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At least 19 recordsLinked to original sources
PubMed2004

Agonist-induced internalization of leukotriene B(4) receptor 1 requires G-protein-coupled receptor kinase 2 but not arrestins.

The leukotriene B(4) (LTB(4)) receptor (BLT1) becomes desensitized upon repeated agonist stimulation. Little is known, however, about BLT1 internalization, which follows desensitization in most G-protein-coupled receptors (GPCR). In the current study, transiently expressed BLT1 readily internalized, after LTB(4) stimulation, in RBL-2H3 cells that express high levels of endogenous GPCR kinase 2 (GRK2) but did not in COS-7 or human embryonic kidney (HEK) 293 cells, which do not overexpress GRK. The internalization of BLT1 could be blocked in RBL-2H3 cells by coexpressing dominant-negative (DN) GRK2 K220R and could be promoted in HEK293 cells by coexpressing wild-type (WT) GRK2. Coexpression of WT or DN nonvisual arrestins had no effect on BLT1 internalization. Moreover, upon stimulation with LTB(4), BLT1 did not induce arrestin-green fluorescence protein redistribution in either cell type, even in the presence of overexpressed GRK2. Coimmunoprecipitation experiments confirmed that BLT1 could associate with GRK2 but not with arrestins. A C-tail-truncated mutant of BLT1 lost the capacity to internalize and associate with GRK2 upon exposure to LTB(4), suggesting that the C-tail was required for receptor internalization and association with GRK2. Taken together, our results indicate that the C terminus of BLT1 plays a pivotal role in receptor internalization and GRK2 association. Moreover, ligand-induced BLT1 internalization is dependent on GRK2 but independent of arrestins. This may allow differential, cell-type-specific signaling in response to LTB(4), depending on GRK expression levels.

Animals↗
PubMed1990

Inhibition of cofactor activity of protein S by a complex of protein S and C4b-binding protein. Evidence for inactive ternary complex formation between protein S, C4b-binding protein, and activated protein C.

To elucidate the mechanism by which C4b-binding protein inhibits the cofactor activity of protein S for anticoagulant-activated protein C, the interactions between protein S, activated protein C, and C4b-binding protein were studied using solid-phase enzyme immunoassays. Both activated protein C and C4b-binding protein bound to protein S fixed to microplate wells. C4b-binding protein did not inhibit the binding of activated protein C to protein S, nor did activated protein C inhibit the binding of C4b-binding protein to protein S. Activated protein C bound to a protein S-C4b-binding protein complex which was cross-linked with a chemical reagent as well as it bound to free protein S. Protein S-C4b-binding protein complex competitively inhibited activated protein C-binding to free protein S and also the cofactor activity of free protein S. Immunoblotting analysis showed ternary complex formation with protein S, C4b-binding protein, and activated protein C in the liquid phase by treatment with the cross-linking reagent. These findings suggest that the protein S-C4b-binding protein complex inhibits the cofactor activity of free protein S probably by inhibition of functionally active protein S-activated protein C complex formation by the apparent competitive formation of an inactive ternary complex with protein S, C4b-binding protein, and activated protein C.

Antibodies, Monoclonal↗
PubMed1991

Sendai virus protein-protein interactions studied by a protein-blotting protein-overlay technique: mapping of domains on NP protein required for binding to P protein.

Proteins from Sendai virus particles and from infected cells were analyzed in a protein-blotting protein-overlay assay for their interaction with in vitro-synthesized, [35S]methionine-labeled viral proteins NP, P, and M. After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transfer onto polyvinylidene difluoride membranes, and renaturation, the immobilized proteins were found to interact specifically with radiolabeled proteins. NP proteins from virus particles and from infected cells retained 35S-P protein equally well. Conversely, P protein from virus particles and from infected cells retained 35S-NP protein. 35S-M protein was retained mainly by NP protein but also by several cellular proteins. To determine the domains on NP protein required for binding to immobilized P protein, a series of truncated and internally deleted 35S-NP proteins was constructed. The only deletion that did not affect binding resides between residues 426 and 497. The carboxyl-terminal 27 residues (positions 498 to 524) contribute significantly to the binding affinity. Removal of 20 residues (positions 225 to 244) in the hydrophobic middle part of NP protein completely abolished its binding to P protein.

Animals↗
PubMed2000

UPA, a universal protein array system for quantitative detection of protein-protein, protein-DNA, protein-RNA and protein-ligand interactions.

Protein-protein interactions have been widely used to study gene expression pathways and may be considered as a new approach to drug discovery. Here I report the development of a universal protein array (UPA) system that provides a sensitive, quantitative, multi-purpose, effective and easy technology to determine not only specific protein-protein interactions, but also specific interactions of proteins with DNA, RNA, ligands and other small chemicals. (i) Since purified proteins are used, the results can be easily interpreted. (ii) UPA can be used multiple times for different targets, making it economically affordable for most laboratories, hospitals and biotechnology companies. (iii) Unlike DNA chips or DNA microarrays, no additional instrumentation is required. (iv) Since the UPA uses active proteins (without denaturation and renaturation), it is more sensitive compared with most existing methods. (v) Because the UPA can analyze hundreds (even thousands on a protein microarray) of proteins in a single experiment, it is a very effective method to screen proteins as drug targets in cancer and other human diseases.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Protein-protein interfaces: architectures and interactions in protein-protein interfaces and in protein cores. Their similarities and differences.

Protein structures generally consist of favorable folding motifs formed by specific arrangements of secondary structure elements. Similar architectures can be adopted by different amino acids sequences, although the details of the structures vary. It has long been known that despite the sequence variability, there is a striking preferential conservation of the hydrophobic character of the amino acids at the buried positions of these folding motifs. Differences in the sizes of the side-chains are accommodated by movements of the secondary structure elements with respect to each other, leading to compact packing. Scanning protein-protein interfaces reveals that similar architectures are also observed at and around their interacting surfaces, with preservation of the hydrophobic character, although not to the same extent. The general forces that determine the origin of the native structures of proteins have been investigated intensively. The major non-bonded forces operating on a protein chain as it folds into a three-dimensional structure are likely to be packing, the hydrophobic effect, and electrostatic interactions. While the substantial hydrophobic forces lead to a compact conformation, they are also nonspecific and cannot serve as a guide to a conformationally unique structure. For the general folding problem, it thus appears that packing is a prime candidate for determining a particular fold. Specific hydrogen-bonding patterns and salt-bridges have also been proposed to play a role. Inspection of protein-protein interfaces reveals that the hallmarks governing single chain protein structures also determine their interactions, suggesting that similar principles underlie protein folding and protein-protein associations. This review focuses on some aspects of protein-protein interfaces, particularly on the architectures and their interactions. These are compared with those present in protein monomers. This task is facilitated by the recently compiled, non-redundant structural dataset of protein-protein interfaces derived from the crystallographic database. In particular, although current view holds that protein-protein interfaces and interactions are similar to those found in the conformations of single-chain proteins, this review brings forth the differences as well. Not only is it logical that such differences would exist, it is these differences that further illuminate protein folding on the one hand and protein-protein recognition on the other. These are also particularly important in considering inhibitor (ligand) design.

Amino Acids↗
PubMed1996

Bifunctional fusion proteins of calmodulin and protein A as affinity ligands in protein purification and in the study of protein-protein interactions.

An affinity chromatography system is described that incorporates a genetically designed bifunctional affinity ligand. The utility of the system in protein purification and in the study of protein-protein interactions is demonstrated by using the interaction between protein A and the heat shock protein DnaK as a model system. The bifunctional affinity ligand was developed by genetically fusing calmodulin (CaM) to protein A (ProtA). The dual functionality of protein A-calmodulin (ProtA-CaM) stems from the molecular recognition properties of the two components of the fusion protein. In particular, CaM serves as the anchoring component by virtue of its binding properties toward phenothiazine. Thus, the ProtA-CaM can be immobilized on a solid support containing phenothiazine from the C-terminal domain of the fusion protein. Protein A is at the N-terminal domain of the fusion protein and serves as the affinity site for DnaK. While DnaK binds specifically to the protein A domain of the bifunctional ligand, it is released upon addition of ATP and under very mild conditions (pH 7.0). In addition to obtaining highly purified DnaK, this system is very rugged in terms of its performance. The proteinaceous bifunctional affinity ligand can be easily removed by addition of EGTA, and fresh ProtA-CaM can be easily reloaded onto the column. This allows for a facile regeneration of the affinity column because the phenothiazine-silica support matrix is stable for long periods of time under a variety of conditions. This study also demonstrates that calmodulin fusions can provide a new approach to study protein-protein interactions. Indeed, the ProtA-CaM fusion protein identified DnaK as a cellular component that interacts with protein A from among the thousands of proteins present in Escherichia coli.

Calmodulin↗
PubMed2003

Accessory proteins for G protein-signaling systems: activators of G protein signaling and other nonreceptor proteins influencing the activation state of G proteins.

Heterotrimeric G proteins are key transducers for signal transfer from outside of the cell. In addition to their regulation by the superfamily of G protein-coupled receptors, many if not all of the subtypes of heterotrimeric G proteins are also regulated by additional accessory proteins that influence guanine nucleotide binding and/or hydrolysis or subunit interactions. Activators of G protein signaling (AGS1-3) refer to a functionally defined group of proteins that activate G protein-signaling systems in the absence of a classical G protein-coupled receptor. AGS and related proteins provide unexpected insights into the regulation of the G protein activation/deactivation cycle and the functional roles of G proteins. These proteins likely play important roles in the generation of signaling complexes, the positioning of signaling proteins within the cell, and in biological roles of G proteins unrelated to a cell surface receptor. As such, these proteins and the concepts advanced with their discovery provide unexpected avenues for therapeutics and understanding disease mechanisms.

Animals↗
PubMed1997

Hydrophobic folding units at protein-protein interfaces: implications to protein folding and to protein-protein association.

A hydrophobic folding unit cutting algorithm, originally developed for dissecting single-chain proteins, has been applied to a dataset of dissimilar two-chain protein-protein interfaces. Rather than consider each individual chain separately, the two-chain complex has been treated as a single chain. The two-chain parsing results presented in this work show hydrophobicity to be a critical attribute of two-state versus three-state protein-protein complexes. The hydrophobic folding units at the interfaces of two-state complexes suggest that the cooperative nature of the two-chain protein folding is the outcome of the hydrophobic effect, similar to its being the driving force in a single-chain folding. In analogy to the protein-folding process, the two-chain, two-state model complex may correspond to the formation of compact, hydrophobic nuclei. On the other hand, the three-state model complex involves binding of already folded monomers, similar to the association of the hydrophobic folding units within a single chain. The similarity between folding entities in protein cores and in two-state protein-protein interfaces, despite the absence of some chain connectivities in the latter, indicates that chain linkage does not necessarily affect the native conformation. This further substantiates the notion that tertiary, non-local interactions play a critical role in protein folding. These compact, hydrophobic, two-chain folding units, derived from structurally dissimilar protein-protein interfaces, provide a rich set of data useful in investigations of the role played by chain connectivity and by tertiary interactions in studies of binding and of folding. Since they are composed of non-contiguous pieces of protein backbones, they may also aid in defining folding nuclei.

Algorithms↗
PubMed2002

Structural basis for ubiquitin-like ISG 15 protein binding to the NS1 protein of influenza B virus: a protein-protein interaction function that is not shared by the corresponding N-terminal domain of the NS1 protein of influenza A virus.

The N-terminal domains of the NS1 protein of influenza B virus (NS1B protein) and the NS1 protein of influenza A virus (NS1A protein) share one function: binding double-stranded RNA (dsRNA). Here we show that the N-terminal domain of the NS1B protein possesses an additional function that is not shared by its NS1A counterpart: binding the ubiquitin-like ISG15 protein that is induced by influenza B virus infection. Homology modeling predicts that the dimeric six-helical N-terminal domain of the NS1B protein differs from its NS1A protein counterpart in containing large loops between helices 1 and 2 (loops 1 and 1') and between helices 2 and 3 (loops 2 and 2'). Mutagenesis establishes that residues located in loop 1/1' together with residues located in polypeptide segment 94-103 form the ISG15 protein-binding site of NS1B protein. Loop 1/1' is not required for dsRNA binding, which instead requires arginine residues R50, R53, R50', and R53' located in antiparallel helices 1 and 1'. Further, we demonstrate that the binding sites for RNA and protein are independent of each other. In particular, ISG15 and dsRNA can bind simultaneously; the binding of the ISG15 protein does not have a detectable effect on the binding of dsRNA, and vice versa.

Amino Acid Sequence↗
PubMed1990

Activation of the serum-response and TPA-response elements by expression of the v-abl protein: comparison of the mode of action of the v-abl protein with those of protein kinase C, cyclic AMP-dependent protein kinase, and the activated c-raf protein.

In the present study, we have compared the mode of action of the v-abl protein in the regulation of gene expression with those of serine/threonine protein kinases such as protein kinase C, cyclic AMP-dependent protein kinase, and the activated c-raf protein, by measuring the transcriptional activity of the serum-response element, the 12-O-tetradecanoylphorbol-13-acetate (TPA)-response element, and the cyclic AMP-response element in NIH3T3 cells transfected with the v-abl gene. The results indicate that the v-abl protein stimulates the serum-response element and the TPA-response element, but not the cyclic AMP-response element, in a manner similar to that of the activated c-raf protein, but different from those of protein kinase C and cyclic AMP-dependent protein kinase.

Blood Physiological Phenomena↗
PubMed2005

The relationship between the flexibility of proteins and their conformational states on forming protein-protein complexes with an application to protein-protein docking.

We investigate the extent to which the conformational fluctuations of proteins in solution reflect the conformational changes that they undergo when they form binary protein-protein complexes. To do this, we study a set of 41 proteins that form such complexes and whose three-dimensional structures are known, both bound in the complex and unbound. We carry out molecular dynamics simulations of each protein, starting from the unbound structure, and analyze the resulting conformational fluctuations in trajectories of 5 ns in length, comparing with the structure in the complex. It is found that fluctuations take some parts of the molecules into regions of conformational space close to the bound state (or give information about it), but at no point in the simulation does each protein as whole sample the complete bound state. Subsequent use of conformations from a clustered MD ensemble in rigid-body docking is nevertheless partially successful when compared to docking the unbound conformations, as long as the unbound conformations are themselves included with the MD conformations and the whole globally rescored. For one key example where sub-domain motion is present, a ribonuclease inhibitor, principal components analysis of the MD was applied and was also able to produce conformations for docking that gave enhanced results compared to the unbound. The most significant finding is that core interface residues show a tendency to be less mobile (by size of fluctuation or entropy) than the rest of the surface even when the other binding partner is absent, and conversely the peripheral interface residues are more mobile. This surprising result, consistent across up to 40 of the 41 proteins, suggests different roles for these regions in protein recognition and binding, and suggests ways that docking algorithms could be improved by treating these regions differently in the docking process.

Cluster Analysis↗
PubMed1983

The protein phosphatases involved in cellular regulation. 4. Classification of two homogeneous myosin light chain phosphatases from smooth muscle as protein phosphatase-2A1 and 2C, and a homogeneous protein phosphatase from reticulocytes active on protein synthesis initiation factor eIF-2 as protein phosphatase-2A2.

Two homogeneous protein phosphatases, termed 'smooth muscle phosphatase-I' and 'smooth muscle phosphatase-II', isolated from turkey gizzard as enzymes active against the 20-kDa light chain of smooth muscle myosin, and a third homogeneous protein phosphatase from rabbit reticulocytes, purified as an enzyme active against protein synthesis initiation factor eIF-2, were classified using the criteria defined by Ingebritsen and Cohen [Eur. J. Biochem. (1983) 132, 255-261]. All three enzymes were type-2 protein phosphatases based on their specificity for the alpha-subunit of phosphorylase kinase and insensitivity to inhibitor-1 and inhibitor-2. The substrate specificities of smooth muscle phosphatase-I and the eIF-2 phosphatase were similar to the catalytic subunit of protein phosphatase-2A. Smooth muscle phosphatase-I could be designated as protein phosphatase-2A1 and eIF-2 phosphatase as protein phosphatase-2A2 on the basis of their subunit compositions. The substrate specificity, dependence of activity on Mg2+ and subunit composition of smooth muscle phosphatase-II allowed its assignment as protein phosphatase-2C.

Animals↗
PubMed2001

Differential conformational requirements for activation of G proteins and the regulatory proteins arrestin and G protein-coupled receptor kinase in the G protein-coupled receptor for parathyroid hormone (PTH)/PTH-related protein.

After stimulation with agonist, G protein-coupled receptors (GPCRs) activate G proteins and become phosphorylated by G protein-coupled receptor kinases (GRKs), and most of them translocate cytosolic arrestin proteins to the cytoplasmic membrane. Agonist-activated GPCRs are specifically phosphorylated by GRKs and are targeted for endocytosis by arrestin proteins, suggesting a connection between GPCR conformational changes and interaction with GRKs and arrestins. Previously, we showed that by substitution of histidine for residues at the cytoplasmic side of helix 3 (H3) and helix 6 (H6) of the parathyroid hormone (PTH) receptor (PTHR), a zinc metal ion-binding site is engineered that prevents PTH-stimulated G(s) activation (Sheikh, S. P., Vilardaga, J.-P., Baranski, T. J., Lichtarge, O., Iiri, T., Meng, E. C., Nissenson, R. A., and Bourne, H. R. (1999) J. Biol. Chem. 274, 17033-17041). These data suggest that relative movements between H3 and H6 are critical for G(s) activation. Does this molecular event play a similar role in activation of GRK and arrestin and in PTHR-mediated G(q) activation? To answer this question, we utilized the two previously described mutant forms of PTHR, H401 and H402, which contain a naturally present histidine residue at position 301 in H3 and a second substituted histidine residue at positions 401 and 402 in H6, respectively. Both mutant receptors showed inhibition of PTH-stimulated inositol phosphate and cAMP generation in the presence of increasing concentrations of Zn(II). However, the mutants showed no Zn(II)-dependent impairment of phosphorylation by GRK-2. Likewise, the mutants were indistinguishable from wild-type PTHR in the ability to translocate beta-arrestins/green fluorescent protein to the cell membrane and were also not affected by sensitivity to Zn(II). These results suggest that agonist-mediated phosphorylation and internalization of PTHR require conformational switches of the receptor distinct from the cAMP and inositol phosphate signaling state. Furthermore, PTHR sequestration does not appear to require G protein activation.

Animals↗
PubMed1996

The gene 59 protein of bacteriophage T4. Characterization of protein-protein interactions with gene 32 protein, the T4 single-stranded DNA binding protein.

The gene 59 protein (gp59) of bacteriophage T4 stimulates the activities of gene 41 protein (gp41), the T4 replicative DNA helicase, by promoting the assembly of gp41 onto single-stranded (ss)-DNA molecules that are covered with cooperatively bound gene 32 protein (gp32). This helicase-ssDNA assembly process, which is important for the reconstitution of the primosome component of the T4 DNA replication fork, appears to require both gp59-gp41 and gp59-gp32 protein-protein interactions. In this study we characterize the physical and functional interactions of gp59 with gp32, the T4 ssDNA-binding protein. Experimental results presented herein indicate: 1) that gp59 binds specifically to both free and ssDNA-bound gp32 molecules; and 2) that in both cases binding involves contacts between gp59 and the acidic C-terminal domain of gp32 (the so-called "A-domain"). We further show that single-stranded DNA molecules coated with (gp32-A), a truncated form of gp32 lacking the A-domain, are refractory to gp59-dependent helicase assembly. The data indicate that specific contacts between gp59 molecules and the A-domains of gp32 molecules are essential for gp59-dependent assembly of gp41 onto gp32-ssDNA complexes. Our results are consistent with a model in which gp59 binds to gp32 molecules within the gp32-ssDNA complex and therein forms a target site for helicase-ssDNA assembly.

Adenosine Triphosphatases↗
PubMed1999

Transcriptional autorepression of Msx1 gene is mediated by interactions of Msx1 protein with a multi-protein transcriptional complex containing TATA-binding protein, Sp1 and cAMP-response-element-binding protein-binding protein (CBP/p300).

The TATA-less murine Msx1 promoter contains two Msx1-binding motifs, located at -568 to -573 and +25 to +30, and is subject to potent autorepression [Takahashi, Guron, Shetty, Matsui and Raghow (1997) J. Biol. Chem. 272, 22667-22678]. To investigate the molecular mechanism by which Msx1 represses the activity of its own promoter, we transfected C2C12 myoblasts with Msx1-promoter-luciferase constructs and assessed reporter gene activity, with and without the exogenous expression of Msx1. We demonstrate that Msx1-mediated autorepression remained unaffected, regardless of the presence or absence of the Msx1 recognition motifs on the promoter. Furthermore, graded exogenous expression of TATA-binding protein (TBP), Sp1 or cAMP-response-element-binding protein-binding protein (CBP/p300) could counteract the autoinhibitory activity of Msx1. Finally, we demonstrate that Msx1 protein can be immunoprecipitated in a multiprotein complex containing TBP, Sp1 and CBP/p300. We hypothesize that the interaction of Msx1 protein with one or more ubiquitous or tissue-restricted transcription factors mediates transcriptional autorepression of the Msx1 gene.

Animals↗
PubMed2000

The yeast mitochondrial transport proteins: new sequences and consensus residues, lack of direct relation between consensus residues and transmembrane helices, expression patterns of the transport protein genes, and protein-protein interactions with other proteins.

Mitochondrial transport proteins (MTP) typically are homodimeric with a 30-kDa subunit with six transmembrane helices. The subunit possesses a sequence motif highly similar to Pro X Asp/Glu X X Lys/Arg X Arg within each of its three similar 10-kDa segments. Four (YNL083W, YFR045W, YPR021C, YDR470C) of the 35 yeast (S. cerevisiae) MTP genes were resequenced since the masses of their proteins deviate significantly from the typical 30 kDa. We now find these four proteins to have 545, 285, 902, and 502 residues, respectively. Together with only four other MTPs, the sequences of YPR021C and YDR470C show substitutions of some of the five residues that are absolutely conserved among the 12 MTPs with identified transport function and 17 other MTPs. We do now find these five consensus residues also in the new sequences of YNL083W and YFR045W. Additional analyses of the 35 yeast MTPs show that the location of transmembrane helix sequences do not correlate with the general consensus residues of the MTP family; protein segments connecting the six transmembrane helices and facing the intermembrane space are not uniformly short (about 20 residues) or long (about 40 residues) when facing the matrix; most MTPs have at least one transmembrane helix for which the sum of the negative hydropathy values of all residues yields a very small negative value, suggesting a membrane location bordering polar faces of other transmembrane helices or a non-transmembrane location. The extra residues of the three large MTPs are hydrophilic and at the N-terminal. The 200-residue N-terminal segment of YNL083W has four putative Ca2+-binding sites. The 500-residue N-terminal segment of YPR021C shows sequence similarity to enzymes of nucleic acid metabolism. cDNA microarray data show that YNL083W is expressed solely during sporulation, while the expressions of YFR045W, YPR021C, and YDR470C are induced by various stress situations. These results also show that the 35 MTP genes are expressed under a rather diverse set of metabolic conditions that may help identify the function of the proteins. Interestingly, yeast two-hybrid screens, that will also be useful in identifying the function of MTPs, indicate that MIR1, AAC3, YOR100C, and YPR011C do interact with non-MTPs.

Amino Acid Sequence↗
PubMed2006

Protein-protein- and protein-RNA-binding properties of the movement protein and VP25 coat protein of Apple latent spherical virus.

To elucidate the mechanism of Apple latent spherical virus (ALSV) movement, various properties of its cell-to-cell movement protein (MP) were analyzed. ELISA and blot overlay assays demonstrated that the MP bound specifically to ALSV virions and in particular to one of the three coat proteins (VP25) but not to the other two coat proteins (VP20 and VP24). Mutational analyses have revealed that the MP contains two domains with independent VP25-binding activity (amino acid residues 1-188 and 189-281). Furthermore, nucleotide-binding experiments showed that the MP and VP25 bound to single-stranded RNA (ssRNA) and ssDNA without any sequence specificity, but these two proteins did not bind to double-stranded RNA (dsRNA) and dsDNA. The MP contains three potentially independent single-stranded nucleic acid-binding domains between amino acid residues 95-188, 189-281 and 277-376. The MP demonstrated cooperative and VP25 demonstrated non-cooperative binding to ssRNA in gel-retardation analyses. The cooperative RNA binding of the MP became non-cooperative when MP and VP25 were tested together in competition binding experiments, even though a sufficient amount of the MP for fully cooperative RNA binding the MP was supplied. The roles of the MP and VP25 interactions and nucleic acid binding activities in ALSV movement are discussed.

Capsid Proteins↗
PubMed1996

Evidence for electron transfer from the nitrogenase iron protein to the molybdenum-iron protein without MgATP hydrolysis: characterization of a tight protein-protein complex.

MgA TP hydrolysis has been proposed to be absolutely required for electron transfer from the nitrogenase iron (Fe) protein to the molybdenum-iron (MoFe) protein. This work presents evidence for primary electron transfer from the Azotobacter vinelandii nitrogenase Fe protein to the MoFe protein in the absence of MgATP hydrolysis. Deletion of an amino acid (Leu 127) in a signal transduction pathway in the Fe protein resulted in an Fe protein conformation resembling the MgATP-bound state. This altered Fe protein (L127delta) was found to bind to the MoFe protein in the absence of MgATP, forming a tight protein complex. Both steady state and stopped-flow transient kinetic measurements suggest that two L127delta Fe proteins bind to one MoFe protein with an extremely high affinity. From pre-steady state kinetic determinations of the rate of complex dissociation, the affinity was found to be at least 350 times tighter than that of the wild-type A. vinelandii nitrogenase complex and at least 20 times tighter than that of the heterologous Clostridium pasteurianum Fe protein-A. vinelandii MoFe protein complex. The L127delta Fe protein-MoFe protein complex was isolated by gel filtration liquid chromatography. Scanning densitometry of an SDS gel of the complex isolated from the gel filtration column revealed a stoichiometry of 1.7 L 127 delta Fe proteins bound per MoFe protein. The L 127 delta Fe protein was found to transfer a single electron from its [4Fe-4S] cluster to the MoFe protein at a rate of 0.2s-1. This compares with the MgATP dependent electron transfer rate of 140 s-1 observed for transfer of an electron from the wild-type Fe protein to the MoFe protein. No substrate reduction (H+ or C2H2) was detected when wild-type MoFe protein was complemented with L 127 delta Fe protein. The MgATP-independent electron transfer from the L 127 delta Fe protein to the MoFe protein required active MoFe protein and was not inhibited by MgADP. EPR spectroscopy of the complex was employed to confirm the electron transfer reaction. These results show that Fe protein in a conformation resembling the MgATP-bound state can transfer at least one electron to the MoFe protein without the need for MgATP hydrolysis.

Acetylene↗