Structural Study of Binding of Flagellin by Toll-Like Receptor 5

In order to predict the binding regions within the complex formedby Toll-like receptor 5 (TLR-5) and flagellin, a complementaryhydropathy between the two proteins was sought. A region commonto the flagellins of Salmonella enterica serovar Typhimurium,Pseudomonas aeruginosa, and Listeria monocytogenes was shownto be hydropathically complementary to the 552-to-561 fragmentof TLR-5, whose sequence is EILDISRNQL. The hydrophobicity profileof this region is shared with flagellins of 377 bacterial speciesout of a total of 723 publicly available sequences. A conformationalanalysis of the predicted binding site of TLR-5, whose structureis still unknown, was carried out with a methodology alreadyapplied to similar problems. To sample the conformations availableto the peptide chain, a plot of the number of conformationsper unit energy interval (density of states) versus energy wasbuilt. Following a theoretical argument, conformations belongingto maxima in this plot were selected. The most stable structureobtained in this search, an ${alpha}$ -helical conformation, was shownto form the electrostatic interactions Glu552-Gln89, Asp555-Arg92,and Arg558-Glu93 with the predicted binding site of the flagellinof S. enterica serovar Typhimurium, formed by the 88-to-97 chainfragment (LQRVRELAVQ), which is likewise ${alpha}$ helical.

INTRODUCTION

Top

Introduction

Binding of bacterial flagellin to Toll-like receptor 5 (TLR-5)has been shown (13) to elicit recognition and response by theinnate immune system. The sequences of the flagellins of Salmonellaenterica serovar Typhimurium (11), Pseudomonas aeruginosa (31),and Listeria monocytogenes (27), for which TLR-5 activity hasbeen demonstrated (13), have similar regions that may be thebinding sites of TLR-5. The same sequence identity, limitedto regions that include those considered important in this work,is extended to hundreds of known (http://us.expasy.org/sprot/sprot-top.html)flagellin sequences but not to all of them. Presumably, forthis subset of flagellins, a common structure of the TLR-5-flagellincomplex may be found.

To accomplish such a study, we made use of a strategy that,in this laboratory (6, 21, 28, 34) and elsewhere (14), has provedits value for the prediction of sequences of putative peptideligands and the location of receptor binding sites. A hypothesis(15) that enables prediction of the structure of a peptide ligandwas also used.

The complementary-hydropathy principle (3, 4, 5, 25) postulatesthe existence of stabilizing interactions between two peptidechains in whose sequences there is a correspondence betweenhydrophobic (hydrophilic) residues in one chain and hydrophilic(hydrophobic) residues in the other chain. According to thisprinciple, two complementary peptide chains may form a complex,even though a pairwise binding of all complementary amino acidresidues is not necessary.

There are now 69 reports of the successful exploitation of complementaryhydropathy for the identification of interacting proteins (14),as opposed to only 3 negative reports (1, 9, 12), 1 of which(5) has already been refuted. Furthermore, the definition throughthis strategy of the binding site of collagenase (28) was subsequentlyconfirmed (19) by site-directed mutagenesis. Also, the predictedsite in the cellular prion molecule to which a putative ligandreceptor would bind (21) was confirmed after the complete characterizationof this molecule (34) by peptide inhibition of binding betweenthe two molecules, as well as by removal (34) of the cellularprion binding site by sire-directed mutagenesis.

In the following discussion, it is shown how a search for complementaryhydropathy between the sequences of TLR-5 and flagellin enablesthe prediction of binding sites. There still remains the problem,however, that although the structure of the flagellin of S.enterica serovar Typhimurium (26) has been determined by X-raydiffraction analysis, the structure of TLR-5 has not been experimentallydetermined.

To determine the structure of the TLR-5 fragment predicted tobind flagellin, we made use of a hypothesis that had alreadybeen applied to similar problems, such as ${alpha}$ -MSH (18) and substrates(17) of oligopeptidase EC 24.15. According to this hypothesis,the structures adopted by peptide chains belong to conformationalfamilies that remain the same in different chemical environments.A transition to a new chemical environment causes a change inthe probabilities of occurrence of the various conformationalfamilies so that, for instance, a conformational family thathas a very low probability in an aqueous solution may have avery high probability in a lipid solution and vice versa.

According to the same hypothesis, a peptide chain and a proteinfragment with the same sequence also occupy the same conformationalfamilies, although with different probabilities. Thus, variousconformational families occupied by the predicted TLR-5 bindingsite may be tested against flagellin.

MATERIALS AND METHODS

Top

Materials and Methods

To find the hydropathically complementary regions that are thepredicted binding sites of TLR-5 and flagellin, the sequenceof TLR-5 (7) was scanned against the sequences of the flagellinsof S. enterica serovar Typhimurium (11), P. aeruginosa (31),and L. monocytogenes (27) with the in-house software HYDROLOG.These sequences were also submitted to a comparison, performedwith the CLUSTALW (30) software, with 723 flagellin sequencesdownloaded from the SwissProt database (http://us.expasy.org/sprot/sprot-top.html).A conformational search (16) of the TLR-5 binding site was thencarried out with five main chain rotamers (A [ ${phi}$ = -57, ${psi}$ = -47],B [ ${phi}$ = -139, ${psi}$ = 135], G [ ${phi}$ = -60, ${psi}$ = -30], D [ ${phi}$ = -90, ${psi}$ = 0], andE [ ${phi}$ = 70, ${psi}$ = -60], where ${phi}$ and ${psi}$ [reference 18 and referencesquoted therein] are main chain dihedral angles) as describedin the Appendix. For the side chain conformations, we made useof the gauche minus, trans, and gauche plus rotamers of theclassification of Ponder and Richards (23) (numerals 1, 2, and3 indicate, respectively, the gauche minus, trans, and gaucheplus rotamers of ${chi}$ ₁). Internal energies were calculated withthe ECEPP/2 (35) force field. A search for complementary featureson the surfaces of the binding site of TLR-5 and the S. entericaserovar Typhimurium flagellin, whose structure (26) was downloadedfrom the Protein Data Bank (2) website, was then carried outwith translations and rotations of the two structures untila maximum score of hydrogen bonds and salt bridges was found.This search was done by hand with the software WEBLAB (providedby Accelrys) and WHAT IF (32). After that, the complex was dockedwith the HEX (24) software by using the parameters listed onpage 11 of the manual (http://www.biochem.abdn.ac.uk/hex). Structuresof the TLR-5-flagellin complexes of L. monocytogenes and P.aeruginosa were obtained with WHAT IF by making point mutationsand structural optimizations in the TLR-5-flagellin complexof S. enterica serovar Typhimurium.

RESULTS AND DISCUSSION

Top

Results and Discussion

Table 1 lists hydropathically complementary fragments belongingto TLR-5 and the flagellins of three bacterial species. Although,as shown in Table 1, many instances of complementary hydropathybetween TLR-5 and the flagellins were found, there is only oneTLR-5 region that, in all three cases, meets the criteria C≥ Cm and L = 10 (C [defined in the equation in the Appendix],Cm [= 0.75], and L are, respectively, the actual and minimumcomplementary hydropathies and the fragment length). It wastherefore concluded that this chain fragment is the bindingsite of TLR-5.

View this table:
[in this window]
[in a new window]
TABLE 1. Regions of the TLR-5 sequence for which a complementary hydropathy with the flagellins of three bacterial species was found^a

In Fig. 1, the resemblance between the hydrophobicity profilesof the predicted binding sites of the three flagellins and theircomplementary hydropathies to the TLR-5 binding site are shown.

View larger version (16K):
[in this window]
[in a new window]
FIG. 1. Hydrophobicity profiles (H, hydrophobicity measured by the Kyte and Doolittle scale [20]; aa, amino acid) of the peptides listed in Table 1. Solid line, TLR-5 binding site; dotted line, peptide I; dashed line, peptide II. The hydrophobicity profiles of peptides I and III are almost indistinguishable.

More specifically, the predicted binding site of TLR-5 is the552-to-561 chain fragment, whose sequence is EILDISRNQL. A comparisonwith 723 flagellin sequences downloaded from the SwissProt (http://us.expasy.org/sprot/sprot-top.html)database showed that 377 flagellin sequences share with theflagellins of Table 1 binding regions having a sequence identityequal to or greater than 60% and hydrophobicity profiles similarto those of the predicted flagellin binding sites. This findingis in accordance with the previous finding (22) that hydropathyprofiles tend to be conserved despite a lower sequence identitylevel. For these flagellins, a common TLR-5-flagellin complexmay exist.

However, an additional difficulty is posed by the nonavailabilityof an experimentally determined structure of TLR-5. Althoughthe structures of TLR-1 and TLR-2 are known (33), there is onlypartial homology among the sequences of TLR-1, TLR-2, and TLR-5and this sequence homology does not encompass the predictedbinding site. Therefore, to carry out the intended study, wecounted only on the sequences of flagellin binding sites, thestructure of S. enterica serovar Typhimurium flagellin, andthe sequence of the TLR-5 binding site.

To cope with the absence of a TLR-5 structure, we did an exhaustivesearch of the conformations of its binding site that generated14,414 different structures. The assumption that this high numberof structures may be classified in a limited (15, 18) numberof conformational families facilitated the, otherwise almostimpossible, task of determining the active structure.

A set of conformational families available to the binding regionsof TLR-5 was thus generated as described in the Appendix. Structuresbelonging to some of these conformational families are representedin Fig. 2.

View larger version (22K):
[in this window]
[in a new window]
FIG. 2. Density-of-states ( ${delta}$ ) versus energy (E, in kilocalories per mole) plot obtained for the 552-to-561 (EILDISRNQL) fragment of TLR-5. The energy was rescaled so that the minimum energy corresponds to zero. Thirteen density-of-states maxima are labeled. Some of the conformations found within these maxima are GGGAAAAAAD3112122121 (I), GGGAAAAAAD3112122111, GGGAAAAAGD3112122111 (II), GGGAAAAAAA3112122111 (III), GGGAAAAABA3112122131 (IV), GDDAAAAAGD1311122121 (V), GDDAAAAAAD1111121111 (VI), DDDGGAAAAG3311121121 (VII), GDDDAAAAAG2113211111 (VIII), DDDAAAAABD1311122131 (IX), GDDDDGAAAD1213111111 (X), DDDAAAAAGD1111231221 (XI), EGABBBBGAA1112221111 (XII), and BBDBGBDABG1311123131 (XIII), where the letters and numerals indicate, respectively, main chain and side chain rotamers, as defined in Materials and Methods. Structures found within three maxima are depicted. Stick representations of the polypeptide chain and ribbon representations of the secondary structure are shown.

In the density-of-states versus energy plot shown in Fig. 2(see the Appendix for details), there are 350 density-of-statesmaxima. It has been argued (15, 16) that each maximum containsthe most stable conformations in the corresponding energy range.Although the number of maxima approaches the number of conformationalfamilies, more than one conformational family may be found withinthe same density-of-states maximum, so that the determinationof conformational families is not a straightforward procedure.

In the present study, however, the condition that TLR-5 formsa stable complex with flagellin considerably restricted thesearch. The most obvious choice, the ground level conformation(lists of rotamers that define chain conformations are shownin the legend to Fig. 2) that in Fig. 2 corresponds to the maximumlabeled I in the density-of-states versus energy plot and toconformation I, was considered first.

Docking (see Materials and Methods) of this ${alpha}$ -helical conformationto the, likewise ${alpha}$ -helical, predicted binding site of flagellinshowed that, as shown in Fig. 3, three electrostatic interactionsmay form. More specifically, these are the TLR-5-flagellin interactionsGlu552-Gln89, Asp555-Arg92, and Arg558-Glu93. Model building(data not shown) showed that the Arg558-Glu (or other residuein position) 93 interaction is maintained even when Glu93 isreplaced with glutamine in the L. monocytogenes flagellin andby aspartate in the P. aeruginosa flagellin.

View larger version (21K):
[in this window]
[in a new window]
FIG. 3. Complex formed between the binding sites of flagellin and TLR-5 predicted by the complementary-hydropathy principle. (A) Flagellin complexed to the binding site of TLR-5. Stick representations of the 87-to-97 and 552-to-561 chain fragments, respectively, are shown. Ribbon representations of the flagellin structure and the TLR-5 binding site are also shown. (B) Stick representation of the binding sites of flagellin (gray) and TLR-5 (black). Hydrogen-bonded side chains are represented by dotted lines.

The same complementary positioning between side chains of TLR-5and flagellin would occur, in principle, in other TLR-5 conformationswhose 552-to-558 fragment has the same conformation of the previouslydescribed structure (GGGAAAA) and whose Glu552, Asp555, andArg558 rotamers are also the same (respectively, 3, 2, and 2).Five such conformations were found within the density-of-statesmaxima labeled I, II, III, and IV in Fig. 2.

On the other hand, TLR-5 conformations distorted in relationto the above-shown conformation would not have the requiredpositioning of side chains. Inspection of the conformationsfound within the density-of-states maxima labeled V to XIIIin Fig. 2 showed that, in every case, the three described electrostaticinteractions cannot form because the side chain positioninghas been disrupted, as a comparison between conformations Iand VIII and I and XII, depicted in Fig. 2, would show. Thesefindings were confirmed by docking (see Materials and Methods)conformations VIII and XII (depicted in Fig. 2) to flagellin.

The binding site of flagellin found in the present study islocated within constant domain D1, which is relatively conserved(8) among different species. Two regions required for TLR-5activation were experimentally (8) identified in this domain.One region, on the N-terminal side, includes amino acids 57to 190 and therefore includes a fragment sequentially homologousto the predicted binding site at positions 88 to 97. The otherregion is on the C-terminal side. It has been shown (8), withthe aid of linker insertion mutagenesis experiments, that withinthis region, the short stretch from 485 to 489 (GAIQN) is necessaryfor interleukin-8 release. A search for complementary hydropathywith the parameters Cm ≥ 0.75 and L = 5 identified the 484-to-488chain fragment (LGAIQ) in the sequence (29) of enteroaggregativeEscherichia coli, which is hydropathically complementary tothe 323-to-327 (KINKI) TLR-5 fragment. These fragments werenot included in this molecular modeling study because the 484-to-489flagellin fragment is not part of the experimentally determined(26) structure and because a search of short (L = 5), hydropathicallycomplementary peptide fragments is too unrestricted and is likelyto yield false positives.

The present study is part of the broader research field of protein-proteininteractions. It has been shown (24) that surface complementarity,i.e., optimal positioning of interacting side chains, is aneffective predictor of complexes formed by proteins. This criterionis in accordance with our calculations that showed that theside chains of Gln89, Arg92, and Glu93 on the surface of flagellinare optimally positioned to interact, respectively, with theside chains of Glu552, Asp555, and Arg558 on the surface ofthe predicted TLR-5 binding site.

Although the proposed TLR-5 and flagellin binding sites arehydropathically complementary, the predicted complex is stabilizedby electrostatic interactions between hydrophilic side chains.In this study, we used an in silico search for complementarypeptides in interacting proteins, combined with conformationalanalysis calculations, to predict the binding sites and thestructure of the complex.

APPENDIX

Top

Appendix

Search for complementary hydropathy. The theoretical conceptsbehind the complementary-hydropathy principle have been welldescribed previously (3, 4, 14, 25). Herein, we describe themethod developed to find complementary ligand and receptor regions.

Amino acid residue hydrophobicities are evaluated with the Kyteand Doolittle (20) scale. The degree of complementary hydropathybetween two peptide fragments of length L is calculated (10)with the equation

where H(i) and H'(i) are the Kyte and Doolittle indices of aminoacid i in each sequence and Abs is absolute value. In the presentstudy, the sequences of TLR-5 and flagellin were examined.

The receptor sequence is scanned against the ligand sequencein order to find regions corresponding to a high degree of complementaryhydropathy, as calculated by the equation above. Important parametersin this search are the window length (L) and the minimum complementarity(Cm). Each i...i + L fragment in the receptor sequence is searchedagainst fragments j...j + L in the ligand and those fragmentsfor which C ≥ Cm are tabulated.

Determination of the conformational families of a peptide ligand.When there is substantial evidence that a certain peptide chainor a region of a longer chain is the binding site or peptideligand that we are looking for but the corresponding structureis not known, we can generate a reasonably complete set of conformationalfamilies and test the binding of each one.

To carry out the search for conformational families, a plotof the density of states ( ${delta}$ , which is the number of conformationsper unit energy interval) versus energy is obtained. It hasbeen argued (15, 16) that minima in this plot correspond tokinetic barriers in the potential energy landscape and maximacorrespond to energy minima that, in turn, correspond to conformationalfamilies. Therefore, the number of conformational families availableto a peptide chain is approached by the number of maxima ina density-of-states versus energy plot.

The density-of-states versus energy plot shown in Fig. 2 includes14,414 conformations of the 552-to-561 region of TLR-5. Theseconformations were generated with a matrix algorithm (16) andthe adoption of a set (see Materials and Methods) of five mainchain and three side chain conformations. The internal energyof each conformation was calculated with the ECEPP/2 (35) forcefield.

FOOTNOTES

* Corresponding author. Mailing address: Fundação Antônio Prudente, Centro de Pesquisas, Rua Prof Antonio Prudente 211, São Paulo, SP 01509-090, Brazil. Phone: 55 11 32725000. Fax: 55 11 32725088. E-mail: sgjacchieri{at}procenio.com.

REFERENCES

Top