Flexible Amino Acid Research Articles

Ab initio study of electronic interaction energies and desolvation energies for dopaminergic ligands in the catechol-O-methyltransferase active site

Catechol-O-methyltransferase (COMT) deactivates dopamine and other catecholic substrates via the methylation of a hydroxyl group. The targeted inhibition of COMT can prevent premature metabolism of both endogenous and exogenous ligands; for example, it can prolong the effectiveness of l-DOPA treatments for Parkinson’s disease. The inhibitory ability of a suite of dopaminergic derivatives against COMT has been studied using electronic interaction energies between each ligand and the enzymatic active site as well as desolvation energies for each ligand. A crystal structure of the COMT active site in complex with a known COMT inhibitor, BIA 8-176, was isolated from the Protein Data Bank (PDB ID: 2CL5). The positions of a suite of dopaminergic derivatives in this same active site were optimized using M062X/6-31G with three different model chemistries: in vacuo with rigid amino acid side chains, with implicit solvation and rigid amino acid side chains, and with implicit solvation and flexible amino acid side chains. Electronic interaction energies between the ligands and the active site residues were calculated for each model using M06L and MP2 with the 6-311+G∗ basis set. Ligands with a nitrile substituent were favored over other substituents in vacuo, but this preference was not retained when the same ligands were optimized with implicit solvation; the preference was regained when using implicit solvation and a relaxed active site. Desolvation energies of the ligands were calculated using a series of hydration shells (n=7, 9, 11 and 13) with M062X and the aug-cc-pvdz, cc-pvdz, and cc-pvtz basis sets. Ligands with carboxyl and nitro substituents exhibited the least favorable desolvation energies, whereas ligands with the nitrile substituents exhibited the most favorable desolvation energies.

Single-Point Mutation with a Rotamer Library Toolkit: Toward Protein Engineering.

Protein engineers have long been hard at work to harness biocatalysts as a natural source of regio-, stereo-, and chemoselectivity in order to carry out chemistry (reactions and/or substrates) not previously achieved with these enzymes. The extreme labor demands and exponential number of mutation combinations have induced computational advances in this domain. The first step in our virtual approach is to predict the correct conformations upon mutation of residues (i.e., rebuilding side chains). For this purpose, we opted for a combination of molecular mechanics and statistical data. In this work, we have developed automated computational tools to extract protein structural information and created conformational libraries for each amino acid dependent on a variable number of parameters (e.g., resolution, flexibility, secondary structure). We have also developed the necessary tool to apply the mutation and optimize the conformation accordingly. For side-chain conformation prediction, we obtained overall average root-mean-square deviations (RMSDs) of 0.91 and 1.01 Å for the 18 flexible natural amino acids within two distinct sets of over 3000 and 1500 side-chain residues, respectively. The commonly used dihedral angle differences were also evaluated and performed worse than the state of the art. These two metrics are also compared. Furthermore, we generated a family-specific library for kinases that produced an average 2% lower RMSD upon side-chain reconstruction and a residue-specific library that yielded a 17% improvement. Ultimately, since our protein engineering outlook involves using our docking software, Fitted/Impacts, we applied our mutation protocol to a benchmarked data set for self- and cross-docking. Our side-chain reconstruction does not hinder our docking software, demonstrating differences in pose prediction accuracy of approximately 2% (RMSD cutoff metric) for a set of over 200 protein/ligand structures. Similarly, when docking to a set of over 100 kinases, side-chain reconstruction (using both general and biased conformation libraries) had minimal detriment to the docking accuracy.

Beyond Natural Limitations: Long‐Range Influence of Non‐Natural Flexible and Rigid β‐Turn Mimetics in a Native β‐Hairpin Motif

Abstractβ‐Turns mediate diverse protein recognition processes, although dihedral angle preferences of canonical amino acids limit accessible β‐turn geometries. Organic synthesis can go beyond these limitations and either increase the mobility of a β‐turn or constrain it inside a bicyclic ring. Nine β‐turn dipeptides are studied here in the isolated β‐hairpin of the miniprotein Foldon, which is only moderately structured in the absence of the native protein environment. The dipeptide mimetics, which vary the backbone flexibility from linear alkyl chains to bicyclic dipeptides (Hot=Tap), are ranked against each other with regard to their hairpin‐stabilizing capacities. NMR‐derived parameters and melting temperatures correlate the backbone rigidity of the β‐turns with the hairpin population. In contrast to the general expectation, highly populated hairpins are not only observed for rigid dipeptides, but also for selected flexible amino acids in the i + 1 and i + 2 positions of the β‐turn.

Effect of Linker Flexibility on the Catalytic Features of a Glycoside Hydrolase Family 45 Endoglucanase from Rhizopus stolonifer.

This study investigated an endoglucanase (EGII) from Rhizopus stolonifer var. reflexus TP-02 that consists of a C-terminal catalytic domain and an N-terminal carbohydrate-binding module joined by a linker rich in glycine, serine, threonine, and alanine. Site-directed mutagenesis was applied to characterize the conformation and dynamics of the linker. Mutants were expressed in Escherichia coli BL21 and purified by Ni-chelating column. Structural analysis indicated that glycine provided flexibility in the enzymatic process. G67P, G91Y, G101Y, G108Y, G109Y, G112P, H61G, H75G, and Y103G were selected on the basis of the results of the bioinformatics and Ramachandran plot analysis for the linker. The catalytic activities of EGII and its mutants on CMC-Na, microcrystalline cellulose (Avicel), and phosphoric acid-swollen celluloses (PASC) showed that flexible amino acids strengthened the activity of the enzyme. It indicated that flexible amino acids could improve the flexibility of the linker. Overall, the linker affected the catalytic efficiency of the endoglucanase in hydrolyzing cellulose chains.

Characterization of bioactive recombinant antimicrobial peptide parasin I fused with human lysozyme expressed in the yeast Pichia pastoris system

Parasin I (PI) is a 19 amino acid peptide with potent antimicrobial activities against a broad spectrum of microorganisms and is a good candidate for development as a novel antimicrobial agent. The objective of this study was to express and characterize a codon optimized parasin I peptide fused with human lysozyme (hLY). A 513bp cDNA fragment encoding the mature hLY protein and parasin I peptide was designed and synthesized according to the codon bias of Pichia pastoris. A 4×Gly flexible amino acid linker with an enterokinase cleavage (DDDDK) was designed to link the PI to the C-terminal of hLY. The codon optimized recombinant hLY-PI was cloned into the pPICZαA vector and expressed in P. pastoris. The over-expressed extracellular rehLY-PI was purified using Ni sepharose affinity column and exhibited a molecular mass of approximately 18kDa. After digested with enterokinase the rehLY-PI protein release its corresponding rehLY and rePI, with molecular mass of 16kDa and 2kDa, respectively, on Tricine-SDS-PAGE. The released rehLY exhibited similar lytical activity against Micrococcus lysodeikticus to its commercial hLY. The digested rehLY-PI product exhibited antimicrobial activities against Bacillus subtilis, Staphylococcus aureus and Escherichia coli, and synergism has been found between the released rePI and rehLY. In conclusion, we successfully optimized a rehLY-PI fusion protein encoding gene and over-expressed the rehLY-PI in P. pastoris. The recombination protein digested with enterokinase released functional hLY and antimicrobial parasin I, which demonstrates a potential for future use as an animal feed additive to partly replace antibiotic.

Formation of c- and z-ions due to preferential cleavage at the N[sbnd]C bond of Xxx-Asp/Asn residues in negative-ion CID of peptides

In collision-induced dissociation (CID) experiments of peptides, the formation of c- and z-ions originating from NCα bond cleavage of the peptide backbone is unusual, whereas b- and y-ions are often observed in positive-ion CID spectra. Here we report that characteristic product c- and z-ions can be observed in negative-ion CID spectra of peptides with Asp and Asn residues. The negative c- and z-ions are mainly generated at the NCα bond of Xxx-Asp and Xxx-Asn residues that are classified as the flexible amino acids in protein chemistry. In the absence of any acidic amino acid residues, negative-ion CID spectra show dominant c-ion peaks originating from cleavage at the NCα bond lying in between the 3rd and 4th residues from the C-terminus of analyte peptides. It is proposed that the NCα bond cleavage leading to the formation of c- and/or z-ions is caused by the negative charge located on the amide nitrogen, which generated with an intra-molecular hydrogen abstraction from the backbone amide regions due to negative-charged acidic functional groups.

Discovery of novel 2,4-diarylaminopyrimidine analogues (DAAPalogues) showing potent inhibitory activities against both wild-type and mutant ALK kinases.

We have developed a series of new 2,4-diarylaminopyrimidine analogues (DAAPalogues) bearing a flexible amino acid side chain, different from the majority of the literature reported ALK inhibitors that often possess a structurally constrained arylpiperazine fragment or its equivalents in the solvent-interaction region. Extensive structural elaboration led to compound 15 possessing IC50 values of 2.7 and 15.3 nM, respectively, in the ALK wild-type and gate-keeper mutant L1196M enzymatic assays. This compound not only showed high proliferative inhibition against ALK-addicted cells across different oncogenic forms but also effectively suppressed several ALK secondary mutant cells, including the gate-keeper L1196M and F1174L. Significant antitumor efficacy was achieved in the ALK-driven SUP-M2 xenograft model.

Flexibility analysis of activity-enhanced mutants of bacteriophage T4 lysozyme

Abstract Enzymes are essential biological molecules and widely used in industry. Therefore, investigating the mechanisms underlying how enzyme activity is changed by mutations may have widespread clinical and industrial applications as well as understanding of enzyme catalysis. In this paper, various reported mutants of bacteriophage T4 lysozyme were analyzed to investigate the relationship between enzyme activity and flexibility. The activity-enhanced mutants of bacteriophage T4 lysozyme demonstrated a tendency for mutations to localize to the edge of helices and to involve substitutions to flexible amino acids such as glutamic acid and aspartic acid. Additionally, the mutated residues were shown to demonstrate increased flexibility in B-factor. These findings suggest that flexibility modulation may be a feasible means to enhance enzyme activity.

Identification of Ligand Specificity Determinants in Lantibiotic Bovicin HJ50 and the Receptor BovK, a Multitransmembrane Histidine Kinase

Lantibiotic bovicin HJ50 is produced by Streptococcus bovis HJ50 and acts as the extracellular signal to autoregulate its own biosynthesis through BovK/R two-component system. Bovicin HJ50 shows a linear N-terminal and glubolar C-terminal structure, and the sensor histidine kinase BovK contains eight transmembrane segments lacking any extensive surface-exposed sensory domain. The signal recognition mechanism between bovicin HJ50 and BovK is still unknown. We performed saturated alanine scanning mutagenesis and other amino acid substitutions on bovicin HJ50 using a semi-in vitro biosynthesis. Results of the mutants inducing activities indicated that several charged and hydrophobic amino acids in ring B of bovicin HJ50, as well as two glycines were key residues to recognize BovK. Circular dichroism analyses indicated that both glycines contributed to bovicin HJ50 structural changes in the membrane. Biotin-labeled bovicin HJ50 could interact with the N-terminal sensor of BovK, and several charged residues and a conserved hydrophobic region in the N-terminal portion of BovK sensor domain were important for interacting with the signal bovicin HJ50. By combining the results, we suggested a mechanism of bovicin HJ50 recognizing and activating BovK mainly through electrostatic and hydrophobic interactions.

Self-assembled dipeptide nanotubes constituted by flexible β-phenylalanine and conformationally constrained α,β-dehydrophenylalanine residues as drug delivery system.

Peptide based self assembled nanostructures have attracted growing interest in recent years due to their numerous potential applications particularly in biomedical sciences. Di-peptide Phe-Phe was shown previously to self-assemble into nanotube like structures. In this work, we studied the affect of peptide backbone length and conformational flexibility on the self assembly process by using two dipeptides based on the Phe-Phe backbone (βPhe-Phe and βPhe-ΔPhe): one containing a flexible βPhe amino acid, and the other containing both a flexible βPhe as well as a backbone constraining ΔPhe (α,β-dehydrophenylalanine) amino acid. Electron microscopy and X-ray diffraction experiments revealed that these new di-peptides can self-assemble into nanotubes having different properties than the native Phe-Phe nanotubes. These nanotubes were stable over a broad range of temperatures and the introduction of non-natural amino acids provided them with stability against the action of nonspecific proteases. Moreover, these dipeptides showed no cytotoxicity towards HeLa and L929 cells, and were able to encapsulate small drug molecules. We further showed that anticancerous drug mitoxantrone was more efficient in killing HeLa and B6F10 cells when entrapped in nanotubes as compared to free mitoxantrone. Therefore, these β-phenylalanine and α,β-dehydrophenylalanine containing dipeptide nanotubes may be useful in the development of biocompatible and proteolytically stable drug delivery vehicles.

Structure of the Fusion Core and Inhibition of Fusion by a Heptad Repeat Peptide Derived from the S Protein of Middle East Respiratory Syndrome Coronavirus

Middle East respiratory syndrome coronavirus (MERS-CoV) recently emerged as a severe worldwide public health concern. The virus is highly pathogenic, manifesting in infected patients with an approximately 50% fatality rate. It is known that the surface spike (S) proteins of coronaviruses mediate receptor recognition and membrane fusion, thereby playing an indispensable role in initiating infection. In this process, heptad repeats 1 and 2 (HR1 and HR2) of the S protein assemble into a complex called the fusion core, which represents a key membrane fusion architecture. To date, however, the MERS-CoV fusion core remains uncharacterized. In this study, we performed a series of biochemical and biophysical analyses characterizing the HR1/HR2 complexes of this novel virus. The HR sequences were variably truncated and then connected with a flexible amino acid linker. In each case, the recombinant protein automatically assembled into a trimer in solution, displaying a typical α-helical structure. One of these trimers was successfully crystallized, and its structure was solved at a resolution of 1.9 Å. A canonical 6-helix bundle, like those reported for other coronaviruses, was revealed, with three HR1 helices forming the central coiled-coil core and three HR2 chains surrounding the core in the HR1 side grooves. This demonstrates that MERS-CoV utilizes a mechanism similar to those of other class I enveloped viruses for membrane fusion. With this notion, we further identified an HR2-based peptide that could potently inhibit MERS-CoV fusion and entry by using a pseudotyped-virus system. These results lay the groundwork for future inhibitory peptidic drug design.

Improved thermostability of a Bacillus subtilis esterase by domain exchange

A moderately thermostable esterase from Geobacillus stearothermophilus (BsteE) and its homolog from Bacillus subtilis (BsubE) show a high structural similarity with more than 95% homology and 74% amino acid identity. Interestingly, their thermal stability differs significantly by 30 °C in their melting temperature. In order to identify the positions that are responsible for this difference, most of the flexible amino acids assumed to confer instability were found to be in the cap region. For this reason, a 30 amino acid long cap domain fragment containing ten differing positions derived from BsteE was incorporated into the homologous gene encoding for the more labile BsubE by spliced overlap-extension PCR. The melting temperature of the two wild-type esterases and the mutant was evaluated by circular dichroism spectroscopy, while the kinetic parameters and the stability were determined with a photometric assay. The cap domain mutant maintained its activity, with a catalytic efficiency more similar to BsteE, while it exhibited an increase of the melting temperature by 4 °C compared to BsubE. Additional point mutations based on the differences of the parent enzymes gave a further increase of the thermostability up to 11 °C compared to BsubE; however, a significant reduction in activity was observed.

New Insight into the SAR of Pyrimido [4,5-b][1,4] Benzothiazines as 15-lipoxygenase Inhibitors.

Recently we reported that the soybean 15-lipoxygenase (SLO) inhibitory activity of pyrimido[4,5-b][l,4]benzothiazines largely depends on the orientation of sulfur atom of thiazine core towards Fe(III)-OH in the active site pocket of the enzyme with subsequent oxidation of sulfur to sulfoxide. In this paper the results of a comparative study on the SLO inhibitory activities of the mentioned compounds using ab initio calculations and docking analyses has been reported. Structure optimization and docking analyses were performed using HyperChem 7.5 and AutoDock Tools 4.0 respectively. Enzyme assessment was reduced using spectrophotometric MBTH-DMAB method. Results : The inhibitory activity of synthetic 2-substituted pyrimido[4,5-b][l,4]benzothiazines against soybean 15-lipoxygenase (SLO) was evaluated and structure activity relationships and binding modes of their 4-H and 4-methyl analogs were studied using docking analysis and ab initio calculations. The results of these studies showed that the lack of 4-methyl substituent in the pyrimido[4,5-b][1,4]benzothiazine molecules greatly reduces their lipoxygenase inhibitory activities and it was also found that the HOMO energy difference between the 4-H and 4-Methyl analogs can be responsible for the observed inhibitory activity reduction. Our molecular modeling studies shows that by using more flexible amino acids during the docking process, more rational results can be obtained. The method of measuring the lipoxygenase activity is also of prime importance for the study of structure activity relationship.

Activity Enhancement of Candida antarctica Lipase B by Flexibility Modulation in Helix Region Surrounding the Active Site

The activity of Candida antarctica lipase B was improved by mutation of the area surrounding the active site. We changed the edges of four helices surrounding the active site to flexible amino acids. Two mutants, V139E and I255E, obtained as a result of Pichia pastoris expression, showed enhanced specific activity of 9.9 and 8.1 U/mg while that of wild type was 2.3 U/mg for p-nitrophenyl caprylate hydrolysis. It was nearly 5.4-fold and 3.5-fold, respectively. The stability of both mutants on organic solvent was slightly decreased but almost similar with that of wild type. In the kinetic assay, k(cat) values were shown as dominant factor for the enhancement of catalytic efficiency, k(cat)/K(m), since it was 4.1-fold and 3.8-fold, respectively.

Gating of GluA2 Receptors is Mediated by a Pivot in the M3 Helix

Closure of the ligand-binding domain (LBD) of glutamate receptor channels opens their gate by a mechanism that is not fully clear. We find that glycine substitutions of pore facing residues in the conserved SYTANLAAF region in the transmembrane M3 helix of GluA2 improve gating. notably, replacing alanine 621 with glycine, two turns of the helix below the gate, resulted in a non-desensitizing channel with significant agonist-independent basal activity and ∼ 36-fold increase in glutamate potency without changes in expression or binding. On GluA2(A621G), the partial agonist kainate acted as a full agonist and the antagonist CNQX acted as a partial agonist. In contrast, a glycine mutation above the channel gate, reduced activity and glutamate potency. Therefore, closure of the LBD opens the channel by pulling apart the M3 helix around a pivot at small flexible amino acids in the pore facing region below the gate, in a mechanism similar to potassium channel gating.

Vibrational absorption and vibrational circular dichroism spectra of leucine in water under different pH conditions: Hydrogen-bonding interactions with water

Vibrational absorption (VA) and vibrational circular dichroism (VCD) spectroscopy have been used to study leucine, a flexible branched-chain amino acid, in aqueous solution. The VA spectra in the range of 1800-1250 cm(-1) of leucine in D(2)O under three representative pHs from strongly acidic (pH = 1), near neutral (pH = 6), to strongly basic (pH = 13), have been measured. The related VCD spectrum has been obtained under near neutral condition. Searches have been carried out to identify the most stable conformers of the Zwitterionic, protonated, and deprotonated forms of leucine in water. The geometry optimization, harmonic frequency calculations, and VA and VCD intensities have been computed at the B3LYP/6-311++G(d,p) level with the implicit polarizable continuum solvation model. While the observed VA spectra under three pHs can be well interpreted with the inclusion of the implicit solvation model, both implicit and explicit solvation models have been found to be crucial for the adequate interpretation of the complex VCD features observed. Molecular dynamics simulations and radial distribution functions have been used to aid the modeling of the leucine-(water)(N) clusters. It has been recognized that the insertion of a water molecule between the COO(-) and NH(3) (+) functional groups in the explicit solvated clusters is critical to reproduce the VCD signatures observed. Furthermore, the inclusion of the implicit bulk water environment has been found to be essential to lock water molecules, which are directly hydrogen bonded to leucine, into the positions expected in solution. The application of the explicit and implicit solvation models simultaneously allows new insights into the hydrogen bonding network surrounding leucine in aqueous solution and the role of the surrounding bulk water in stabilizing such hydrogen-bonding network.

Construction of a novel selection system for endoglucanases exhibiting carbohydrate-binding modules optimized for biomass using yeast cell-surface engineering

To permit direct cellulose degradation and ethanol fermentation, Saccharomyces cerevisiae BY4741 (Δsed1) codisplaying 3 cellulases (Trichoderma reesei endoglucanase II [EG], T. reesei cellobiohydrolase II [CBH], and Aspergillus aculeatus β-glucosidase I [BG]) was constructed by yeast cell-surface engineering. The EG used in this study consists of a family 1 carbohydrate-binding module (CBM) and a catalytic module. A comparison with family 1 CBMs revealed conserved amino acid residues and flexible amino acid residues. The flexible amino acid residues were at positions 18, 23, 26, and 27, through which the degrading activity for various cellulose structures in each biomass may have been optimized. To select the optimal combination of CBMs of EGs, a yeast mixture with comprehensively mutated CBM was constructed. The mixture consisted of yeasts codisplaying EG with mutated CBMs, in which 4 flexible residues were comprehensively mutated, CBH, and BG. The yeast mixture was inoculated in selection medium with newspaper as the sole carbon source. The surviving yeast consisted of RTSH yeast (the mutant sequence of CBM: N18R, S23T, S26S, and T27H) and wild-type yeast (CBM was the original) in a ratio of 1:46. The mixture (1 RTSH yeast and 46 wild-type yeasts) had a fermentation activity that was 1.5-fold higher than that of wild-type yeast alone in the early phase of saccharification and fermentation, which indicates that the yeast mixture with comprehensively mutated CBM could be used to select the optimal combination of CBMs suitable for the cellulose of each biomass.

TAP-SSL5 FUNCTIONS AS AN ANTICOAGULANT AND ANTI-INFLAMMATORY PROTEIN TO INHIBIT THROMBOSIS IN RAT AND ATTENUATE ATHEROSCLEROSIS IN APOE-KNOCKOUT MOUSE

ObjectivesThis study aims to inhibit the inflammatory response and thrombosis in atherosclerotic lesions, by using the staphylococcal superantigen-like protein-5 (SSL5) to inhibit neutrophil activation, and tick anticoagulant peptide (TAP) to...

Structural Basis for the Thermostability of Sulfur Oxygenase Reductases

The thermostability of three sulfur oxygenase reductases (SORs) was investigated from thermoacidophilic achaea Acidianus tengchongensis (SORAT) and Sulfolobus tokodaii (SORST) as well as the moderately thermophilic bacterium Acidithiobacillus sp. SM-1 (SORSB). The optimal temperatures for catalyzing sulfur oxidation were 80 °C (SORAT), 85 °C (SORST), and 70 °C (SORSB), respectively. The half-lives of the three SORs at their optimal catalytic conditions were 100 min (SORAT), 58 min (SORST), and 37 min (SORSB). In order to reveal the structural basis of the thermostability of these SORs, three-dimensional structural models of them were generated by homology modeling using the previously reported high-resolution X-ray structure of SORAA (from Acidianus ambivalens) as a template. The results suggest that thermostability was dependent on: (a) high number of the charged amino acid glutamic acid and the flexible amino acid proline, (b) low number of the thermolabile amino acid glutamine, (c) increased number of ion pairs, (d) decreased ratio of hydrophobic accessible solvent surface area (ASA) to charged ASA, and (e) increased volumes of the cavity. The number of cavities and the number of hydrogen bonds did not significantly affect the thermostability of SORs, whereas the cavity volumes increased as the thermal stability increased.

Isolation and characterization of human polyreactive pneumococcal polysaccharide antibodies

Natural antibodies serve as the body’s first line of defense against pneumococcal challenge. Polyreactive human pneumococcal polysaccharide IgG antibodies have not been extensively studied. We analyzed human polyreactive antibodies that bind multiple pneumococcal polysaccharides, including PPS14 and PPS23F. These antibodies were isolated from single pneumococcal polysaccharide specific B cells allowing for the analysis of human immunoglobulins with natively paired variable regions. Although isolated individually, these antibodies demonstrated similar characteristics. Most antibodies possessed a variable light chain with a CDR3 length made up of nine amino acids and relatively high number of flexible amino acids in combined VH/VL. While these antibodies were polyreactive and structurally alike, kinetic analysis revealed unique KD values. Variable chains are responsible for antigen recognition whereas antibody fine specificity is affected by isotype structure. To investigate the contribution of the constant region of these isotypes and their effect on antibody avidity to pneumococcal polysaccharide, the polyreactive variable regions were expressed as IgG1 or IgG2 and subjected to kinetic analysis. The IgG1 antibodies uniformly had a stronger avidity to PPS14 and PPS23F compared to IgG2. To further document the importance of the constant region in antibody avidity and fine specificity, analysis of antibody F(ab)’2 fragment binding to PPS14 and PPS23F resulted in similar KD values. These studies suggest that antigen recognition by polyreactive antibodies is determined by a conserved variable light chain CDR3 length and longer, more flexible variable heavy CDR3s when compared to pneumococcal polysaccha-ride-specific sequences while differences in specific avidities are modulated by antibody isotype.