1

Crystal structure at 1.5 Å resolution of Pyrus pyrifolia pistil

ribonuclease responsible for gametophytic self-incompatibility*

Takanori Matsuura‡, Hiroaki Sakai‡, Masaki Unno‡, Koh Ida†, Mamoru Sato†,

Fumio Sakiyama¶, and Shigemi Norioka‡§

From the ‡Institute for Protein Research, Osaka University, Suita, Osaka 565-

0871, Japan, †Graduate School of Integrated Science, Yokohama City University,

Tsurumi-ku, Yokohama 230-0045, Japan, and ¶International Buddhist University,

Habikino, Osaka-538-8501, Japan

§Corresponding author:

Shigemi Norioka

Institute for Protein Research, Osaka University,

3-2 Yamadaoka, Suita, Osaka 565-0871, Japan

Tel. +81-6-6879-8618 Fax. +81-6-6879-8619

Email. norioka@protein.osaka-u.ac.jp

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on September 27, 2001 as Manuscript M107617200 by guest on M

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Running Title

Crystal Structure of Pyrus pyrifolia S3-RNase

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SUMMARY

The crystal structure of the Pyrus pyrifolia pistil ribonuclease (S3-RNase)

responsible for gametophytic self-incompatibility was determined at 1.5 Å

resolution. It consists of eight helices and seven β-strands, and its folding

topology is typical of RNase T2 family enzymes. Based on a structural

comparison of S3-RNase with RNase Rh, a fungal RNase T2 family enzyme, the

active site residues of S3-RNase assigned were His33 and His88 as catalysts and

Glu84 and Lys87 as stabilizers of an intermediate in the transition state.

Moreover, amino acid residues that constitute substrate binding sites of the two

RNases could be superimposed geometrically. A hypervariable (HV) region

that has an S-allele-specific sequence comprises a long loop and short α-helix.

This region is far from the active site cleft, exposed on the molecule’s surface,

and positively charged. Four positively selected (PS) regions, in which the

number of nonsynonymous substitutions exceeds that of synonymous ones, are

located on either side of the active site cleft, and accessible to solvent. These

structural features suggest that the HV or PS regions may interact with a pollen

S-gene product(s) to recognize self and non-self pollen.

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INTRODUCTION

Many flowering plants have a self-incompatibility system which recognizes

self or non-self between the pistil and pollen (tube) after pollination and

suppresses growth of the self-pollen tube to prevent self-fertilization (1,2).

Gametophytic self-incompatibility (GSI)1 is controlled genetically by a single

locus (S-locus) with multiple alleles (1,2). When a pollen grain lands on a

stigma of the pistil, a process which discriminates as to whether an S-allele of

the pollen matches one of the two S-alleles of the pistil takes place. The pollen

grain germinates on the stigma and grows into the style toward the embryo. If

its S-allele matches one of the two S-alleles of the pistil, pollen tube growth is

arrested in the style, and no fertilization takes place. In solanaceous,

scrophulariaceous, and rosaceous plants that have GSI, the pistil glycoproteins

that cosegregate with the S-alleles have been identified as ribonucleases of the

RNase T2 family (S-RNase) (3). McClure et al. reported that pollen rRNA is

degraded after self-pollination but not after cross-pollination and suggested that

GSI expression is mediated by degradation of the pollen rRNA of self-pollen

tubes by S-RNase, leading to depletion of protein biosynthesis and the eventual

arrest of tube growth (4). S-RNase has been confirmed necessary for GSI from

results of gain-of-function and loss-of-function experiments on transgenic plants

of solanaceous species (5,6). Transgenic experiments also have shown that

the RNase activity of S-RNase is necessary for GSI (7), that in petunia the

carbohydrate moiety is not responsible for GSI (8), and that mutant S-RNase

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which has lost RNase activity acts as a dominant negative for GSI (9).

Based on these findings, two models have been proposed to explain S-

allele-specific inhibition of pollen tube growth; the receptor and inhibitor models

(10). In the receptor model, the pollen S-gene product(s) is the receptor that

incorporates the S-RNase that matches the pollen S-allele. In the inhibitor

model, S-RNase enters the pollen tube nonspecifically and is inhibited, except

for the enzyme that has the same pollen S-allele. Recent,

immunocytochemical research has shown that S-RNase enters and is

accumulated in the cytoplasm of all pollen tube haplotypes without S-allele

specificity, experimental support for the inhibitor model (11). The GSI

mechanism, however, is still not clear because no pollen S-gene product(s) that

interacts with S-RNase and causes self and non-self recognition between the

pollen and pistil has yet been found.

Amino acid sequence analyses of solanaceous S-RNases have identified two

hypervariable regions, HVa and HVb, with extremely high levels of divergence

between allelic sequences; candidates for sequences that would be recognized

as S-allele-specific by pollen (12). In fact, the S-allele specificity of the

Solanum chacoense S11-RNase was changed to the S13-allele by swapping the

HVa and HVb domains (13), and mutation in the HVa and HVb regions of S11-

RNase produced a new S-RNase with dual specificity, S11- and S13-alleles (14).

In contrast, only one hypervariable region, (HV)1, corresponding to the HVa of

the solanaceous S-RNase, has been detected in sequences of rosaceous S-

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RNases (15,16), the HVb region apparently being deleted (16,17). This

structural feature suggests that the recognition mechanism of S-allele specificity

may differ for solanaceous and rosaceous S-RNases.

Knowledge of the three-dimensional structure is required for further

investigation of the recognition mechanism of rosaceous S-RNases at the

molecular level. We therefore made a three-dimensional structural analysis of

the S-RNases from Pyrus pyrifolia (Japanese pear), a member of the Rosaceae.

Seven S-RNases (S1 – S7-RNase) have been identified in and purified from the

styles of Pyrus pyrifolia (15,18). From these we chose S3-RNase for this X-ray

crystallographic analysis because it has only two N-glycosylation sites (19), and

its amino acid sequence is highly homologous to that of Pyrus pyrifolia S5-RNase

(95.5% identity) (15). Identification of the three-dimensional structures of the

S3- and S5-RNases will pave the way for a structural comparison that provides

high resolution information on molecular recognition between these S-RNases

and the pollen S-gene product(s). We report the crystal structure of Pyrus

pyrifolia S3-RNase at 1.5 Å resolution and discuss the recognition site(s) present

on S3-RNase and the structural basis of its enzymatic activity. This is the first

report on the three-dimensional structure of a rosaceous S-RNase.

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EXPERIMENTAL PROCEDURES

Crystallization and Data collection— Pyrus pyrifolia S3-RNase was purified

and crystallized as described previously (20). The crystals belong to the P21

space group with unit cell dimensions of a=45.65, b=52.59, c=47.57 Å, and

β=106.45° (20). Diffraction data were collected with an image plate detector

Raxis IV (Rigaku) at the beam line of BL40B2 at SPring-8 to 1.5 Å resolution.

Image data were processed by the programs DENZO and SCALEPACK (21)

(Table I). Multiple isomorphous replacement was used to determine the crystal

structure of S3-RNase. Two ethylmercurithiosalicylic acid derivatives (Hg1,

Hg1’), a mercury (II) ammonium thiocyanate derivative (Hg2), and a lead (II)

acetate derivative (Pb) were prepared by soaking the crystals in heavy atom

reagents (Table I). All diffraction data on the derivative crystals were collected

at the Photon Factory with a CCD detector Quantum 4R (ADSC) at the beam

line of BL6A and processed by the MOSFLM (22) and SCALA (23) programs.

Crystal structure determination— Patterson maps were automatically

interpreted by the program SOLVE (24) to locate the heavy atom coordinates of

each derivative. By use of the solution coordinates, MIR phase calculation was

done at 2.5 Å resolution by the maximum-likelihood refinement program SHARP

(25) (Table I). The experimental MIR phases were improved by the DM (23)

and wARP (26) programs. First, the phases at 2.5 Å resolution gradually were

extended to 1.7 Å resolution by DM with solvent flattening and histogram

matching. The phases obtained were further refined and extended to 1.5 Å

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resolution by weighted averaging of multiple-refined dummy atomic models

implemented in wARP. This technique for averaging the structure factors was

very effective for improving the phases and provided substantial improvement in

the resulting electron density map. The subsequent main chain trace was done

by wARP. Based on the automatically traced main chain coordinates, 200

amino acid residues were successfully built by program O (27). X-ray

crystallographic refinement was done by the CNS program (28). One cycle of

simulated annealing refinement followed by positional and temperature factor

refinements was repeated several times. Throughout the refinements bulk

solvent correction was applied, and the resolution range gradually was extended

from 2.3 to 1.5 Å. At each stage of crystallographic convergence, the model

was carefully checked and rebuilt into the simulated annealed omit map

calculated by CNS. Solvent water molecules were picked up from a difference

Fourier map by use of the automated scripts implemented in CNS. The lower

cut off level for picking up the solvent water molecules in the residual electron

densities was set at 3σ. High-resolution model coordinates of sugar chains

were obtained from the Uppsala web server, HIC-Up (29). The crystal structure

was refined to the respective R- and free R-factors of 17.2% and 20.2% in the

resolution range of 500-1.5 Å. The final model has 1643 protein atoms, 59

sugar atoms, and 266 water atoms. A Ramachandran plot from the program

PROCHECK (30) shows that 91.5% of the residues are in the most favorable

region, 8.5% in the additional allowed region, and none in the generally allowed

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and disallowed regions. The coordinates have been deposited in the Protein

Data Bank with the accession code 1IQQ.

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RESULTS

Overall Structure— The crystal structure of Pyrus pyrifolia S3-RNase was

determined by the multiple isomorphous replacement (MIR) and refined to an R-

factor of 17.2% at 1.5 Å resolution. Table I summarizes the data collected and

gives the final refinement statistics. The geometry of the current model is such

that the root mean square deviations (rmsds) from the ideal values are 0.022 Å

for bond length and 2.02° for bond angles.

A stereo view of the three-dimensional structure of S3-RNase is shown in Fig.

1. The molecule’s size is approximately 40 Å x 50 Å x 30 Å. This protein has

an α + β type structure consisting of eight helices (six α-helices [α1, α2, α4, and

α6-α8], two 310-helices [α3 and α5]), and seven β-strands (β1-β7) (Fig. 1a).

The folding topology of its main chain is very similar to the topologies of the

RNase T2 family enzymes: the RNase Rh from Rhizopus niveus (31,32), RNase

LE from cultured tomato cells (33), and RNase MC1 from bitter gourd seeds (34),

all of whose tertiary structures have been determined (Fig. 1b). Two secondary

structural element characteristics are present in S3-RNase; the length of the α2-

helix is shorter than the lengths of the other RNases, and a very short 310-helix

(α3), Arg74-Lys76, not seen in the RNase T2 family, is present (Fig. 2). The

electron density map for S3-RNase clearly shows four disulfide bridges: Cys15-

Cys22, Cys48-Cys91, Cys155-Cys193, and Cys170-Cys181. The topologies of these

bridges are the same as those of the Pyrus pyrifolia S4-RNase and Nicotiana

alata S2-RNase determined by mass spectrometry of fragmented peptides

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(35,36).

Catalytic Site— The structure of S3-RNase was compared in detail with that

of RNase Rh to search for catalytic site residues, because many RNase Rh

studies on the structure-function relationship of this RNase T2 family enzyme

have been done by chemical modification (37-39), site-directed mutagenesis

(40-45), and X-ray crystallographic analysis (31,32). The main chain

frameworks, including the catalytic P1 site residues of RNase Rh (β2 and α4),

were well superimposable on those of S3-RNase (Fig. 1b), indicative that their P1

site topologies are similar. The P1 site is here defined according to the

nomenclature of the subsites of RNase A given by Richards and Wyckoff (46).

As shown in Fig. 3a, the His33 and His88 of S3-RNase were superimposable

geometrically on the general acid and base catalysts His(46) and His(109) of RNase

Rh (amino acids numbered in parentheses are those of RNase Rh) (32,38,41),

suggesting that His33 and His88 are the catalysts in S3-RNase. Glu84 and Lys87

are the respective counterparts of the Glu(105) and Lys(108) of RNase Rh which are

considered to stabilize a pentacovalent intermediate of substrate RNA in the

transition state (32,33,42-44). Glu84 and Lys87 therefore may have the same

respective roles as Glu(105) and Lys(108). Trp36 is the counterpart of the Trp(49) of

RNase Rh (Fig. 3a) which functions in the fixation of catalytically important

Glu(105) and His(109). Trp(49)Nε1 forms a hydrogen bond with the γ-carboxyl group

of Glu(105), and the indole ring of Trp(49) has a partial stacking interaction with the

imidazole ring of His(109) (32). In S3-RNase, a similar stacking interaction

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between Trp36 and His88 is possible because the distance between the indole

ring of Trp36 and the imidazole ring of His88 is about 3.50 Å, and both rings are

nearly parallel (Fig. 3a). Trp36Nε1 cannot, however, interact directly with Glu84Oε1

by hydrogen bonding because the distance between the two atoms is 4.59 Å.

Possibly, they may interact via a water molecule. Indeed, a water molecule that

can hydrogen bond to both Trp36Nε1 and Glu84Oε1 is present in the P1 pocket (Fig.

3a).

Substrate Binding Sites— Two pockets, which correspond to the base

binding B1 and B2 sites of RNase Rh (32,33), are present on either side of the P1

site in S3-RNase (Fig. 1c). These B1 and B2 sites also have the nomenclature

given by Richards and Wyckoff (46). The amino acid residues that constitute

these sites could be superimposed geometrically on S3-RNase and RNase Rh

(Figs. 3b and 3c).

As for the B1 site of S3-RNase, Trp36, Ser38, and Asp44 are the respective

counterparts of the Trp(49), Asp(51), and Tyr(57) of RNase Rh (Fig. 3b). The Trp(49)

of RNase Rh has two important functions; to fix the active side residues

(described above) and to recognize the base of the substrate RNA by an

aromatic π-π stacking interaction together with Tyr(57) (called double-sided

stacking) (32,33). Trp36 may bind to the base of the substrate in the same

manner as Trp(49), but the position corresponding to Tyr(57) is occupied by Asp44,

consequently double-sided stacking recognition is impossible in S3-RNase (Fig.

3b). The base therefore probably is recognized at the B1 site of S3-RNase by a

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single-sided stacking interaction with Trp36, unlike RNase Rh and RNase LE

(32,33). X-ray crystallographic data on the RNase Rh / 2’-AMP complex shows

that Asp(51) contributes to the adenylic acid preference at the B1 site of RNase Rh

by hydrogen bonding to the adenine base of 2’-AMP (32,33). The position

corresponding to Asp(51), however, is occupied by Ser38 in S3-RNase (Fig. 3b).

Experiments on substrate specificity done with dinucleotide monophosphate

showed that the base specificity at the B1 site of S3-RNase is wider than that of

RNase Rh (data not shown). This extended base specificity may be due to

substitution of Asp(51) with Ser38, to single-sided stacking with Trp36, or both.

The B2 site of S3-RNase seems to be comprised of Gln9, Pro69, Asn70, Val71,

Phe72, and Phe80 (Fig. 3c), which geometrically correspond to the Gln(32), Pro(92),

Ser(93), Asn(94), Gln(95), and Phe(101) of RNase Rh that have been identified as

components of the B2 site based on X-ray crystallographic data for the complex

with d(ApC) (33). In RNase Rh, the base is thought to be recognized at the B2

site by an aromatic stacking interaction with Phe(101) and van der Waals contact

with the side-chains of Asn(94) and Gln(95) (33). Similar recognition should occur

at the B2 site of S3-RNase because Val71, Phe72, and Phe80, respectively, can be

superimposed geometrically on Asn(94), Gln(95), and Phe(101) (Fig. 3c).

Hypervariable Region— One hypervariable (HV)1 region with an allele-

specific sequence is present in the rosaceous S-RNases (Fig. 4a) and is a

candidate for the recognition site of pollen S-gene product(s) (15). The HV

region of S3-RNase (Pro49-Gln63) is made up of a loop (Pro49-Glu57) and half of an

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α2-helix (Lys58-Gln63) and is exposed on the molecule’s surface (Figs. 1a and 4b).

Although the exposed loop appears to be somewhat flexible, three of its nine

amino acid residues (Asn52, Arg54, and Arg56) interact with the amino acid

residues inside the molecule by hydrogen bonding (Fig. 5). Asn52Nδ2 is

hydrogen bonded to the carbonyl group of Asp97 directly, and Asn52Oδ1 to the

amide of Glu99 via a water molecule. The δ-guanidino group of Arg54 forms

water-mediated hydrogen bonds individually with Asn100Nδ2 and Asp97Oδ1. The

δ-guanidino group of Arg56 directly forms three hydrogen bonds with Asp82Oδ1,

Glu99Oε1, and Glu61Oε1 (Fig. 5).

In contrast, the half of the α2-helix (Lys58-Gln63), located within the HV region,

has six amino acid residues. Of these, the side-chains of Leu60 and Glu61

interact with other amino acids (Fig. 5). The side-chain of Leu60 is packed into a

hydrophobic space composed of the side-chains of Leu64, Asn100, Phe103, and

Ile107 and the main chain of Arg56-Glu57. The Oε1 and Oε2 of Glu61 respectively

are hydrogen bonded to Arg56Nε and Asn78Nδ2 (Fig. 5). These described

interactions may fix and stabilize the conformations of the loop and helix; i.e., the

HV region.

This HV region is comprised of 15 amino acid residues, ten of which, Pro49,

Ile50, Lys51, Ile53, Lys55, Glu57, Lys58, Leu59, His62, and Gln63 are widely exposed to

solvent. The side-chains of Ile53, Lys55, Glu57, Lys58, Leu59 and His62, in particular,

prominently extend into the solvent (Fig. 5). Given that the HV region is in

contact with the pollen factor(s), these residues may have importance in pollen

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factor binding.

Positively Selected Regions— Recognition sites in some proteins (e.g., the

major histocompatibility complex (MHC) [47-49], antigenic surface proteins of

parasites and viruses (49), and acrosomal proteins of the abalone [50,51]) are

reported to be regions in which the number of nonsynonymous nucleotide

substitutions (dN)1 exceeds that of synonymous substitutions (dS)1, and positive

selection probably takes place in these regions. Window analysis of the dS and

dN in rosaceous S-RNases detected four regions with an excess of dN over dS,

in which positive selection may operate (PS1-PS4) (Fig.4) (52). PS1 nearly

duplicates the HV region. It is interesting that the four positively selected (PS)1

regions were detected by window analysis of the rosaceous S-RNase genes but

that there is only a single region with an allele-specific amino acid sequence.

PS1 (HV) and PS2, as well as PS3 and PS4 respectively are close in the three-

dimensional structure of S3-RNase, on either side of the active site cleft

(including the P1, B1, and B2 sites) and accessible to solvent (Figs. 1c and 4b).

Sugar Chains— Pyrus pyrifolia S3-RNase has two potential N-glycosylation

sites, Asn18 and Asn116 (Fig. 1a). The Asn18 site is specific to the S3- and S5-

RNases, whereas the Asn116 site is conserved among all the resaceous S-

RNases (Fig. 4a). A two-dimensional sugar map and mass spectrometry of

fragmented peptides showed that both sites have heterogeneous N-glycans,

Asn18 mainly glycosylated by a chitobiose (GlcNAcβ1-4GlcNAc) and Asn116

primarily occupied by a xylomannose type sugar chain (19). The core structure,

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Manβ1-4GlcNAcβ1-4GlcNAc, of the xylomannose type sugar chain at Asn116,

clearly visible in the difference Fourier map, was modeled to fit the densities

(data not shown). No clear electron densities corresponding to the chitobiose

at Asn18 and the mannose and xylose moieties of the sugar chain at Asn116 were

observed, evidence that their conformations are highly disordered. As Asn18

and Asn116 respectively are located at the end of and on the opposite side of the

active site cleft (Figs.1a and 1c), the S3-RNase sugar chains probably are not

involved in its enzymatic properties and the recognition of self and non-self.

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DISCUSSION

The structure of the active site (the catalytic P1 site and substrate binding B1

and B2 sites) of S3-RNase is typical of the structures of the RNase T2 family

enzymes. Probably, His33 and His88 function as general acid and base catalysts,

and Glu84 and Lys87 stabilize the pentacovalent intermediate in the transition

state. One marked difference between the P1 sites of S3-RNase and RNase Rh

is that the imidazole ring of His33 is rotated about 90° from that of His(46) (Fig. 3a),

even though the distance between His33Nε2 and His88Nε2 (7.15 Å) is nearly equal

to that between His(46)Nε2 and His(109)Nε2 (6.71 Å) (32). As S3-RNase has

ribonuclease activity (20), rotation of the ring of His33 is not crucial for its catalytic

activity. Another difference is that the His(104) of RNase Rh, which is expected to

bind to the phosphate group of the substrate RNA (32,41), is replaced by Lys83 in

S3-RNase (Fig. 3a). As the ε-amino group of Lys83 extends outside the P1 site

and is about 8 Å from the imidazole ring of His(104), it is unlikely that it interacts

with the phosphate group of the substrate unless a large induced fit occurs on

the side-chain of Lys83 during catalysis. A more detailed X-ray crystallographic

study of the S3-RNase complex with nucleotides is required to clarify how the

rotation of the imidazole ring of His33 and substitution of His(104) for Lys83 affect the

ribonuclease activity of S3-RNase.

Although it has not been shown experimentally what the in vivo substrate of

S-RNase during the GSI reaction is, it must be pollen (tube) rRNA according to

the receptor and inhibitor models (10). As the overall shape of the active site of

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S3-RNase is very similar to the active sites of the other RNase T2 family enzymes

(except for a few amino acid substitutions), S3-RNase is not likely to have a strict

substrate specificity corresponding to the S-alleles. Actually, S3-RNase can

hydrolyze dinucleotide monophosphate nonspecifically (data not shown),

therefore it is reasonable that S-allele specificity in the inhibition of pollen tube

growth is not expressed due to the enzyme’s restricted substrate specificity but

to its interaction with the pollen S-gene product(s); consistent with either the

receptor or inhibitor model.

Domain swapping experiments proved that in the solanaceous S-RNases,

two hypervariable regions, HVa and HVb, essentially are responsible for S-

allele-specific pollen recognition (13,14). Only one hypervariable (HV) region

appears to be present in the rosaceous S-RNases, and it most likely is a

recognition site for the pollen factor(s) (Fig.4a). As compared with the

sequences of the solanaceous S-RNases (16,17), the HVa and HVb regions

correspond respectively to the HV region and vicinity of the α3-helix (Arg74-Lys76),

tentatively called the HVb’, of S3-RNase. As HVb’ is much shorter than HVb, it

has not been identified as an allele-specific sequence (Fig.4a). The three-

dimensional structure around the α3-helix therefore also is assumed to differ in

the solanaceous and rosaceous S-RNases.

The HV region of S3-RNase is located on the molecule’s surface, easily

accessible to external molecules such as the pollen factor(s). As shown in Fig.

1b, the main chain frameworks of RNase T2 family enzymes, including S3-RNase,

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superimpose well on one another; in particular their core structures composed of

three α-helices (α4, α6 and α7) and four β-strands (β1, β2, β4 and β5). The

backbone structures, however, are notably very variable in what corresponds to

the HV region (Fig. 1b and 4b) even though it is composed of the same

secondary elements, a loop and an α-helix, as in all the RNases (Fig. 2). This

suggests that the HV region of S3-RNase has an allele-specific conformation.

Five basic amino acids, Lys51, Arg54, Lys55, Arg56, and Lys58 and two acidic

amino acids, Glu57 and Glu61, are present in the HV region of S3-RNase (Fig. 4a).

Although they seem to form a positively or negatively charged cluster on the

molecule’s surface, no such cluster was clearly detected because the side

chains of the basic or acidic amino acids are oriented in different directions (Figs.

5 and 6). As the δ-guanidino groups of Arg54 and Arg56 are neutralized by the

formation of hydrogen bonds, the surface of HV carries a weak rather than

strong positive charge (Figs. 5 and 6). If the other six Pyrus pyrifplia S-RNases

(S1, S2, and S4~S7) have the same main chain framework as S3-RNase, their HV

regions also would similarly have a weak positive charge, which might be

important for binding to the pollen factor(s) (Figs. 4 and 5).

PS regions with an excess of dN over dS, like the HV region a candidate for

the binding site to the pollen factor(s), are located on either side of the active site

cleft (Fig. 4b). Although there is no marked charge cluster in any of these

regions, PS1 (HV), PS2, and PS3 are hydrophilic and weakly basic (Fig. 6). In

contrast, PS4 is neutral and hydrophobic in spite of its exposure to solvent.

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Why the rosaceous S-RNase has been positively selected on such a wide area

of its molecular surface is not clear, but location topology of the PS regions

suggests that S3-RNase simultaneously interacts with multiple pollen factors and

the substrate RNA.

The amino acid sequence identity between the Pyrus pyrifolia S3- and S5-

RNases is 95.5%, and there are only nine substitutions in their 200 amino acid

residues, all concentrated on the N-terminal half of S3-RNase (Fig. 4a). Two of

the nine substitutions, Lys(Arg)51 and His(Pro)62, are the most likely to contribute

to recognition between the two alleles because they are located on the

molecular surface of the HV region (amino acids in parentheses are those of S5-

RNase) (Fig. 4b). The side-chain of Lys(Arg)51 is fully exposed to solvent and

therefore has no interaction with the other residues (Fig. 5). In contrast,

His(Pro)62 are close to Glu(Ala)65 in the three-dimensional structure although at

separate locations in the primary structure. His62Nε2 forms a water-mediated

hydrogen bond with Glu65Oε1 and both are appreciably exposed to solvent (Fig.

5). Two possibilities are suggested as to how the pollen factor(s) discriminates

S3-RNase from S5-RNase. The pollen factor(s) must come in contact with Lys51,

the site consisting of His62 and Glu65, or both in order to recognize the difference

between the S3- and S5-RNases. Or, substitution of His62 for Pro produces a

large conformational change in the α2-helix and the pollen factor(s) recognize

such a conformational change rather than the amino acid substitutions. In

general, substitution for proline tends to bend the helix structure (53). An X-ray

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crystallographic study of Pyrus pyrifolia S5-RNase is in progress in our laboratory.

If the three-dimensional structure of S5-RNase can be determined, a detailed

structural comparison can be made of the two S-Rnases which should shed light

on the recognition mechanism that operates between S3- and S5-alleles.

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Acknowledgments

We thank Prof. T. Tsukihara of the Institute for Protein Research, Osaka

University for his helpful advice on the crystallization and the X-ray

crystallographic analysis. We also thank K. Miura of SPring-8, Hyogo and N.

Igarashi of the Photon Factory, Ibaragi, for their useful advice on the data

collection.

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Footnotes

*This work was supported in part by a Grant-in-Aid for Special Research on

Priority Areas (B) (11238204) from the Ministry of Education, Culture, Sports,

Science and Technology, Japan.

The atomic coordinates and structure factors (code 1IQQ) have been deposited

in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics,

Rutgers University, New Brunswick, NJ (http://www.rcsb.org/pdb).

1The abbreviations used are GSI, gametophytic self-incompatibility; HV,

hypervariable; PS, positively selected; dN, the number of nonsynonymous

nucleotide substitutions; dS, the number of synonymous nucleotide

substitutions.

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Figure Legends

Figure 1. Stereo diagram showing the overall structure of Pyrus pyrifolia S3-

RNase. (a) Stereo ribbon diagram of S3-RNsae. N-Glycosylated asparagines,

N18 and N116, are shown as ball-and-stick models. (b) Stereo diagram

showing the superposition of RNase T2 family enzymes. S3-RNase is in red,

RNase Rh in cyan (32), RNase LE in yellow (33), and RNase MC1 in green (34).

(c) Stereo drawing of the space filling model of S3-RNase. Amino acid residues

that constitute the P1, B1, and B2 sites (46) respectively are colored red, blue,

and green. Orientations of the S3-RNase molecules in figures (a), (b) and (c)

are the same. Figures (a) and (b) were prepared by the programs MOLSCRIPT

(54) and RASTER3D (55), figure (c) by the program GRASP (56).

Figure 2. Sequence alignment of RNase T2 family enzymes and their

secondary structural elements. Amino acid residue numbering is based on that

for Pyrus pyrifolia S3-RNase. Amino acid residues that form secondary

structures are the α-helix (red), 310-helix (red, slanted), and β-strand (blue).

The secondary S3-RNase structures were defined by the program DSSP (57).

Figure 3. Superposition of the active sites of Pyrus pyrifolia S3-RNase and

RNase Rh. (a), Catalytic P1 site; (b), base-binding B1 site; (c), base-binding B2

site. Names of the P1, B1, and B2 sites are based on the nomenclature of the

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subsites of RNase A (46). The amino acid residues of S3-RNase and RNase

Rh respectively are shown as ball-and-stick and wireframe constructions. The

figures were prepared by the program MOLSCRIPT (54).

Figure 4. Location of the HV and PS regions on the primary and tertiary

structures of Pyrus pyrifolia S3-RNase. (a) Sequence alignment of rosaceae S-

RNases; P. pyrifolia, Japanese pear; M. domestica, apple. HV and PS regions

are represented aligned as colored bars: HV (blue), HVb’ (black), PS1 (red), PS2

(yellow), PS3 (green), PS4 (purple). Amino acid residues that are the same as

those of S3-RNase are shaded, and positions of the amino acid substitutions

between S3- and S5-RNases are indicated by the symbol # over the alignment.

Numbering is the same that for S3-RNase. Potential N-glycosylation sites are in

red with white letters. (b) Location of HV and PS regions in the tertiary structure

of S3-RNase. These regions are in the same colors as in Figure 4(a); HV (blue),

PS1 (red), PS2 (yellow), PS3 (green), PS4 (purple). The substituted amino

acids in the S3- and S5-RNases are labeled and shown as ball-and-stick

constructions. The S3-RNase molecule orientation is the same as in Fig. 1.

This figure was prepared by the program GRASP (56).

Figure 5. Stereo diagram of the HV and PS2 regions of Pyrus pyrifolia S3-

RNase. The main chains of the HV and PS2 regions are shown as thick wires,

respectively in blue and yellow. A water molecule is indicated by a pink ball and

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a hydrogen bond by a dotted pink line. This figure was prepared by the

program MOLSCRIPT (54).

Figure 6. Electrostatic surface potential of the HV and PS regions of Pyrus

pyrifolia S3-RNase. (a) Surfaces of the HV (PS1) and PS2 regions; (b) surfaces

of the PS3 and PS4 regions. The potential displayed represents a range of -10

to +10 kBT, red being negative and blue positive. This figure was prepared by

the program GRASP (56).

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Table I

Data collection and refinement statistics for Pyrus pyrifolia S3-RNase

Rmerge = ∑|Iobs-<I>|/∑Iobs

Riso = ∑||Fdeliv|-|Fnative||/∑|Fnative|, Diffraction data were processed by DENZO &

SCALEPACK (20) (native) or MOSFLM (21) & SCALA (22) (derivative).

Rcullis = ∑||Fhobs|-|Fhcalc||/∑|Fhobs| for centric reflections, where Fhobs and Fhcalc are the

observed and calculated heavy atom structure factor amplitudes.

Phasing power = rms(|Fh|/E), where.Fh is the heavy atom structure factor amplitude,

and E the residual lack of closure error.

Figure of merit = |F(hkl)best|/|F(hkl) |

R-factor = ∑||Fobs|-|Fcalc||/∑|Fobs|

Diffraction data statisticsData set Native Hg1 Hg1’ Hg2 PbResolution (Å) 1.5 2.5 2.5 2.5 2.5Completeness (%) 96.3 99.7 100.0 98.2 99.7Unique reflection 33441 7483 7536 7450 7667Rmerge 5.7 8.5 10.1 5.2 11.5Riso 0.125 0.131 0.320 0.181Phasing statisticsRcullis 0.88 0.92 0.85 0.97Phasing power(Centrics/Acentrics)

0.97/0.96 1.18/1.45 1.64/1.68 1.04/0.9

Figure of merit(Centrics/Acentrics)

0.57/0.66

Refinement statisticsResolution range (Å) 500-1.5R-factor (%) 17.2Free- R-factor (%) 20.2Rms deviation from ideality Bond distance (Å) 0.022 Bond angles (º) 2.02

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(b)

(c)

12 5 4

7

1

3

2

6

4

5

6

8

3

7

N116

N18

12 5 4

7

1

3

2

6

4

5

6

8

3

7

N116

N18

(a)

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1 10 20 30 40

* * * * *

11

2S3 YDYFQFTQQYQLAVCNSNRT---LCKDPPDKLFTVHGLWPSNM

LE AKDFDFFYFVQQWPGSYCDTKQSCCYPTTGKPAADFGIHGLWPNNN

MC1 FDSFWFVQQWPPAVCSFQKS--GSCPGSGLRTFTIHGLWPQQ-

Rh SSCSSTALSCSNSANSDTCCSPEYGLVVLNMQWAPGYG-------------PDNAFTLHGLWPDKC

50 60 70 80 90

* * * * *

23

45

S3 VGPD-PSK-C-P-IKNI--RKREKL----LEHQLEIIWPNVFD-RTKNNLFWDKEWMKHGSCGYPT

LE DGTY-PSN-CDPNSPYD--QSQISD----LISSMQQNWPTLACPSGSGSTFWSHEWEKHGTCAESV

MC1 SGTSLT-N-C-PGSPFD--ITKISH----LQSQLNTLWPNVLR--ANNQQFWSHEWTKHGTCSEST

Rh SGAYAPSGGCDSNRASSSIASVIKSKDSSLYNSMLTYWPSNQG---NNNVFWSHEWSKHGTCVSTY

100 110 120 130 140 150

* * * * * *

67

38

S3 ID----------NENHYFETVIKMYISKKQNVSRILSKAKIEPDGKKRALLDIENAIRNGADNKKP

LE LT----------NQHAYFKKALDLKNQID--LLSILQGADIHPDGESYDLVNIRNAIKSAIG-YTP

MC1 F-----------NQAAYFKLAVDMRNNYD--IIGALRPHAAGPNGRTKSRQAIKGFLKAKFG-KFP

Rh DPDCYDNYEEGEDIVDYFQKAMDLRSQYN---VYKAFSSNGITPGGTYTATEMQSAIESYFG-AKA

160 170 180 190 200

* * * * *

45

67

S3

KLKCQKK--GTTTELVEITLCSDKSG---EHFIDCPHPFEPISPHYCPTNNIKY

LE

WIQCNVD-QSGNSQLYQVYICVDGSG---SSLIECPIF----PGGKCGTSIEFPTF

MC1

GLRCRTDPQTKVSYLVQVVACFAQDG---STLIDCTRD-------TCGANFIF

Rh

KIDCSS------GTLSDVALYFYVRGRDTYVITDALST------GSCSGDVEYPTK

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(c)

(b)

(a)

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1 10 20 30 40 50 60 70 80 90 * * * * * * * * * * # # # # # # # # #P. pyrifolia S3 YDYFQFTQQYQLAVCNSNRTLCKDPPDKLFTVHGLWPSNMVGPDPSKCPIKNIRK--REKLLEHQLEIIWPNVFDRTKNNLFWDKEWMKHGSCG-YPTIDNE S5 YDYFQFTQQYQLAVCNSNRTPCKDPPDKLFTVHGLWPSSMAGPDPSNCPIRNIRK--REKLLEPQLAIIWPNVFDRTKNKLFWDKEWMKHGTCG-YPTIDNE S1 YDYFQFTQQYQPAVCNSNPTPCNDPTDKLFTVHGLWPSNRNGPDPEKCKTTALNSQK-IGNMTAQLEIIWPNVLNRSDHVGFWEKEWIKHGTCG-YPTIKDD S2 ARYDYFQFTQQYQQAFCNSNPTPCKDPPDKLFTVHGLWPSTKVGRDPEYCKTKRYRK---IQRLEPQLEIIWPNVSDRKANRGFWRKQWYKHGSCA-SPALPNQ S4 FDYFQFTQQYQPAVCNSNPTPCNDPTDKLFTVHGLWPSNRNGPDPEKCKTTTMNSQK-IGNMTAQLEIIWPNVLNRSDHVGFWEREWLKHGTCG-YPTIKDD S6 YNYFQFTQQYQPAVCNSNPTPCKDPPDKLFTVHGLWPSNDVGDDPIYCKNKTIKSQQ-IGNLTAQLIIIWPNVLDRTDHVGFWNRQWNKHGSCGKAPTIKDE S7 YDYFQFTQQYQPAVCNSKPTPCKDPPDKLFTVHGLWPSNLNGPHPENCTNATVNPHR-IKNIQAQLKIIWPNVLDRTNHVGFWNKQWIKHGSCG-YPAIMNDM. domestica S2 YDYFQFTQQYQPAACNSNPTPCKDPPDKLFTVHGLWPSNMNRSELFNCSSSNVTYAK-IQNIRTQLEMIWPNVFNRKNHLGFWNREWNKHGACG-YPTIRND S3 VKFDYFQFTQQYQPAVCSSNPTPCKDPPDKLFTVHGLWPSNVNGSDPKKCKTTILNPQT-ITNLTAQLEIIWPNVLNRKAHARFWRKQWRKHGTCG-YPTIADD S4 FDYFQFTQQYQPAVCNFvPTPCKDPTDKLFTVHGLWPSNNVGGDPESCKIRNHRT--RAKALEPQLEIIWPNVLDRTNHTAFWRRQWIKHGTCG-YPTIQNE S7 YDYFQFTQQYQLAACNSKPIPCKDPPDKLFTVHGLWPSDSNGHDPVNCSKSTVDAQK-LGNLTTQLEIIWPNVYNRTDHISFWDKQWNKHGTCG-HPTIMND S9 YDYFQFTQQYQPAVCHFNPTPCRDPPDKLFTVHGLWPSNSSGNDPIYCKNTTMNSTK-IANLTARLEIIWPNVLDRTDHITFWNKQWNKHGSCG-HPAIQND

HV HVb' PS1 PS2

100 110 120 130 140 150 160 170 180 190 200 * * * * * * * * * * *

P. pyrifolia S3 NHYFETVIKMYISKKQNVSRILSKAKIEPDGKKRALLDIENAIRNGADNKKPKLKCQKK-GTTTELVEITLCSDKSGEHFIDCPHPFEPISPHYCPTNNIKY S5 NHYFETVIKMYISKKQNVSRILSKAKIEPDGKKRALLDIENAIRNGADNKKPKLKCQKK-GTTTELVEITLCSDKSGEHFIDCPHPFEPISPHYCPTNNIKY S1 MHYLQTVIRMYITQKQNVSAILSKAAIQPNGTNRPLVDIENAIRRGTNNTKPKFKCQKNTRTTTELVEVTLCSDRDLKKFINCPHGPPQGSRFSCPSS-VQY S2 KHYFETVIRMFLAEKQNVSRILSMATIEPEGKNRTLLEIQNAIRAGTTNMIPKLKCQKV-NGMTELVEVTLCHDSNLTQFINCPRPLPQASPYFCPIDDIQY S4 MHYLKTVIKMYITQKQNVSAILSKATIQPNGNNRSLVDIENAIRSGNNNTKPKFKCQKNTRTTTELVEVTLCSNRDLTKFINCPHGPPKGSRYFCPAN-VKY S6 MHYFKTVIKMYITQKQNVSEILSRAKIEPEGKIRRRDDIINAIRLGTKDKKPKLKCQKN-NQTTELVEITICSDRNLTQFIDCPRSSFKGSPFHCPTNHILY S7 THYFQTVINMYITQKQNVSEILSKAKIEPLGIQRPLVHIENAIRNSTNNKKPKFKCQKN-SGVTELVEVGLCSDGSLTQFRNCPHPPP-GSPYLCPAD-VKYM. domestica S2 LHYFQTVIKMYITQKQNVSDILSKAKIEPDGNIRTQKEIVDAIRKGIHGKEPNLKCQKN-TQMTELVEVTLCSDGNLKQFIDCPHHFPNGSRHNCPTNHILY S3 MHYFSTVIEMYITKKQNVSEILSKAKIKPEKKFRTRDDIVNAISQSIDYKKPKLKCKNN-NQITELVEVGLCSDNNLTQFIDCPRPFPQGSPFFCPTNNIQY S4 NDYFETVVKMYITEKQNVSKILSNAKIEPDGIKRTLADLEIAIRSGTDNKKPKFKCQKK-RRVTELVEVTLCSDKNRAHFIDCPNPFQPGSPYLCPTNNILY S7 IHYFQTVIKMYITQKQNVSKILSRAKIEPEGKPRKQVDIVNAIRKGTNDKEPKLKCQKN-NQVTELVEVTLCSNRNLTGFINCPRHIPNGSRYSCPTKNILY S9 MHYLQTVIKMYITQKQNVSEILSKAKIEPVGKFRTQKEIEKAIRKGTNNKEPKLKCQKN-SQRTELVEVTLCSDRNLNQFIDCPRPILNGSRYYCPTNNILY

PS3 PS4

(a)

(b)

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HV

(P

S1)

PS

2

PS

4

PS

3

(a)

(b)

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Sakiyama and Shigemi NoriokaTakanori Matsuura, Hiroaki Sakai, Masaki Unno, Koh Ida, Mamoru Sato, Fumio

responsible for gametophytic self-incompatibilityCrystal structure at 1.5 A resolution of Pyrus pyrifolia pistil ribonuclease

published online September 27, 2001J. Biol. Chem. 

  10.1074/jbc.M107617200Access the most updated version of this article at doi:

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