Leucine rich repeats (LRRs) are present in over 100,000 proteins from viruses to eukaryotes. The LRRs are 20–30 residues long and occur in tandem. LRRs form parallel stacks of short β-strands and then assume a super helical arrangement called a solenoid structure. Individual LRRs are separated into highly conserved segment (HCS) with the consensus of LxxLx- LxxNxL and variable segment (VS). Eight classes have been recognized. Bacterial LRRs are short and characterized by two prolines in the VS; the consensus is xxLPxLPxx with Nine residues (N-subtype) and xxLPxxLPxx with Ten residues (T-subtype). Bacterial LRRs are contained in type III secretion system effectors such as YopM, IpaH3/9.8, SspH1/2, and SlrP from bacteria. Some LRRs in decorin, fribromodulin, TLR8/9, and FLRT2/3 from vertebrate also contain the motifs. In order to understand structural features of bacterial LRRs, we performed both secondary structures assignments using four programs—DSSP-PPII, PROSS, SEGNO, and XTLSSTR—and HELFIT analyses (calculating helix axis, pitch, radius, residues per turn, and handedness), based on the atomic coordinates of their crystal structures. The N-subtype VS adopts a left handed polyproline II helix (PPII) with four, five or six residues and a type I β-turn at the C -terminal side. Thus, the N-subtype is characterized by a super secondary structure consisting of a PPII and a β-turn. In contrast, the T-subtype VS prefers two separate PPIIs with two or three and two residues. The HELFIT analysis indicates that the type I β-turn is a right handed helix. The HELFIT analysis determines three unit vectors of the helix axes of PPII (P), β-turn (B), and LRR domain (A). Three structural parameters using these three helix axes are suggested to characterize the super secondary structure and the LRR domain. Keywords Bacterial leucine rich repeat family · Polyproline II helix · Type I β-turn · Super secondary structure · Helical parameters · Helix axis · Vector analysis Abbreviations LRR Leucine rich repeat PPII Polyproline II helix Electronic supplementary material The online version of this HCS Highly conserved segment article (https ://doi.org/10.1007/s1093 0-018-9767-9) contains supplementary material, which is available to authorized users. * Purevjav Enkhbayar Institute of Physics and Technology, Mongolian Academy firstname.lastname@example.org of Sciences, Enkhtaivan avenue 54B, Ulaanbaatar 210651, Mongolia * Norio Matsushima email@example.com Hokubu Rinsho Co., Ltd, Sapporo 060-0061, Japan Institute of Tandem Repeats, Sapporo 004-0882, Japan Laboratory of Bioinformatics and Systems Biology, Department of Information and Computer Science, School Department of Biology, University of Virginia, of Engineering and Applied Sciences, National University Charlottesville 22904, USA of Mongolia, Ulaanbaatar 14201, Mongolia Sapporo Medical University, Sapporo 060-8556, Japan Department of Physics, School of Mathematics and Natural Sciences, Mongolian National University of Education, Ulaanbaatar 210648, Mongolia Vol.:(0123456789) 1 3 224 D. Batkhishig et al. VS Variable segment 3 -helix, and an extended conformation or a tandem C α-Carbon arrangement of β-turns [1, 6]. Their secondary structures CC Cysteine containing on the convex side are connected to the strands forming the P Helix pitch β-sheet on its concave side by two loops . One of the R Helix radius loops is an “ascending loop” which links the C-terminus N Number of repeat unit/residue per turn in helix of the HCS to the N-terminus of the VS. The other is a δz Rise per repeat unit/residue in helix “descending loop” which links the C-terminus of the VS to ΔΦ Rotation angle per repeat unit/residue in helix the N-terminus of the HCS of the following unit. Each LRR n R epeat number of leucine rich repeat domain contains a concave surface, a convex surface, an PDB Protein data bank ascending surface, and a descending surface on the opposite RMSD Root mean square deviation side. LRR domains are involved in direct interaction with TLR Toll like receptor proteins (including hormones) or ligands (including nucleic SLRP Small leucine rich repeat proteoglycan protein acid, lipid, lipo-polysaccharide, and plant steroid hormones) FLRT Fibronectin leucine rich repeat transmembrane . LRR domains can engage structurally various proteins protein or ligands using different surfaces of the LRR domains [1 , CTD C-terminal repeat domain of the large subunit of 5]. RNA polymerase II LRR proteins participate in the plant immune response POL II RNA polymerase II and in the mammalian innate immune response [1–6]. They are also involved in a broad range of functions including apoptosis, autophagy, ubiquitin related processes, nuclear 1 Introduction mRNA transport, and neuronal development [1, 8]. Plant LRR proteins, many of which involve kinases and other Leucine rich repeats (LRRs) are unusually rich in leucine receptor like proteins, act as signal amplifiers in tissue [1–6]. The LRRs are composed of 20–30 residues stretches damage, in symbiotic relationships, and in developmental and repeat in tandem. The published repeat numbers range processes [1, 9]. from 2 to 97. LRRs have been reported in over 100,000 pro- Furthermore, LRR proteins are contained in the type III teins from viruses to eukaryotes. secretion system of many gram-negative bacterial patho- LRR units are divided into a highly conserved segment gens. The LRR proteins called effectors are delivered into (HCS) and a variable segment (VS) . Eight classes of the cytosol of animal or plant cells . Consequently, these LRRs have been recognized . Matsushima and Kretsinger effectors enable the bacteria to avoid the immune response recently proposed twenty-three types of LRRs . Their of the infected organism by modulating cell functions of the grouping is based mainly on the difference of the VS parts. host. The effector proteins include YopM from the bubonic The eight classes are RI-like, cysteine containing (CC), plague bacterium, Yersinia pestis, and SspH1, SspH2, and SDS22-like, IRREKO, bacterial, plant specific, typical, and SlrP from Salmonella enterica, and IpaH3 and IpaH9.8 from TpLRR. Shigella flexneri . These effectors are bacterial LRR proteins The HCS part consists of an 11 or 12 residue stretch, . The LRR domain of SspH1 directly interacts with PKN1 LxxLxLxx(N/C)(x/-)L, in which “L” is Leu, Ile, Val, or Phe, . “N” is Asn, Thr, Ser, or Cys, “C” is Cys, Ser or Asn, “x” is Bacterial LRRs are characterized by two Leu Pro any amino acid, and “−” is a deletion. Three residues at posi- sequences in the VS; the consensus is xxLPxLPxx with Nine tions 3–5 in the underlined residues form a short β-strand [4, residues (N-subtype) and xxLPxxLPxx with Ten residues 6]. These β-strands stack parallel; they have the pattern of (T-subtype) where “L” is Leu, Val, or Ile and “x” is pre- H-bonding (N–H → O=C), and then these tandem repeats dominantly occupied by small residues such as Thr, Ser, or of LRRs assume their super helical arrangements. The LRRs Gly. T-subtype is seen in Salmonella SlrP. Moreover, LRRs fold into a horse shoe, a right handed or left handed helix, in the subfamily of toll-like receptors (TLR7, TLR8, and or a prism shape . Conserved hydrophobic residues such TLR9), the small leucine rich repeat proteoglycan (SLRP) as leucine, valine, isoleucine, or phenylalanine in the con- family including fibromodulin, decorin and biglycan, and sensus sequences of LRRs contribute to the hydrophobic the fibronectin leucine rich repeat transmembrane family cores. Capping structures that shield the hydrophobic core (FLRT) contains N- or T-subtype . The LRRs consist of of the first LRR unit at the N -terminus and/or the last unit tandem repeats of a super motif of STT or ST in which “S” at the C-terminus are observed in most of the known LRR is Bacterial and “T” is Typical [12–14]. We called this the structures [1, 5]. STT class . Characteristic of each LRR class, the VS parts adopt PPIIs are known to be observed frequently in proline a variety of secondary structures including the α-helix, rich regions [15–19]. The PPIIs are characterized by the 1 3 Super Secondary Structure Consisting of a Polyproline II Helix and a β-Turn in Leucine Rich Repeats… 225 backbone dihedral angles (Φ, Ψ) of (− 75°, 145°) [20–29]. 2 Methods The PPIIs have helical parameters: 2.9 residues per turn, a pitch value of 8.7 Å per turn, and a helix radius of 1.33 Å. 2.1 Structure Data The assignment of PPII is not done in the widely used pro- grams such as DSSP  and STRIDE . Consequently, We collected the structure data of proteins containing PPIIs in newly solved protein structures are not registered bacterial LRRs from the PDB. We performed sequence in protein data bank (PDB) . Now there are some tools alignments in LRR proteins from the PDB by LRRpred for assigning PPII number—DSSP-PPII , PROSS , that recognizes and aligns LRR motifs that predict the SEGNO , XTLSSTR , and ASSP . repeat number and “phasing” of LRRs with greater reli- Super secondary structures with several adjacent ele- ability  and identified bacterial LRR based on the ments of a secondary structure are also observed in protein consensus sequence. Bacteria LRR proteins are YopM, structures [36, 37]. Examples include β-hairpins, α-helix SspH1, SspH2, SlrP, IpaH3, IpaH9.8, TLR8, TLR9, fibro- hairpins, and β–α–β motifs. Adzhubei and Sternburg  modulin, decorin, biglycan, FLRT2, and FLRT3 (Table 1) identified super secondary structures consisting of PPII [39, 42–56]. LRRs in YopM, SspH1, SspH2, SlrP, IpaH3, and α-helix and of 3 -helix and PPII. Kumar and Bansal and IpaH9.8 belong to bacterial LRR class, while TLR8,  also identified those consisting of β-strand and PPII, TLR9, fibromodulin, decorin, biglycan, FLRT2, and of β-strand, PPII, and α-helix, and of β-strand, PPII, and FLRT3 belong to the STT class (Table 1). Eighteen PDB β-strand. files solved at resolution ≤ 3.4 Å were used; the sequence Evdokimov et al.  noted that the VS parts in the identity of the 18 different chains shows that the maximum YopM LRRs adopt 3 -helices. Matsushima et al. [6, 7], is 48% and the average is 7% (Supplementary Table S1). Bella et al. , and Park et al.  proposed that the VS The structure data of mouse FLRT2 at resolution 4.0 and parts in the bacterial LRR adopt left handed polyproline 6.0 Å were not used for analyses. II helices (PPII). A review article by Adzhubei et al.  noted PPIIs in LRRs. However, it appears that PPII in LRR structures has not yet been well characterized based on the 2.2 Secondary Structures Analysis consensus sequence. Structural data of proteins containing bacterial LRRs have increased. The crystal structures of Secondary structures assignments were made from the YopM, SspH1, SspH2, SlrP, IpaH3, and IpaH9.8 have been atomic coordinates of the LRR structures using four pro- determined [39, 42–46]. The structures of TLR8, TLR9, grams—DSSP-PPII , PROSS , SEGNO , and fibromodulin, decorin, biglycan, FLRT2, and FLRT3 are XTLSSTR . The assignment of the DSSP program is also available [47–56]. based on the identification of precise hydrogen bond pat- The purpose of this study is to understand structural fea- terns corresponding to regular secondary structures . tures of bacterial LRRs. We performed both the secondary In DSSP-PPII based on DSSP, PPII are assigned solely in structures analyses using secondary structures prediction the coil region for at least two consecutive residues in coil programs of DSSP-PPII, PROSS, SEGNO, and XTLSSTR with Φ = − 75° ± 29° and Ψ = +145° ± 29°. The PROSS and the HELFIT analyses that calculate helix axis, helix program assigns secondary structures, based mainly on pitch, helix radius, repeat/residue number per turn, based Φ and Ψ dihedral angles. SEGNO utilizes the Φ and Ψ on the atomic coordinates of the crystal structures . dihedral angles coupled with other angles. XTLSSTR This present analysis demonstrates that the N-subtype VS uses two angles and three distances. DSSP-PPII, PROSS, adopts PPII consisting of 4–6 residues and type I β-turn at and XTLSSTR assign β-turns; while SEGNO does not. the C-terminal side. Thus, the VS part is characterized by The secondary assignments were performed using the super secondary structure consisting of PPII and a β-turn. In PolyprOnline web interface . Types of β-turn were contrast, the T-subtype VS frequently prefers two separate also identified by the programs of PROMOTIF  and PPIIs consisting of two or three and of two residues. The STRIDE . Furthermore, the root-mean-square devia- HELFIT analysis indicates that the type I β-turn is a right tion (RMSD) of the VS part between within the N-subtype handed helix and consequently determines three unit vectors and within the T-subtype and between these two subtypes of the helix axes of PPII (P), β-turn (B), and LRR domain using the coordinates of the backbone atoms of each resi- (A). We propose three structural parameters which are due were evaluated by the CHIMERA program . two angles between the two helix axes of PPII and β-turn, between the two helix axes of PPII and LRR domain, and between the helix axis of LRR domain and the vector prod- uct of P × B. These three angles are suggested to character- ize the super secondary structure and the LRR domain. 1 3 226 D. Batkhishig et al. Table 1 Known structures of proteins containing bacterial LRRs Number of LRR class Protein name Repeat num- Number of Number of PDBID Chains Resolution (Å) protein ber of LRR N-subtype T-subtype 1 Bacterial Y. pestis yopM 16 (16) 13 0 1JL5 A 2.10 Bacterial Y. pestis yopM 16 (16) 13 0 1G9U A 2.35 2 Bacterial Y. entercocolitica yopM 21 (20) 19 0 4OW2 A,B,C,D 3.20 3 Bacterial S. enterica SspH1 10 (9) 6 1 4NKH A,B,C,D,E,F 2.75 Bacterial S. enterica SspH1 10 (9) 6 1 4NKG A,C 2.90 4 Bacterial S. enterica SspH2 13 (13) 10 1 3G06 A 1.90 5 Bacterial S. flexneri ipaH3 9 (9) 5 1 3CVR A 2.80 6 Bacterial S. flexneri ipa9.8 8 (8) 5 1 5B0N A,B 1.80 Bacterial S. flexneri ipa9.8 8 (8) 5 1 5B0T A 2.00 7 Bacterial S. enterica SlrP 12 (12) 0 10 4PUF A,B 3.30 8 STT Human fibromodulin 13 (12) 2 2 5MX0 A,B 2.21 9 STT Horse TLR9 27 (27) 1 1 3WPC A,B 1.60 STT Horse TLR9 27 (27) 1 1 3WPB A 2.40 STT Horse TLR9 27 (27) 1 1 3WPD A 2.75 10 STT Bovine TLR9 27 (27) 1 1 3WPE A 2.38 11 STT Mouse TLR9 27 (27) 1 1 3WPF A 1.96 STT Mouse TLR9 27 (27) 1 1 3WPG A 2.25 STT Mouse TLR9 27 (27) 1 1 3WPI A 2.25 STT Mouse TLR9 27 (27) 1 1 3WPH A 2.35 12 STT Human TLR8 27 (27) 0 3 3WN4 A 1.81 STT Human TLR8 27 (27) 0 3 3W3J A,B 2.00 STT Human TLR8 27 (27) 0 3 3W3N A,B 2.10 STT Human TLR8 27 (27) 0 3 3W3G A,B 2.30 STT Human TLR8 27 (27) 0 3 3W3K A,B 2.30 STT Human TLR8 27 (27) 0 3 3W3L A,B,C,D 2.33 STT Human TLR8 27 (27) 0 3 3W3M A 2.70 13 STT Bovine decorin 12 (12) 0 3 1XKU A 2.15 STT Bovine decorin 12 (12) 0 3 1XEC A,B 2.30 STT Bovine decorin 12 (12) 0 3 1XCD A 2.31 14 STT Bovine biglycan 12 (12) 0 3 2FT3 A,B,C,D,E,F 3.40 15 STT Human TLRT2 13 (13) 0 3 4V2D A 2.50 16 STT Mouse TLRT2 13 (13) 0 3 5FTT B,F 3.40 17 STT Human TLRT3 13 (13) 0 3 5CMP A,B,C,D 2.60 18 STT Mouse FLRT3 13 (13) 0 3 4V2E A,B 2.50 STT Mouse FLRT3 12 (12) 0 3 2YEB B,F 3.19 The number in the parentheses indicates the number of variable segment of LRRs 2.3 HELFIT Analysis the helix (ΔΦ = 360°/N). Moreover, HELFIT gives rmsd: where d is the closest distance from the data point to the We have developed a total least squares program for fitting trace of the helix. a helix to data points—HELFIT . A helix consisting 1∕ 2 of n repeat units may be characterized by helix axis, helix rmsd = the minimum of d N (1) pitch (P), helix radius (R), and number of repeat units/ 1/2 residue per turn (N). HELFIT computes these parameters Here p = rmsd / (n − 1) gives the regularity of heli- in which the helix axis is represented by the unit vector. cal structures independent of the number of data points These parameters also yield the rise per repeat unit/resi- or helix length. The criterion for regular PPII helices is due (Δz = P/N) and the rotation per repeat unit/residue in p ≤ 0.10 Å. This same test is used for α-helices, ω-helices, 1 3 Super Secondary Structure Consisting of a Polyproline II Helix and a β-Turn in Leucine Rich Repeats… 227 and 3 -helices in proteins [63, 64]. The HELFIT analysis 3 Results requires only four data points: the coordinates of α-carbon (Cα) of each residue. LRRs form a β-strand of three resi- 3.1 Two Subtypes of Consensus Sequences dues at positions 3–5 in the HCS part. Thus, in LRRs, the C coordinates of the consensus leucine residue at position Bacterial LRR is 20 or 21 residues long and is classified four in individual LRR repeat units are used. The repeat into two subtypes. The N-subtype has the VS consensus number of individual LRR domains was defined as the num- of xxLPxLPxx with nine residues and the T-subtype has ber that participates in the parallel β-sheet. This definition xxLPxxLPxx with ten residues where “L” is Leu, Val, or Ile means that the first LRR is sometimes contained in the cap- and “x” is predominantly occupied by small residues such ping structures. β-Turns consist of four amino acid residues as Thr, Ser, or Gly (Fig. 1). Thus bacterial LRR is charac- (labelled i, i + 1, i + 2, and i + 3). We also estimate the heli- terized by two Leu Pro sequences in the VS parts; although cal parameters of β-turns using the C coordinates of each variable VS that lack one of the two conserved prolines is residue. observed. The characteristics are not seen in other LRR The HELFIT analysis indicates that the β-turn is regarded classes. For examples, ribonuclease inhibitors and Nod-like as a right handed helix, as noted later. Consequently, HELFIT receptors contain RI-like LRRs which of the consensus is determines three unit vectors of the helix axes of LRR domain LxxLxLxx(N/C)xLxxxgoxxLxxoLxxzxxx with typically 28 (A), PPII (P), and type I β-turn (B). We estimate three struc- or 29 residues . tural parameters. One is the angle between the two helix axes The N-subtype appears sixty-three times in the known of PPII and β-turn (Ω ). The second is the angle between the structures (Table 1). The VS consensus is xxLPxLPxx, as two helix axes of PPII and LRR domain (Ω ). The third is the expected, in which “x” positions at the N- and C-terminal angle between the helix axis of LRR domain and the vector sides are frequently occupied by relatively small residues product of P × B. (Ω ). The three angles of Ω , Ω , and Ω are such as Thr or Ser; while the central “x” position is rich in 3 1 2 3 represented by the following equations. Glu. Fifty-two of the sixty-three VS are completely consist- ent with the consensus (Fig. 1a). The remaining VSs are ⋅ = cosΩ (2) xxLxxLPxx in YopM; the conserved Pro at position four is replaced by Cys or Ser. The T-subtype appears forty-three ⋅ = cosΩ (3) times (Table 1). The conserved Leu at position three in the ( × ) ⋅ = × cos Ω 3 (4) VS consensus is frequently occupied by other hydrophobic residues such as Val or Ile (Fig. 1b). Fig. 1 Consensus sequence of variable segment (VS) of bacte- rial LRRs in the known struc- tures. a N-subtype; b T-subtype. The graphical sequence diagrams were generated with WebLogo , representing T E S sixty-three LRR units for the 1 S A G AE P L N T L Q KA D P E N-subtype and forty-three for K I EL I CVCV R R CMNAD F QT ENAE STP VS the T-subtype 23 45 67 89 1T P G S L E SE N K D T T ET PSA KK I DR G D GAN RA K S I RQ F MQ Y MP V VSV 12 34 56 78 910 1 3 bits bits M 228 D. Batkhishig et al. underlined residues of LxxLxLxxNxL. In addition, the 3.2 Secondary Structures assignment indicates that the VS parts are rich in PPII con- formations. However, the secondary structures show a dif- The assignment of PPII patterns differs among the four pro- grams. Bacterial LRR proteins form not only monomers ference between the two subtypes. At least one of the four programs of secondary structures but also homo-dimers, -tetramers, and -hexamers in crys- tals (Table 1). The PPII patterns assigned also differ among assignment indicates that the N-subtype VS adopts PPIIs consisting of four, five or six residues (Fig. 2a; Supplemen- their individual molecules. We therefore analyzed all chains of the known structures. tary Table S2). Four, five or six residue PPIIs are observed in the underlined residues of xxLPxLPxx, xxLPxLPxx, The four programs for secondary structures assignment indicate that the HCS parts adopt short β-strands in three and xxLPxLPxx, respectively. For example, all four N-subtype ProteinSspH2 SspH2SspH2 SspH2SspH2 LRR2 LRR3 LRR4 LRR5 LRR6 VS sequence TSLPALPPE TSLPVLPPG THLPALPSG TSLPVLPPG ASLPALPSE DSSP-PPII S--PPPPTT S----PPTT ----PPPTT S--PPPPTTS--PPPPTT PROSS ---PPPPTT ---P-PPTT -EEEPPPTT ---PPPPTT ---PPPPTT SEGNO --PPPPp-- --PPPPp-- --PPPPp-- --PPPPp----PPPPp-- XTLSSTR EEePPPPTT EEePPPPTT EEePPPPTT EEePPPPTT EEePPPPTT ProteinSspH2 SspH2SspH2 SspH2SspH2 LRR7 LRR8 LRR9 LRR10LRR11 VS sequence TSLPMLPSG ASLPTLPSE TSLPALPSG TSLPVLPSE TSLPMLPSG DSSP-PPII S--PPPPTT S---PPPTT SS-PPPPTTS---PPPTT S---PPPTT PROSS ---PPPPTT ----PPPTT ---PPPPTT ----PPPTT ----PPPTT SEGNO --PPPPp-- --PPPPp-- -pPPPPp-- --PPPPp-- --PPPPp-- XTLSSTR EEePPPPTT EEePPPPTT EEePPPPTT ---PPPPTT EEePPPPTT T-subtype ProteinSspH2 Bovine TLR9Human TLR8 Human TLR8Human TLR8 LRR1 LRR7 LRR1 LRR4 LRR7 VS sequence TTLPDCLPAH TTVPRSLPPS QEVPQTVGKY PQIPSGLPES SHVPPKLPSS DSSP-PPII S---S---TT SSPPSSPPTT SSPPS-PPTT SSPPTTPPTT SSPPS-PPTT PROSS ---P---PTT --PPP-PPTT --PPTTPPTT --PPTTPPTT --PPTTPPTT SEGNO -----pPp-- -pPPp--------------- --PPp----- -pPPp----- XTLSSTR EEe-EEeNNN --PPPPPPTT ------PpTT ---Pp-PpTT EePPPPPPTT Fig. 2 Secondary structure assignment of the variable segment (VS) TLR9 (LRR7) (PDB ID:3WPC_A) and Human TLR8 (LRR1, LRR4, of representative bacterial LRRs by the four programs (DSSP-PPII, and LRR7) (PDB ID: 3WN4_A). A one letter code is used to rep- PROSS, SEGNO, and XLTSSTR). a N-subtype, SspH2 (PDB ID: resent a specific conformation; P and p, PPII; E and e, β-strand; T, 3G06_A). b T-subtype, SspH2 (LRR1) (PDB ID: 3G06_A), Bovine β-turn; N, non-hydrogen-bonded β-turn; H, α-helix; and S, bend 1 3 Super Secondary Structure Consisting of a Polyproline II Helix and a β-Turn in Leucine Rich Repeats… 229 program assign PPII in underlined residues of KKLPDL- N-subtype VS. It appears that the structure of the T-subtype PLS (LRR7) in Y. pestis YopM (n = 12) [3G06_A] (Fig. 2a). is more variable. The variable VS of xxLxxxLPxx frequently adopt four or five PPIIs. The HELFIT analysis demonstrated that all of the PPIIs assigned are definitely left handed polyproline 3.3 Helical Parameters of PPIIs, Type I β‑turns, helices, as noted later. and LRR Domains In addition, the programs identified β-turns at the C -ter- minal side in the VS parts. The sequences in the underlined The number of assigned PPII of which the helix length residues of xxLPxLPxxLxxLxLxxNxL adopt β-turns; the is longer than three residues increase in order of DSSP- second conserved Pro corresponds to the residue, i, of PPII < PROSS < XLTSSTR < SEGNO (Table 3). SEGNO β-turns. The types are distinguished by the Φ, Ψ, angles assigns longer PPIIs. Four residue PPIIs are regular and of residues i + 1 and i + 2. The average Cα (i)−Cα (i + 3) thus are a near ideal form (Table 2). All of five and six is 5.48 (0.22 Å); the numbers in parenthesis are standard residue PPIIs are irregular. The helix regularity decreases deviations; they are reasonable . The Φ, Ψ angles of with increasing helix length. The deviation of helix param- residues i + 1 and i + 2 of the β-turns have the average angles eters from ideal values increases with increasing helix of Φ = − 60.7 (8.8°), Ψ = − 22.6 (8.4°), Φ = − 94.0 length. The helix irregularities in the five and six resi- i+1 i+1 i+2 (13.8°), Ψ = 3.4 (14.0°); the numbers in parentheses are due PPIIs are mostly due to larger deviations of the Φ, Ψ i+2 standard deviations. These values are close to − 60°, − 30° angles from ideal: − 75°, 145° in the first and/or second and − 90°, 0° which define the type I β-turns. The β-turns residues of N-terminal side in the sequence of xLPxLP. assignments by PROMOTIF  and STRIDE  give the The sequence of VP(A/R)LP in TLR9 (LRR4), which same results and indicate that most of the β-turns are type I corresponds to the underlined residues of xxLPxLPxx, (Table 2). Types IV and VIII rarely appear. Also very rarely adopt five residue PPIIs with large helix irregularity; β-turns are not assigned. p = 0.41–0.47 Å. In this case, the irregularity comes In the T-subtype VS PPIIs assigned may be divided into from large deviation of the Φ, Ψ angles of the conserved three patterns. Many VSs adopt two separate PPIIs with Leu at the C-terminal side. Taking account of the helical two or three and two residues, which are observed in the parameters, the five residue PPIIs are regarded as a highly underlined residues of xxLPxxLPxx and xxLPxxLPxx deformed form. Consequently, the HELFIT analysis dem- (Fig. 2b; Supplementary Table S2). The second is one onstrated that all four, five, and six residue PPIIs assigned PPIIs with three or four residues in the underlined residues by the secondary structures analyses are regular or irregu- of xxLPxxLPxx, xxLPxxLPxx or xxLPxxLPxx. The third lar left handed polyproline helices. pattern is six residue PPII in the underlined residues of In the T-subtype only the XLTSSTR program identifies xxLPxxLPxx, which are seen in decorin, TLR9, and TLR8 long PPIIs with six residues in LRR7 of horse/bovine TLR9, by the XLTSSTR program. Moreover, the C-terminal two LRR4 of human TLR8, and LRR4 of decorin, as noted. The residues of the T-subtype VS are assigned to adopt mostly p values are very large; p = 0.38–0.50 Å (Table 3). The high type I β-turn as does that of the N-subtype VS. irregularity comes from the Φ, Ψ angle of any residue at The average RMSD of the VS parts of the N-subtypes and position six in the T-subtype VS consensus of xxLPxxLPxx; of the T-subtypes is 0.589 (0.315 Å) and 0.993 (0.337 Å), the Φ, Ψ angles are in regions of a left handed α-helix. In respectively; all Bacterial VSs show the RMSD of 1.087 addition, the helical parameters deviate highly from those (0.549 Å). of ideal form (Table 3). The six residue PPIIs assigned are In conclusion the N-subtype VS is characterized by not recognized as a PPII. Alternatively, the SEGNO pro- a super secondary structure consisting of PPII with four, gram identifies regular, four residue PPIIs in the underlined five, or six residues and a type I β-turn (Fig. 3), while the residues of xxLPxxLPxx (Supplementary Table S2). Thus, T-subtype VS strongly prefers one or two separate PPIIs the six residue PPIIs may be divided into two separate PPIIs and adopts a type I β-turn at the C-terminal side as does the with three and two residues. This supports the conclusion by the secondary structure assignment that the T-subtype VS contains two separate PPIIs. Table 2 Types of β-turns at the C-terminal side in the bacterial LRRs The average helix parameters of the type I β-turns are; P = 6.11→ 6.14 Å, N = 3.65 →3.68 residues/turn, R = 2.26 DSSP-II PROSS XLTSSTR →2.28 Å, and Δz = 1.68 → 1.69 Å (Table 4). It appears that Type I 66 87 137 the helical parameters are close to those of α-helix as it has Type IV 5 5 5 P = 5.4 Å, N = 3.6 residues/turn, R = 2.4 Å, and Δz = 1.5 Å. Type VIII 1 0 1 The average p value is 0.02→ 0.03 Å. The HELFIT analysis Total 72 92 143 1 3 230 D. Batkhishig et al. Convex surface Ascending loop Descending loop Concave surface Ala Val Pro Pro Pro Pro Ser Leu Leu 261 138 Pro Val Gly Ser Leu Leu Fig. 3 Super secondary structure consisting of a PPII and a β-turn sequence PVLP adopt PPII and the PPGL adopt type I β-turn. The in bacterial LRRs. a Secondary structures. Left panel. Sequence sequence PVLP is a part of the LRR3 VS which correspond to the 244-LRTLEVSGNQLTSLPVLPPGLLELSIFSNPL-274 in SspH2 underlined residues of xxLPxLPxx (the consensus of the N-type (LRR3 and HCV of LRR4) (PDB ID: 3G06_A). Right panel. VS). The sequence PVLP adopts four residue PPII and the PPGL 125-LEELNLSYNGITTVPALPSSLVSLILSRTNI-155 in bovine adopts type I β-turn. Right panel. Sequence 138-VPALPSSL-145 in TLR9 (LRR4 and HCS of LRR5) (PDB ID:3WPC_A). Blue arrows bovine TLR9 (PDB ID: 3WPC_A). The sequence VPALP is a part represent β-strands, green ribbons PPIIs, and red tubes β-turns. of the LRR4 VS which correspond to the underlined residues of b Super secondary structure (HELFIT). Left panel. Sequence xxLPxLPxx. The sequence VPALP forms five residue PPII and the 258-PVLPPGL-264 in SspH2 (PDB ID: 3G06_A). The sequence sequence PPSL forms one type I β-turn. Best fitted lines by HELFIT PVLP is a part of the LRR3 VS which correspond to the under- are colored green for PPIIs and red for β-turns, and α-carbons grey. lined residues of xxLPxLPxx (the consensus of the N-type VS. The (Color figure online) indicates that these type I β-turns form a regular, right D is the average C (i) – C (i + 1) dist ance between α α handed helix. adjacent repeats—i and i + 1; D corresponds to the inter- The helix parameters of LRR domains were determined strand distance. Equation 5 gives D = 4.97 ± 0.10 Å; this for IpaH9.8 (n = 8), SspH1, (n = 10), SspH2 (n = 13), Y. allows the formation of hydrogen bonds between parallel pestis YopM (n = 16), Y. enterocolitica YopM (n = 21), and strands. This circle plot shows that the helix pitch, P, and SlrP (n = 12). The bacterial LRR domains are represented rise per turn, Δz, of bacterial LRR is comparable to those by a right handed helix (Table 5). The helix parameters of SDS22-like and Plant specific LRRs; while it is larger range over: P = 47.3 → 115 Å, N = 28.4 → 41.3 units/turn, than those of RI-like and CC LRRs . R = 18.9 → 24.6 Å, Δz = 1.67 → 3.46 Å, and ΔΦ = 8.7° → 12.7°; p = 0.03 → 0.19 Å. Figure 4 shows a plot of 2·R·sin (ΔΦ/2) versus Δz. The 3.4 Geometrical Analysis values fall on a circle with radius D (circle plot) . D is a function of Δz, ΔΦ, and R . Figure 5 shows the frequency distributions of three angles of Ω , Ω , and Ω . The Ω angle shows an asymmetrical 1 2 3 1 1∕2 distribution (Fig. 5b). The Ω angle ranges from 70° to ΔΦ 1 D = 2R sin + (Δz) (5) 120°; the average value is ∼ 103° (Table 4). The Ω and Ω 2 2 3 have the average values of ∼ 33° and ∼ 99°, respectively. 1 3 Super Secondary Structure Consisting of a Polyproline II Helix and a β-Turn in Leucine Rich Repeats… 231 Table 3 Helix parameters of PPIIs in bacterial LRRs a a a a 3 a Programs Number of PPII P (Å) N R (Å) Δz (Å) p (Å) V (Å ) PPII ‒ ‒ 8.96 2.99 1.36 3.00 ‒ 17.41 PPII ‒ ‒ 8.69 2.90 1.33 3.00 ‒ 16.70 PPII ‒ ‒ 8.58 2.99 1.45 2.87 ‒ 18.95 PPII ‒ ‒ 8.62 3.08 1.52 2.80 ‒ 20.30 Four residue PPII DSSP-PPII 50 8.61 (0.40) 2.82 (0.13) 1.25 (0.05) 3.06 (0.05) 0.10 (0.03) 15.07 (1.07) PROSS 67 8.52 (0.51) 2.78 (0.21) 1.23 (0.09) 3.07 (0.08) 0.10 (0.04) 14.70 (1.74) SEGNO 21 8.78 (0.31) 3.05 (0.10) 1.47 (0.05) 2.87 (0.05) 0.05 (0.04) 19.49 (0.05) XLTSSTR 130 8.45 (0.38) 2.75 (0.15) 1.23 (0.07) 3.07 (0.07) 0.10 (0.04) 14.49 (1.49) Five residue PPII DSSP-PPII 49 9.83 (0.50) 3.54 (0.25) 1.53 (0.11) 2.78 (0.10) 0.26 (0.04) 20.50 (2.39) PROSS 32 9.94 (0.43) 3.64 (0.16) 1.58 (0.06) 2.73 (0.05) 0.27 (0.03) 21.46 (1.39) SEGNO 132 10.05 (0.37) 3.65 (0.15) 1.57 (0.07) 2.75 (0.07) 0.27 (0.04) 21.38 (1.62) XLTSSTR 43 10.17 (1.22) 3.54 (0.39) 1.47 (0.16) 2.88 (0.18) 0.29 (0.09) 19.39 (3.10) Six residue PPII SEGNO 21 10.45 (1.21) 3.82 (0.50) 1.50 (0.09) 2.74 (0.07) 0.30 (0.04) 19.56 (2.18) XLTSSTR 14 13.91 (1.64) 6.10 (0.71) 2.40 (0.09) 2.28 (0.05) 0.45 (0.04) 41.36 (2.92) The number in the parentheses indicates standard deviations a 2 P helix pitch, N residue number per turn, R helix radius, Δz helix rise per turn; Vc = πR (Δz) (Φ, Ψ) = (− 75, 150) by Jha  (Φ, Ψ) = (− 75, 145) by Hopfinger  (Φ, Ψ) = (− 65, 140) by Adzhubei  (Φ, Ψ) = (− 60, 140) by Schulz and Schirmer  a variable N-subtype VS of QKIPKVSEK; the structure of 4 Discussion human chondroadherin which forms tetramers in crystal has been determined at 2.1 Å resolution . Their secondary 4.1 Structural Role of PPIIs in Bacterial LRRs structure assignment sometimes shows no PPII or only short PPII of two or three residues. These observations indicate The backbone dihedral angles (Φ, Ψ) of ideal PPII is (− 75°, that two conserved prolines in the N-subtype VS parts are 145°) . Other dihedral angles has been also proposed; strongly required for the super secondary structure consist- (Φ, Ψ) = (− 75°, 140°), (− 65°, 145°), and (− 60°, 140°) ing of PPII and β-turn. (Table 3) [26, 67, 68]. It appears that four residue PPIIs in It may be significant that Src tyrosine kinases SH3 proteins are a near ideal form with (Φ, Ψ) = (− 75°, 145°) domain binds to short proline rich sequence of xPxxP that or (− 75°, 140°). forms PPII . This sequence is very similar to LPxLP in The secondary structure assignment and the HELFIT the N-subtype. analysis indicate that the N-subtype VS adopts one stretch of PPII of four, five, or six residues. In contrast, the T-sub- type prefers two separate PPIIs consisting two or three and 4.2 Super Secondary Structure of two residues. Consequently, there is a clear difference in the PPII patterns between the two subtypes. The two The present analyses demonstrate that the N-subtype adopts hydrophobic residues in the VS part of the two subtypes a super secondary structure consisting of a PPII and a β-turn. are concentrated on the side that is oriented toward the Ananthanarayanan et al.  first described this super sec- hydrophobic core as well as other conserved hydrophobic ondary structure. Tandem repeats of the super secondary residues in the HCS part (Fig. 6). This structural restriction structure form a novel helical structure called the polypro- makes the difference. The assignments by the four programs line, β-turn helix . This structure is observed in tandem show different PPII patterns in most cases. This observa- repeats of the hepta-peptide, YSPSPSPS, in the C-terminal tion mainly comes from high flexibility of PPII due to no repeat domain (CTD) of the large subunit of RNA poly- intrachain hydrogen bond. merase II (POL II) [73–75]. Many factors involved in RNA The variable N-type VSs are seen in YopM— processing bind the CTD . RSLCDLPPS and SGLSELPPN (Supplementary Table S2). The VSs of RI-like and CC LRRs adopt an α-helical The first repeat of twelve LRRs in chondroadherin is also conformation (β–α structural units). Typical LRR VSs 1 3 232 D. Batkhishig et al. prefer tandem β-turns. The SDS22-like LRR VSs strongly prefer 3 -helix (β-3 ). The Plant specific LRR structural 10 10 unit is β-β-3 . The occurrence of β-turns at the C-ter- minal sides of the VS parts are also observed in Plant specific, SDS22-like, and Typical LRRs as well as bacte- rial LRR . Consequently, a super secondary structure consisting of 3 -helix and β-turns is present in Plant spe- cific and SDS22-like LRRs. The unique super secondary structures consisting of β-turns and PPII, and of β-turns and 3 -helix should be recognized as structural elements in proteins. Here we propose a structural parameter that character- izes the super secondary structure; the parameter is the angle between the two helix axes of PPII and type I β-turn (Ω ). Its average value is ∼ 103°. The Ω angle also helps 1 1 to characterize other super secondary structures including those consisting of PPII and an α-helix, and of PPII and a 3 -helix. 4.3 Solenoid Structure of Bacterial LRR Domains The circle plot of bacterial LRR is comparable to those of SDS22-like and Plant specific LRR; while it differs from those of RI-like and CC LRRs (Fig. 4). We recently calcu- lated helical parameters of 642 LRRs of known structures of 114 proteins by the HELFIT program . The results indicate that the helical parameters are influenced by the structures of the ascending loops rather than of the descend- ing loops, helical elements on the convex face, and the uni- formity of parallel strand stacking on the concave face [1, 7]. The helix radius of PPII is the smallest between α-helix, 3 -helix, π-helix, ω-helix, and PPII. This partly contrib- utes to a relatively large helix pitch for the bacterial LRR domains. We determined the Ω angle between the helix axes of PPII and of the bacterial LRR domain. The VSs of SDS22- like, Plant-specific, CC, and RI-like adopt 3 -helix or α-helix instead of PPII. The comparison of the Ω angle with the angles between their helices and LRR domains may identify fundamental features of individual LRR classes. 4.4 The PPII Assignment by the Four Programs In many methods for assignments of secondary structures from atomic coordinates, the termini of the segments are frequently ill-defined and it is difficult to decide unambigu- ously which residues at the edge of the segments have to be included . In this study the PPII pattern assigned also differs between the four programs in most cases. The number of four, five, or six residue PPIIs assigned is larger in SEGNO and XLTSSTR than in DSSP-PPII and PROSS. However, the HELFIT analyses indicate that all PPIIs 1 3 Table 4 Helix parameters of β-turn and three structural parameters in bacterial LRRs Programs Number of second- Helical parameters of the β-turns Structural parameters ary structure a a a a 3 a b b b b P (Å) N R (Å) Δz (Å) p (Å) V (Å ) Ω (°) Ω (°) Ω (°) c 1 2 3 DSSP-PPII 72 6.14 (0.32) 3.68 (0.23) 2.28 (0.15) 1.68 (0.16) 0.03 (0.02) 16.36 (2.16) 99.8 (9.0) 30.6 (6.9) 100.6 (8.2) PROSS 92 6.11 (0.34) 3.66 (0.22) 2.26 (0.14) 1.68 (0.15) 0.03 (0.02) 26.90 (1.65) 101.3 (10.2) 30.8 (7.1) 99.9 (7.6) XLTSSTR 143 6.14 (0.31) 3.65 (0.22) 2.26 (0.14) 1.69 (0.14) 0.03 (0.02) 26.93 (1.58) 103.7 (8.1) 33.4 (6.5) 99.3 (8.9) The number in the parentheses indicates standard deviations a 2 P helix pitch, N residue number per turn, R helix radius; Δz helix rise per turn; V = πR (Δz) Ω is the angle between the two helixes of the PPII and the β-turn; Ω is the angle between the two helixes of the PPII and the LRR domain; Ω is the angle between the helix of the LRR 1 2 3 domain and the vector product of P×B in which P and B is the unit vectors of the helices of the PPII and the β-turn, respectively Super Secondary Structure Consisting of a Polyproline II Helix and a β-Turn in Leucine Rich Repeats… 233 Table 5 Helix parameters of a a a a a a a Protein n P(Å) N R(Å) Δz (Å) ΔΦ (°) p(Å) PDB_chanis LRR domains in bacteria LRR proteins 1 IpaH9.8 8 114.94 33.19 18.87 3.46 10.85 0.14 5B0N_A IpaH9.8 8 111.48 34.21 20.48 3.26 10.52 0.13 5B0N_B 2 SspH2 13 76.72 30.57 21.57 2.51 11.78 0.03 3G06 3 YopM 16 47.31 28.37 20.58 1.67 12.69 0.08 1JL5A 4 YopM 21 71.27 30.35 21.70 2.35 11.86 0.12 4OW2_A YopM 21 67.16 30.46 22.10 2.20 11.82 0.14 4OW2_B YopM 21 71.42 29.75 21.09 2.40 12.10 0.08 4OW2_C YopM 21 70.50 29.58 20.99 2.38 12.17 0.09 4OW2_D 5 SspH1 10 84.44 34.74 23.69 2.43 10.36 0.14 4NKH_A SspH1 10 88.78 35.95 24.31 2.47 10.01 0.13 4NKH_B SspH1 10 83.55 35.39 24.63 2.36 10.17 0.13 4NKH_C SspH1 10 89.67 34.39 23.14 2.61 10.47 0.16 4NKH_D SspH1 10 103.20 32.90 20.47 2.50 10.94 0.16 4NKH_E SspH1 10 83.66 41.32 29.48 2.02 8.71 0.19 4NKH_F SspH1 10 89.67 33.55 22.10 2.67 10.73 0.16 4NKG_A SspH1 10 87.89 30.75 19.83 2.86 11.71 0.15 4NKG_C 6 IpaH3 9 89.22 35.25 24.13 2.53 10.21 0.07 3CVR 7 slrP 12 70.03 34.93 25.34 2.00 10.31 0.11 4PUF_A slrP 12 54.07 35.31 26.44 1.53 10.20 0.15 4PUF_B n repeat number of LRRs, P helix pitch, N residue number per turn, R helix radius, Δz helix rise per turn, ΔΦ rotation per repeat unit, p helix regularity assigned are unambiguously regular or irregular left handed polyproline helices with only a few exceptions. The com- bination of the secondary structure assignment programs (SEGNO and XLTSSTR) and the HELFIT analysis is useful for PPII assignment in proteins. 5 Conclusions The present study shows that the N-subtype bacterial LRRs are characterized by a unique super secondary structures consisting of PPII helices and a β-turn. In contrast, the T-subtype VS prefers two separate PPIIs with two or three or with only two residues. The type I β-turns can be regarded as regular, right handed helices. We propose three important structural parameters: the three angles between the two helix axes of PPII and β-turn, between two helix axes of PPII and LRR domain, and between the helix axis ΔΦ 2Rsin( ) () of LRR domain and the vector product of P × B. These three angles characterize the super secondary structure and the LRR domain. Fig. 4 The correlation of Δz and 2·R·sin(ΔΦ/2) in the helix param- eters of LRR domains in the seven bacterial LRR proteins 1 3 Δ z () ---------------- 234 D. Batkhishig et al. → → P B 60 40 30 30 20 30 15 15 5 5 0 0 0 85 90 95 100105 110115 120 20 25 30 35 40 45 50 55 75 80 85 90 95 100105 110115 120125 Ω (°) Ω (°) Ω (°) 1 2 Fig. 5 Geometrical analysis of super secondary structure. a The definition of the three angles of Ω Ω , and Ω . b Frequency distributions of the 1, 2 3 Ω Ω , and Ω angles (by the XLTSSTR program) 1, 2 3 247 249 Leu Val 204 206 Leu Leu Leu 201 Leu Leu 260 Leu Leu Leu Val Leu Fig. 6 Hydrophobic cores in the N-subtype (a) and T-subtype 211-LTHLSLKYNNLTTVPRSLPPS-221 (LRR7) in bovine TLR9 (b) of bacterial LRR. a Sequence 244-LRTLEVSGNQLT- (PDB ID:3WPC_A). Green is α-carbon, red oxygen, and blue nitro- SLPVLPPG-263 (LRR3) in SspH2 (PDB ID: 3G06_A). b Sequence gen. (Color figure online) Open Access This article is distributed under the terms of the Crea- Funding This study was funded by National University of tive Commons Attribution 4.0 International License (http://creat iveco Mongolia (FELLOWSHIP GRANT-P2016-1173) (to P. E.) mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- tion, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Compliance with Ethical Standards Conflict of interest All authors declare that they have no conflicts of interest. 1 3 ---------------- Frequency Frequency Frequency Super Secondary Structure Consisting of a Polyproline II Helix and a β-Turn in Leucine Rich Repeats… 235 21. Cubellis MV, Caillez F, Blundell TL, Lovell SC (2005) Proper- References ties of polyproline II, a secondary structure element implicated in protein–protein interactions. Proteins 58:880‒892 1. Matsushima N, Kretsinger RH (2016) Leucine rich repeats: 22. Berisio R, Loguercio S, De Simone A, Zagari A, Vitagliano L sequences, structures, ligand—interactions, and evolution. LAM- (2006) Polyproline helices in protein structures: a statistical sur- BERT Academic Publishing, Saarbrücken vey. Protein Pept Lett 13:847‒854 2. Kobe B, Deisenhofer J (1994) The leucine-rich repeat: a versatile 23. Barlow DJ, Thornton JM (1988) Helix geometry in proteins. J Mol binding motif. Trends Biochem Sci 19:415–421 Biol 201:601–619 3. Kobe B, Kajava AV (2001) The leucine-rich repeat as a protein 24. Creamer TP, Campbell MN (2012) Determinants of the poly- recognition motif. Curr Opin Struct Biol 1:725–732 proline II helix from modeling studies. Adv Protein Chem 4. Matsushima N, Tachi N, Kuroki Y, Enkhbayar P, Osaki M, 62:263–282 Kamiya M, Kretsinger RH (2005) Structural analysis of leucine- 25. Hopfinger A (2012) Conformational properties of macromol- rich-repeat variants in proteins associated with human diseases. ecules. Elsevier, Amsterdam Cell Mol Life Sci 62:2771–2791 26. Schulz GE, Schirmer RH (1979) Principles of protein structure. 5. Bella J, Hindle KL, McEwan PA, Lovell SC (2008) The leucine- Springer, Berlin rich repeat structure. Cell Mol Life Sci 65:2307–2333 27. Kumar P, Bansal M (2016) Structural and functional analy- 6. Matsushima N, Enkhbayar P, Kamiya M, Osaki M, Kretsinger RH ses of PolyProline-II helices in globular proteins. J Struct Biol (2005) Leucine-rich repeats (LRRs): structure, function, evolution 196:414‒425 and interaction with ligands. Drug Design Rev 2:305–322 28. Kabsch W, Sander C (1983) Dictionary of protein secondary struc- 7. Enkhbayar P, Miyashita H, Kretsinger RH, Matsushima N (2014) ture: pattern recognition of hydrogen-bonded and geometrical fea- Helical parameters and correlations of Tandem Leucine rich tures. Biopolymers 22:2577–2637 repeats in proteins. J Proteom Bioinform 7:139–150 29. Frishman D, Argos P (1995) Knowledge-based protein secondary 8. Ng AC, Eisenberg JM, Heath RJ, Huett A, Robinson CM, Nau structure assignment. Proteins 23:566–579 GJ, Xavier RJ (2011) Human leucine-rich repeat proteins: a 30. Berman HM, Kleywegt GJ, Nakamura H, Markley JL (2014) genome-wide bioinformatic categorization and functional anal- The protein data bank archive as an open data resource. J Com- ysis in innate immunity. Proc Natl Acad Sci USA 108(Suppl put Aided Mol Des 28:1009‒1014 1):4631–4638 31. Mansiaux Y, Joseph AP, Gelly JC, de Brevern AG (2011) 9. Tör M, Lotze MT, Holton N (2009) Receptor-mediated signal- Assignment of PolyProline II conformation and analysis of ling in plants: molecular patterns and programmes. J Exp Bot sequence-structure relationship. PLoS ONE 6:e18401 60:3645–3654 32. Srinivasan R, Rose GD (1999) A physical basis for protein sec- 10. Coburn B, Sekirov I, Finlay BB (2007) Type III secretion systems ondary structure. Proc Natl Acad Sci USA 96:14258‒14263 and disease. Clin Microbiol Rev 20:535–549 33. Cubellis MV, Cailliez F, Lovell SC (2005) Secondary structure 11. Haraga A, Miller SI (2006) A Salmonella type III secretion effec- assignment that accurately reflects physical and evolutionary tor interacts with the mammalian serine/threonine protein kinase characteristics. BMC Bioinform 6(Suppl 4):S8 PKN1. Cell Microbiol 8:837–846 34. King SM, Johnson WC (1999) Assigning secondary structure 12. Matsushima N, Ohyanagi T, Tanaka T, Kretsinger RH (2000) from protein coordinate data. Proteins 35:313–320 Super-motifs and evolution of tandem leucine-rich repeats within 35. Kumar P, Bansal M (2005) Identification of local variations the small proteoglycans-biglycan, decorin, lumican, fibromodu- within secondary structures of proteins. Acta Crystallogr lin, PRELP, keratocan, osteoadherin, epiphycan, and osteoglycin. 71:1077–1086 Proteins 38:210–225 36. Richards FM, Kundrot CE (1988) Identification of structural 13. Matsushima N, Kamiya M, Suzuki N, Tanaka T (2000) Super- motifs from protein coordinate data: secondary structure and r fi st- motifs of leucine-rich repeats (LRRs) proteins. Genome Inform level supersecondary structure. Proteins 3:71–84 11:343–345 37. Chiang YS, Gelfand TI, Kister AE, Gelfand IM (2007) New clas- 14. Matsushima N, Tanaka T, Enkhbayar P, Mikami T, Taga M, sification of supersecondary structures of sandwich-like proteins Yamada K, Kuroki Y (2007) Comparative sequence analysis of uncovers strict patterns of strand assemblage. Proteins 68:915–921 leucine-rich repeats (LRRs) within vertebrate toll-like receptors. 38. Adzhubei AA, Sternberg MJ (1994) Conservation of polyproline BMC Genom 8:24–143 II helices in homologous proteins: implications for structure pre- 15. Siligardi G, Drak AF (1995) The importance of extended confor- diction by model building. Protein Sci 3:2395–2410 mations and, in particular, the PII conformation for the molecular 39. Evdokimov AG, Anderson DE, Routzahn KM, Waugh DS (2001) recognition of peptides. Biopolymers 37:281–292 Unusual molecular architecture of the Yersinia pestis cytotoxin 16. MacArthur MW, Thornton JM (1991) Influence of proline resi- YopM: a leucine-rich repeat protein with the shortest repeating dues on protein conformation. J Mol Biol 218:397–412 unit. J Mol Biol 312:807–821 17. Rath A, Davidson AR, Deber CM (2005) The structure of 40. Park H, Huxley-Jones J, Boot-Handford RP, Bishop PN, Attwood “unstructured” regions in peptides and proteins: role of the poly- TK, Bella J (2008) LRRCE: a leucine-rich repeat cysteine capping proline II helix in protein folding and recognition. Biopolymers motif unique to the chordate lineage. BMC Genom 9:599 80:179‒185 41. Adzhubei AA, Sternberg MJ, Makarov AA (2013) Polypro- 18. Vitagliano L, Berisio R, Mastrangelo A, Mazzarella L, Zagari line-II helix in proteins: structure and function. J Mol Biol A (2001) Preferred proline puckerings in cis and trans pep- 425:2100–2132 tide groups: implications for collagen stability. Protein Sci 42. Keszei AF, Tang X, McCormick C, Zeqiraj E, Rohde JR, Tyers M, 10:2627–2632 Sicheri F (2014) Structure of an SspH1-PKN1 complex reveals the 19. Adzhubei AA, Sternberg MJ (1993) Left-handed polyproline basis for host substrate recognition and mechanism of activation II helices commonly occur in globular proteins. J Mol Biol for a bacterial E3 ubiquitin ligase. Mol Cell Biol 34:362‒373 229:472–493 43. Quezada CM, Hicks SW, Galan JE, Stebbins CE (2009) A fam- 20. Stapley BJ, Creamer TP (1993) A survey of left-handed polypro- ily of Salmonella virulence factors functions as a distinct class line II helices. Protein Sci 8:587‒595 of autoregulated E3 ubiquitin ligases. Proc Natl Acad Sci USA 106:4864‒4869 1 3 236 D. Batkhishig et al. 44. Zhu Y, Li H, Hu L, Wang J, Zhou Y, Pang Z, Liu L, Shao F (2008) 61. Heinig M, Frishman D (2004) STRIDE: a web server for sec- Structure of a Shigella effector reveals a new class of ubiquitin ondary structure assignment from known atomic coordinates of ligases. Nat Struct Mol Biol 15:1302‒1308 proteins. Nucleic Acids Res 32(WebServer issue):W500–W5022 45. Takagi K, Kim M, Sasakawa C, Mizushima T (2016) Crystal 62. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt structure of the substrate-recognition domain of the Shigella E3 DM, Meng EC, Ferrin TE (2004) UCSF Chimera—a visualiza- ligase IpaH9.8. Acta Crystallogr 72:269‒275 tion system for exploratory research and analysis. J Comput Chem 46. Zouhir S, Bernal-Bayard J, Cordero-Alba M, Cardenal-Munoz E, 25:1605–1612 Guimaraes B, Lazar N, Ramos-Morales F, Nessler S (2014) The 63. Enkhbayar P, Hikichi K, Osaki M, Kretsinger RH, Matsushima structure of the Slrp-Trx1 complex sheds light on the autoinhibi- N (2006) 3(10)-helices in proteins are parahelices. Proteins tion a of the type III secretion system effectors of the NEL family. 64:691‒699 Biochem J 464:135‒144 64. Enkhbayar P, Boldgiv B, Matsushima N (2010) Omega-Helices 47. Kokatla HP, Sil D, Tanji H, Ohto U, Malladi SS, Fox LM, Shimizu in proteins. Protein J 29:242‒249 T, David SA (2014) Structure-based design of novel human Toll- 65. Matsushima N, Miyashita H, Enkhbayar P, Kretsinger RH (2015) like receptor 8 agonists. ChemMedChem 9:719‒723 Comparative geometrical analysis of leucine-rich repeat structures 48. Ohto U, Shibata T, Tanji H, Ishida H, Krayukhina E, Uchiy- in the nod-like and toll-like receptors in vertebrate innate immu- ama S, Miyake K, Shimizu T (2015) Structural basis of CpG nity. Biomolecules 5:1955–1978 and inhibitory DNA recognition by Toll-like receptor 9. Nature 66. Wilmot CM, Thornton JM (1988) Analysis and prediction of the 520:702‒705 different types of beta-turn in proteins. J Mol Biol 203:221‒232 49. Tanji H, Ohto U, Shibata T, Miyake K, Shimizu T (2013) Struc- 67. Jha AK, Colubri A, Zaman MH, Koide S, Sosnick TR, Freed KF tural reorganization of the toll-like receptor 8 dimer induced by (2005) Helix, sheet, and polyproline II frequencies and strong agonistic ligands. Science 339:1426–1429 nearest neighbor effects in a restricted coil library. Biochemistry 50. Paracuellos P, Kalamajski S, Bonna A, Bihan D, Farndale RW, 44:9691–9702 Hohenester E (2017) Structural and functional analysis of two 68. Adzhubei AA, Eisenmenger F, Tumanyan VG, Zinke M, Brodzin- small leucine-rich repeat proteoglycans, fibromodulin and chon- ski S, Esipova NG (1987) Approaching a complete classification droadherin. Matrix Biol 63:106–116 of protein secondary structure. J Biomol Struct Dyn 5:689–704 51. Scott PG, McEwan PA, Dodd CM, Bergmann EM, Bishop PN, 69. Rämisch S, Pramhed A, Tillgren V, Aspberg A, Logan DT (2017) Bella J (2004) Crystal structure of the dimeric protein core of Crystal structure of human chondroadherin: solving a difficult decorin, the archetypal small leucine-rich repeat proteoglycan. molecular-replacement problem using de novo models. Acta Crys- Proc Natl Acad Sci USA 101:15633‒15638 tallogr 73:53–63 52. Scott PG, Dodd CM, Bergmann EM, Sheehan JK, Bishop PN 70. Kay BK, Williamson MP, Sudol M (2000) The importance of (2006) Crystal structure of the biglycan dimer and evidence that being proline: the interaction of proline-rich motifs in signaling dimerization is essential for folding and stability of class I small proteins with their cognate domains. FASEB J 14:231–241 leucine-rich repeat proteoglycans. J Biol Chem 281:13324–13332 71. Ananthanarayanan VS, Soman KV, Ramakrishnan C (1987) A 53. Seiradake E, del Toro D, Nagel D, Cop F, Härtl R, Ruff T, Seyit- novel supersecondary structure in globular proteins comprising Bremer G, Harlos K, Border EC, Acker-Palmer A, Jones EY, the collagen-like helix and beta-turn. J Mol Biol 198:705‒709 Klein R (2014) FLRT structure: balancing repulsion and cell adhe- 72. Matsushima N, Creutz CE, Kretsinger RH (1990) Polyproline, sion in cortical and vascular development. Neuron 84:370–385 beta-turn helices. Novel secondary structures proposed for the 54. Lu YC, Nazarko OV, Sando R 3rd, Salzman GS, Südhof TC, Araç tandem repeats within rhodopsin, synaptophysin, synexin, gliadin, D (2015) Structural basis of latrophilin-FLRT-UNC5 interaction RNA polymerase II, hordein, and gluten. Proteins 7:125‒155 in cell adhesion. Structure 23:1678–1691 73. Meredith GD, Chang WH, Li Y, Bushnell DA, Darst SA, Korn- 55. Ranaivoson FM, Liu Q, Martini F, Bergami F, von Daake S, Li S, berg RD (1996) The C-terminal domain revealed in the structure Lee D, Demeler B, Hendrickson WA, Comoletti D (2015) Struc- of RNA polymerase II. J Mol Biol 258:413–419 tural and mechanistic insights into the latrophilin3-FLRT3 com- 74. Kumaki Y, Matsushima N, Yoshida H, Nitta K, Hikichi K (2001) plex that mediates glutamatergic synapse development. Structure Structure of the YSPTSPS repeat containing two SPXX motifs 23:1665–1677 in the CTD of RNA polymerase II: NMR studies of cyclic model 56. Jackson VA, Mehmood S, Chavent M, Roversi P, Carrasquero peptides reveal that the SPTS turn is more stable than SPSY in M, Del Toro D, Seyit-Bremer G, Ranaivoson FM, Comoletti D, water. Biochim Biophys Acta 1548:81–93 Sansom MS, Robinson CV, Klein R, Seiradake E (2016) Super- 75. Cramer P, Bushnell DA, Kornberg RD (2001) Structural basis complexes of adhesion GPCRs and neural guidance receptors. Nat of transcription: RNA polymerase II at 2.8 angstrom resolution. Commun 17:11184 Science 292:1863–1876 57. Enkhbayar P, Damdinsuren S, Osaki M, Matsushima N (2008) 76. Hsin JP, Manley JL (2012) The RNA polymerase II CTD HELFIT: helix fitting by a total least squares method. Comput coordinates transcription and RNA processing. Genes Dev Biol Chem 32:307‒310 26:2119–2137 58. Matsushima N, Miyashita H, Mikami T, Yamada K (2011) A new 77. Martin J, Letellier G, Marin A, Taly JF, de Brevern AG, Gibrat method for the identification of leucine-rich repeats by incorporat- JF (2005) Protein secondary structure assignment revisited: a ing protein secondary structure prediction. Tuteja R (ed) Bioinfor- detailed analysis of different assignment methods. BMC Struct matics: genome bioinformatics and computational biology. NOVA Biol 5:17 Sience Pulishers, Hauppauge, pp 61–88 78. Crooks GE, Hon G, Chandonia JM, Brenner SE (2004) WebLogo: 59. Chebrek R, Leonard S, de Brevern AG, Gelly JC (2014) Poly- a sequence logo generator. Genome Res 14:1188–1190 prOnline: polyproline helix II and secondary structure assignment database. Database 2014:bau102 60. Hutchinson EG, Thornton JM (1996) PROMOTIF—a program to identify and analyze structural motifs in proteins. Protein Sci 5:212–220 1 3
The Protein Journal – Springer Journals
Published: Apr 12, 2018
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