Selective ligand recognition by a diversity-generating retroelement variable protein

Diversity-generating retroelements (DGRs) recognize novel ligands through massive protein sequence variation, a property shared uniquely with the adaptive immune response. Little is known about how recognition is achieved by DGR variable proteins. Here, we present the structure of the Bordetella bacteriophage DGR variable protein major tropism determinant (Mtd) bound to the receptor pertactin, revealing remarkable adaptability in the static binding sites of Mtd. Despite large dissimilarities in ligand binding mode, principles underlying selective recognition were strikingly conserved between Mtd and immunoreceptors. Central to this was the differential amplification of binding strengths by avidity (i.e., multivalency), which not only relaxed the demand for optimal complementarity between Mtd and pertactin but also enhanced distinctions among binding events to provide selectivity. A quantitatively similar balance between complementarity and avidity was observed for Bordetella bacteriophage DGR as occurs in the immune system, suggesting that variable repertoires operate under a narrow set of conditions to recognize novel ligands.     

Selective Ligand Recognition by a Diversity-Generating Retroelement Variable Protein

Diversity-generating retroelements (DGRs) recognize novel ligands through massive protein sequence variation, a property shared uniquely with the adaptive immune response. Little is known about how recognition is achieved by DGR variable proteins. Here, we present the structure of the Bordetella bacteriophage DGR variable protein major tropism determinant (Mtd) bound to the receptor pertactin, revealing remarkable adaptability in the static binding sites of Mtd. Despite large dissimilarities in ligand binding mode, principles underlying selective recognition were strikingly conserved between Mtd and immunoreceptors. Central to this was the differential amplification of binding strengths by avidity (i.e., multivalency), which not only relaxed the demand for optimal complementarity between Mtd and pertactin but also enhanced distinctions among binding events to provide selectivity. A quantitatively similar balance between complementarity and avidity was observed for Bordetella bacteriophage DGR as occurs in the immune system, suggesting that variable repertoires operate under a narrow set of conditions to recognize novel ligands.     

Revealing the set of mutually correlated positions for the protein families of immunoglobulin fold

In this study, I explain the observation that a rather limited number of residues (about 10) establishes the immunoglobulin fold for the sequences of about 100 residues. Immunoglobulin fold proteins (IgF) comprise SCOP protein superfamilies with rather different functions and with less than 10% sequence identity; their alignment can be accomplished only taking into account the 3D structure. Therefore, I believe that discovering the additional common features of the sequences is necessary to explain the existence of a common fold for these SCOP superfamilies. We propose a method for analysis of pair-wise interconnections between residues of the multiple sequence alignment which helps us to reveal the set of mutually correlated positions, inherent to almost every superfamily of this protein fold. Hence, the set of constant positions (comprising the hydrophobic common core) and the set of variable but mutually correlated ones can serve as a basis of having the common 3D structure for rather distinct protein sequences.     

Expression of an exogenous peptide epitope genetically engineered in the variable domain of an immunoglobulin: implications for antibody and peptide folding.

Immunoglobulins bind antigens and express individual antigenic specificities mainly through residues located in hypervariable loops of their N-terminal domains. Hypervariable loops are kept in place by a molecular scaffold organized in a sandwich-like structure with two beta-sheets stabilized by a disulfide bridge (the immunoglobulin fold). This structural feature, together with the possibility of obtaining high level expression, extracellular secretion, easy purification and stability of the protein product, render immunoglobulin an ideal 'molecular vehicle' for the expression of exogenous peptides. Here we report on the engineering of an immunoglobulin expressing an exogenous epitope, the repetitive tetrapeptide Asn-Ala-Asn-Pro (NANP)3. By recombinant DNA techniques, we inserted three copies of the tetrapeptide (NANP)3 in the third hypervariable loop (D region) of an immunoglobulin heavy chain variable domain. We show that the engineered antibody was properly assembled and secreted. A panel of polyclonal and monoclonal antibodies, including anti-synthetic peptides and anti-(NANP)n antibodies, were used to study the molecular configuration of the engineered domain's surface. The results indicate that (i) the exogenous sequence did not appreciably alter the overall fold of the variable domain; and (ii) the inserted epitope folded with a configuration immunologically similar to the one assumed in the native protein, suggesting that short- and medium- rather than long-range interactions stabilized the structure of the (NANP)3 peptide in the folded protein. We propose this system for the expression of peptidic sequences, and their structural and functional analysis.     

Immunoglobulin fold and tandem repeat structures in proteoglycan N-terminal domains and link protein

Detailed primary sequence and secondary structure analyses are reported for the hyaluronate binding region (G1 domain) and link protein of proteoglycan aggregates. These are based on six full or partial sequences from the chicken, pig, human, rat and bovine proteins. Determinations of a full pig and a partial human link protein sequence are reported in the Appendix. Five sequences at the N terminus in both proteins were compared with the structures of 11 variable immunoglobulin (Ig) fold domains for which crystal structures are available. Despite only modest sequence homology, a clear alignment could be proposed. Analysis of this shows that the equivalents of the first and second hypervariable segments are now significantly longer, and both proteins have N-terminal extensions that are up to 23 residues in length. Secondary structure predictions showed that these sequences could be identified with available crystal structures for the variable Ig fold. However the hydrophobic residues involved in interactions between the light and heavy chains in Igs are replaced by hydrophilic charged groups in both proteins. These results imply that both proteins are members of the Ig superfamily, but exhibit structural differences distinct from other members of this superfamily for which crystal structures are known. The proteoglycan tandem repeat (PTR) is a repeat of 99 residues that is found twice in the amino acid sequence of link protein and the proteoglycan G1 domain adjacent to the Ig fold, and also twice in the proteoglycan G2 domain. A total of 16 PTRs was available for analysis. Compositional analyses show that these are positively charged if these originate from link protein, and negatively charged if from the G1 or G2 domains. The 16 Robson secondary structure predictions for the PTRs were averaged to improve the statistics of the prediction, and checked by comparison with Chou-Fasman calculations. A strong alpha-helix prediction was found at residues 13 to 25, and several beta-strands were predicted. The overall content is 18% alpha-helix and 28% beta-sheet, with 44% of the remaining sequence being predicted as turns. These analyses show that both the proteoglycan G1 domain and link protein are constructed from two distinct globular components, which may provide the two functional roles of these proteins in proteoglycan aggregation.     

Conservation of the C-type lectin fold for accommodating massive sequence variation in archaeal diversity-generating retroelements

Background

Diversity-generating retroelements (DGRs) provide organisms with a unique means for adaptation to a dynamic environment through massive protein sequence variation. The potential scope of this variation exceeds that of the vertebrate adaptive immune system. DGRs were known to exist only in viruses and bacteria until their recent discovery in archaea belonging to the ‘microbial dark matter’, specifically in organisms closely related to Nanoarchaeota. However, Nanoarchaeota DGR variable proteins were unassignable to known protein folds and apparently unrelated to characterized DGR variable proteins.

Results

To address the issue of how Nanoarchaeota DGR variable proteins accommodate massive sequence variation, we determined the 2.52 Å resolution limit crystal structure of one such protein, AvpA, which revealed a C-type lectin (CLec)-fold that organizes a putative ligand-binding site that is capable of accommodating 10¹³ sequences. This fold is surprisingly reminiscent of the CLec-folds of viral and bacterial DGR variable protein, but differs sufficiently to define a new CLec-fold subclass, which is consistent with early divergence between bacterial and archaeal DGRs. The structure also enabled identification of a group of AvpA-like proteins in multiple putative DGRs from uncultivated archaea. These variable proteins may aid Nanoarchaeota and these uncultivated archaea in symbiotic relationships.

Conclusions

Our results have uncovered the widespread conservation of the CLec-fold in viruses, bacteria, and archaea for accommodating massive sequence variation. In addition, to our knowledge, this is the first report of an archaeal CLec-fold protein.

     

Combining sites of bacterial fimbriae

The few known crystal structures of receptor-binding domains of fimbrial tip adhesins, FimH, PapGII, and F17G, tell us that each of these structures is unique and surprising. Despite little to no sequence identity, common to them all is their variable immunoglobulin (Ig)-fold. Nevertheless, their glycan-binding sites have evolved in different locations onto this similar scaffold, and with distinct, highly specific binding properties. Difficult to capture is the often dominant role played by the fimbrial shaft in host cell recognition and biofilm formation. The major pilin FaeG, building up the shaft of F4 fimbriae, also harbors the carbohydrate receptor-binding property and has thereto an enlarged Ig-domain, with the insertion of two beta-strands and two alpha-helices. Bordetella and CFA/I fimbriae combine a tip adhesin with major subunit adhesins. Still other fimbriae incorporate a specialized invasin at the very tip of polyadhesive fibers for uptake of bacteria in cells of the immune system and host epithelia. Finally, glycan recognition by fimbrial adhesins has often been found to coincide with the binding of cell-surface integrins and components of the extracellular matrix, such as collagen IV and laminin.     

Structure of human POFUT2: insights into thrombospondin type 1 repeat fold and O-fucosylation

Protein O-fucosylation is a post-translational modification found on serine/threonine residues of thrombospondin type 1 repeats (TSR). The fucose transfer is catalysed by the enzyme protein O-fucosyltransferase 2 (POFUT2) and 440 human proteins contain the TSR consensus sequence for POFUT2-dependent fucosylation. To better understand O-fucosylation on TSR, we carried out a structural and functional analysis of human POFUT2 and its TSR substrate. Crystal structures of POFUT2 reveal a variation of the classical GT-B fold and identify sugar donor and TSR acceptor binding sites. Structural findings are correlated with steady-state kinetic measurements of wild-type and mutant POFUT2 and TSR and give insight into the catalytic mechanism and substrate specificity. By using an artificial mini-TSR substrate, we show that specificity is not primarily encoded in the TSR protein sequence but rather in the unusual 3D structure of a small part of the TSR. Our findings uncover that recognition of distinct conserved 3D fold motifs can be used as a mechanism to achieve substrate specificity by enzymes modifying completely folded proteins of very wide sequence diversity and biological function.     

Conservation of folding and stability within a protein family: the tyrosine corner as an evolutionary cul-de-sac

What are the selective pressures on protein sequences during evolution? Amino acid residues may be highly conserved for functional or structural (stability) reasons. Theoretical studies have proposed that residues involved in the folding nucleus may also be highly conserved. To test this we are using an experimental "fold approach" to the study of protein folding. This compares the folding and stability of a number of proteins that share the same fold, but have no common amino acid sequence or biological activity. The fold selected for this study is the immunoglobulin-like beta-sandwich fold, which is a fold that has no specifically conserved function. Four model proteins are used from two distinct superfamilies that share the immunoglobulin-like fold, the fibronectin type III and immunoglobulin superfamilies. Here, the fold approach and protein engineering are used to question the role of a highly conserved tyrosine in the "tyrosine corner" motif that is found ubiquitously and exclusively in Greek key proteins. In the four model beta-sandwich proteins characterised here, the tyrosine is the only residue that is absolutely conserved at equivalent sites. By mutating this position to phenylalanine, we show that the tyrosine hydroxyl is not required to nucleate folding in the immunoglobulin superfamily, whereas it is involved to some extent in early structure formation in the fibronectin type III superfamily. The tyrosine corner is important for stability, mutation to phenylalanine costs between 1.5 and 3 kcal mol(-1). We propose that the high level of conservation of the tyrosine is related to the structural restraints of the loop connecting the beta-sheets, representing an evolutionary "cul-de-sac".     

CCR2 and CCR5 receptor-binding properties of herpesvirus-8 vMIP-II based on sequence analysis and its solution structure.

Human herpesvirus-8 (HHV-8) is the infectious agent responsible for Kaposi's sarcoma and encodes a protein, macrophage inflammatory protein-II (vMIP-II), which shows sequence similarity to the human CC chemokines. vMIP-II has broad receptor specificity that crosses chemokine receptor subfamilies, and inhibits HIV-1 viral entry mediated by numerous chemokine receptors. In this study, the solution structure of chemically synthesized vMIP-II was determined by nuclear magnetic resonance. The protein is a monomer and possesses the chemokine fold consisting of a flexible N-terminus, three antiparallel beta strands, and a C-terminal alpha helix. Except for the N-terminal residues (residues 1-13) and the last two C-terminal residues (residues 73-74), the structure of vMIP-II is well-defined, exhibiting average rmsd of 0.35 and 0.90 A for the backbone heavy atoms and all heavy atoms of residues 14-72, respectively. Taking into account the sequence differences between the various CC chemokines and comparing their three-dimensional structures allows us to implicate residues that influence the quaternary structure and receptor binding and activation of these proteins in solution. The analysis of the sequence and three-dimensional structure of vMIP-II indicates the presence of epitopes involved in binding two receptors CCR2 and CCR5. We propose that vMIP-II was initially specific for CCR5 and acquired receptor-binding properties to CCR2 and other chemokine receptors.     

Target site recognition by a diversity-generating retroelement

Diversity-generating retroelements (DGRs) are in vivo sequence diversification machines that are widely distributed in bacterial, phage, and plasmid genomes. They function to introduce vast amounts of targeted diversity into protein-encoding DNA sequences via mutagenic homing. Adenine residues are converted to random nucleotides in a retrotransposition process from a donor template repeat (TR) to a recipient variable repeat (VR). Using the Bordetella bacteriophage BPP-1 element as a prototype, we have characterized requirements for DGR target site function. Although sequences upstream of VR are dispensable, a 24 bp sequence immediately downstream of VR, which contains short inverted repeats, is required for efficient retrohoming. The inverted repeats form a hairpin or cruciform structure and mutational analysis demonstrated that, while the structure of the stem is important, its sequence can vary. In contrast, the loop has a sequence-dependent function. Structure-specific nuclease digestion confirmed the existence of a DNA hairpin/cruciform, and marker coconversion assays demonstrated that it influences the efficiency, but not the site of cDNA integration. Comparisons with other phage DGRs suggested that similar structures are a conserved feature of target sequences. Using a kanamycin resistance determinant as a reporter, we found that transplantation of the IMH and hairpin/cruciform-forming region was sufficient to target the DGR diversification machinery to a heterologous gene. In addition to furthering our understanding of DGR retrohoming, our results suggest that DGRs may provide unique tools for directed protein evolution via in vivo DNA diversification.     

The immunoglobulin fold family: sequence analysis and 3D structure comparisons.

Fifty-two 3D structures of Ig-like domains covering the immunoglobulin fold family (IgFF) were compared and classified according to the conservation of their secondary structures. Members of the IgFF are distantly related proteins or evolutionarily unrelated proteins with a similar fold, the Ig fold. In this paper, a multiple structural alignment of the conserved common core is described and the correlation between corresponding sequences is discussed. While the members of the IgFF exhibit wide heterogeneity in terms of tissue and species distribution or functional implications, the 3D structures of these domains are far more conserved than their sequences. We define topologically equivalent residues in the Ig-like domains, describe the hydrophobic common cores and discuss the presence of additional strands. The disulfide bridges, not necessary for the stability of the Ig fold, may have an effect on the compactness of the domains. Based upon sequence and structure analysis, we propose the introduction of two new subtypes (C3 and C4) to the previous classifications, in addition to a new global structural classification. The very low mean sequence identity between subgroups of the IgFF suggests the occurrence of both divergent and convergent evolutionary processes, explaining the wide diversity of the superfamily. Finally, this review suggest that hydrophobic residues constituting the common hydrophobic cores are important clues to explain how highly divergent sequences can adopt a similar fold.     

Molecular modeling of the multidomain structures of the proteoglycan binding region and the link protein of cartilage by neutron and synchrotron X-ray scattering

The interaction of proteoglycan monomers with hyaluronate in cartilage is mediated by a globular binding region at the N-terminus of the proteoglycan monomer; this interaction is stabilized by link protein. Sequences show that both the binding region (27% carbohydrate) and the link protein (6% carbohydrate) contain an immunoglobulin (Ig) fold domain and two proteoglycan tandem repeat (PTR) domains. Both proteins were investigated by neutron and synchrotron X-ray solution scattering, in which nonspecific aggregate formation was reduced by the use of citraconylation to modify surface lysine residues. The neutron and X-ray radius of gyration RG of native and citraconylated binding region is 5.1 nm, and the cross-sectional RG (RXS) is 1.9-2.0 nm. No neutron contrast dependence of the RG values was observed; however, a large contrast dependence was seen for the RXS values which is attributed to the high carbohydrate content of the binding region. The neutron RG for citraconylated link protein is 2.9 nm, its RXS is 0.8 nm, and these data are also independent of the neutron contrast. The scattering curves of binding region and link protein were modeled using small spheres. Both protein structures were defined initially by the representation of one domain by a crystal structure for a variable Ig fold and a fixed volume for the two PTR domains calculated from sequence data. The final models showed that the different dimensions and neutron contrast properties of binding region compared to link protein could be attributed to an extended glycosylated C-terminal peptide with extended carbohydrate structures in the binding region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunoglobulin heavy chain gene organization and complexity in the skate, Raja erinacea

Immunoglobulin heavy chain genes from Raja erinacea have been isolated by cross hybridization with probes derived from the immunoglobulin genes of Heterodontus francisci (horned shark), a representative of a different elasmobranch order. Heavy chain variable (VH), diversity (DH) and joining (JH) segments are linked closely to constant region (CH) exons, as has been described in another elasmobranch. The nucleotide sequence homology of VH gene segments within Raja and between different elasmobranch species is high, suggesting that members of this phylogenetic subclass may share one VH family. The organization of immunoglobulin genes segments is diverse; both VD-J and VD-DJ joined genes have been detected in the genome of non-lymphoid cells. JH segment sequence diversity is high, in contrast to that seen in a related elasmobranch. These data suggest that the clustered V-D-J-C form of immunoglobulin heavy chain organization, including germline joined components, may occur in all subclasses of elasmobranchs. While variation in VH gene structure is limited, gene organization appears to be diverse.     

Enantioselective high-performance liquid chromatography determination of methadone enantiomers and its major metabolite in human biological fluids using a new derivatized cyclodextrin-bonded phase

The simultaneous determination of methadone (Mtd) enantiomers and its major metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), in human urine and serum by enantioselective HPLC using a new Cyclobond I-2000 RSP column is described. After alkaline extraction from urine or serum with estazolam as an internal standard, Mtd enantiomers and its metabolite (EDDP) are separated on the previous column with reversed-mobile phase and detected at 210 nm. Peak resolutions are about 2.0 for Mtd enantiomers. The relative standard deviations (R.S.D.) of Mtd and EDDP standards are between 0.5 and 4.5%. Most drugs of abuse are shown not to interfere with this technique. The method has been applied to study levels of each Mtd enantiomer and of its racemic metabolite in urine and serum of patients under maintenance treatment for opiate dependence. In urine, R-(−)-Mtd levels are always higher (about 2±0.5-fold_ than those of S-(+)-Mtd and in most cases, metabolite concentrations are greater than those of global Mtd enantiomers. However, the R-(−) enantiomer levels of residual drug in serum of some patients were lower than those of its antipode. This method is suitable for pharmacokinetic and toxicological studies of Mtd enantiomers and its major metabolite in biological fluids.     

Importance of secondary structural specificity determinants in protein folding: insertion of a native beta-sheet sequence into an alpha-helical coiled-coil

To examine how a short secondary structural element derived from a native protein folds when in a different protein environment, we inserted an 11-residue beta-sheet segment (cassette) from human immunoglobulin fold, Fab new, into an alpha-helical coiled-coil host protein (cassette holder). This de novo design protein model, the structural cassette mutagenesis (SCM) model, allows us to study protein folding principles involving both short- and long-range interactions that affect secondary structure stability and conformation. In this study, we address whether the insertion of this beta-sheet cassette into the alpha-helical coiled-coil protein would result in conformational change nucleated by the long-range tertiary stabilization of the coiled-coil, therefore overriding the local propensity of the cassette to form beta-sheet, observed in its native immunoglobulin fold. The results showed that not only did the nucleating helices of the coiled-coil on either end of the cassette fail to nucleate the beta-sheet cassette to fold with an alpha-helical conformation, but also the entire chimeric protein became a random coil. We identified two determinants in this cassette that prevented coiled-coil formation: (1) a tandem dipeptide NN motif at the N-terminal of the beta-sheet cassette, and (2) the hydrophilic Ser residue, which would be buried in the hydrophobic core if the coiled-coil structure were to fold. By amino acid substitution of these helix disruptive residues, that is, either the replacement of the NN motif with high helical propensity Ala residues or the substitution of Ser with Leu to enhance hydrophobicity, we were able to convert the random coil chimeric protein into a fully folded alpha-helical coiled-coil. We hypothesized that this NN motif is a "secondary structural specificity determinant" which is very selective for one type of secondary structure and may prevent neighboring residues from adopting an alternate protein fold. These sequences with secondary structural specificity determinants have very strong local propensity to fold into a specific secondary structure and may affect overall protein folding by acting as a folding initiation site.     

Conformation of amyloid fibrils of beta2-microglobulin probed by tryptophan mutagenesis

Beta2-microglobulin (beta2-m), a protein responsible for dialysis-related amyloidosis, adopts an immunoglobulin domain fold in its native state. Although beta2-m has Trp residues at positions 60 and 95, both are located near the surface of the domain. Hence, beta2-m does not have a conserved Trp common to other immunoglobulin domains, which is buried in close proximity to the disulfide bond. To study the structure of amyloid fibrils in relation to their native fold, we prepared a series of Trp mutants. Trp60 and Trp95 were both replaced with Phe, and a single Trp was introduced at various positions. Among various mutants, W39-beta2-m, in which a Trp was introduced at the position corresponding to the conserved Trp, exhibited a remarkable quenching of fluorescence in the native state, as observed for other immunoglobulin domains. An x-ray structural analysis revealed that W39-beta2-m assumes the native fold with Trp39 located in the vicinity of the disulfide bond. Comparison of the fluorescence spectra of various mutants for the native and fibrillar forms indicated that, while the Trp residues introduced in the middle of the beta2-m sequence tend to be buried in the fibrils, those located in the C-terminal region are more exposed. In addition, the fluorescence spectra of fibrils prepared at pH 2.5 and 7.0 revealed a large difference in the fluorescence intensity for W60-beta2-m, implying a major structural difference between them.     

Aplysia attractin: biophysical characterization and modeling of a water-borne pheromone.

Attractin, a 58-residue protein secreted by the mollusk Aplysia californica, stimulates sexually mature animals to approach egg cordons. Attractin from five different Aplysia species are approximately 40% identical in sequence. Recombinant attractin, expressed in insect cells and purified by reverse-phase high-performance liquid chromatography (RP-HPLC), is active in a bioassay using A. brasiliana; its circular dichroism (CD) spectrum indicates a predominantly alpha-helical structure. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) characterization of proteolytic fragments identified disulfide bonds between the six conserved cysteines (I-VI, II-V, III-IV, where the Roman numeral indicates the order of occurrence in the primary sequence). Attractin has no significant similarity to any other sequence in the database. The protozoan Euplotes pheromones were selected by fold recognition as possible templates. These diverse proteins have three alpha-helices, with six cysteine residues disulfide-bonded in a different pattern from attractin. Model structures with good stereochemical parameters were prepared using the EXDIS/DIAMOD/FANTOM program suite and constraints based on sequence alignments with the Euplotes templates and the attractin disulfide bonds. A potential receptor-binding site is suggested based on these data. Future structural characterization of attractin will be needed to confirm these models.