Streptococcal beta protein has separate binding sites for human factor H and IgA-Fc.

The group B streptococcus (GBS) is the most important cause of life-threatening bacterial infections in newborn infants. Protective immunity to GBS infection is elicited by several surface proteins, one of which, the beta protein, is known to bind human IgA-Fc. Here, we show that the beta protein also binds human factor H (FH), a negative regulator of complement activation. Absorption experiments with whole human plasma demonstrated binding of FH to a GBS strain expressing beta protein but not to an isogenic beta-negative mutant. This binding was due to a direct interaction between beta and FH, as shown by experiments with purified proteins. Inhibition tests and studies with beta fragments demonstrated that FH and IgA-Fc bind to separate and nonoverlapping regions in beta. Heparin, a known ligand for FH, specifically inhibited the binding between beta and FH, suggesting that FH has overlapping binding sites for beta and heparin. Bacteria-bound FH retained its complement regulatory activity, implying that beta-expressing GBS may use bound FH to evade complement attack. The finding that beta protein binds FH adds to a growing list of interactions between human pathogens and complement regulatory proteins, supporting the notion that these interactions are of general importance in bacterial pathogenesis.     

Human interstitial retinoid-binding protein. Gene structure and primary structure

Interstitial retinoid-binding protein (IRBP) is synthesized and secreted by rod photoreceptor cells into the interphotoreceptor matrix and is known to bind retinoids and fatty acids. We have used cDNA clones encoding human IRBP to isolate a 15-kilobase genomic fragment that encompasses the complete human IRBP gene. The IRBP gene spans more than 11 kilobases and is interrupted by three introns, all of which are positioned near the 3'-end of the coding sequence. The 3741-base pair coding region of IRBP appears to have been generated by quadruplication of an approximately 900 base pair long ancestral gene. The deduced amino acid sequence predicts a mature protein of 1,230 residues (calculated molecular weight 133,000). The protein sequence can be aligned into four homologous segments, each consisting of about 300 residues. Sequence similarity between segments is as high as 60% when conservative substitutions are taken into account. Two putative N-linked glycosylation sites are located in highly conserved domains in the center of the first and second segment of IRBP. A domain consisting of 41 residues at the COOH-terminal end of the third segment has 15 matching residues (38%) with an intradiscal loop of rhodopsin, a retinal-binding protein in rod photoreceptors.     

G protein antagonists. A novel hydrophobic peptide competes with receptor for G protein binding.

A substance P (SP) analog, [D-Pro4,D-Trp7,9,10] SP4-11, is known to inhibit the actions of various structurally unrelated messenger molecules as well as SP. Our studies on the effects of this peptide on the regulation of purified G proteins by receptor showed that at least some of the biological effects of the peptide can be explained by the ability of the peptide to block the activation of G proteins by receptors. Here we report that a novel truncated SP-related peptide, pGlu-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH2, inhibited the activation of G(i) or G(o) by M2 muscarinic cholinergic receptor (M2 mAChR) or of Gs by beta-adrenergic receptor in the reconstituted phospholipid vesicles, assayed by receptor-promoted GTP hydrolysis. The inhibition by the peptide was apparently reversible and competitive with respect to receptor binding to G proteins; the inhibition could be overcome by increasing the concentration of receptor in the vesicles and was not altered by changes in the concentration of G protein. The competing effects of the peptide were used to analyze the effect of agonist on receptor-G protein interaction. The concentration change of muscarinic agonist did not alter the inhibitory effects of the peptide on M2 mAChR-promoted GTPase by G(o), which is consistent with the idea that agonist increases the regulatory efficiency of the receptor but does not alter its affinity for G proteins. This new group of compounds (G protein antagonists) is a promising tool to study receptor-G protein interaction quantitatively.     

Conformational stability and binding properties of porcine odorant binding protein.

Apparently homogeneous odorant binding protein purified from pig nasal mucosa (pOBP) exhibited subunit molecular masses of 17 223, 17 447, and 17 689 (major component) Da as estimated by ESI/MS. According to gel filtration, this protein, its truncated forms, and/or its variants are homodimeric under physiologic conditions (pH 6-7, 0.1 M NaCl). The dimer if monomer equilibrium shifts toward a prevalent monomeric form at pH <4.5. Velocity sedimentation reveals a monomeric state of OBP at both pH 7.2 and 3.5, indicating a pressure-induced dissociation of the homodimer. High-sensitivity differential scanning calorimetry (HS-DSC) shows that the unfolding transition of pOBP is reversible at neutral pH. It is characterized by the transition temperature of 69.23 degrees C and an enthalpy of 391.1 kJ/mol per monomer. The transition heat capacity curve of pOBP is well-approximated by the two-state model on the level of subunit, indicating that the two monomers behave independently. Isothermal titration calorimetry (ITC) shows that at physiological pH pOBP binds 2-isobutyl-3-methoxypyrazine (IBMP) and 3,7-dimethyloctan-1-ol (DMO) with association constants of 3.19 x 10(6) and 4.94 x 10(6) M(-)(1) and enthalpies of -97.2 and -87.8 kJ/mol, respectively. The binding stoichiometry of both ligands is nearly one molecule of ligand per homodimer of pOBP. The interaction of pOBP with both ligands is enthalpically driven with an unfavorable change of entropy. The binding affinity of pOBP with IBMP does not change significantly at acidic pH, while the binding stoichiometry is nearly halved. According to HS-DSC data, the interaction with IBMP and DMO leads to a substantial stabilization of the pOBP folded structure, which is manifested by the increase in the unfolding temperature and enthalpy. The calorimetric data allow us to conclude that the mechanism of binding of the studied odorants to pOBP is not dominated by a hydrophobic effect related to any change in the hydration state of protein and ligand groups but, most likely, is driven by polar and van der Waals interactions.     

G蛋白亚单位基因家族研究进展

G蛋白由α、β、γ三个亚单位组成异源三聚体。目前已发现16个α、6个β和12个γ基因。G蛋白亚单位基因家族相当保守并且原始,几乎所有G蛋白基因外显子-内含子连接均遵从GT-AG规则,并且各亚单位基因编码区内含子结构和位置显示出很高的保守性。多数G蛋白基因具有持家基因的特点。G蛋白基因在基因组中的分布存在着丛集的倾向,有5对α基因呈二联串连排列。     

Energy-optimized structure of antifreeze protein and its binding mechanism.

A combination of Monte Carlo simulated annealing and energy minimization was utilized to determine the conformation of the antifreeze protein from the fish winter flounder. It was found from the energy-optimized structure that the hydroxyl groups of its four threonine residues, i.e. Thr2, Thr13, Thr24, Thr35, are aligned on almost the same line parallel to the helix axis and separated successively by 16.1, 16.0 and 16.2 A, respectively, very close to the 16.6 A repeat spacing along [0112] in ice. Based on such a space match, a zipper-like model is proposed to elucidate the binding mechanism of the antifreeze protein to ice crystals. According to the current model, the antifreeze protein may bind to an ice nucleation structure in a zipper-like fashion through hydrogen bonding of the hydroxyl groups of these four Thr residues to the oxygen atoms along the [0112] direction in ice lattice, subsequently stopping or retarding the growth of ice pyramidal planes so as to depress the freeze point. The calculated results and the binding mechanism thus derived accord with recent experimental observations. The mechanistic implications derived from such a special antifreeze molecule might be generally applied to elucidate the structure-function relationship of other antifreeze proteins with the following two common features: (1) recurrence of a Thr residue (or any other polar amino acid residue whose side-chain can form a hydrogen bond with water) in an 11-amino-acid period along the sequence concerned; and (2) a high percentage of Ala residue component therein. Further experiments are suggested to test the ice binding model.     

Crystal structure and binding properties of the Serratia marcescens chitin-binding protein CBP21

Chitin proteins are commonly found in bacteria that utilize chitin as a source of energy. CBP21 is a chitin-binding protein from Serratia marcescens, a Gram-negative soil bacterium capable of efficient chitin degradation. When grown on chitin, S. marcescens secretes large amounts of CBP21, along with chitin-degrading enzymes. In an attempt to understand the molecular mechanism of CBP21 action, we have determined its crystal structure at 1.55 angstroms resolution. This is the first structure to be solved of a family 33 carbohydrate-binding module. The structure reveals a "budded" fibronectin type III fold consisting of two beta-sheets, arranged as a beta-sheet sandwich, with a 65-residue "bud" consisting of three short helices, located between beta-strands 1 and 2. Remarkably, conserved aromatic residues that have been suggested previously to play a role in chitin binding were mainly found in the interior of the protein, seemingly incapable of interacting with chitin, whereas the structure revealed a surface patch of highly conserved, mainly hydrophilic residues. The roles of six of these conserved surface-exposed residues (Tyr-54, Glu-55, Glu-60, His-114, Asp-182, and Asn-185) were probed by site-directed mutagenesis and subsequent binding studies. All single point mutations lowered the affinity of CBP21 for beta-chitin, as shown by 3-8-fold increases in the apparent binding constant. Thus, binding of CBP21 to chitin seems to be mediated primarily by conserved, solvent-exposed, polar side chains.     

Gene for an immunoglobulin-binding protein from a group G streptococcus.

The gene (spg) for an immunoglobulin G (IgG)-binding protein from a Streptococcus clinical isolate of Lancefield group G was cloned and expressed in Escherichia coli. The complete nucleotide sequence of the gene and 5'-flanking sequences was determined. The DNA sequence includes an open reading frame which encodes a hypothetical protein of 448 amino acid residues (Mr = 47,595). The 5' end of this open reading frame encodes a sequence resembling a typical secretion signal sequence, and the remainder of the encoded protein has features reminiscent of staphylococcal protein A and of streptococcal M6 protein, including repeated sequences and a similar C-terminal structure. Aside from this C-terminal structure, the encoded protein has little direct amino acid sequence homology to either protein A or M6 protein. In E. coli, the cloned gene directs the synthesis of a protein which binds to immunoglobulins, including rabbit immunoglobulin, goat IgG, and human IgG3(lambda). Its binding properties are similar to those of the protein G described by Bj?rck and Kronvall (L. Bj?rck and G. Kronvall, J. Immunol. 133:969-974, 1984), a type III Fc receptor from a group G streptococcus.     

Crystal structure and immunoglobulin G binding properties of the human major histocompatibility complex-related Fc receptor(,)

The neonatal Fc receptor (FcRn) performs two distinct but related functions: transport of maternal immunoglobulin G (IgG) to pre- or neonatal mammals, thus providing passive immunity, and protection of IgG from normal serum protein catabolism. FcRn is related to class I MHC proteins but lacks a functional peptide binding groove. The crystal structure of human FcRn has been determined at 2.7 A resolution and compared to the previously described structure of rat FcRn [Burmeister et al. (1994) Nature 372, 336-343] and to the structures of MHC and MHC-related proteins. Human FcRn is structurally similar to the rat receptor but does not form receptor dimers in the crystals as observed in crystals of rat FcRn. The interaction between human FcRn and IgG was characterized by determining the binding stoichiometry using equilibrium gel filtration and by deriving binding affinities for the different human IgG subclasses using a surface plasmon resonance assay. Like rat and mouse FcRn, human FcRn interacts with IgG with a 2:1 receptor:ligand stoichiometry. The binding of human FcRn to the four human IgG subclasses shows subclass and allotype variations but no clear subclass affinity differences that correlate with serum half-lives. The structure of human FcRn and studies of its ligand binding are relevant to current efforts to use FcRn-mediated regulation of IgG half-life in serum to increase the lifetimes of antibody-based therapeutics.     

Retinoblastoma protein binding properties are dependent on 4 cysteine residues in the protein binding pocket.

The retinoblastoma gene product (pRB) participates in regulating mammalian cell replication. The mechanism responsible for pRB's growth regulatory activity is uncertain. However, pRB is known to bind viral transforming proteins including the papilloma virus E7 protein, cellular proteins, and DNA. pRB contains a critical domain termed the "binding pocket" which is required for binding activities. This binding pocket contains 8 cysteine residues. A naturally occurring mutation affecting one of these cysteines is known to eliminate pRB's protein and DNA binding activities. To investigate the cysteine residues in pRB's binding pocket, each residue was mutated to alanine, phenylalanine, or serine. These mutant genes were used to prepare pRBs harboring specific amino acid substitutions. Individual mutations at positions 407, 553, 666, and 706 depressed pRB binding to E7 protein, DNA, and a conformation-specific anti-pRB antibody, XZ133. Combinations of these inhibitory mutations exhibited additive inhibitory effects on pRB's binding properties. Mutations at positions 438, 489, 590, 712, and 853 did not affect pRB binding to E7 protein, DNA, or the XZ133 antibody. Combination of these five neutral mutations yielded a pRB species with full E7 protein, DNA, and XZ133 binding activities. These studies indicate that the cysteine residues at positions 407, 553, 666, and 706 contribute to the E7 protein and DNA binding properties of pRB and appear to do so by maintaining pRB's normal conformation.     

Interphotoreceptor retinoid-binding protein. Gene characterization, protein repeat structure, and its evolution

The gene for bovine interphotoreceptor retinoid-binding protein (IRBP) has been cloned, and its nucleotide sequence has been determined. The IRBP gene is about 11.6 kilobase pairs (kb) and contains four exons and three introns. It transcribed into a large mRNA of approximately 6.4 kb and translated into a large protein of 145,000 daltons. To prove the identity of the genomic clone, we determined the protein sequence of several tryptic and cyanogen bromide fragments of purified bovine IRBP protein and localized them in the protein predicted from its nucleotide sequence. There is a 4-fold repeat structure in the protein sequence with 30-40% sequence identity and many conservative substitutions between any two of the four protein repeats. The third and fourth repeats are the most similar pair. All three of the introns in the IRBP gene fall in the fourth protein repeat. Two of the exons, the first and the fourth, are large, 3173 and 2447 bases, respectively. The introns are each about 1.5-2.2 kb long. The human IRBP gene has a sequence that is similar to one of the introns from the bovine gene. The unexpected gene structure and protein repeat structure in the bovine gene lead us to propose a model for the evolution of the IRBP gene.     

Rapid evolution of peptide and protein binding properties in vitro.

A significant bottleneck in protein engineering arises from the problem of identifying particular molecules with new functions from a potentially enormous range of peptide or protein variants. Two areas of emerging technology, phage display and multiple peptide synthesis, provide new means of screening huge libraries in vitro for novel binding properties. This review is also published in Current Opinion in Structural Biology 1992, 2:597-604.