Screening of cyclic peptide phage libraries identifies ligands that bind streptavidin with high affinities.

The screening of combinatorial peptide libraries has emerged as an important tool in the discovery of novel substrates or ligands for enzyme and receptor targets. For example, screening linear peptide libraries using streptavidin as a model receptor system has previously identified many low-affinity peptide ligands, all of which contain the common motif His-Pro-Gln (HPQ). We reasoned that constraining the conformational freedom of linear peptides by cyclization in a library would yield peptide ligands of increased affinity. Three different cyclic peptide libraries were constructed in an M13 phage display system as N-terminal pIII protein fusions. The random peptide sequences were flanked by two cysteine residues, which allows efficient disulfide bond formation and cyclization during phage assembly. These cyclic peptide libraries were screened with streptavidin as the model receptor system. Many sequences, all of which contained the motif His-Pro-Gln (HPQ), were discovered, and in the preceding paper, the structures of complexes of streptavidin-bound cyclic and linear peptides are described (Katz, 1995). Analysis of binding kinetics and affinities demonstrated that the conformationally constrained cyclic peptides bound streptavidin with affinities up to 3 orders of magnitude higher than linear peptides identified in previous library screens. These results demonstrate the potential of screening conformationally constrained peptide libraries for high-affinity novel receptor ligands or enzyme substrates.

Purification and characterization of the periplasmic domain of the aspartate chemoreceptor.

In order to facilitate biochemical studies of cell-surface receptors, a plasmid allowing the expression of the periplasmic domain of the aspartate receptor from Salmonella typhimurium as a soluble periplasmic protein has been constructed. This 18-kDa protein is exported to the periplasm, where it may be extracted by mild osmotic lysis. This isolated domain behaves as a normal, soluble protein and has been purified to homogeneity by standard techniques. The purified periplasmic domain binds aspartate with a kD similar to that of the full-length receptor, and the binding occurs with negative cooperativity, i.e. the binding of one molecule of aspartate induces a conformational change that interferes with the binding of the second aspartate. Unlike the full-length receptor, the periplasmic domain undergoes a protein concentration- and aspartate-dependent monomer-dimer equilibrium. At low protein concentrations and in the absence of aspartate, the protein is monomeric. At higher protein concentrations or in the presence of saturating aspartate, the protein is dimeric. Two charge variants of the protein have been identified on native polyacrylamide gels. The more acidic form is blocked to Edman degradation, indicating that modification of the amino terminus of this protein can occur after cleavage of the signal peptide in the periplasm.

Refined structures of the ligand-binding domain of the aspartate receptor from Salmonella typhimurium.

The aspartate receptor is a transmembrane-signalling protein that mediates chemotaxis behaviour in bacteria. Aspartate receptors in Salmonella typhimurium and Escherichia coli exist as dimers of two subunits in the presence as well as in the absence of aspartate. We have previously reported the three-dimensional structures of the external ligand-binding domain of the S. typhimurium aspartate receptor with and without bound aspartate. The external or periplasmic region of the aspartate receptor is a dimer of four-alpha-helical bundle subunits; a single aspartate molecule binds to one of two sites residing at the subunit interface, increasing the affinity of the subunits for one another. Here we report the results of a detailed analysis of the aspartate receptor ligand-binding domain structure (residues 25 to 188). The dimer interface between the twofold related subunits consists primarily of contacts mediated by the side-chains of the N-terminal helix of each four-alpha-helical bundle subunit. The N-terminal helices pack approximately 20 degrees from parallel as an approximate coiled-coil super-secondary structure. We have refined aspartate receptor ligand-binding domain structures in the presence and in the absence of a bound aromatic compound, 1,10-phenanthroline, to 2.2 A and 2.3 A resolution, respectively, as well as crystal structures in the presence of specifically bound Au(I), Hg(II) and Pt(IV) complex ions at 2.4 A, 3.0 A and 3.3 A resolution, respectively. The possible biological relevance of the aromatic ligand-binding site and the metal ion-binding sites is discussed. The dimer of four-alpha-helical bundle subunits composing the periplasmic region of the S. typhimurium aspartate receptor provides a basis for understanding the results of mutational analyses performed on related chemotaxis transmembrane receptors. The crystal structure analysis provides an explanation for the way in which mutations in the E. coli aspartate receptor affect its binding to the periplasmic maltose-binding protein and how mutations in the more distantly related E. coli Trg chemotaxis receptor affect its binding to the periplasmic ribose and glucose-galactose binding proteins.

Three-dimensional structures of the ligand-binding domain of the bacterial aspartate receptor with and without a ligand.

The three-dimensional structure of an active, disulfide cross-linked dimer of the ligand-binding domain of the Salmonella typhimurium aspartate receptor and that of an aspartate complex have been determined by x-ray crystallographic methods at 2.4 and 2.0 angstrom (A) resolution, respectively. A single subunit is a four-alpha-helix bundle with two long amino-terminal and carboxyl-terminal helices and two shorter helices that form a cylinder 20 A in diameter and more than 70 A long. The two subunits in the disulfide-bonded dimer are related by a crystallographic twofold axis in the apo structure, but by a noncrystallographic twofold axis in the aspartate complex structure. The latter structure reveals that the ligand binding site is located more than 60 A from the presumed membrane surface and is at the interface of the two subunits. Aspartate binds between two alpha helices from one subunit and one alpha helix from the other in a highly charged pocket formed by three arginines. The comparison of the apo and aspartate complex structures shows only small structural changes in the individual subunits, except for one loop region that is disordered, but the subunits appear to change orientation relative to each other. The structures of the two forms of this protein provide a step toward understanding the mechanisms of transmembrane signaling.

Intrasubunit signal transduction by the aspartate chemoreceptor.

Receptors that transmit signals across cell membranes are typically composed of multiple subunits. To test whether subunit interactions are required for transmembrane signaling by the bacterial aspartate receptor, dimers were constructed with (i) two full-length subunits, (ii) one full-length subunit and one subunit lacking the cytoplasmic domain, or (iii) one full-length subunit and one subunit lacking both the cytoplasmic and the transmembrane domains. Methylation of the cytoplasmic domain of all three receptor constructs was stimulated by the binding of aspartate. These findings demonstrate that transmembrane signaling does not require interactions between cytoplasmic or transmembrane domains of adjacent subunits and suggest that signaling occurs via conformational changes transduced through a single subunit.

Crystallization and preliminary X-ray diffraction study of the ligand-binding domain of the bacterial chemotaxis-mediating aspartate receptor of Salmonella typhimurium.

The periplasmic domain of the aspartate chemotaxis receptor from Salmonella typhimurium has been crystallized in the presence and absence of bound aspartate. Both crystal forms were grown by precipitation with lithium sulfate and diffract to 1.8 A resolution. The aspartate receptor structure is believed to be prototypical of a large class of receptors including those for polypeptide growth factor hormones as well as those for small chemotaxis-affector molecules such as aspartate and serine.

The amino terminus of the aspartate chemoreceptor is formylmethionine.

The amino terminus of the Salmonella typhimurium aspartate receptor has been identified as formylmethionine by mass spectral analysis of the amino-terminal tryptic peptide. Purification and analysis of the blocked amino-terminal peptide was facilitated by the use of a mutant aspartate receptor which has a cysteine residue at position 3. The sequence of this peptide confirms the translational start site predicted from the nucleotide sequence of the tar gene. Furthermore, in vivo labeling experiments reveal that the formyl group is present on chemotaxis receptors produced at wild-type levels in Escherichia coli, indicating that the presence of the formyl group is not a consequence of over-production of the receptor. The stability of the amino-terminal formyl group on the receptor may be a consequence of the membrane localization of the receptor and the dependence of this localization on the membrane transport machinery of the cell.

Structure of a bacterial sensory receptor. A site-directed sulfhydryl study.

Cysteines are substituted at six positions in the aspartate receptor, and these mutant proteins are used to investigate three major facets of receptor structure. 1) The surface of the receptor is examined through measurement of the rate constants for chemical modification of the cysteines by aqueous reagents. Different positions exhibit a range of accessibility (for example, Cys-128 most exposed, Cys-36 most buried). 2) The transmembrane structure of the receptor is determined by reaction of the cysteines with a membrane-impermeant reagent. 3) The spatial proximities in the folded structure of specific pairs of cysteines are investigated by disulfide bond formation. These studies illustrate the usefulness of site-directed sulfhydryl chemistry in the analysis of protein structure.

Site-directed cross-linking. Establishing the dimeric structure of the aspartate receptor of bacterial chemotaxis.

Cysteine residues introduced at specific locations in the aspartate receptor of Salmonella typhimurium provide anchor points for cross-linking and serve as chemical markers for structural studies of this oligomeric receptor. These markers have been used to measure the rate of subunit exchange between oligomeric receptors and to show that ligand binding inhibits this exchange. The cysteine-containing receptors can be oxidatively cross-linked to completion within the oligomeric receptor, indicating that the receptor has an even number of subunits. Based on this observation, a technique has been developed that can be used to determine the oligomeric structure of proteins under a variety of experimental conditions. The technique involves the measurement of the effect of dilution by "cysteineless" receptor subunits on cross-linking and reveals that the aspartate receptor is dimeric in detergent solution, in a mixed-micelle system, and in reconstituted membrane vesicles. Binding of aspartate does not change the oligomeric structure of the receptor, indicating that transmembrane signaling occurs within an oligomeric receptor of constant size.

Noninducibility of cytochrome P-450 in the earthworm Dendrobaena veneta.

Cytochrome P-450 has been measured in the earthworm Dendrobaena veneta (Rosa) in a direct spectrophotometric procedure. The P-450 was found not in the dense microsomal fraction, but in the less dense overlying fraction often referred to as buffy coat. Earthworm P-450 was not induced by 3-methylcholanthrene or phenobarbitol.