Molecular dynamics of the 5-HT1a receptor and ligands

A 3-D model of the human 5-HT_1a receptor was constructed fromits amino acid sequence by computer graphics techniques, molecularmechanics calculations and molecular dynamics simulations. Themodel has seven -helical membrane spanning segments, which forma central core containing a putative ligand binding site. Electrostaticpotentials 1.4 Å outside the water accessible surfacewere mainly negative on the synaptic side of the receptor modeland at the postulated ligand binding site, and positive in thecytoplasmic domains. The negative electrostatic potentials aroundthe synaptic domains indicate that positively charged ligandsare attracted to the receptor by electrostatic forces. Moleculardynamics simulations of the receptor model with serotonin, ipsapirone,R(–)-methiothepin or S(+)- methiothepin in the centralcore suggested that up to 22 different amino acid residues mayform a ligand binding pocket, and contribute to the specificityof ligand recognition and binding.     

Homology modelling of rat kallikrein rK9, a member of the tissue kallikrein family: implications for substrate specificity and inhibitor binding

The rat kallikrein rK9 is one of the six members of the rattissue kallikrein family isolated to date. It is 84% identicalto rK2 (tonin), and both proteinases are thought to have vasoconstrictiveproperties. Recently we have shown that rK9 and rK2 have distinctsubstrate specificities and sensitivities to inhibitors, despitetheir similar sequences. Unlike all other mammalian kallikrein-relatedproteinases, rK9 is resistant to inhibition by aprotinin. Wehave developed a 3-D model of rK9, based on the known X-raystructures of rK2, porcine kallikrein and bovine trypsin, toidentify the structural features underlying this functionaldiversity. The final rK9 model is structurally similar to rK2,but variable regions surrounding the active site differ quitemarkedly from the reference proteins. The kallikrein loop, whichdiffers from that in porcine kallikrein by a seven-residue insertion,has been generated de novo and subjected to simulated annealingto assess its influence on the restricted substrate specificityof these proteinases. The proposed conformation of the specificitypocket in rK9 differs from that of other serine proteinases,but it can still accommodate both aromatic and basic amino acidside chains at the substrate P₁ position, thus explaining thedual chymotrypsin and trypsin-like activity of rK9. The electrostaticpotentials of rK9 and aprotinin were calculated using the finitedifference Poisson–Boltzmann method. They indicated alarge positive region near the active site of rK9 not foundin related proteinases because of positively charged residuesat positions 61 and 65 in rK9. They generate a positive region,which overlaps a positive region in aprotinin, and may preventaprotinin binding. A single mutation in aprotinin is suggestedthat might allow kallikrein rK9 inhibition by aprotinin. Thismodel contributes significantly to our understanding of thestructure-function relationships among proteinases of the tissuekallikrein family.     

Computer modelling of the interaction between human choriogonadotropin and its receptor

Primary structural homology between the hormone binding siteof the LH/CG receptor and the enzyme binding site of chymotrypsininhibitor has been identified. This has led to the applicationof a knowledge-based approach of molecular modelling to describethe interaction of choriogonadotropin (CG) with the LH/CG receptor.A tertiary structural model for the mode of recognition betweenthe hormone and the receptor has been proposed. As in othersuch processes at the molecular level, the recognition betweenCG and its receptor is mediated through non-covalent interactions.The specificity of recognition is achieved by complementarityin van der Waals surfaces, hydrogen bonding and non-polar associations.The model shows nine hydrogen bonds between the hormone andthe receptor involving polar side chains as well as backboneamine and carbonyl groups. A hydropbobic cluster involving sidechain groups at the interface is also important in stabilizationof the intermolecular interactions     

Dimeric character of a basic phospholipase A2 from cobra venom: experimental and modelling study

When it is gel filtered on Sephadex in the absence of calciumions, basic phospholipase A₂ from Naja nigricollis venom elutesas a dimer. In order to study the possibility of this dimerizationfrom a structural point of view, three-dimensional models ofboth monomeric and dimeric N. nigricollis phospholipases A havebeen graphically built on the basis of homologies with the phospholipasesA₂ from pancreatic bovine and Crotalus atrox venom. The buildingof a duneric model is made possible by the deletion of a particularloop of the bovine structure. The predicted models of N. nigricollisphospholipase A₂ have been checked using molecular mechanicsand molecular dynamics techniques according to a suitable protocolwhich has been developed starting from refined X-ray structuresof phospholipases A₂ as the test case. The observed stabilityof the dimeric model, in the absence of calcium, agrees withthe hypothesis of the dimerization of the basic phospholipaseA Particularly, Arg31, which replaces the hydrophobic residuepresent in pancreatic bovine and C.atrox venom phospholipasesA₂, contributes to this stability.     

Molecular dynamics simulation of fungal cellulose-binding domains: differences in molecular rigidity but a preserved cellulose binding surface

A total of 23 fungal cellulose-binding domain (CBD) sequenceswere aligned. Structural models of the cellulosebinding domainof an exoglucanase (CBHII) and of three endoglucanases (EGI,EGII and EGV) from Trichoderma reesei cellulases were homologymodelled based on the NMR structure of the fungal cellobiohydrolaseCBHI, from the same organism. The completed models and the knownstructure of the CBHI cellulose-binding domain were refinedby molecular dynamics simulations in water. All four modelswere found to be very similar to the structure of the CBHI cellulose-bindingdomain and sequence comparison indicated that in general thethree-dimensional structures of fungal cellulose-binding domainsare very similar. In all the CBDs studied, two disulphide bridgesapparently stabilize the polypeptide fold. From the models,an additional disulphide bridge was predicted in EGI and CBHII,and in eight further CBDs from other organisms. Three highlyconserved aromatic residues on the hydrophilic side of the wedgemake this surface flat This surface is expected to make contactwith the substrate. Three invariant amino acids, Gln7, Asn29and Gln34, on this flat face are in suitable positions for hydrogenbonding with the cellulose surface. Analysis of the differencesin the protein surface properties indicated that the endoglucanasestend to be more hydrophilic than the exoglucanases. The largeststructural variation was found around positions 12-16. The fungalCBD sequences are discussed in relation to variations in functionand pH dependence. Comparison of the modelled structures withexperimental binding data for the CBHI and EGI allowed the formulationof a qualitative relationship to cellulose affinity. This relationshipwas used to predict the cellulose affinities for 21 CBDs.     

Computer simulations of signal transduction mechanism in {alpha}1B-adrenergic and m3-muscarinic receptors

Molecular dynamices simulations of the hamster _1Badrenergicand the rat m3-muscarinic seven-helix bundle receptor modelshave been carried out. The free, agonist-bound and antagonist-boundforms have been considered. Moreover, three mutant forms ofthe m3-muscarinic recep-tor (N507A, N507D and N507S) have alsobeen simulated; among these, the N507S mutant shows a constitutiveactivity. A comparative structural/dynamics analysis has beenperformed to elucidate (i) the perturbations induced by thefunctionally different ligands upon binding to their targetreceptor, (ii) the features of the three single-point mutantswith respect to the receptor wild type and (iii) the propertiesshared by the agonist-boundforms of the _1B-adrenergic receptorand the m3-muscarinic receptor and by the constitutively activemutant N507S. The consistency obtained between the structuralrearrangement of the transmembrane seven-helix bundle modelsconsidered, and the experimental pharmacological efficaciesof the ligands and of the mutants, constitute an important validationof the 3-D models obtained and allow the inference of the mechanismof ligand- or mutation-induced receptor activation at the molecularlevel.     

Molecular modeling of coiled-coil {alpha}-tropomyosin: analysis of staggered and in register helix--helix interactions

In register and staggered models of tropomyosin coiled-coilwere built from X-ray C coordinates and refined via moleculardynamics. The two models show similar structural features withthe X-ray structure of GCN4 leucine zipper. Empirical energeticmethods used to compare the in register and staggered modelsindicate that both are equally probable. The two models havesimilar profiles of solvation free energy of folding for residuesat positions a and d of the repeating heptad, indicating thatresidues at these positions are as well buried in an in registerstructure as in a staggered one. Neither the in register northe 14 residues staggered structure can be ruled out based onhydrophobic or e–g' (g–e') electrostatic interactionswhich are not able to distinguish between the two models andare therefore not selective. However, the eg–b'c' electrostaticinteractions, although smaller in magnitude, are in favor ofthe in register model. Furthermore, analysis of hydrophobicand electrostatic interactions along the tropomyosin sequenceshows that bulky residues in positions a and d prevent the formationof inter-chain salt bridges.     

Molecular dynamics simulations of phospholipases A2

An extensive molecular dynamics study of phospholipases A₂ frompancreatic bovine and Crotalus atrox venom has shown that thewell-conserved homologous core of the phospholipases A₂, includingthe so called catalytic network, is very stable during the courseof the calculations. The fluctuations which occur are locatedin segments which have significantly different three-dimensionalconformations in the two phospholipases A₂ studied, suggestingthat a particularly stable core conformation gives rise to alarge homologous family of similar three-dimensional structure.The calcium ion, which exhibits a crucial structural role inthe monomeric phospholipases A₂, appears not to be requiredto stabilize the C.atrox dimer. Moreover, the behaviour of thedimeric structure during the dynamics raises the question ofa possible dissociation of the two subunits into functionalmonomers.     

Characterization of the interactions of a bifunctional inhibitor with {alpha}-thrombin by molecular modelling and peptide synthesis

A potent thrombin inhibitor, [D-Phe⁴⁵, Arg⁴⁷] hirudin 45-65,that contains an active site-directed sequence D-Phe-Pro-Arg-Pro,an exosite specific fragment hirudin 55-65 (H55-65) and a linkerportion hirudin 49-54, was designed based on the hirudin sequence[DiMaio et al. (1990) J. Biol. Chem., 265, 21698-21798]. A three-dimensionalmodel of the complex between the B-chain of human thrombin andthe inhibitor [D-Phe45, Arg47⁴⁵, Arg⁴⁷] hirudin 45-65 was constructedusing molecular modelling starting from the X-ray Ca coordinatesof the thrombin-hirudin complex and the NMR-derived structureof the thrombin-bound hirudin 55 - 65. The contribution of theH49 -54 fragment to the thrombin - inhibitor interaction wasdeduced by examining a series of analogs containing single glycinesubstitution and analogs with reduced number of residues withinthe linker. The results were consistent with the molecular modellingobservations i.e. the H49-54 fragment serves the role of a spacerin the binding interaction and could be replaced by four glycineresidues. The studies on the interaction of the exosite-directedportion of the inhibitor with thrombin using a series of syntheticH55 -65 analogs demonstrated that residues AspH55 to ProH60play a major role in binding to human thrombin where the sidechains of PheH56, IleH59 and GluH57 showed critical contributions.Molecular modelling suggested that these side chains may contributeto inter- and intramolecular hydrophobic and electrostatic interactions,respectively.     

A model for the C5a receptor and for T its interaction with the ligand

A model of the C5a receptor was built based on the assumptionthat the seven membrane-spanning helices of known inward/outwarddirection are in an arrangement roughly similar to that in bacteriorhodopsin.Guidelines for the positioning of the helices were cysteinepairing, ‘ridges into grooves’ interdigitation ofside chains and aromatic cluster formation. The chain segmentsprotruding from the membrane are too short for folding intoan independent ectodomain. The only longer segment (179–202)is tied down in its centre onto the membrane by a disulphidebridge and, thereby, made into two short loops as well. Ideasof the interaction of the C5a receptor with its ligand werederived mainly from the search for accommodation of the functionallyessential arginine residues 40 and 74 of C5a. Asp82 is the onlycharged residue in a pocket {small tilde}20 A below the receptorsurface and is conserved in the rhodopsin superfamily. It commendsitself for binding Arg74 which is the tip of the flexible C-terminalchain of C5a, and rules out Arg40 in the structurally well-definedpart of the molecule. The latter may bind to Glul80 at the bottomof a more shallow pocket which happens to resemble the substrate-bindingsite of trypsin.     

Molecular modelling study of antigen binding to oxazolone-specific antibodies: the Ox1 idiotypic IgG and its mature variant with increased affinity to 2-phenyloxazolone

Structural models of the variable domains of the murine anti-2-phenyloxazoloneIgG (Oxl idiotype) and its somatic variant, which has higheraffinity to the hapten 2-phenyloxazolone, were constructed bycomputer-aided model building using known structures of highlyhomologous immunoglobulins as templates. Molecular dynamicssimulations were used to dock the hapten between the V_L andV_H domains. The hapten is predicted to bind to slightly differentsites in the two models. Hypotheses concerning the role of anumber of preferred mutations in anti-oxazolone variants arepresented. These can be tested by mutagenesis and crystallography.In particular, the higher binding affinities of the differentantibody variants are shown to correlate with better complementarityof electrostatics. The molecular dynamic simulations also suggestthat two mobile tryptophans at the mouth of the pocket may playan important role in the binding of hapten.     

Modelling of the disulphide-swapped isomer of human insulin-like growth factor-1: implications for receptor binding

Insulin-like growth factor-1 (IGF-1) is a serum protein whichunexpectedly folds to yield two stable tertiary structures withdifferent disulphide connectivities; native IGF-1 [18–61,6–48,47–52]and IGF-1 swap [18–61,6–47, 48–52]. Here we demonstratein detail the biological properties of recombinant human nativeIGF-1 and IGF-1 swap secreted from Saccharomyces cerevisiae.IGF-1 swap had a ~30 fold loss in affinity for the IGF-1 receptoroverexpressed on BHK cells compared with native IGF-1.The parallelincrease in dose required to induce negative cooperativity togetherwith the parallel loss in mitogenicity in NIH 3T3 cells impliesthat disruption of the IGF-1 receptor binding interaction ratherthan restriction of a post-binding conformational change isresponsible for the reduction in biological activity of IGF-1swap. Interestingly, the affinity of IGF-1 swap for the insulinreceptor was ~200 fold lower than that of native IGF-1 indicatingthat the binding surface complementary to the insulin receptor(or the ability to attain it) is disturbed to a greater extentthan that to the IGF-1 receptor. A 1.0 ns high-temperature moleculardynamics study of the local energy landscape of IGF-1 swap resultedin uncoiling of the first A-region -helix and a rearrangementin the relative orientation of the A- and B-regions. The modelof IGF-1 swap is structurally homologous to the NMR structureof insulin swap and CD spectra consistent with the model arepresented. However, in the model of IGF-1 swap the C-regionhas filled the space where the first A-region -helix has uncoiledand this may be hindering interaction of Val44 with the secondinsulin receptor binding pocket.     

Homology modelling of the Lactococcus lactis leader peptidase NisP and its interaction with the precursor of the lantibiotic nisin

A model is presented for the 3-D structure of the catalyticdomain of the putative leader peptidase NisP of Lactococcuslactis, and the interaction with its specific substrate, theprecursor of the lantibiotic nisin. This homology model is basedon the crystal structures of subtilisin BPN' and thermitasein complex with the inhibitor eglin. Predictions are made ofthe general protein fold, inserted loops, Ca²⁺ binding sites,aromatic interactions and electrostatic interactions of NisP.Cleavage of the leader peptide from precursor nisin by NisPis the last step in maturation of nisin. A detailed predictionof the substrate binding site attempts to explain the basisof specificity of NisP for precursor nisin. Specific acidicresidues in the SI subsite of the substrate binding region ofNisP appear to be of particular importance for electrostaticinteraction with the PI Arg residue of precursor nisin afterwhich cleavage occurs. The hydrophobic S4 subsite of NisP mayalso contribute to substrate binding as it does in subtilisins.Predictions of enzyme-substrate interaction were tested by proteinengineering of precursor nisin and determining susceptibilityof mutant precursors to cleavage by NisP. An unusual propertyof NisP predicted from this catalytic domain model is a surfacepatch near the substrate binding region which is extremely richin aromatic residues. It may be involved in binding to the cellmembrane or to hydrophobic membrane proteins, or it may serveas the recognition and binding region for the modified, hydrophobicC-terminal segment of precursor nisin. Similar predictions forthe tertiary structure and substrate binding are made for thehighly homologous protein EpiP, the putative leader peptidasefor the lantibiotic epidermin from Staphylococcus epidermidis,but EpiP lacks the aromatic patch. Based on these models, proteinengineering can be employed not only to test the predicted enzyme-substrateinteractions, but also to design lantibiotic leader peptidaseswith a desired specificity.     

Molecular modelling of Staphylococcal {delta}-toxin ion channels by restrained molecular dynamics

-Toxin is a 26-residue channel-forming peptide from Staphylococcusaureus which forms an amphipathic -helix in a membrane environment.Channel formation in planar bilayers suggests that an averageof six -toxin helices self-assemble to form transbilayer pores.Molecular models for channels formed by -toxin and by a syntheticanalogue have been generated using a simulated annealing protocolapplied via restrained molecular dynamics. These models areanalysed in terms of the predicted geometric and energetic propertiesof the transbilayer pores. Pore radius calculations of the modelsdemonstrate that rings of channel-lining residues contributea series of constrictions along the pore. Electrostatic propertiesof the pores are determined both by pore-lining charged sidechains and by the aligned helix dipoles of the parallel helixbundle. Molecular dynamics simulations (100 ps) of -toxin modelscontaining intra-pore water were performed. Analysis of theresultant dynamics trajectories further supports the proposalthat alternative conformations of pore-constricting side chainsmay be responsible for the observed conductance heterogeneityof -toxin ion channels.     

Enhancing the thermal stability of mitochondrial cytochrome b5 by introducing a structural motif characteristic of the less stable microsomal isoform

Outer mitochondrial membrane cytochrome b5 (OM b5) is the most thermostable cytochrome b5 isoform presently known. Herein, we show that OM b5 thermal stability is substantially enhanced by swapping an apparently invariant motif in its heme-independent folding core with the corresponding motif characteristic of its less stable evolutionary relative, microsomal cytochrome b5 (Mc b5). The motif swap involved replacing two residues, Arg15 with His and Glu20 with Ser, thereby introducing a Glu11-His15-Ser20 H-bonding triad on the protein surface along with a His15/Trp22 pi-stacking interaction. The ferric and ferrous forms of the OM b5 R15H/E20S double mutant have thermal denaturation midpoints (Tm values) of approximately 93 degrees C and approximately 104 degrees C, respectively. A 15 degrees C increase in apoprotein Tm plays a key role in the holoprotein thermal stability enhancement, and is achieved by one of the most common natural mechanisms for stabilization of thermophilic versus mesophilic proteins: raising the unfolding free energy along the entire stability curve.     

Free energy perturbation studies on binding of A-74704 and its diester analog to HIV-1 protease

Free energy simulations have been employed to rationalize thebinding differences between A-74704, a pseudo C₂- symmetricinhibitor of HIV-1 protease and its diester analog. The diesteranalog inhibitor, which misses two hydrogen bonds with the enzymeactive site, is surprisingly only 10-fold weaker. The calculatedfree energy difference of 1.7 ± 0.6 kcal/mol is in agreementwith the experimental result. Further, the simulations showthat such a small difference in binding free energies is dueto (1) weaker hydrogen bond interactions between the two (P₁and P₁) NH groups of A-74704 with Gly27/Gly27' carbonyls ofthe enzyme and (2) the higher desolvation free energy of A-74704compared with its ester analog. The results of these calculationsand their implications for design of HIV-1 protease inhibitorsare discussed.     

Predicted ligand interactions for 3',5'-cyclic nucleotide-gated channel binding sites: comparison of retina and olfactory binding site models

Cyclic nucleotide-gated channels (CNGC) open in response tothe binding of 3',5'-cyclic nucleotides. Members of the CNGCfamily vary as much as 100-fold in their ability to respondto cAMP and cGMP. Molecular models of the nucleotide bindingdomains of the bovine retina and catfish and rat olfactory CNGCswere built from the crystal structure of cAMP bound to catabolitegene activator protein (CAP) with AMMP, a program for molecularmechanics and dynamics. The nucleotide conformation can be predictedfrom the number of strong and weak interactions between thepurine ring and the binding site. The amino acids predictedto be important for determining the nucleotide affinity andspecificity are residues 61, 83 (mediated through a water molecule),119 and 127 (CAP sequence numbers) which interact with the purinering. These residues also dictate the conformation of the ligandin the binding pocket cGMP is preferentially bound in the synconformation in bovine retina, bovine olfactory and rat olfactoryCNGCs due to Thr83, while either conformation can bind in catfisholfactory CNGC. cAMP is predicted to bind either in syn or anticonformation, depending on the interaction with residue 119:the anti conformation is preferentially bound in olfactory CNGCs.     

The expression of bovine microsomal cytochrome b5 in Escherichia coli and a study of the solution structure and stability of variant proteins

The DNA sequence of bovine microsomal cytochrome b₅ has beenamplified from a liver cDNA library using a polymerase chainreaction. The amplified cDNA when cloned into plasmids thatsupport the high-level production of cytochrome b_s in E.colileads to protein overexpression and results in cell coloniesbearing a strong red colouration. Using cassette mutagenesis,truncated versions of the cytochrome b₅ cDNA have been madethat encode the first 90 amino acid residues (Ala1-Lys90), thefirst 104 amino acids (Ala1-Ser104) and the complete protein(Ala1-Asnl33). The location of the overexpressed cytochromebs within prokaryotic cells is dependent on the overall lengthof the protein. Expression of the Ala-Lys90 and Alal-SerlO4variants leads to a location in the cytoplasmic phase of thebacteria whereas the whole protein, Alal-Asnl33, is found withinthe bacterial membrane fraction. The last 30 residues of cytochromebs therefore contain all of the necessary information to insertthe protein into E.coli membranes. The solubility of the Alal-SerlO4variant permits the solution structure and stability of thisprotein to be measured using 1- and 2-D ¹HNMR methods and electronicspectroscopy. 1-D NMR studies show that the chemical shiftsof the haem and haem ligand resonances of the Alal - Ser 104variant exhibit only very slight perturbations to their magneticmicroenvlronments when compared with the tryptic fragment offerricytochrome b₅. These results indicate an arrangement ofresidues in the haem pocket that is very similar in both theAlal-Ser 104 variant and the tryptic fragment and by 2-D NMRit is shown that this similarity extends to the conformationsof the poly peptide backbone and side chains. Electronic spectroscopyof this variant shows absorbance maxima for the Soret peaksat 423 run (reduced) and 413 nm (oxidized). From absorbancespectra the relative thermal stabilities of the Alal-Ser 104variant and the tryptic fragment were measured. In the oxidizedstate the Ala1 - Ser104 variant denatures in a single cooperativetransition with a midpoint temperature (T_m of 73°C thatis significantly higher than that of ‘tryptic’ ferricytochromebs. The reduced form of the protein shows increased transitiontemperatures (T_m 78°C) reflected in the values of H_m, S_mand (G) of 420 kj/mol, 1096 J/mol/K and 12.38 kj/mol respectively,estimated for this variant. The increased stability of the Alal-SerlO4variant and other recombinant forms of cytochrome b_s is correlatedwith the presence of additional residues at the N- and C-termini.The subtle differences in reactivity, stability and targetingbetween variant forms of cytochrome bs and the tryptic fragmentare discussed in terms of the overall structure of the protein.     

Streptomyces aminopeptidase P: biochemical characterization and insight into the roles of its N-terminal domain

We purified and characterized the aminopeptidase P from Streptomyces costaricanus TH-4 (thAPP). This enzyme has a tetramer structure, a metal-ion preference toward Zn, broad substrate specificity and a narrow pH dependency for activity. The primary structure of thAPP, respectively, exhibits 91% and 65% identity with those of two other APPs-APP I and APP II-from Streptomyces lividans (slAPP I and slAPP II). We next overexpressed the genes encoding thAPP and slAPP II in Escherichia coli and characterized them. Two differences were apparent in their properties: slAPP II formed a dimer, whereas thAPP formed a tetramer; also, the alkaline side pKa for the catalytic action of slAPP II is higher than that of thAPP. Investigation using chimeras of both enzymes revealed that the N-terminal domain is associated with the determination of pKa values for catalytic action and quaternary structure.     

Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains

We cloned 17 different PCR fragments encoding V_H genes of camel(Camelus dromedarius). These clones were derived from the camelheavy chain immunoglobulins lacking the light chain counterpartof normal immunoglobulins. Insight into the camel V_H sequencesand structure may help the development of single domain antibodies.The most remarkable difference in the camel V_H, consistent withthe absence of the V_L interaction, is the substitution of theconserved Leu45 by an Arg or Cys. Another noteworthy substitutionis the Leu11 to Ser. This amino acid normally interacts withthe C_H1 domain, a domain missing in the camel heavy chain immunoglobulins.The nature of these substitutions agrees with the increasedsolubility behavior of an isolated camel V_H domain. The V_H domainsof the camels are also characterized by a long CDR3, possiblycompensating for the absence of the V_L contacts with the antigen.The CDR3 lacks the salt bridge between Arg94 and Asp101. However,the frequent occurrence of additional Cys residues in both theCDR1 and CDR3 might lead to the formation of a second internaldisulfide bridge, thereby stabilizing the CDR structure as inthe DAW antibody. Within CDRs of the camel V_H domains we observea broad size distribution and a different amino acid patterncompared with the mouse or human V_H. Therefore the camel hypervariableregions might adopt structures which differ substantially fromthe known canonical structures, thereby increasing the repertoireof the camel antigen binding sites within a V_H.