Common features of the conformations of antigen-binding loops in immunoglobulins and application to modeling loop conformations.

Using database screening techniques we have examined the relationship between antigen-binding loops in immunoglobulins, and regions of similar conformation in other protein families. The conformations of most antigen-binding loops are not unique to immunoglobulins. But in many cases, the geometrical relationship between the loop and the peptides flanking it differs between the immunoglobulins and other structures with the same loop. We assess model building by data base screening, compared with that based on canonical structures.     

Structural determinants of the conformations of medium-sized loops in proteins

Loops are integral components of protein structures, providing links between elements of secondary structure, and in many cases contributing to catalytic and binding sites. The conformations of short loops are now understood to depend primarily on their amino acid sequences. In contrast, the structural determinants of longer loops involve hydrogen-bonding and packing interactions within the loop and with other parts of the protein. By searching solved protein structures for regions similar in main chain conformation to the antigen-binding loops in immunoglobulins, we identified medium-sized loops of similar structure in unrelated proteins, and compared the determinants of their conformations. For loops that form compact substructures the major determinant of the conformation is the formation of hydrogen bonds to inward-pointing main chain atoms. For loops that have more extended conformations, the major determinant of their structure is the packing of a particular residue or residues against the rest of the protein. The following picture emerges: Medium-sized loops of similar conformation are stabilized by similar interactions. The groups that interact with the loop have very similar spatial dispositions with respect to the loop. However, the residues that provide these interactions may arise from dissimilar parts of the protein: The conformation of the loop requires certain interactions that the protein may provide in a variety of ways.     

Intrinsic flexibility of snRNA hairpin loops facilitates protein binding

Stem–loop II of U1 snRNA and Stem–loop IV of U2 snRNA typically have 10 or 11 nucleotides in their loops. The fluorescent nucleobase 2-aminopurine was used as a substitute for the adenines in each loop to probe the local and global structures and dynamics of these unusually long loops. Using steady-state and time-resolved fluorescence, we find that, while the bases in the loops are stacked, they are able to undergo significant local motion on the picosecond/nanosecond timescale. In addition, the loops have a global conformational change at low temperatures that occurs on the microsecond timescale, as determined using laser T-jump experiments. Nucleobase and loop motions are present at temperatures far below the melting temperature of the hairpin stem, which may facilitate the conformational change required for specific protein binding to these RNA loops.     

Destabilizing loop swaps in the CDRs of an immunoglobulin VL domain.

It is generally believed that loop regions in globular proteins, and particularly hypervariable loops in immunoglobulins, can accommodate a wide variety of sequence changes without jeopardizing protein structure or stability. We show here, however, that novel sequences introduced within complementarity determining regions (CDRs) 1 and 3 of the immunoglobulin variable domain REI VL can significantly diminish the stability of the native state of this protein. Besides their implications for the general role of loops in the stability of globular proteins, these results suggest previously unrecognized stability constraints on the variability of CDRs that may impact efforts to engineer new and improved activities into antibodies.     

New computational method for prediction of interacting protein loop regions

Flexible loop regions of proteins play a crucial role in many biological functions such as protein–ligand recognition, enzymatic catalysis, and protein–protein association. To date, most computational methods that predict the conformational states of loops only focus on individual loop regions. However, loop regions are often spatially in close proximity to one another and their mutual interactions stabilize their conformations. We have developed a new method, titled CorLps, capable of simultaneously predicting such interacting loop regions. First, an ensemble of individual loop conformations is generated for each loop region. The members of the individual ensembles are combined and are accepted or rejected based on a steric clash filter. After a subsequent side‐chain optimization step, the resulting conformations of the interacting loops are ranked by the statistical scoring function DFIRE that originated from protein structure prediction. Our results show that predicting interacting loops with CorLps is superior to sequential prediction of the two interacting loop regions, and our method is comparable in accuracy to single loop predictions. Furthermore, improved predictive accuracy of the top‐ranked solution is achieved for 12‐residue length loop regions by diversifying the initial pool of individual loop conformations using a quality threshold clustering algorithm. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Epitope specificity of murine and human bactericidal antibodies against PorA P1.7,16 induced with experimental meningococcal group B vaccines

Synthetic peptides derived from the predicted loops 1 and 4 of meningococcal PorA, sero-subtype P1.7,16, were used to study the epitope specificity of murine and human PorA P1.7,16 bactericidal antibodies. The predicted loops 1 and 4 are surface exposed and carry in their apices the sero-subtype epitopes P1.7 (loop 1) or P1.16 (loop 4), respectively. Peptides were synthesized as mono- and multimeric peptides. Murine monoclonal and polyclonal antibodies were induced with meningococcal whole cell preparations. Polyclonal antibodies were evoked in volunteers after one immunization with 50 μg or 100 μg protein of a hexavalent meningococcal PorA vesicle vaccine. The induction of PorA antibodies was determined in ELISA using purified PorA P1.7,16. The epitope specificity of anti-PorA antibodies for both murine and human antibodies could be demonstrated by direct peptide ELISA using overlapping multimeric peptides almost spanning the entire loops 1 or 4 of the protein. The capacity of peptides to inhibit the bactericidal activity of murine and human antibodies was investigated using meningococcal strain H44/76 (B:15:P1.7,16) as a target strain. Bactericidal activities could be inhibited with both monomeric and multimeric peptides derived from epitopes P1.7 and P1.16.     

Modified method of obtaining washings of the large intestine for determining coproantibodies and immunoglobulins in the dynamics of acute dysentery]

The study of the washings obtained during rectoscopy from the mucous membrane of the large intestine with the use of a special attachment for a rectoscope revealed that this method had a number of advantages when compared with the study of coprofiltrates; in particular, this method allowed to obtain pure secretions of the mucous membrane free of fecal admixtures and to concentrate the gamma globulin fraction. The study of the dynamics of coproantibody secretion in acute dysentery revealed that coproantibodies were most frequently detected on the second week of the disease (reaching 85.7%), while immunoglobulins occurred in the protein fraction of the washings with almost the same frequency during the whole period of the disease.     

Ab initio structure prediction of the antibody hypervariable H3 loop

Antibodies have the capability of binding a wide range of antigens due to the diversity of the six loops constituting the complementarity determining region (CDR). Among the six loops, the H3 loop is the most diverse in structure, length, and sequence identity. Prediction of the three‐dimensional structures of antibodies, especially the CDR loops, is an important step in the computational design and engineering of novel antibodies for improved affinity and specificity. Although it has been demonstrated that the conformation of the five non‐H3 loops can be accurately predicted by comparing their sequences against databases of canonical loop conformations, no such connection has been established for H3 loops. In this work, we present the results for ab initio structure prediction of the H3 loop using conformational sampling and energy calculations with the program Prime on a dataset of 53 loops ranging in length from 4 to 22 residues. When the prediction is performed in the crystal environment and including symmetry mates, the median backbone root mean square deviation (RMSD) is 0.5 Å to the crystal structure, with 91% of cases having an RMSD of less than 2.0 Å. When the prediction is performed in a noncrystallographic environment, where the scaffold is constructed by swapping the H3 loops between homologous antibodies, 70% of cases have an RMSD below 2.0 Å. These results show promise for ab initio loop predictions applied to modeling of antibodies. © 2012 Wiley Periodicals, Inc.     

Impact of cofactor-binding loop mutations on thermotolerance and activity of E. coli transketolase

Improvement of thermostability in engineered enzymes can allow biocatalysis on substrates with poor aqueous solubility. Denaturation of the cofactor-binding loops of Escherichia coli transketolase (TK) was previously linked to the loss of enzyme activity under conditions of high pH or urea. Incubation at temperatures just below the thermal melting transition, above which the protein aggregates, was also found to anneal the enzyme to give an increased specific activity. The potential role of cofactor-binding loop instability in this process remained unclear. In this work, the two cofactor-binding loops (residues 185–192 and 382–392) were progressively mutated towards the equivalent sequence from the thermostable Thermus thermophilus TK and variants assessed for their impact on both thermostability and activity. Cofactor-binding loop 2 variants had detrimental effects on specific activity at elevated temperatures, whereas the H192P mutation in cofactor-binding loop 1 resulted in a two-fold improved stability to inactivation at elevated temperatures, and increased the critical onset temperature for aggregation. The specific activity of H192P was 3-fold and 19-fold higher than that for wild-type at 60 °C and 65 °C respectively, and also remained 2.7-4 fold higher after re-cooling from pre-incubations at either 55 °C or 60 °C for 1 h. Interestingly, H192P was also 2-times more active than wild-type TK at 25 °C. Optimal activity was achieved at 60 °C for H192P compared to 55 °C for wild type. These results show that cofactor-binding loop 1, plays a pivotal role in partial denaturation and aggregation at elevated temperatures. Furthermore, a single rigidifying mutation within this loop can significantly improve the enzyme specific activity, as well as the stability to thermal denaturation and aggregation, to give an increased temperature optimum for activity.     

A single-domain antibody fragment in complex with RNase A: non-canonical loop structures and nanomolar affinity using two CDR loops

BACKGROUND: Camelid serum contains a large fraction of functional heavy-chain antibodies - homodimers of heavy chains without light chains. The variable domains of these heavy-chain antibodies (VHH) have a long complementarity determining region 3 (CDR3) loop that compensates for the absence of the antigen-binding loops of the variable light chains (VL). In the case of the VHH fragment cAb-Lys3, part of the 24 amino acid long CDR3 loop protrudes from the antigen-binding surface and inserts into the active-site cleft of its antigen, rendering cAb-Lys3 a competitive enzyme inhibitor. RESULTS: A dromedary VHH with specificity for bovine RNase A, cAb-RN05, has a short CDR3 loop of 12 amino acids and is not a competitive enzyme inhibitor. The structure of the cAb-RN05-RNase A complex has been solved at 2.8 A. The VHH scaffold architecture is close to that of a human VH (variable heavy chain). The structure of the antigen-binding hypervariable 1 loop (H1) of both cAb-RN05 and cAb-Lys3 differ from the known canonical structures; in addition these H1 loops resemble each other. The CDR3 provides an antigen-binding surface and shields the face of the domain that interacts with VL in conventional antibodies. CONCLUSIONS: VHHs adopt the common immunoglobulin fold of variable domains, but the antigen-binding loops deviate from the predicted canonical structure. We define a new canonical structure for the H1 loop of immunoglobulins, with cAb-RN05 and cAb-Lys3 as reference structures. This new loop structure might also occur in human or mouse VH domains. Surprisingly, only two loops are involved in antigen recognition; the CDR2 does not participate. Nevertheless, the antigen binding occurs with nanomolar affinities because of a preferential usage of mainchain atoms for antigen interaction.     

Immobilization of Chloroperoxidase on Aminopropyl-Glass

Chloroperoxidase (CPO) purified from Caldariomyces fumago CMI 89362 was covalently bound to aminopropyl-glass by using a modification of an established method. Acid-washed glass was derivatized by using aminopropyltriethoxysilane, and the enzyme was ionically bound at low ionic strength. Further treatment with glutaraldehyde covalently linked the enzyme to the glass beads in an active form. No elution of bound activity from glass beads could be detected with a variety of washings. The loading of enzyme protein to the glass beads was highest, 100 mg of CPO per g of glass, at high reaction ratios of CPO to glass, but the specific activity of the immobilized enzyme was highest, 36% of theoretical, at low enzyme-to-carrier ratios. No differences in the properties of the soluble and immobilized enzymes could be detected by a number of criteria: their pH-activity and pH-stability profiles were similar, as were their thermal stabilities. After five uses, the immobilized enzyme retained full activity between pH 6.0 and 6.7.     

Sequence Structure of van der Waals Locks in Proteins

Abstract

Recent works has suggested that proteins in early evolution have gone through a stage of closed loop elements with a typical contour size of 25–35 residues. These closed loops are still the elementary protein units to these days, and can be used to spell out protein sequence/structure relationship through a relatively small number of protein prototypes. In this study we aimed to identify the sequences that are used to lock the loop ends to one another, and to show how an extensive dictionary of such locking pairs can be created using positional correlation data from a large proteome database, and structural data from PDB databases. Such a dictionary can be used in reconstructing the evolutionary pathway the modern proteins have gone through, and in identifying closed loop elements in modern proteins with yet unknown 3D structure.     

Simultaneous modeling of multiple loops in proteins.

The most reliable methods for predicting protein structure are by way of homologous extension, using structural information from a closely related protein, or by "threading" through a set of predefined protein folds ("inverse folding"). Both sets of methods provide a model for the core of the protein--the structurally conserved secondary structures. Due to the large variability both in sequence and size of the loops that connect these secondary structures, they generally cannot be modeled using these techniques. Loop-closure algorithms are aimed at predicting loop structures, given their end-to-end distance. Various such algorithms have been described, and all have been tested by predicting the structure of a single loop in a known protein. In this paper we propose a method, which is based on the bond-scaling-relaxation loop-closure algorithm, for simultaneously predicting the structures of multiple loops, and demonstrate that, for two spatially close loops, simultaneous closure invariably leads to more accurate predictions than sequential closure. The accuracy of the predictions obtained for pairs of loops in the size range of 5-7 residues each is comparable to that obtained by other methods, when predicting the structures of single loops: the RMS deviations from the native conformations of various test cases modeled are approximately 0.6-1.7 A for backbone atoms and 1.1-3.3 A for all-atoms.     

Humoral factors of local and systemic immunity in the multiple peroral administration of a corpuscular pertussis vaccine

Multiple oral immunization with pertussis corpuscular vaccine was shown to lead to the considerable stimulation of local and systemic humoral immunity. The data on the titers of specific and normal secretory antibodies, on the levels of IgA in washings from the oral cavity, the small intestine and the lungs, on the titers of agglutinins and hemagglutinins in the blood serum, as well as on the morpho-functional transformation of the mucous membrane and the associated lymphoid tissue in the digestive tract, are presented in their dynamics. Specific pertussis antibodies in high titers were detected in both intestinal and pulmonary washings. The multiple administration of the vaccine did not produce pathological changes in internal organs.     

Determination of the secondary structure of group II bulge loops using the fluorescent probe 2-aminopurine

Eleven RNA hairpins containing 2-aminopurine (2-AP) in either base-paired or single nucleotide bulge loop positions were optically melted in 1 M NaCl; and, the thermodynamic parameters ΔH°, ΔS°, ΔG°₃₇, and T_M for each hairpin were determined. Substitution of 2-AP for an A (adenosine) at a bulge position (where either the 2-AP or A is the bulge) in the stem of a hairpin, does not affect the stability of the hairpin. For group II bulge loops such as AA/U, where there is ambiguity as to which of the A residues is paired with the U, hairpins with 2-AP substituted for either the 5′ or 3′ position in the hairpin stem have similar stability. Fluorescent melts were performed to monitor the environment of the 2-AP. When the 2-AP was located distal to the hairpin loop on either the 5′ or 3′ side of the hairpin stem, the change in fluorescent intensity upon heating was indicative of an unpaired nucleotide. A database of phylogenetically determined RNA secondary structures was examined to explore the presence of naturally occurring bulge loops embedded within a hairpin stem. The distribution of bulge loops is discussed and related to the stability of hairpin structures.     

Classification of common functional loops of kinase super-families

A structural classification of loops has been obtained from a set of 141 protein structures classified as kinases. A total of 1813 loops was classified into 133 subclasses (9 betabeta(links), 15 betabeta(hairpins), 31 alpha-alpha, 46 alpha-beta and 32 beta-alpha). Functional information and specific features relating subclasses and function were included in the classification. Functional loops such as the P-loop (shared by different folds) or the Gly-rich-loop, among others, were classified into structural motifs. As a result, a common mechanism of catalysis and substrate binding was proved for most kinases. Additionally, the multiple-alignment of loop sequences made within each subclass was shown to be useful for comparative modeling of kinase loops. The classification is summarized in a kinase loop database located at http://sbi.imim.es/archki.     

Antibody binding loop insertions as diversity elements

In the use of non-antibody proteins as affinity reagents, diversity has generally been derived from oligonucleotide-encoded random amino acids. Although specific binders of high-affinity have been selected from such libraries, random oligonucleotides often encode stop codons and amino acid combinations that affect protein folding. Recently it has been shown that specific antibody binding loops grafted into heterologous proteins can confer the specific antibody binding activity to the created chimeric protein. In this paper, we examine the use of such antibody binding loops as diversity elements. We first show that we are able to graft a lysozyme-binding antibody loop into green fluorescent protein (GFP), creating a fluorescent protein with lysozyme-binding activity. Subsequently we have developed a PCR method to harvest random binding loops from antibodies and insert them at predefined sites in any protein, using GFP as an example. The majority of such GFP chimeras remain fluorescent, indicating that binding loops do not disrupt folding. This method can be adapted to the creation of other nucleic acid libraries where diversity is flanked by regions of relative sequence conservation, and its availability sets the stage for the use of antibody loop libraries as diversity elements for selection experiments.     

The nature of the turn in omega loops of proteins

An analysis of Omega loops in a nonredundant set of protein structures from the Protein Data Bank has been carried out to determine the nature of the "turn elements" present. Because Omega loops essentially reverse their direction in three-dimensional space, this analysis was made with respect to four turn elements identified as (1) Gly; (2) Pro; (3) a residue with alpha-helical phi,psi angles, termed a helical residue; and (4) a cis peptide. A set of 1079 Omega loops from a set of 680 proteins were used for the analysis. Apart from other criteria that define Omega loops, the selection of an Omega loop from a cluster of loops is based on an exposure index. In this study, analyses have been made with two sets of data: (1) Omega loops arising from a minimum exposure index indicative of a less exposed loop (xmin set) and (2) Omega loops with a maximum exposure index indicative of a relatively exposed loop (xmax set). Overall residue preferences and positional preferences have been examined. Positions of the turn elements for Omega loops of varying length have also been studied. Specific positional preferences are observed for particular turn elements with regard to the length of Omega loops. Analysis in terms of the turn elements can provide guidelines for modeling of loops in proteins. Apart from Pro, which has the natural tendency to form cis peptide bonds, a higher occurrence of non-Pro cis peptide bonds is observed. Torsion angles in Omega loops also indicate the occurrence of a large number of residues with helical phi,psi angles, necessary for the turn in the loop structures.