid sequences of the two subunits were deduced from the nucleotide sequences. The methanogenic alpha (578 amino acid residues) and beta (459 amino acid residues) subunits were highly homologous to the large and small subunits, respectively, of vacuolar H+-ATPases; 52% of the residues of the methanogenic alpha subunit were identical with those of the large subunit of vacuolar enzyme of carrot or Neurospora crassa, respectively, and 59, 60, and 59% of the residues of the methanogenic beta subunit were identical with those of the small subunits of N. crassa, Arabidopsis thaliana, and Sacharomyces cerevisiae, respectively. The methanogenic subunits were also highly homologous to the corresponding subunits of Sulfolobus acidocaldarius ATPase. The methanogenic alpha and beta subunits showed 22 and 24% identities with the beta and the alpha subunits of Escherichia coli F1, respectively. Furthermore, important amino acid residues identified genetically in the E. coli enzyme were conserved in the methanogenic enzyme. This sequence conservation suggests that vacuolar, F1, methanogenic, and S. acidocaldarius ATPases were derived from a common ancestral enzyme.     

Distinct roles for the alpha and beta subunits in the functions of integrin alphaMbeta2

Integrin alphaMbeta2 (Mac-1, CD11b/CD18) is a noncovalently linked heterodimer of alphaM and beta2 subunits on the surface of leukocytes, where it plays a pivotal role in the adhesion and migration of these cells. Using HEK293 cells expressing alphaMbeta2 or the individual constituent chains on their surface, we analyzed the contributions of the alphaM or beta2 subunits to functional responses mediated by the integrin. In cells expressing only alphaM or beta2, the individual subunits were not associated with the endogenous integrins of the cells, and other partners for the subunits were not detected by surface labeling and immunoprecipitation under a variety of conditions. The alphaM cells mediated adhesion and spreading on a series of alphaMbeta2 ligands (fibrinogen, Factor X, iC3b, ICAM-1 (intercellular adhesion molecule-1), and denatured ovalbumin) but could not support cell migration to any of these. The spreading of the alphaM cells suggested an unanticipated linkage of this subunit to the cytoskeleton. The beta2 cells supported migration and attachment but not spreading on a subset of the alphaMbeta2 ligands. The heterodimeric receptor and its individual subunits were purified from the cells by affinity chromatography and recapitulated the ligand binding properties of the corresponding cell lines. These data indicate that each subunit of alphaMbeta2 contributes distinct properties to alphaMbeta2 and that, in most but not all cases, the response of the integrin is a composite of the functions of its individual subunits.