1472-6807-9-46, bioinformatyka, artykuly

//-->.pos {position:absolute; z-index: 0; left: 0px; top: 0px;}BMC Structural BiologyResearch articleBioMedCentralOpen AccessSequence and structural analysis of the Asp-box motif and Asp-boxbeta-propellers; a widespread propeller-type characteristic of theVps10 domain family and several glycoside hydrolase familiesEsben M Quistgaard1,2and Søren S Thirup*1Address:1MIND Centre, Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10C, DK 8000 Århus C, Denmark and2Department of Medical Biochemistry and Biophysics, Karolinska Institute, 17177 Stockholm, SwedenEmail: Esben M Quistgaard - eq@mb.au.dk; Søren S Thirup* - sth@mb.au.dk* Corresponding authorPublished: 13 July 2009BMC Structural Biology2009,9:46doi:10.1186/1472-6807-9-46Received: 14 May 2009Accepted: 13 July 2009This article is available from: http://www.biomedcentral.com/1472-6807/9/46© 2009 Quistgaard and Thirup; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.AbstractBackground:The Asp-box is a short sequence and structure motif that folds as a well-definedβ-hairpin. It is present in different folds, but occurs most prominently as repeats inβ-propellers.Asp-boxβ-propellersare known to be characteristically irregular and to occur in many medicallyimportant proteins, most of which are glycosidase enzymes, but they are otherwise not wellcharacterized and are only rarely treated as a distinctβ-propellerfamily. We have analyzed thesequence, structure, function and occurrence of the Asp-box and s-Asp-box -a related shortervariant, and provide a comprehensive classification and computational analysis of the Asp-boxβ-propeller family.Results:We find that all conserved residues of the Asp-box support its structure, whereas theresidues in variable positions are generally used for other purposes. The Asp-box clearly has astructural role inβ-propellersand is highly unlikely to be involved in ligand binding. Sequenceanalysis of the Asp-boxβ-propellerfamily reveals it to be very widespread especially in bacteria andsuggests a wide functional range. Disregarding the Asp-boxes, sequence conservation of thepropeller blades is very low, but a distinct pattern of residues with specific properties have beenidentified. Interestingly, Asp-boxes are occasionally found very close to other propeller-associatedrepeats in extensive mixed-motif stretches, which strongly suggests the existence of a novel classof hybridβ-propellers.Structural analysis reveals that the top and bottom faces of Asp-boxβ-propellers have striking and consistently different loop properties; the bottom is structurallyconserved whereas the top shows great structural variation. Interestingly, only the top face is usedfor functional purposes in known structures. A structural analysis of the 10-bladedβ-propellerfold,which has so far only been observed in the Asp-box family, reveals that the inner strands of theblades are unusually far apart, which explains the surprisingly large diameter of the central tunnelof sortilin.Conclusion:We have provided new insight into the structure and function of the Asp-box motifand of Asp-boxβ-propellers,and expect that the classification and analysis presented here willprove helpful in interpreting future data on Asp-box proteins in general and on Asp-boxβ-propellers in particular.Page 1 of 18(page number not for citation purposes)BMC Structural Biology2009,9:46http://www.biomedcentral.com/1472-6807/9/46BackgroundThe Asp-box is a phylogenetically ubiquitous sequenceand structure motif. It was first described as a short repeatmotif with consensus sequence S-X-D-X-G-X-T-W distin-guishing bacterial from influenza sialidases or neuramin-idases (the two terms are equivalent) [1]. For this reasonit is also known as the bacterial neuraminidase repeat(BNR repeat). However, it is now clear that it is neitherlimited to sialidases nor to bacteria and we therefore pre-fer the term Asp-box, which is also the more prevalentterm in the literature. The structure ofSalmonella typhimu-riumLT2 sialidase [2] was the first structure to be deter-mined of a bacterial sialidase or indeed any glycosidehydrolase family 33 (GH33) protein; bacterial, eukaryoticand some related viral sialidases all belong to this family.This structure revealed that the GH33 catalytic domainadopts a 6-bladedβ-propellerfold with the Asp-boxesspanning the loop between the two outer strands, strand3 and 4, of the four-stranded 'up-and-down' propellerblades [2], and that the Asp-box itself adopts aβ-hairpinfold [2,3]. Structures of GH33 sialidases, trans-sialidasesor sialidase-like proteins have since been determinedfrom a variety of organisms including bacteria [2,4-9],trypanosomes [10,11], leech [12] and man [13]. Asp-boxrepeats have furthermore been observed in equivalentpositions in the structures of several otherβ-propellerdomains including the 6-bladedβ-propellerof the bacte-riophage K1F sialidase (GH58 family) [3,14], the tandem7-bladedβ-propellersof GH74 hydrolases [15-17] andthe 10-bladedβ-propellersof the Vps10-domain (Vps10-D) receptors, which stand out from all other structurallycharacterized Asp-boxβ-propellersby not being carbohy-drate active enzymes [18,19]. The Asp-box is however notrestricted to theβ-propellerfold, but also occurs in thejelly-roll subrepeats of reelin [3,20], as singlet in theimmuno-globulin-like (Ig-like) C-terminal domains ofchitobiase (GH20 family) and sulfite oxidase and as sin-glet in the central 'up-and-down'β-sheetof the microbialribonuclease fold [3,21]. Furthermore, a shorter variant ofthe Asp-box, missing the conserved glycine, has recentlybeen identified. This motif occurs in the arabinase/levan-sucrase/invertase group of 5-bladedβ-propellersand insome carbohydrate binding modules [15]. The Asp-box isjust one among several repeats that are found in specificpositions inβ-propellersand defines a particular propel-ler family. Others include for example the WD40, kelch,YWTD (LDL receptor class B), PQQ (tryptophan dockingmotif), NHL and RCC1 repeats, which seem to generallyfunction in folding and/or stabilization of the propellers.Such functions may also apply to the Asp-box [5,22], butfunctions in carbohydrate binding or secretion have alsobeen proposed [2,3]. The Asp-boxβ-propellerfamilystands out from otherβ-propellerfamilies in at least threeways; firstly Asp-boxβ-propellersare unusually irregular[23], secondly Asp-boxes are often missing in several andindeed sometimes most of the blades e.g. the 6-bladedβ-propeller of bacteriophage K1F sialidase has just two Asp-boxes [14], and thirdly it is so far the onlyβ-propellerfamily that encompass a 10-bladed fold. The considerablestructural work carried out on Asp-box glycosidasesreflects a fundamental interest in the mechanisms of theseenzymes, but implications in diseases has also been amotivating factor. Bacterial sialidases serve parasitic nutri-tional functions for several pathogens and may act directlyas virulence factors in some diseases e.g. cholera, gas gan-grene, septiacemia, meningitis and cystic fibrosis [24],trypanosomal sialidases are crucial for the life-cycles ofthe parasitic species causing sleeping sickness and Chagasdisease [25,26], human Neu3 sialidase is a potential targetfor cancer treatment [27], Neu1, Neu3 and Neu4 may finduse in cancer diagnosis [28] and mutations in Neu1 arethe cause of the lysosomal storage disease sialidosis [29].Structural insight into the Vps10-D family has justrecently been obtained by the structure determination ofsortilin [19], but more structures will probably follow,since these proteins are now emerging as important play-ers for central cellular functions in sorting and signalingand are firmly linked to several human disorders e.g.SorLA is implicated in Alzheimers disease, SorCS1 in type2 diabetes and sortilin in age and trauma-induced neuro-nal cell death [30]. An examination of the structure andoccurrence of the Asp-box as well as a discussion of itsevolution and function has been published previously byCopley et al. [3]. However, at that time the only knownAsp-boxβ-propellerswere a number of sialdiases. Onlyvery recently has it been realized, that Asp-boxes are alsofound in severalβ-propellersnot belonging to the siali-dase group [15]. Here we provide an updated and consid-erably more detailed analysis of the structure and functionof the Asp-box motif and a much needed classificationand comprehensive analysis of the Asp-boxβ-propellerfamily.Results and DiscussionSequence and structure of the Asp-boxFrom the full Pfam BNR alignment, which is based on5236 sequences, we find that the Asp-box consensussequence can be expressed as X-X-(S/t)-X-(D/n)-X-G-X-(T/s)-(W/f/y)-X where capital letters represent the most con-served residue in a given position, small letters representmore or less commonly seen alternatives and X signifiesvariable positions. The additional variable residues at thebeginning and end of the motif are included to indicatethat they too are structurally conserved. Positions 3, 5, 7and 10 are all strongly conserved (JalView conservationscores of 9–10 for the full Pfam BNR alignment), whereasthe T/s of the 9thposition is only partly conserved (JalViewscore 4). The most preferred residues in positions 2, 6 and8 are R, G and K respectively, but they were omitted fromthe expression due to very weak conservation (JalViewPage 2 of 18(page number not for citation purposes)BMC Structural Biology2009,9:46http://www.biomedcentral.com/1472-6807/9/46score of 2). The Asp-box forms a structurally conservedhairpin loop, which is delineated by the two main chainhydrogen bonds between the residues at position 2 and11, typically leaving the remaining residues to constitutea type 8:8β-hairpin[31]. However, since the main chaincarbonyl group at position 9 points into the loop towardsthe amides at positions 4 and 5 the loop can occasionallybe classified differently (Figure 1A). The structural conser-vation is reflected in the distribution of phi and psi angles,and two standardβ-turns[32] are easily recognized; a typeI' turn at position 5–8 and a type VIII turn at position 7–10 of the motif (Figure 1E). The loop structure is furtherstabilized by an intricate network of hydrogen bondsinvolving the conserved residues of the Asp-box motif anda water molecule in a conserved position (Figure 1A). Thewater molecule is coordinated by the main chain amide inthe 4thand the main chain carbonyl in the 9thposition andmost often also the side chain hydroxyl in position 9when a threonine or serine is found here (although not inthe example shown in Figure 1A). The hydrogen bondingnetwork, though slightly variable, is in general constitutedby the following interactions; the conserved hydroxylgroup at position 3 forms a hydrogen bond to the mainchain amide at position 6, the conserved D/n is withinhydrogen bonding distance of several main chain amidesand also hydrogen bonds to the side chain hydroxyl of theS/t in position 3 as well as T/s if present in position 9. TheCβ of S/t in position 3 forms Van der Waal contacts to theside chain of the conserved aromatic residue in position10 and when this is conserved as a tryptophan, which isstrongly favored, a hydrogen bond is generally formedbetween the Nξ1 atom and the main chain carbonyl of theglycine in position 7. The conserved residues thus clearlycontribute to maintaining the fold. Divergence from theconsensus sequence is tolerated to some extent, but noother residues than D/n are found in position 5 and nonon-aromatic residues are found in position 10 in anyknown structures. Furthermore, rare examples are foundof Asp-boxes that conform to the consensus sequence butare distorted in structure. Two such examples are the Asp-box in blade 5 ofM. viridifacienssialidase [5] and the Asp-box in blade 6 of sortilin. In the case of sortilin, the con-served aspartate in position 5 is forced into an unusualposition by forming ionic interactions with an arginineside chain of the interacting 10cc-b domain, which leadsto a marked change in the loop structure. A motif thatappears to be derived from the Asp-box has recently beenidentified. It lacks the conserved G and so conforms to thesimplified consensus S/t-X-D/n-X-X-X-W/f/y [15] (Figure1B). The structures of these motifs, which we name s-Asp-boxes, s is for short, are conserved and overall similar tothat of the regular Asp-box in positioning of the conservedside chains and the structurally conserved water molecule(Figure 1D). The absence of the glycine in position 7 iscompensated for by changes in the psi-angle of the twopreceding residues and in the phi angle of position 8. As aresult of this, no standardβ-turnsare recognized in the s-Asp-box, though the traces of the two loops in the Ram-achandran plot are similar (Figure 1E–F). Due to theshorter loop, the hairpin of the s-Asp-box is typically clas-sified as a type 7:7β-hairpin.Occurrence of Asp-boxes in different foldsWe have performed a structure-based search for the Asp-box motif in the pdb database and find that it in additionto the already known occurrences listed in the introduc-tion, also occurs as repeats in the 7-bladedβ-propellerofthe functionally uncharacterized bacterial YP_299179.1protein labeled 'glycosyl hydrolase' in the NCBI database,as singlet in the 5-bladedβ-propellerof inulinase (GH32family) and not surprisingly as singlet in the Ig-like dimer-ization domains of bacterial sulfite dehydrogenase andnitrate reductase, both of which are related to the dimeri-zation domain of eukaryotic sulfite oxidase, which isalready known to contain a single Asp-box [3]. We con-clude that Asp-boxes occur in 5-bladed (rare occurrence inGH32), 6-bladed (GH33, GH58), 7-bladed (GH74,'YP_299179.1 family') and 10-bladed (Vps10-D)β-pro-pellers, in theβ-sandwichfolds of sulfite oxidase, somestructurally related enzymes, in reelin and in chitobiase(GH20 family), and finally in the microbial ribonucleasedomain characteristic of barnase, binase, RNase Sa, Sa2and Sa3. A structure-based search has also been performedfor the s-Asp-box variant. This motif is found as repeats orsinglet in the 5-bladedβ-propellersof the GH32 andGH43 families and as singlet in theβ-sandwichfold ofmany family 32 carbohydrate binding modules (CMB32domains), which is in good agreement with previousobservations [15]. In addition it occurs in the F5/F8 typeC domain of neuropilins and coagulation factors V andVIII, which is not surprising, since these domains adoptthe same fold as the CBM32 domain. Finally it occurs inthe centralβ-sandwichof the PqqB coenzyme PQQ syn-thesis protein and in the anti-parallelβ-sheetof the N-ter-minal domain of type III pantothenate kinase (type IIIPanK) fromThermotoga maritima.The fold of the latterbelongs to the widespread Ribonuclease H-like family[33], which does not generally encompass an s-Asp-box.Indeed an s-Asp-box is even missing in type III PanK fromBacillus anthracisand it therefore seems likely that it hasevolved relatively recent inThermotoga maritimaby chanceconvergence.Structural contexts and functions of Asp-boxes in non-propeller foldsIn Ig-like and jelly-rollβ-sandwichdomains the Asp-boxis rather surface exposed and is often involved in support-ing domain-domain interactions. The Asp-box bendsaway from the sandwich interface and the conserved aro-matic residue in position 10 is found on the outer face ofPage 3 of 18(page number not for citation purposes)BMC Structural Biology2009,9:46http://www.biomedcentral.com/1472-6807/9/46Figure 1(see legend on next page)Page 4 of 18(page number not for citation purposes)BMC Structural Biology2009,9:46http://www.biomedcentral.com/1472-6807/9/46Figure 1of the Asp-box andStructure(see previous page)short-Asp-box motifsStructure of the Asp-box and short-Asp-box motifs.A. The main chain of the Asp-box of blade 1 of 2bf6 and side chainsin highly conserved positions are shown as sticks. The structurally conserved water molecule is shown as a sphere. Colorationis by atom type. Hydrogen bonds involving the four shown side chains and the water molecule are represented by dotted blacklines. B. Multiple structural alignment of fifteen Asp-boxes and four s-Asp-boxes. The sequences are labeled with protein name,pdb code, blade number (b) when extracted fromβ-propellersand propeller number (p) when extracted from a tandemβ-propeller. The conserved S, D, G and W are shown in light blue. The vertical green and orange bars mark regular Asp-boxesand s-Asp-boxes respectively. C. Structural overlay of the Asp-boxes in (B). The main chain traces are shown as green ribbons,conserved side chains as sticks colored by atom type, and the conserved water molecules are shown as spheres. D. Same as in(C) but the short Asp-boxes are included. All Asp-boxes are uniformly green and the short-Asp-boxes are colored magenta,yellow, blue and red in order of appearance in the alignment in (B). E. Ramachandran plot for the residues SXDXGXXW of theAsp-box. The blue trace shows average values for phi and psi calculated for the 15 asp-boxes listed in (B). The positions of theconserved residues are indicated by their one letter abbreviation. The trace of standard type I' (red) and type VIII (blue) turnsare shown for reference. F. Ramachandran plot for residues SXDXXXW of the s-asp-box. The orange trace shows averagevalues of phi and psi calculated for the 4 s-asp-boxes listed in (B).one of the twoβ-sheetswhere it interacts quite extensivelywith several other side chains belonging to the same sheet.A C-terminal Ig-like domain with a single Asp-box isfound in both sulphite oxidase and chitobiase. In sulfiteoxidase the Asp-box contributes to the homodimer inter-action face (Figure 2A), but chitobiase is a monomer andit is not clear if the Asp-box plays any functional role forthis protein, although it may be noted that it is involvedin crystal packing of 1qba. Reelin is a large protein con-taining several so called reelin repeats, which are compactdomains consisting of three modules; an EGF sub-domainand twoβ-sandwichor jelly-roll sub-domains called sub-repeats A and B, which both contains a single Asp-box(Figure 2B). As has been reported previously, the Asp-boxin subrepeat B seems to ensure that the three individualmodules form a single compact entity by binding to sub-repeat A of the same repeat [20]. We find however, thatthe Asp-box in subrepeat A also seems to support struc-tural integrity. It contributes to the interface between tworeelin repeats by interacting with subrepeat B of the pre-ceding repeat and may thus support the rodlike super-structure of the protein. In bacterial ribonucleases the sin-gle Asp-box is found in a surface exposed position in thecentralβ-sheet(Figure 2C) and has several functions; H85and Y86 of RNase Sa representing positions 6 and 7 of theAsp-box are involved in binding of the nucleotide [34]and the histidine in position 6 is also directly involved incatalysis [35]. In addition the tyrosines in Asp-box posi-tions 1, 2 and 7 of RNase Sa (Y80, Y81, Y86) form hydro-gen bonds, that have been shown to significantlyinfluence the stability of the protein [36].Structural contexts and functions of Asp-boxes inβ-propellersInβ-propellersAsp-boxes are invariantly found in theloops between strand 3 and 4 of the sheets/blades and arerather surface exposed (Figure 2D). They always form con-tacts to the previous blade or at least to the loop connect-ing the blade to the previous blade and additionalstabilizing contacts are often formed to the succeedingblade as well. The distance between blades and the actualinteractions formed are quite variable, excepting that theconserved aromatic residue in position 10 almost alwaysforms van der Waals interactions to the preceding bladeand/or to the loop connecting the blade to the precedingblade. Further common interactions are from positions 6and 8 to the preceding blade, from position 1 to the loopconnecting the blade to the preceding blade and frompositions 2 and more rarely 4 to the succeeding blade. Ithas previously been proposed that the PQQ repeat/tryp-tophan docking motif is related to the Asp-box since thismotif also has a conserved aromatic residue in the begin-ning of strand 4, which is likewise involved in blade toblade interactions [5]. We find however that the loopbetween strand 3 and 4 is not overall similar in the twomotifs, and furthermore the conserved aromatic residueof the PQQ motif points in the opposite direction andthus interacts with the succeeding rather than the preced-ing blade.Nonetheless we can conclude that the repeats of Asp-boxβ-propellersappear to have a structural function in medi-ating blade to blade interactions reminiscent of other pro-peller-associated repeats. This notion is furthermore inagreement with mutational analysis carried out on theClostridium perifringensNanH sialidase, since mutatingconserved Asp-box positions in this enzyme resulted indelayed or abolished secretion and reduced enzymaticactivity indicative of misfolding [22].Furthermore, the W240R mutation in position 10 of aconserved Asp-box in human Neu1 sialidase causes type IIsialidosis [37], and although this was hypothesized to becaused by altered surface properties, destabilization ormisfolding of the enzyme seems to be valid alternativeexplanations. However, the fact that Asp-boxes are oftenPage 5 of 18(page number not for citation purposes)

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