in central and northern 
 europe as well as in north america a significant 
 proportion of patients who suffer from birch pollen allergy develop 
 intolerance to certain kinds of fruits and vegetables . such birch pollen - related food allergies are the result 
 of initial sensitization to the major birch pollen allergen , bet v 
 1 , and subsequent immunologic cross - reactivity of the bet v 1-specific 
 ige antibodies with structurally homologous food proteins . among the 
 most frequent triggers of birch pollen - related food allergies are 
 apples , with > 70% of all individuals that are sensitized to birch 
 pollen developing allergic reactions when consuming apples . symptoms typically occur locally at the site 
 of food contact and within minutes after apple consumption , including 
 itching and swelling of the lips , tongue , and throat ( oral allergic 
 syndromes , oas ) . frequently , allergic 
 patients can also exhibit symptoms of food - induced rhinoconjunctivitis 
 and dyspnea . in apples ( malus domestica ) , the 
 major allergen that is responsible for birch pollen - related food allergies 
 is the 17.5 kda protein mal d 1 . mal d 
 1 belongs to group 10 of pathogenesis - related ( pr ) proteins that are 
 activated in plants in response to different kinds of stress . the concentration of mal d 1 in apples is highly 
 dependent on the cultivar and also influenced by various biotic and 
 abiotic factors , storage conditions , and storage duration . typically , 130 g of mal d 1 per gram of fresh apple 
 ( accounting for up to 7% of total soluble protein ) is present directly 
 after harvest . after storage , these values can 
 rise to values exceeding 100 g mal d 1 per gram of apple . although mal d 1 has been found in both the pulp and peel of apples , 
 higher concentrations are present in the peel . on 
 the basis of this observation , and because mal d 1 appears to be up - regulated 
 upon biotic stress , it has been speculated that this protein may play 
 a role in plant defense response to pathological situations . mal d 1 is encoded by a multigene family , 
 and a number of isoforms 
 of mal d 1 have been identified to date , which are clustered into 
 four groups on the basis of their dna sequence similarities , that 
 is , mal d 1.01 , mal d 1.02 , mal d 1.03 , and mal d 1.04 . pcr screening and mass spectrometric studies 
 showed that mal d 1 isoforms are not cultivar specific and that mixtures 
 of isoforms are present in apple fruits . along with mal d 1.02 , and depending on the cultivar , isoforms from 
 the mal d 1.01 cluster are by far the most abundant isoforms found 
 in apples . within the mal d 1.01 cluster , immunologic investigations of naturally 
 occurring mal d 1 isoforms revealed only small differences of their 
 ige binding capacities and it appears that divergent allergenicities 
 of apple strains are predominantly determined by different mal d 1 
 expression levels . whereas the 
 immunological properties of mal d 1 suggest that this 
 protein has a three - dimensional structure and ige binding epitopes 
 that are similar to those of bet v 1 and other members of the pr-10 
 protein family , experimental structural data for mal d 1 have not 
 been available to date . as a first step toward structural characterization , 
 we recently assigned the nmr backbone and side chain h , c , and n chemical shifts of the isoform mal 
 d 1.0101 . golden 
 delicious , were the first isoforms for which the dna sequence 
 was determined and are identical at the amino acid level . here the dna of mal d 1.0101 ( genbank nucleotide 
 code x83672 , protein code caa58646 ) was cloned into the expression vector pet28b 
 by using the restriction sites ncoi and xhoi . construct integrity was ensured by 
 dna sequencing ( microsynth ag , balgach , switzerland ) , and the protein 
 was expressed in escherichia coli bl21 
 star ( de3 ) . mal d 1.0101 was purified by anion exchange and size exclusion 
 chromatography as described in detail elsewhere . the mass and the amino acid sequence of purified mal d 
 1.0101 were confirmed by mass spectrometry using a 7 t fourier transform 
 ion cyclotron resonance ( ft - icr ) mass spectrometer ( bruker daltonics , 
 bremen , germany ) with an attached electrospray ionization ( esi ) source . protein concentrations for nmr spectroscopic experiments for structure 
 determination were 0.5 mm for n / c - labeled 
 and 0.8 mm for n - labeled samples in 91% h2o/9% d2o ( v / v ) at ph 6.9 , 10 mm sodium phosphate , and 
 7 or 11.2 mm l - ascorbic acid , respectively . all nmr experiments 
 were carried out at 298 k , using either a 500 mhz agilent directdrive 
 spectrometer ( agilent technologies , santa clara , ca , usa ) equipped 
 with a room temperature probe or a 600 mhz bruker avance ii+ spectrometer 
 ( bruker biospin , karlsruhe , germany ) equipped with a prodigy cryoprobe . nmr resonance assignments of mal d 1.0101 were made using standard 
 triple - resonance methods and were deposited 
 at the biological magnetic resonance data bank ( bmrb ) under accession 
 no . three - dimensional n and c edited 
 noesy - hsqc experiments ( mixing times of 150 ms ) were recorded for 
 derivation of distance restraints . nmr data were processed using nmrpipe and analyzed with ccpnmr . for measuring protein translational diffusion , we 
 employed a stimulated 
 echo pulsed field gradient nmr experiment . experimental details were identical to those reported for bet v 
 1 . for the determination of the hydrodynamic 
 radius of mal d 1.0101 , we used dioxane as a standard reference under 
 identical buffer conditions , assuming a hydrodynamic radius of 2.12 
  . structure calculations were performed 
 with the program xplor - nih 2.42 using a simulated annealing 
 protocol . an initial structural model was generated with cs - rosetta using the bmrb cs - rosetta server . a total of 2079 distance restraints were obtained 
 from 3d n and c edited noesy - hsqc spectra . noe values were converted on the basis of peak intensities into distances 
 with upper limits of 3.0  ( strong ) , 4.0  ( medium ) , 5.0 
  ( weak ) , and 6.0  ( very weak ) . dihedral angle restraints 
 were predicted using talos+ and cs - rosetta . in all regular secondary structure elements 
 hydrogen bonds were included for backbone amide protons , if the n edited noesy - hsqc spectra did not show a water exchange 
 cross peak . of 100 generated structures , the 20 lowest energy structures 
 were picked and further refined in explicit solvent with the amber14 
 simulation package using pmemd.cuda and the amber force field 99sb - ildn . each structure was soaked into a truncated octahedral 
 solvent box of tip3p water molecules with a minimum wall distance 
 of 10  . for the refinement , hydrogen atoms and water molecules 
 were minimized with fixed heavy atoms . the temperature was increased 
 from 0 to 300 k , where the structures were simulated using the noe 
 distance restraints , minimized again , and validated using the protein 
 structure validation software ( psvs ) suite ( table 1 ) . the coordinates 
 of the mal d 1.0101 structures were deposited in the protein data 
 bank under the accession code number 5mmu . calculated 
 for all residues , using 
 sum over r. largest violation among all 20 reported 
 structures . the three - dimensional 
 structure of mal d 1.0101 consists of a curved , 
 seven - stranded antiparallel -sheet ( 1-7 ) embracing 
 a long helix at the c - terminus of the protein ( 3 ) and two consecutive 
 short helices ( 1 , 2 ) ( figure 1 ) . the edges of the -sheet are formed 
 by strands 1 and 2 , which are connected by helices 1 
 and 2 that form a v - shaped support for the c - terminal part 
 of helix 3 . in total , 35% -sheet 
 and ca . 25% helical structure , agreeing well with secondary structure 
 estimates from infrared and circular dichroism . as in other 
 proteins from the pr-10 family , strands 2 and 3 are 
 connected by a glycine - rich loop motif ( gly46-asn47-gly48-gly49-pro50-gly51 ) . together , these structural elements create the large internal cavity 
 that is typical for the canonical pr-10 fold . from figure 1b it is evident that in our 
 nmr structural ensemble of mal d 1 , secondary structure elements are 
 very well - defined and conformationally homogeneous in all 20 structural 
 models . only slightly elevated levels of conformational heterogeneity 
 are observed for some of the solvent - exposed loops that connect secondary 
 structure elements and the c - terminus of the protein . nmr solution structure 
 of the major apple allergen mal d 1.0101 
 ( pdb accession code 5mmu ) . secondary 
 structure elements are labeled 1 ( val2ser11 ) , 2 
 ( gln40glu45 ) , 3 ( ile53thr57 ) , 4 ( tyr66ile74 ) , 
 5 ( ser80gly88 ) , 6 ( glu96val105 ) , 7 
 ( ser111thr121 ) , 1 ( pro15val23 ) , 2 ( ala26ile33 ) , 
 3 ( lys128asp152 ) . ( b ) backbone overlay 
 of the ensemble of the 20 lowest energy structures of mal d 1.0101 . secondary structure elements are colored from red ( n - terminus ) to 
 purple ( c - terminus ) . a peculiar feature of the pr-10 fold is the large internal 
 cavity . 
 in mal d 1.0101 , the volume of this cavity is ca . 2230  , which is comparable in size to those 
 of other pr-10 proteins . as found in the 
 birch pollen allergen bet v 1 and other homologous food allergens , 
 in mal d 1 the majority of amino acids that form the surface of the 
 cavity are hydrophobic ( figure 2 ) . a large proportion of the inner cavity surface is formed 
 by amino acid residues in the -sheet whose hydrophobic side 
 chains are located at the protein interior ( ile56 ( 3 ) , val67 
 ( 4 ) , ile71 ( 4 ) , tyr81 ( 5 ) , tyr83 ( 5 ) , 
 leu85 ( 5 ) , ile98 ( 6 ) , tyr100 ( 6 ) , ile113 ( 7 ) ) 
 along with inward - pointing residues in the long amphiphilic helix 
 3 ( val132 , val134 , ala139 , leu142 , phe143 , ile146 ) , the two 
 short helices 1 ( phe22 , val23 ) and 3 ( ala26 , ile30 ) , 
 and loop regions ( ile38 , phe58 , tyr64 , ala90 ) . in addition , a few 
 polar and charged side chains are located at the inside of the molecule 
 and form part of the cavity surface , such as asp27 ( 2 ) , his69 
 ( 4 ) , ser115 ( 7 ) , and lys138 ( 3 ) , so that the 
 cavity itself is actually amphiphilic , as noted before for the major 
 birch pollen allergen , bet v 1 . in crystal 
 structures of other pr-10 proteins the cavity is occupied by water , 
 amphiphilic ligand molecules , or components of the crystallization 
 buffer . for mal d 1 , it is currently not 
 known whether ligands bind specifically to the cavity or what the 
 biological function of ligand binding could be . ( a ) internal cavity of 
 mal d 1.0101 , colored according to the lipophilic 
 potential as implemented in moe , where 
 hydrophilic regions are colored in blue and lipophilic regions are 
 colored in yellow . ( b ) surface representation of the lowest energy 
 solution structure of mal d 1.0101 . the two amphiphilic entrances 
 to the internal cavity are indicated as 1 ( between the n - terminal 
 end of helix 3 and the loops connecting strands 34 
 and 56 ) and 2 ( between the edge of the 
 -sheet and the c - terminal end of helix 3 ) . the internal cavity in mal d 1 can be reached by 
 two openings ( figure 2 ) . one entrance to 
 the protein interior , 1 , is shaped by residues in the n - terminal 
 half of helix 3 ( his131 , val134 ) along with the loops connecting 
 strands 34 ( gln63 , tyr64 ) and strands 56 
 ( asp89 ) . together , these amino acids create an amphiphilic access 
 route to the protein interior . a second amphiphilic entrance , 2 , 
 is present at the edge of the -sheet between helix 3 
 ( lys136 , his140 , lys144 , and glu147 ) and strand 1 ( asn7 , phe9 , 
 and ser 11 ) . in the nmr solution structures of mal d 1 this access 
 route entries 
 to the internal cavity at similar locations have also been described 
 for other members of the pr-10 protein family . figure 3 shows 
 a 
 comparison of mal d 1 with bet v 1 and birch pollen - related food allergens 
 from the pr-10 family whose structures have been determined so far . 
 despite the fact that sequence identities between these proteins are 
 only slightly higher than 50% in some cases , their three - dimensional 
 structures are generally very similar , with backbone rmsd values for 
 secondary structures typically below 2  . in light of the observed immunologic cross - reactivity between 
 mal d 1 and the major birch pollen allergen , bet v 1 the backbone 
 rmsd between mal d 1.0101 and the hyperallergenic isoform bet v 1.0101 
 ( 61% sequence identity ) of the birch pollen allergen is 2.13  
 ( 1.70  for secondary structure elements ) . of note , mal d 1 and 
 bet v 1 differ in length by one amino acid , and divergent presumptions 
 have been made about the location of the gap in mal d 1 . on the basis 
 of sequence alignments of pr-10 food allergens it has been proposed 
 that either the loop right before or right after strand 7 is one residue shorter in mal d 1 . our solution 
 structure shows that the loop right before strand 7 is the 
 one that is shorter in mal d 1.0101 ( glu96val105 
 in both mal d 1.0101 and bet v 1.0101 ) and 7 ( ser111thr121 
 in mal d 1.0101 and ser112thr122 in bet v 1.0101 ) occupy identical 
 positions and have equal hydrogen bonding patterns in the antiparallel 
 -sheets of these proteins . they are connected via loops consisting 
 of four residues ( cys - gly - ser - gly in mal d 1 ) and five residues ( thr - pro - asp - gly - gly 
 in bet v 1 ) , respectively , which produces a small structural difference 
 in these loop segments between the two proteins . ( a ) overlay of the lowest energy structure of mal d 1.0101 ( green , 
 pdb accession code 5mmu ) with the structures of the major birch pollen allergen bet v 1.0101 
 ( blue , 4a88 ) , the carrot allergen dau c 1.0103 ( orange , 2wql ) , the 
 celery allergen api g 1.0101 ( gray , 2bk0 ) , the soybean allergen gly 
 m 4.0101 ( yellow , 2k7h ) , the strawberry allergen fra a 1e ( red , 2lpx ) , 
 and the cherry allergen pru av 1.0101 ( purple , 1e09 ) . amino acids are marked with asterisks ( identical ) , 
 colons ( conserved ) , and dots ( semiconserved ) . mal d 1 is known to have a tendency for cysteine - mediated 
 dimerization , 
 as shown for the isoform mal d 1.0108 by nonreducing gel electrophoresis 
 and size exclusion chromatography . like 
 mal d 1.0108 , the isoform mal d 1.0101 contains a single cysteine 
 residue , cys107 . in the three - dimensional solution structure of mal 
 d 1.0101 cys107 is located at the c - terminal tip of strand 7 , 
 with its side chain oriented toward the protein surface . to probe 
 the oligomerization state of mal d 1.0101 under the conditions that 
 we employed for nmr structure determination ( ph 6.9 , 10 mm sodium 
 phosphate , 14 mol equiv of l - ascorbic acid , 298 k ) we performed 
 pulsed - field - gradient nmr diffusion experiments . we obtained a value 
 of 21.6  0.8  for the hydrodynamic radius of mal d 1.0101 , 
 which is comparable to the hydrodynamic radius of monomeric bet v 
 1.0101 ( 20.1  ) under similar experimental conditions . this is consistent with our observation that , 
 using the same buffer , mal d 1.0101 elutes from a size exclusion column 
 with a retention time that is virtually identical to that of bet v 
 1.0101 . these results were further verified by ft - icr mass spectrometry , 
 which shows that mal d 1.0101 does not form dimers or higher order 
 aggregates . the nmr solution structure of mal d 1 shows that 
 this protein consists 
 of a highly curved antiparallel -sheet and three -helices 
 forming a large internal cavity , very similar in fashion to other 
 pr-10 proteins . this is in agreement with 
 the observed immunologic cross - reactivity between mal d 1 and the 
 major birch pollen allergen , bet v 1 , as well as other food allergens 
 from the pr-10 protein family . in most patients bet 
 v 1 is the sensitizing agent , whereas bet v 1-specific ige antibodies 
 subsequently cross - react with mal d 1 and elicit an allergic response , 
 as reflected by the clinical observation that apple allergy develops 
 only after the onset of birch pollinosis . along these lines , cross - inhibition experiments of mal d 1 
 using 
 sera from apple - allergic patients showed that mal d 1 shares ige epitopes 
 with the major birch pollen allergen , bet v 1 . from 
 a structural perspective , limited information about the exact nature 
 of binding epitopes of mal d 1 and bet v 1 is available . detailed 
 structural information about a sequentially discontinuous ( i.e. , conformational ) 
 b - cell epitope in bet v 1 was obtained by cocrystallizing the particular 
 isoform bet v 1.0112 with an antigen - binding fragment ( fab ) derived 
 from the murine monoclonal igg antibody bv16 . this epitope is formed by the segment between glu42 and thr52 ( including 
 the glycine - rich loop motif between strands 2 and 3 ) , 
 along with arg70 , asp72 , his76 , ile86 , and lys97 of bet v 1 , covering 
 approximately 10% ( 900  ) of the entire protein 
 surface . binding of bv16 to this epitope measurably reduces serum 
 ige interactions , indicating that ige and monoclonal igg bv16 compete 
 for overlapping binding surfaces on bet v 1 . moreover , mutation of a central residue ( glu45ser ) significantly 
 reduced the ige binding capacity of bet v 1 , confirming the significance 
 of this particular epitope for interactions with ige . figure 4a shows 
 the molecular interaction surface that corresponds with the bv16 epitope 
 in the apple allergen . in mal d 1 these residues form a contiguous 
 surface patch along with a somewhat distal residue ( glu76 ) , similar 
 in shape and size to the bv16 epitope of bet v 1 . moreover , the contributing 
 amino acids are largely conserved between mal d 1 and bet v 1 . thirteen 
 of the 16 amino acids in the bv16 epitope are identical , whereas only 
 3 residues are different in mal d 1.0101 and bet v 1.0101 ( figure 4b ) . these data thus 
 provide a structural rationale for the observed allergic cross - reactivity 
 between birch pollen and apple allergens . interestingly , mutational 
 studies indicate that the ability of mal d 1 to bind serum ige from 
 patients with birch pollen allergies can be increased by increasing 
 the similarity of the bv16 epitope in mal d 1 to that of bet v 1 , 
 indicating that these amino acids are indeed involved in binding of 
 bet v 1 specific to mal d 1 . amino acid residues that 
 correspond to the molecular interaction surface between monoclonal 
 igg bv16 and bet v 1.0112 ( residues glu42thr52 , arg70 , asp72 , 
 glu76 , ile86 , and lys97 in mal d 1.0101 ) are colored in blue . amino acid positions that were shown to be crucial 
 for ige recognition of mal d 1 in mutational analyses ( thr10 , ile30 , 
 thr57 , ser111 , thr112 , and ile113 ) are shown in green ( ile30 and ile113 
 are located in the protein interior and do not contribute to the surface ) . ( b ) amino acid similarities between bet v 1.0101 and mal d 1.0101 
 using a color gradient from lilac ( highly similar ) to teal ( highly 
 dissimilar ) . epitope residues that are different between bet v 1.0101 
 and mal d 1.0101 are labeled . similarities were calculated on the 
 basis of substitution matrix scores ( blosum62 ) as implemented in moe . it is likely that mal d 1 contains more than a single conformational 
 epitope . a number of amino acid positions 
 that are relevant for ige recognition have been identified by mutational 
 analysis . for a five - point mutant of mal d 1.0108 
 ( thr10pro , ile30val , thr57 asn , thr112cys , 
 and ile113val ) a markedly reduced capacity for binding mal 
 d 1-specific ige was found in vitro . skin 
 prick tests in apple - allergic patients comparing wild - type mal d 1 
 with the five - point mutant further showed a significantly lower ability 
 of the mutant protein to induce skin reactions in vivo . further experiments showed that the t - cell recognition 
 level of wild - type mal d 1 is conserved in the five - point mutant . because these five amino acids are likely involved 
 in ige interactions not only in mal d 1 but also in bet v 1 , they 
 could well be part of common cross - reacting epitopes in these two 
 allergens . this is corroborated by mutational 
 studies , which showed that peptide stretches encompassing these residues 
 are indeed involved in immunological cross - reactivity between mal 
 d 1 and bet v 1 . in addition , in an independent 
 study , ser111 was identified as being essential for ige binding to 
 mal d 1 , and a ser111cys mutation resulted in significantly 
 reduced affinity for ige in immunoblotting experiments . figure 4a shows 
 that these six residues are fairly dispersed on the protein surface 
 of mal d 1 and that neither of these amino acids overlaps with the 
 bv16 epitope . amino acids thr10 , ser111 , and thr112 form a common 
 patch on the protein surface , whereas thr57 is located approximately 
 3739  away and close to the bv16 epitope . considering 
 that an epitope of typical size ( 600900  ) and circular shape would have 
 an arc length of 2834  on the mal d 1 surface , residues 
 thr10 , ser111 , and thr112 are probably too far away from thr57 to 
 be part of a common binding epitope . the remaining two residues , ile30 
 and ile113 , do not reach the protein surface in mal d 1 . whereas ile113 
 is close in space to the thr10-ser111-thr112 patch , its hydrophobic 
 side - chain is pointing toward the interior of the protein , where it 
 participates in a small hydrophobic core located at the inner end 
 of the proteins cavity ( between helices 1 and 3 
 and the -sheet ) . residue ile30 is also located in the protein 
 interior with its aliphatic side chain forming part of the internal 
 cavity and does not contribute to the protein surface . of note , 
 because the loop between strands 6 and 7 
 is shorter by one residue in mal d 1 than in bet v 1 , ser111 and thr112 
 of 7 in mal d 1 occupy the 7 positions of ser112 and 
 ile113 in bet v 1 . the surface patch formed by thr10 , ser111 , and 
 thr112 in mal d 1 thus appears to be less hydrophobic than the corresponding 
 surface patch in bet v 1 ( thr10 , ser112 , and ile113 ) . as a matter 
 of fact , also the protein surface surrounding these three residues 
 displays a considerably lower level of similarity between mal d 1.0101 
 and bet v 1.0101 than other parts of the protein surface , as can be 
 seen in figure 4b . this might in part be responsible for the different ige binding properties 
 of these allergens . it has been noted , on the other hand , that epitope 
 coincidence between bet v 1 and mal d 1 may be limited , as exemplified by a recent study describing 
 the isolation of human ige binding to bet v 1 but not to mal d 1 . moreover , different mal d 1 isoforms contain 
 amino acid substitutions within potential ige interaction surfaces , suggesting that they may influence the immunologic 
 reaction . it is clear that high - resolution structural data provide 
 the basis 
 to determine and compare structural details of ( cross - reactive ) binding 
 epitopes in allergenic proteins . in addition , grafting of conformational 
 epitopes by transferring stretches of residues between homologous 
 allergens has become a valuable experimental tool . epitope grafting 
 was used to characterize the role of the bv16 epitope in mal d 1 by 
 recreating this epitope on the mal d 1 surface , confirming its importance 
 for ige binding and cross - reactivity with bet v 1 . in an orthogonal approach , several mal d 1 stretches encompassing 
 residues that are crucial for ige binding were transferred to bet 
 v 1 to investigate the role of these structural segments for cross - reactivity , and chimeras of bet v 1 and mal d 1 were created 
 to map the epitope of a human monoclonal ige , which was isolated from 
 a phage library , to the c - terminus of bet v 1 . in addition , epitope grafting provides access to chimeric 
 allergens with fine - tuned antigenic properties , such as reduced ige 
 binding capacitites , for molecule - based allergy diagnosis and specific 
 immunotherapy . knowledge of the structural 
 details of these allergens elements is required to generate correctly 
 folded chimeras , because transfer of ( partly ) mismatching stretches 
 of secondary structure between different allergens may well be the 
 reason for a loss of protein fold and , consequently , reduction of 
 ige - binding capacities . the three - dimensional 
 structure of mal d 1.0101 presented here provides the biophysical 
 basis for elucidating the molecular details of immunological cross - reactivity 
 in great detail .
more than 70% of birch pollen - allergic 
 patients develop allergic 
 cross - reactions to the major allergen found in apple fruits ( malus domestica ) , the 17.5 kda protein mal d 1 . 
 allergic 
 reactions against this protein result from initial sensitization to 
 the major allergen from birch pollen , bet v 1 . 
 immunologic cross - reactivity 
 of bet v 1-specific ige antibodies with mal d 1 after apple consumption 
 can subsequently provoke severe oral allergic syndromes . 
 this study 
 presents the three - dimensional nmr solution structure of mal d 1 ( isoform 
 mal d 1.0101 , initially cloned from 
 granny smith apples ) . 
 this protein is composed of a seven - stranded antiparallel -sheet 
 and three -helices that form a large internal cavity , similar 
 to bet v 1 and other cross - reactive food allergens . 
 the mal d 1 structure 
 provides the basis for elucidating the details of allergic cross - reactivity 
 between birch pollen and apple allergens on a molecular level .