Sequence and structural analysis of the Asp-box motif and Asp-box beta-propellers; a widespread propeller-type characteristic of.docx

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1、BMC Structural Biology。BioMedCentralOpen AccessReceived: 14 May 2021Accepted: 13 July 2021Research articleSequence and structural analysis of the Asp-box motif and Asp-box beta-propellers; a widespread propeller-type characteristic of the Vps10 domain family and several glycoside hydrolase families

2、Esben M Quistgaardi,2 and Soren S Thirup*iAddress: 1MIND Centre, Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10C, DK 8000 Arhus C, Denmark and 2Department of Medical Biochemistry and Biophysics, Karolinska Institute, 17177 Stockholm, SwedenEmail: Esben M Quistgaard - eqmb

3、.au.dk; Soren S Thirup* - sthmb.au.dk* Corresponding authorPublished: 13 July 2021BMC Structural Biology 2021, doi: 10.1186/1472-6807-9-46帮 article is available from: :/ biomedcentral /1472-6807/9/46 2021 Quistgaard and Thirup; licensee BioMed Central Ltd.This is an Open Access article distributed u

4、nder the terms of the Creative Commons Attribution License ( :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

5、folds as a well-defined - hairpin. It is present in different folds, but occurs most prominently as repeats in -propellers. Asp- box -propellers are known to be characteristically irregular and to occur in many medically important proteins, most of which are glycosidase enzymes, but they are otherwi

6、se not well characterized and are only rarely treated as a distinct -propeller family. We have analyzed the sequence, structure, function and occurrence of the Asp-box and s-Asp-box -a related shorter variant, and provide a comprehensive classification and computational analysis of the Asp-box - pro

7、peller family.Results: We find that all conserved residues of the Asp-box support its structure, whereas the residues in variable positions are generally used for other purposes. The Asp-box clearly has a structural role in -propellers and is highly unlikely to be involved in ligand binding. Sequenc

8、e analysis of the Asp-box -propeller family reveals it to be very widespread especially in bacteria and suggests a wide functional range. Disregarding the Asp-boxes, sequence conservation of the propeller blades is very low, but a distinct pattern of residues with specific properties have been ident

9、ified. Interestingly, Asp-boxes are occasionally found very close to other propeller-associated repeats in extensive mixed-motif stretches, which strongly suggests the existence of a novel class of hybrid -propellers. Structural analysis reveals that the top and bottom faces of Asp-box - propellers

10、have striking and consistently different loop properties; the bottom is structurally conserved whereas the top shows great structural variation. Interestingly, only the top face is used for functional purposes in known structures. A structural analysis of the 10-bladed -propeller fold, which has so

11、far only been observed in the Asp-box family, reveals that the inner strands of the blades are unusually far apart, which explains the surprisingly large diameter of the central tunnel of sortilin.Conclusion: We have provided new insight into the structure and function of the Asp-box motif and of As

12、p-box -propellers, and expect that the classification and analysis presented here will prove helpful in interpreting future data on Asp-box proteins in general and on Asp-box - propellers in particular.Page 1 of 18(page number not for citation purposes)Table 2: Overview of all known structures of -p

13、ropellers containing Asp-box or s-Asp-box motifs (Continued)Not knownYP_299179.1Ralstonia eutropha752, 3,4,0-13b7f(2.20 A)Vps10 domainSortilinHomo sapiens*1091,2, 3, 4, 5, 6, 7, 8,90-13f6k (2.00 A)GH32Exo-inulinaseAspergillus niger*51221,331y4w (1755A)InvertaseThermotoga maritima50-31,2,321w2t (1.87

14、 A)Cell-wall invertaseArabidopsis thaliana*50-21,352ac1 (2.15 A)FructosidaseCichorium intybus50-21.351st8 (2.35 A)GH43XylosidaseBacillus halodurans50-1111yrz(2.Q0 A)XylosidaseBacillus subtilis50-1111yif (1.80 A)ArabinaseBacillus subtilis50-1111uv4 (1.50 A)ArabinaseCellvibrio japonicus50-1131gyh_(1.8

15、9 A)XylosidaseClostridium acetobutylicum50-1121y7b(1.60 A)XylosidaseGeobacillus stearothermophilus50-1142exh (1788A)ArabinaseGeobacillus thermodenitrificans50-1111wl7 (1.90 A)Xylosidase/ara- binofuranosidaseSelenomonas ruminantium50-1113c2u(1.3Q A)There are currently 76 known structures with Asp-box

16、 -propeller domains. These have 2-9 Asp-boxes and represent 16 different proteins and 5 different protein families. There are furthermore 29 known structures with 1-3 s-Asp-box motifs, which represent 12 different proteins and 2 families of 5-bladed -propellers. Most known structures are from bacter

17、ia (unmarked), but there are also several from eukaryotes (species marked by *) and one from a bacteriophage (marked by t).BMC Structural Biology 2021, 9:46:/ biomedcentral /1472-6807/9/46cal modeling of Asp-box -propellers of structurally uncharacterized families.Putative hybrid -propellersAmong th

18、e yet uncharacterized putative Asp-box -propellers a heterogeneous group of putative hybrid -propellers deserves specific mentioning. Hybrid -propeller domains containing more than one type of propeller- associated repeats are rare in known structures, yet Asp- boxes are found in between other prope

19、ller-associatedIt is thus not surprising that a structural alignment including all blades of the same set of proteins reveals that the hydrophobic positions are conserved in all the blades, regardless if they contain an Asp-box or not (see additional file 2: Suppl_Figure1.pdf for figure). It may how

20、ever be noted that these patterns can probably be readily recognized by fold recognition programs that employ secondary structure algorithms and sensitive sequence profile matching, and thus be useful for evaluating if an uncharacterized Asp-box repeat protein adopts a -pro-repeats in several sequen

21、ces. Examples are a putativepeller fold or like reelin adopts an alternative fold. Map-archeal kelch hybrid (InterPro accession A3CWT3) and some putative bacterial PQQ (e.g. Q3KK02) and Reg_Prop (e.g. A9KRI6) hybrids. The Reg_prop motif is related to the WD40 and PQQ repeats and is according to Pfam

22、 believed to promote a -propeller fold, although this has not yet been shown. Interestingly, it seems that inping out the conserved residues in the structures clearly reveals the likely reasons for why they are conserved; the hydrophobic residues basically make up the hydrophobic core, and as previo

23、usly described the Asp-boxes mediate stabilizing blade to blade interactions at the outer rim (Figure 6A). The overlay resulting from the structuralsome cases, Asp-boxes are even found in the same bladesalignment reveals that the two ends of the blades andas other propeller-associated repeats. The p

24、ositioning of thereby also the top and bottom faces of the propeller i.e. the Asp-box and Reg_prop motifs in the sequence of the the faces that comprise the N-terminal and C-terminal YP_001558799.1 hypothetical protein from Clostridium parts of the inner strands respectively, have very distinct phyt

25、ofermentans (A9KRI6) suggests the presence of severaroperties. The end comprising loop1-2 (the loophybrid blades as the two motifs follow each other in immediate or almost immediate succession with often just three amino acids in between (Figure 4). Such hybridblade -propellers have to our knowledge

26、 neither been described nor predicted before. A recombination event involving just two strands does not seem particularly likely and the blades of such propellers may thus represent ancestral units that have since diversified into blades retaining just one or the other repeat. Alternatively, they ma

27、y reflect that some propeller-associated repeats have evolved more than once e.g. the Reg_prop motif may in one case have evolved in a blade already containing an Asp-box.Structural alignment of Asp-box -propeller bladesA multiple structural alignment was made of all blades containing an Asp-box fro

28、m ten representative Asp-box - propellers (Figure 5). The Asp-box itself is clearly the most well-conserved feature of these blades, but there are also several conserved hydrophobic positions; a doublet in strand 1, a triplet in the beginning of strand 2 and a doublet in strand 3. These residues and

29、 some surrounding residues in the first three strands are furthermore characterized by having a strong propensity for forming - strands. Conversely, there is in between the strands, a high incidence of residues with strong propensity for forming turn structure i.e. G, P, S, D and N 32. The conserved

30、 hydrophobic positions overlaps a set of positions previously identified as being generally conserved in -propellers 23 and the observed conservation of hydrophobic positions and the pattern of strand and loop propensities do therefore not appear to represent specific properties of the Asp-box famil

31、y, but rather of -propellers in general.between strand 1 and 2) and loop3-4 is very well-defined, whereas the opposite end comprising loop2-3 is extremely variable (Figure 6B). The strands generally overlay fairly well although with some exceptions, but there is a marked variation in lengths, the ou

32、ter strands are sometimes reduced to loop structure and the inner strands may encompass various insertions, e.g. a signature of the GH33 family is a -bulge in the inner strand of blade 3, which we find to conform to the consensus sequence G-X- G-X-G. It is these features that underlie the previously

33、 noted distinctive irregularity of members of the Asp-box -propeller family as compared to other -propellers 23. The conserved positions of the Asp-box are restricted to just one loop, whereas they are scattered over longer regions in most other propeller-associated repeats. This probably leaves mor

34、e room for structural variation in Asp- box -propellers than in other families and could thus at least partly explain why they contain more irregularities. Interestingly, the set of residues involved in ligand binding and catalysis in all known Asp-box glycosidases are located in the variable loop2-

35、3 on the top face of the propeller. In sortilin it appears that ligand binding generally occurs in the tunnel rather than at the loops of one of the two faces 19, but two hydrophobic loops that strongly protrude from the structure are expected to be of functional significance, and these loops are in

36、deed found on the top face. It thus seems, that the variable top face is generally used for functional activities, whereas the well- defined bottom face comprising the Asp-boxes is generally used for supporting the structural integrity of the fold. In new structures of Asp-box -propellers, we theref

37、ore recommend that focus should be on the top face in any attempts to identify functionally important residues. It isPage 11 of 18(page number not for citation purposes)BMC Structural Biology 2021, 9:46:/ biomedcentral /1472-6807/9/46Figure 4Putative hybrid -propeller. The YP_001558799.1 hypothetica

38、l protein from Clostridium phytofermentans (Interpro accession A9KRI6) is an example of a putative Asp-box/Reg_prop hybrid -propeller. A single blade (blade 2) of a fold recognition model is shown along with a schematic of the sequence. The putative hybrid propeller boundaries suggested by fold reco

39、gnition are marked by stippled purple frames and the likely boundaries of the blades are indicated by vertical lines. The Reg_Prop repeats are colored pink, the Asp-box repeats are light blue and residues not belonging to any of these motifs are grey. The structural model is in cartoon representatio

40、n with the side chain of the tryptophan in the 10th position of the Asp-box (W111) shown in sticks. It is based on 2cn2 and is probably rather inaccurate in most respects (sequence id. 15%), but it illustrates well how the two motifs are likely to be arranged in special hybrid propeller blades. The

41、fourth strand is missing in the modeled blade, which may appear to indicate that the model is erroneous in this area, but a lack of the fourth strand is indeed quite commonly observed for Asp-box -propeller blades.beyond the scope of this paper to systematically investigate if this structural and fu

42、nctional distinction between the two faces also applies to other families of -propellers, but it should be mentioned that most -propellers bind ligands at the top face 44, although binding can also occur at the bottom face 45 or at the side of the domain 46,47. Phasing by molecular replacement can b

43、e challenging for Asp-box -propellers e.g. phases for the M. viridifaciens GH33 sialidase could not be obtained using the GH33 sialidase structures from V. Cholera or S. typh-imurium as search models 5. There are now so many known structures of GH33 proteins, that phasing by molecular replacement is

44、 a routine matter for members of this family, but Asp-box -propellers that are slightly more distantly related to potential templates, can still prove challenging. For such cases, we recommend that the search models should include the strands and Asp-boxes, whereas loop2-3 should be removed complete

45、ly from all blades. Furthermore, the side chains of the conserved Asp- box residues should not be trimmed. Finally we suggestPage 12 of 18(page number not for citation purposes)BMC Structural Biology 2021, 9:46:/ biomedcentral /1472-6807/9/46e tri?” “ma”*phHhXU5fM0 X*M4VvHM0e9rW9 MMr.lj KMW.y2 HMM.4

46、TKXF j -faAMM4e，2y 4IWe4WeK333l207”A2024A22222s；A2eA232w2tAz22K7;l7*2l2tM222l222MB*B-A .“,3 jab iu ”2,MMMrerWCVWMMlrVMV 了 “1JMAWe3 d*oeMMe.2t ， i， ，g ”1， J* *MK O ，454 * - 4t5。9 0x321o38KXFigure 5Multiple structural alignment of -propeller blades containing Asp-box repeats. This alignment was made u

47、sing all blades with an Asp-box from ten representative structures: Sortilin from man (3f6k), cytoplasmic sialidase Neu2 from man (1so7), sialidase from S. typhimurium (3sil), sialidase from M. viridifaciens (1w8o), intramolecular trans-sialidase from the leech M. decora (2sli), trans-sialidase from the trypasnosome parasite T. cruzi (1ms9), endosialidase from