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G F Ames

Publications and source records attributed to G F Ames.

At least 37 records · Page 2Linked to original sources
PubMed1993

Three-dimensional structures of the periplasmic lysine/arginine/ornithine-binding protein with and without a ligand.

Many proteins exhibit a large-scale movement of rigid globular domains. Among these, bacterial periplasmic binding proteins involved in substrate transport, or transport and chemotaxis, can be used as prototypes for understanding the mechanism of the movement. Such movements have been found to be associated with specific functions, such as substrate binding, catalysis, and recognition by other biomolecules. We have determined the three-dimensional structures of the lysine/arginine/ornithine-binding protein (LAO) from Salmonella typhimurium with and without lysine by x-ray crystallographic methods at 1.8- and 1.9-A resolution, respectively. The structures are composed of two lobes held together by two short connecting strands. The two lobes are far apart in the unliganded structure, but in contact with each other in the lysine-liganded structure. The large movement of the lobes is a consequence of a 52 degrees rotation of a single backbone torsion angle in the first connecting strand and of distributed smaller changes of three backbone torsion angles of the second connecting strand. The absence of contact between the lysine and the connecting strands suggests that the ligand does not induce the conformational change directly. We instead propose that the unliganded protein undergoes a dynamic change between an "open" and a "closed" conformation and that the role of the ligand is to stabilize the closed conformation. We discuss the nature of a surface area which might be recognized by the membrane-bound complex of these amino acids transport systems.

Amino Acid Sequence↗
PubMed1993

The ATP-binding component of a prokaryotic traffic ATPase is exposed to the periplasmic (external) surface.

The membrane-bound complex of bacterial periplasmic permeases consists of two hydrophobic integral membrane proteins and two copies of a hydrophilic ATP-binding protein. The ATP-binding proteins from all periplasmic permeases display a high level of sequence similarity and are referred to as "conserved components." The conserved component from the histidine permease, HisP, has been postulated on the basis of genetic evidence to be accessible at the exterior membrane surface, in contrast to the commonly postulated association with the interior membrane surface as peripheral membrane proteins. We have used proteolysis and biotinylation of membrane vesicles to show that HisP is accessible to these reagents at the external surface and that this orientation depends on the presence of the two hydrophobic components, HisQ and HisM. Several binding-protein-independent hisP mutants are shown to produce HisP proteins that are more susceptible to proteases from the external membrane surface. Since the hydrophilic component is well conserved also in a group of eukaryotic transporters, which together with many prokaryotic systems form the superfamily of traffic ATPases, this insight about its membrane topology has general implications for understanding the molecular mechanism of action of this large superfamily, which includes the cystic fibrosis transmembrane conductance regulator and multidrug-resistance proteins.

ATP-Binding Cassette Transporters↗
PubMed1993

ATP-dependent transport systems in bacteria and humans: relevance to cystic fibrosis and multidrug resistance.

The prokaryotic permeases are members of a superfamily of membrane transporters called traffic ATPases, which includes the medically important eukaryotic multidrug resistance (MDR) protein and cystic fibrosis transmembrane regulator (CFTR). Members of this superfamily have extensive sequence and structural similarity, in particular in an ATP-binding motif, and are believed to use ATP to energize translocation of substrates across biological membranes. The prokaryotic histidine permease is well-characterized and serves as a convenient model system. In this review, we highlight some of the biochemical and molecular biological approaches used to study the functional and architectural organization of this permease and relate the results of these approaches to what is known about other traffic ATPases. We have identified specific regions that we believe critical for the function of the histidine permease and propose that the corresponding regions in the eukaryotic traffic ATPases are also important for their function. In light of the fact that CFTR (and possibly the MDR protein) is an ion channel, we compare the properties of channels and transporters; in addition, we discuss the possibility that other members of the traffic ATPases may also have channel-like activity.

ATP-Binding Cassette Transporters↗
PubMed1992

Purification and characterization of the periplasmic lysine-, arginine-, ornithine-binding protein (LAO) from Salmonella typhimurium.

The lysine-, arginine-, ornithine-binding protein (LAO) from Salmonella typhimurium has been purified to homogeneity and characterized. The dissociation constants (KD) were determined by equilibrium dialysis assay to be 14, 15, and 29 nM for L-arginine, L-lysine, and L-ornithine respectively. L-Histidine was found to be a relatively good ligand (KD, 500 nM). Methods have been developed for the separation of liganded from unliganded LAO, for the estimation of bound ligand, and for unliganding LAO. Liganded and unliganded LAO are shown to have distinct UV spectra. The UV spectrum also varies with the nature of the substrate. Inhibition studies with substrate analogs yielded information useful for understanding the nature of the ligand-binding pocket.

Arginine↗
PubMed1992

Crystallization and preliminary X-ray studies of the liganded lysine, arginine, ornithine-binding protein from Salmonella typhimurium.

The periplasmic binding protein LAO from Salmonella typhimurium, which is involved in lysine, arginine and ornithine transport, has been crystallized together with one of its ligands, arginine (LAO-Arg). Preliminary X-ray diffraction studies of LAO-Arg crystal show that it belongs to the orthorhombic space group P2(1)2(1)2(1) and has the unit cell dimensions of a = 37.65 A, b = 59.45 A, c = 115.91 A. Crystals of the LAO-Arg complex diffract beyond 2.0 A resolution.

Bacterial Proteins↗
PubMed1992

Topology of the hydrophobic membrane-bound components of the histidine periplasmic permease. Comparison with other members of the family.

The membrane-bound complex of periplasmic permeases comprises two hydrophobic proteins which have been hypothesized to be integral membrane-spaninning proteins. We have investigated the topological organization of the hydrophobic components of the Salmonella typhimurium histidine permease, HisQ and HisM. Both proteins are digested by trypsin and proteinase K when either inside-out or right-side-out membrane vesicles are used. Therefore, these proteins are exposed to both surfaces of the membrane. Digestion with carboxypeptidase and binding studies with antibodies directed against the carboxyl terminus of HisQ and HisM have localized their carboxyl termini to the inside surface of the cytoplasmic membrane. Aminopeptidase digestion suggests periplasmic localization of their amino termini. Alkaline phosphatase fusions to HisQ and HisM indicate the existence of five spanners in both proteins. The periodicity and orientation of spanners and loops in HisQ and HisM match those of the five carboxyl-terminal spanners of MalF, the only other hydrophobic component of the periplasmic permeases for which topological information is available. An alignment of the sequences of all known hydrophobic components of periplasmic permeases is presented which indicates clear conservation of secondary structure and some conservation of primary sequence. The structural conservation of the components is discussed, and a role for a hydrophilic loop containing a conserved sequence (the EAA loop) is proposed.

ATP-Binding Cassette Transporters↗
PubMed1992

ATP-dependent bacterial transporters and cystic fibrosis: analogy between channels and transporters.

The traffic ATPases superfamily includes known transporters, both prokaryotic and eukaryotic, including the medically important proteins, P-glycoprotein, and the cystic fibrosis gene product (CFTR), which is known to be a Cl- channel. The structure and mechanism of action of the best-studied members of the superfamily, the periplasmic permeases, are described and related to that of CFTR and eukaryotic traffic ATPases in general. The contention is put forward that the distinction between the architecture and mechanisms of action of channels and transporters is blurred.

Adenosine Triphosphate↗
PubMed1992

Physical mapping of repetitive extragenic palindromic sequences in Escherichia coli and phylogenetic distribution among Escherichia coli strains and other enteric bacteria.

Repetitive extragenic palindromic (REP) sequences are highly conserved inverted repeat sequences originally discovered in Escherichia coli and Salmonella typhimurium. We have physically mapped these sequences in the E. coli genome by using Southern hybridization of an ordered phage bank of E. coli (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987) with generic REP probes derived from the REP consensus sequence. The set of REP probe-hybridizing clones was correlated with a set of clones expected to contain REP sequences on the basis of computer searches. We also show that a generic REP probe can be used in Southern hybridization to analyze genomic DNA digested with restriction enzymes to determine genetic relatedness among natural isolates of E. coli. A search for these sequences in other members of the family Enterobacteriaceae shows a consistent correlation between both the number of occurrences and the hybridization strength and genealogical relationship.

Base Sequence↗
PubMed1991

Crystal structure of the lysine-, arginine-, ornithine-binding protein (LAO) from Salmonella typhimurium at 2.7-A resolution.

A wide variety of sugars, amino acids, peptides, and inorganic ions are transported into bacteria by periplasmic transport systems consisting of substrate-specific receptors (binding proteins) and membrane-bound protein complexes. The crystal structure of the lysine-, arginine-, ornithine-binding protein (LAO) at 2.7-A resolution shows that the molecule has a bi-lobal structure and that its topological structure is different from other amino acid-binding proteins but is similar to the sulfate-binding protein and maltose-binding protein. High sequence homology between LAO and the histidine-binding protein (HisJ) and the fact that LAO and HisJ share the same membrane-bound protein complex allow one to define functional regions responsible for the ligand binding and for the interaction with the membrane complex.

Amino Acid Sequence↗
PubMed1991

Structure-function analysis of the histidine permease and comparison with cystic fibrosis mutations.

Traffic ATPases constitute a superfamily of transporters that include prokaryotic permeases and medically important eukaryotic proteins, such as the multidrug resistance P-glycoprotein and the cystic fibrosis gene product. We present a structure-function analysis of a member of this superfamily, the prokaryotic histidine permease, using mutations generated both in vitro and in vivo, and assaying several biochemical functions. The analysis supports a previously predicted structural model and allows the assignment of specific functions to several predicted structural features. Mutations in the secondary structure features which form the nucleotide-binding pocket in general cause the loss of ATP binding activity. Mutations in the helical domain retain ATP binding activity. Several mutations have been identified which may affect the signaling mechanism between ATP hydrolysis and membrane translocation. We relate our findings to those emerging from the recent biochemical and genetic analyses of cystic fibrosis mutations.

ATP-Binding Cassette Transporters↗
PubMed1991

Binding protein-independent histidine permease mutants. Uncoupling of ATP hydrolysis from transmembrane signaling.

Periplasmic permeases consist of a substrate-binding receptor, located in the periplasm, and a membrane-bound complex composed of two integral membrane proteins and two nucleotide-binding proteins. The receptor interacts with the membrane-bound complex, which, upon receiving this signal, is postulated to hydrolyze ATP and translocate the substrate. We show that a class of mutations in the membrane-bound complex of the histidine permease, which allow transport in the absence of the substrate-binding protein, hydrolyze ATP independently from any signal. The data are compatible with the notion that cross-membrane signaling between the liganded periplasmic receptor and the cytoplasmic ATP-binding sites initiates conformational changes leading to ATP hydrolysis and substrate translocation.

ATP-Binding Cassette Transporters↗
PubMed1991

The membrane-bound proteins of periplasmic permeases form a complex. Identification of the histidine permease HisQMP complex.

The membrane-bound proteins of periplasmic transport systems have been hypothesized to form a complex with relatively little experimental support. Here we present experimental evidence that HisQ, HisM, and HisP, the membrane-bound proteins of the periplasmic histidine transport system of Salmonella typhimurium, form such a complex. We have developed antibodies specific to each of these proteins to aid in their characterization. Extractions with urea, alkaline pH, or Triton X-114 show that HisQ and HisM are integral membrane proteins. By these tests HisP displays an unusual behavior, being associated with the membrane whether or not HisQ and HisM are present and despite its hydrophilic sequence. However, the nature of HisPs interaction with the membrane is shown to vary depending on the presence of HisQ and HisM. In their absence, HisP is somewhat peripherally associated with the membrane, while in their presence it binds much more tightly, indicating that it forms a complex in association with HisQ and HisM. This is demonstrated by the coimmunoprecipitation of all three proteins by antibodies directed against any one of them. Chemical cross-linking allowed the characterization of the subunit stoichiometry of the complex as two HisPs to one HisQ and one HisM. Within this complex all three proteins probably contact each other and the two HisPs form a dimer. We hypothesize that HisQ and HisM with their multiple membrane-spanning segments form a "channel" within which the HisP subunits are located.

ATP-Binding Cassette Transporters↗
PubMed1991

Use of exonuclease for rapid polymerase-chain-reaction-based in vitro mutagenesis.

We describe a simple strategy for improving site-specific mutagenesis. We have combined the polymerase chain reaction (PCR) with a phage lambda exonuclease (Exo lambda) treatment to produce mutated fragments larger than 2.5 kb. The applicability of this approach has been proven with two overlapping mutated primers. The procedure has also been made more cost-effective by the use of a single mutated primer, which is referred to as SMP-PCR procedure. The entire procedure of kinasing the primer, amplification by PCR, Exo lambda digestion and second step of PCR can be performed in less than 6 h. We have used this approach to generate a number of mutations in the Salmonella typhimurium hisP gene of the histidine transport operon.

Bacteriophage lambda↗
PubMed1991

Structural model of the nucleotide-binding conserved component of periplasmic permeases.

The amino acid sequences of 17 bacterial membrane proteins that are components of periplasmic permeases and function in the uptake of a variety of small molecules and ions are highly homologous to each other and contain sequence motifs characteristic of nucleotide-binding proteins. These proteins are known to bind ATP and are postulated to be the energy-coupling components of the permeases. Several medically important eukaryotic proteins, including the multidrug-resistance transporters and the protein encoded by the cystic fibrosis gene, are also homologous to this family. By multiple sequence alignment of these 17 proteins, the consensus sequence, secondary structure, and surface exposure were predicted. The secondary structural motifs that are conserved among nucleotide-binding proteins were identified in adenylate kinase, p21ras, and elongation factor Tu by superposition of their known tertiary structures. The equivalent secondary structural elements in the predicted conserved component were located. These, together with sequence information, served as guides for alignment with adenylate kinase. A model for the structure of the ATP-binding domain of the permease proteins is proposed by analogy to the adenylate kinase structure. The characteristics of several permease mutations and biochemical data lend support to the model.

Adenylate Kinase↗