J. Mol. Biol. (1992) 227. 4933509

Structure of Porin Refined at l-8 A Resolution Manfred

S. Weiss and Georg E. Schulz

Institut fiir Organische Chemie und Biochemie der Universitiit Albert&r. 21, D-7800 Freiburg im Breisgau, Germany (Received 1 April

1992; accepted 29 May

1992)

The crystal structure of porin from Rhodobacter capsulatus has been refined using the simulated annealing method. The final model consists of all 301 amino acid residues well obeying standard geometry, three calcium ions, 274 solvent molecules, three detergent molecules and one unknown ligand modeled as a detergent molecule. The final crystallographic R-factor is l&ST/, based on 42,851 independent’ reflections in the resolution range 10 to 1.8 A. The model is described in detail. Keywords:

porin; membrane channel: X-ray diffraction;

1. Introduction Porins are integral membrane proteins that are found in the outer membrane of Gram-negative bacteria, mitochondria and chloroplasts. They act as molecular sieves to ensure the unhindered diffusion of nutrients into the periplasmic space while protecting t,he cell from hostile substances like degrading enzymes or bile salts. Exclusion limits are tvpically around 600 Da. Porins are usually trimeric with subunit sizes ranging from 30 to 50 kDa. They are very stable towards sodium dodecylsulfate, high t,emperature or extreme pH values (Nikaido 8r Vaara. 1985; Renz 61.Rauer. 1988; Jap & Walian. 1990). It has been demonstrated spectroscopically that porins contain mainly P-pleated sheet with a content of around 60% (Sabedryk et al., 1988). Most other integral membra,ne proteins consist, of x-helices. Struct’ural studies of two-dimensional porin crystals using an electron microscope have been reported by Engel et al. (1985), Sass et al. (1989): Rachel et al. (1990) and Jap et al. (1991). Porin GmpF from /Cs&erichia coli was the first membrane protein to yield three-dimensional X-ray-grade cryst’als (Garavito & Rosenbusch. 1980). The first three-dimensional structure of a porin was obt’ained from unrelated crystals of a porin from a different source, the phototrophic bacterium Rh.odobacter capsulatus (Weiss ef al., 1989, 1990, 199ta,h). This porin is a homotrimer with 301 amino acid residues per subunit (Schiltz et al., 1991), the structure of which has now been refined to convergence. 2. Materials and Methods (a)

(‘rystals

Initially, porin from R. cupsulatus strain 37b4 crystallized in space group R3 yielding crystals that diffract to

Rhodobactercapsulatus

2.8 A (1 A = 0.1 nm) resolution (crystal form A. R;estel et al., 1989). Here, we report on crystals that diffract t.o 18 !I resolution (cryst.al form B, Kreusch et at., 1991) and belong also to space group R3 with unit, cell axes aher = 92.3 A and chex= 1462 A. Crystal forms R and A are closely related: the cell axes of form B are slightly shrunk with respect to form A (Aahex = 3.0 A, Achex = 66 A), and the a,verage structure factor amplitude difference between both crystal forms is as low as R, = 349a in t’he resolution range xi to 5.9 A. Both crystal forms contain 1 subunit/ asymmetric unit. For data collection. the crystals were handled at room temperature in reservoir solution, which was 20 rnM-tris(hydroxymethyl)aminomethane HCI at’ pH 7.2. 300 mM-LiCl, 060/;, (w/v) C,E,t. 30”!?,, (w/v) polyethylene glycol 600. 3 mM-?u’aKj.

dative form B data were collected from I crystal on a 4-circle diffractometer (Siemens/Pu’icolet. model Pd,) to a resolution of 3.6 a. The measurement was done in 4 shells following the method of Thieme et al. (1981). A second data set was collected from another crystal on an image plate detector to 1.8 A resolution at the EMBL beam line X31 (DESY. Hamburg), using a wavelength of 1.009 A. The total measuring time was 15 h. The data were reduced using the MOSFLM/IMAGES program package (Evans, 1987). All 195;183 reflection intensities caollected on the image plate were merged to yield a final data set of 42,823 unique reflections in the resolution range 20 to I.8 .h with an overall R,,, of 6.7qb on intensities. The data were 98% complete. Intensity data were convert,ed to structure factors using the program TRUSCATE (French & Wilson. 1978). The synchrotron data were then merged with the 4 shells of diffractometer data (resolution limits 59 A. t Abbreviations used: CsE,. n-octyltetraoxyethylene: K-factor. crystallographic temperature factor: r.m.s.. root-mean-square: u, standard deviation; SFOFC-map. electron density map based on coefficients (PWIFbhS- ~F,,I,) exp ~~,alc according to Read ( 1986).

Figure 1. Wilson plot of crystal form B data. The dotted line rorresponds to an overall temperature factor of 26 8’.

4.7 !I. 4.0 a and 3.6 A) showing f&-factors of 3.9?,o. 4.2 (+,. 4.4% and 4.7 ‘&, respectively. The final data set consisted of 43.147 reflect,ions and was 990/;, complete. The Wilson (1949) plot of these data in Fig. I is linear down to the very end, indicating that. even at high resolution the dat.a quality is acceptable. This is rorroborated by the cornparativelg low K,,, values at high resolution (see Fig. 3(a)). At 1% A resolution there were still more than 40”1,, of the reflections larger than 30. The average temperature factSor estimated from the Wilson plot is 26 A2.

was derived f+om the twist, of the 16.stranded /I- barrtbl. H.S the right-handed twist clearly predominates ill I)rotrin st,ruc!.turrs (Schulz B: Schirmer. 1979). The% amincj acid sequence was guessed from the electron density This preliminary model was then subjected to several rounds ot simulated annealing refinement using the program SI’LOR (Briinger et al.. 1987). The protoc,ol used fix refinement was very similar to that reported hy St,ehle ct al. (1991). The resulting model in crystal form ;\ contained 310 instead of the real 301 residues (Table 1 ). The chain tracing in the barrel region was ~lc;~r. some ambiguities remained in t.hr large loops at t.htB rough rr~d of the barrel (\Veiss c!t (11.. 1990). When crystal form H became available. permitting data collection out to 1.8 A resolut.ion (Kreusch it nl.. 1991). t,he (furrent. model was transferred to the almost isomorphnus qvstal form B, and the refinement was continued. First., the model location was adjusted by a rigid hod! refinement at 6 if. Several structure refinement. ,ounds were carried nut at 3.6 B (diffractometer dat’a). 30 A. 2.4 a and 2.1 A resolution. After each round the model was scrutinized by difference Fourier maps with co&cients (mF, - DF,) exp ia, (Read. 1986) and rna.ps based on (2mP0- /IF,) exp ice, (SFOF(‘-map). Occasionally omit maps (Hhat, & (lohen. 1984) were used. Only thts most ob\:ious changes were int,rodur(bd. The first. segnlrants of the real sequence could be fitted at a stag:ta &t*n the Il-fact,or rrbached 300’o. Subseyuenti~. the whoIt. ~rcluen~~ (Schi1t.z ct ~1.1..1991 ) was incorporated. the restrlut.ion limit was inr:rvased to 1% ‘4 and the R-fact.or rracthed 22”, (Table I ). Tn the last, rounds of the refinement \vt’ added non-peptide atoms. The assignments started with c*al(~ium ~_ ions and wat,er molrculw. anti wrv la&r tcctendrd tcj detergent molec~ules. Water molrc*ules with elec+,r,on drns~-

Table 2 Q!unlity

(c) Barting

model,

model

building

a,nd r&emrnt

The structure was initially solvkd bv multiple isomorphous replacement and solvent flatten& in crystal form :I (Weiss et nl.. 1989. 1990). The resulting electron density maps at 3 4 resolution were used for chain tracing. first. in a minimap and lat,er on a graphics system (model PS300, Evans & Sutherland. tJ.S.A.). The chirality of the model

Table 1 Course

of structural annealing

Resolution Ztound range (8)

I?, (9b)

15-3.1 15-3.1 I O-3.0

390 262

IO-36

252

G.-Z.,

rejknement program

53.7

simulated

Commrnt

Starting value 276 residues. poly-Ala model 310 residues. X-ray sequence. 71 solvent molecules. last round in cry&l form A Model from round 18 without solvent molecules, first. round in crystal form U

Z+U

313

residues. SeCp”“Cl?

80 residues

as real

IO-l.8

22.0

IO-I.8

18%

301 residues, complete real sequence, 161 solvent rr10lecules and 2 calcium ions Final model including calcium ions. solvent, detergent and ligand molecules (see the text)

finul

model

(‘rystallographie R-factor (‘!,,)t Sumher of reflections K~esolutivn rangr (-4) t ,ompleteriess (‘L;,) r.tn.s. drviationh frclm standard geometr,v bond tmgths (A) bond angirs (deg.) dihedrn.1 a.ngks ((leg ) improper ;tnglrs (dep.) Nrlmber

using the X PLOR

of the

of non-hydrcqrrr

protein water calcium ions detergent total Avrragr W-factor (.A*) all atoms protein atoms main~chain atoms calcium iuns u ater n~c~lrculeh$ detergent molec:ule~II

18% for all data 42.X5 I l0~M.X !I!) f H 1I :i 2.x 37.1 I.1

atoms

i?lC,$ 2744

3 w “.5X!) :i2 20 2li “(I .io 61

Pork

Structuw

495

26

Figure 2. Quality Arg24-SerWArg26.

of the final BFOFC-map at 1.8a resolution as exemplified by the electron density of the segment The contours are at 2.0~. llot,e tha,t the guanidinium groups are at van der Waals’ distance.

ties below lo in the dFOFC-map were routinel,v deleted after each round. The final model consists of all 301 amino acid residues with 2219 non-hydrogen atoms, 274 water molecules. 3 calcium ions. 3 detergent molecules and 1 unknown ligand modeled as a detergent’ molecule. The quality of t)he model is specified in Table 2. Part of the final electron density map is depicted in Fig. 2. Among the 301 amino acid residues. 3 assumed alternative side-chain conformations, which were individually refined. The final temperature factors of the calcium ions corresponded well with the liganding atoms, confirming the calcium assignment. Among t)he 274 modeled water molecules, 34 were at the non-polar surface within 5 A of the next protein atom, but with no direct or indirect hydrogen bond to the polypeptide. Most likely, they represent preferential binding sites of single methvlene kroups of detergent molerules. the remaining parts of which have no ordered position in the c*ryst)als and can therefore not be seen in an X-ray analysis. These fragments were treated as water molecules, but they were labeled in order to distinguish them from the real water molecules.

(d) A nisotropic

packing

order

After finishing the refinement we checked t’he observed structure factor amplitudes Bibs for anisotropic scattering. For this purpose. we performed an anisotropic R-factor refinement of Fobs UWHLS EIcalc using the respective XPLOR routine. The procedure converged after 25 cycles. The resulting anisotropic R-factors (Table 2) show, for instance, that at 2 ,+%resolution the average jibs for reflections around the ahex and chex axes are 140/b below and 407, above thr isotropic average. respectively. This indicates that parking contacts and molecule rigidity corn bine to give good ordering along the S-fold axis (memhra,ne normal) and worse ordering perpendicular to this axis. The 6 added parameters rrduced the R-factor from 18.6% to 17.3 “/;, for t,he 42,851 reflections in t,he resolution range 10 to I.8 I%. Although t,his drop is noteworthy, we refrained from incorporating the anisotropic correction into our final modf4

(e) Model

accuracy

Following Luzzati (1952). the co-ordinat,e error was estimated using the R-factor plot given in Fig. 3(a). The resulting value is about 0.23 .%. Below 5 .A resolution. the Il-fartor increases because disordered solvent was omitted: above 2 X. the R-factor increase reflects the limited quality of the data as represented by the &,,,,, value. For another co-ordinate error estimate (Read. 1986). we calculated o, as a fun&on of resolution (Fig. 3(b)). The slope of this plot yields an error estimate of 0.24 A. which agrees well with the Luzzati estimate. Model accuracy was further checked by plotting the real space R-factor as calculated wibh program 0-RSF of Jones rt al. (1991) as a function of the residue position as depicted in Fig. 3(c). The plot shows 3 local maxima at positions 160. 191 and 288. These are all in loops connecting b-strands that have also the highest t.emperature factors (see Fig. 9). Apart from these peaks, thr real space R-factor is in the range observed for reasonably well-refined structures. the average value is 189’?, for all atoms and 17.50/, for main-chain atoms only. The qualit) of t)he model can also be judged from the distribution of main-chain and side-chain dihedral angles as discxussed below (see Fig. 4).

3. Results and Discussion (a)

Main-chain

conforwlation

A scatter plot of the main-chain dihedral angles (c#I,$) with marked glycine residues is depict,ed in Figure 4(a). As expect’ed, the highest population is found in the allowed /?-sheet region around (- 135”, + 150”) which is near to the regular antiparallel b-sheet conformation. Also populated is the cc-helical region around ( - 60”) - 40”) and t’he bridge region around (-90”,0”). Six out of 261 non-glycine residues are in the left-handed m-helix region around (+60”, +40”). among them are four Asp and one Glu. Most of the glycine residues are in regions

60

2sinB/X

CA-‘)

(a) -180

b

,o -120

-60

0 Q (deg.

60

/

I

120

)

(a)

0.02

0.04 sin28/X2

0.06

O-08

240

(?I

lb)

120

60

0

60

120

180 x, (deg.

240

300

1

(b) I

0

4

50

100

150

Residue

200

250

300

number (cl

Figure 3. Accuracy of the final porin model. (a) R-factor (thick line) as a function of resolution for all reflections. From the lines of constant error, we estimate a co-ordinate error of O-23 A (Luzzati, 1952). The Rsymvalue of the synchrotron data set (thin line) is given for comparison. (b) The dependence of the structure quality index eraon resolution according to Read (1986). The dotted line fitt,ing to the central region shows a co-ordinate error of’ @24 A. (c) Residual real space R-factor plot’ as calculated with program 0-RSF of Jones et al. (1991) accounting for all atoms of a given residue. The maxima are labeled, fi-&rands are indicated by bars.

forbidden for residues with side-chains. None of the ,5 prolines has a cis-peptide bond. For hydrogen bond assignments within the main chain, we used the list from XPLOR applying the 3.5 A donor. . acceptor distance criterion wit’h

Figure 4. Chain conformation. (a) Scatter plot of mainchain torsion angles ($J,$) f or all 261 non-glycine residues (e) and for 38 glycine residues (0). Non-glycine residues in the left-handed u-helix region near (+60”,+40”) art’ Asp93 and Asp108 involved in Ca’+-binding, as well as Glu109. AspllS, Asp216 and Thr256. Among the 38 glycine positions, 24 are forbidden for residues with sidechains. (b) Scatter plot of side-chain torsion angles (x1,x2) for leucine (A) and isoleucinr (0) residues.

S-T? . 0 angles above 120”. The secondary struct’ure assignment grouped all residues t,hat wert involved in appropriate hydrogen bonds. The lowe& N-fi . . . 0 angle turned out as 133”. Residues at the borders of secondary structure elements with only one hydrogen bond were excluded if the dihedral angle at the hydrogen-bonded peptide group was outside the respective secondary structure region. The secondary structures are given in Figure 6, which also lists the small corrections of an earlier stage of t’he assignment at an intermediate

Pork

Structure

497

10

20 30 40 50 60 70 I I I I I I I EVKLSGDARnaVnYNGDDISSRSRVLF~SGTTDSGLEFGASF~~SVGAETGEffi~LSGAFGKIENGDALGASEALFGDLYEVGYTDLDDRGGN EEEEEEEEEEEEEEETTEEEEEEEEEEEEEEEEEETT EEEEEEEETT HHHHH EEEEEEETTEEEEEEE EEEEEEEEEEEEE SS EEEEEEEEEEEEEEEE TT EEEEEEEGGGHHHHTTTSSSEEEEEETTEEEEEES ___ __ ___ Pl 03 01 P4 85

80

110

120 I

130

140

I

I

150 I

160 I

170 I

S

SSBTTSS

BTTB

EEEEEEEETTEEEEEEE ___ ___ 86

210

B

08

230

I

240

I

270

I

PI2

a3

Pl3

280

I

EEEEEEEEEEEEEETTEEEEEEEEEEEETT EEEEEEEEEEEEEE EEEEEEEEEEEEEEETTEEEEEEEEEEEETTTEEEEEEEEEEEEEEETTEEEEEEEEEE

SS

I

PlO

YADGELDRDFARAVFDLTPVAAAATAVDH~YGLSVDSTFGAT~GG~QVLDIDTIDD~YYGLGASYDLGGGASIV06IMNDLPNSD~ADLGVKFKF EEEEEE HHHHHHH EEEEEEEHHHHHHHTT --

200

EEEEEEEEEEEETTEEEEEE EEEEEEEEEEEETTEEEEEE

TT S TTTS

260

I

Pll

290

TTEEEEEEEEEEE

301

I

I

014

S

I

P9

250

I

TT TTS

190 I

EEEEEEEEEEETTEEEEEEEEEEE

P7

220

I

SBSTTSS

I

TT TTTT

S

180 I

100 I

HHHHHHH B HHHHHH _I_ a2

P2

DIPYLTGDERLTAEDNPVLLYTYSAGAFSVMSNS~~GETSEDDAQE~V~YTFGNY~GLGYEKIDSPDTA~NEQLE~IAKFGAT~~Y TT TT EEEEEEEETTEEEEEEEE TT EEEEEEEEEEETTEEEEEEEEEEE

90 I

TT SSS

I EEEEEEEEEE EEEEEEEEE 016

PI5

Figure 5. Secondary structure assignments in porin from R. capsulatus. Upper line, amino acid sequence (Schiltz et al., 1991); second line, visual assignment based on the final structure using the distance criterion D A I35 A for hydrogen bonds. As compared to Weiss et al. (1991a), strands 81, fi2,84, 85 and /?15 were extended by residues 15, 18,59, 74 and 285, respectively; /I14 by residues 270-271, and helix a2 by residue 83. Moreover, hydrogen-bonded turns were assigned to positions 16-17, 47-48, 273-274 and 287-288; third line, assignment using the program DSSP of Kabsch & Sander (1983) and their nomenclature: E, P-sheet; H, a-helix; G, 3 ,,-helix; B, bridge; S, bend without hydrogen bond: T, isolated turn with (i:i+n). n = 3,4,5 hydrogen bond; fourth line. assigned names of b-strands and cc-helices.

secondary structures, which is in the usual range observed with water-soluble proteins.

formations are more frequent than t or g’ As usual, however, the g+ conformation dominates for serine. No residue type showed significant deviations from the staggered positions. The overall x1 averages for the g+, t and g-- conformations are 60”, 182” and 299”, respectively, which is very close t,o the ideal positions. In the (x1,x2) scatter plot for Leu and Ile residues in Figure 4(b), the isoleucine residues show a strong preference for the g-t conformation. The leucine residues cluster at g t as well as at tg+. Four residues are outside these two regions: Ile2.57 assumes the rarely observed g-g- conformation; Leu165, Leu265 and Ile281 have conformations with one eclipsed dihedral angle. These four residues are in the P-barrel with their side-chains pointing to the outside, i.e. to the non-polar interior of the membrane. The observed eclipsed positions of Leu165 and Leu265 could be an artefact, because the average B-factors of their side-chain atoms are as high as 55 A2 and 51 A2, respectively. Side-chain atoms involved in polar interactions are listed in Table 4. All four salt-bridges are very

The

topology

of the 16-stranded

/?-barrel

is very

connected

t’o next

neighbors.

A representation

of

this barrel including all hydrogen bonds is given in Figure

6

(see

also

Fig.

12(a)).

The

three

short

x-helical segments of five, seven and seven residues are all at the rough end of the barrel (top end of Figs 6 and 12(a)). Molecular replacement results with crystals of several other porins (Pauptit et al., 1991) indicate that this 16stranded p-barrel is common among many porins, although no overall sequence homology can be observed. The average (4,1/1) angles of the 146 internal residues of the /?-barrel are (- 139”, + 149”), which is near to the antiparallel B-pleated sheet position. According to McLachlan (1979), the shear number of the barrel is + 20. The average ($,$) angles of the a-helical residues is ( -65”, -38”). The average X . . 0 hydrogen bond distances are 2.98 A in the P-barrel (161 hydrogen bonds of Fig. 6, o = 0.14 A) bonds, and 3.10 b in a-helices (7 hydrogen CJ= 0.15 A). The 17 reverse turns are displayed as arrow plots in Figure 7. Remarkable are the four type II’ reverse turns all starting with glycine residues, required for the (i+ I)-position, at ( + 70”, - 120”). These turns are at the smooth end of the barrel (bottom end of Figs 6 and 12(a)); they

maxima

plot (see

coincide

simple, all /?-strands being antiparallel and all being

with

of the B-factor

refinement’ (Weiss et al., 1991a). The assigned secondary structures are in general agreement with the output of program DSSP (Kabsch & Sander, 1983). The main portion of the structure is a 16stranded a-barrel containing 178 residues (59%). Further I9 residues (6%) are in a-helices and 34 residues (11%) in reverse turns. Thus, altogether 231 out, of 301 residues (77%) are involved in

Fig. 9). (b) Side-chain

coyformations

and

interactions

The distribution of the side-chain dihedral angles x1 for all residue types except Gly, Ala and Pro is given

local

in Table

along

3. As commonly

the

chain.

Two

observed,

of them

are

g- con-

between

neighbors or next neighbors along the sequence, a third (7. .26) is between lateral neighbors at the inside

of the

P-barrel

(Fig.

6). Strong

side-chain

hydrogen bonds occur in the long loop between /I5 and

/?6 (residues

75 to 117)

that’

forms

the

pore

Figure 6. Sketch of the complete p-harrel of porin. The first strand is given twice. The S 0 distances of all hytlrogm honds are st’ated in E\. The numbers of residues with side-chains pointing to the outer surfare of the barrel are given in bold fare. Tt should be mentioned that residues 47. 127, 160, 194, 242, 255 and 274 of this Figure are assigned to turns in Fig. 5.

eyelet inside the barrel. Among them are 7 of the I6 strongest ones (Table 4) holding this loop tightly at its place. The shortest observed distance is 2.50 A between the carboxylate groups of Glu168 and Glu185 at the inner surface of the barrel. This contact must be mediated by a proton forming a strong hydrogen bond. In the course of the analysis, residues Metl3, Ser50 and Va1152 turned out to assume more than one conformation. The partial occupancies of these conformations were refined (Table 5). For Ser50 at the subunit interface, the OG atom alternates between two hydrogen bonds: a 2.68 A bond to Ala47 0 in the conformation 1 and several longer hydrogen bonds (involving Ala47’ 0, Ala47 0. Va151 N, Wat543 and Wat543’) in conformation 2. In contrast’, no obvious reason for the multiple conformations of Met13 and Va1152 can be found.

Met13 Val152

points into the interior is located at the non-polar (c) The external

of the pore and outer surfacr.

protrusion

Residues 205 to 229 and 252 to 260 form a small globule protruding from the barrel at’ its rough end. which faces the external space (see below). As shown in Figure 8, this globule consists of helix x3. the upper ends of strands /I1 1, p12, /?13 and 014, and a long loop around residue 220. This loop is stabilized by hydrogen bonds between the guanidinium group of Arg208 at one hand and Val220 0, 41a222 0. Ala223 0 and Thr225 OGl at the other. Arg208 is backed up by a salt-bridge from Asp209. Both residues are in helix ~3. The chain segment arching over helix a3 has the peculiar sequence -Thr-ProVal-Ala-Ala-Ala-Ala-Thr-Ala-. This architecture is

Pork

Structure

499

Table 3

for

L3atistic.s

side-chain

Number ---~ Total g+t

Side-chain type Asn .4sp Ary Gin (:lu His Ilr Len Lys M&g: I’he Sel$ Thr TOP Tyr Valjj

x 34 7 :I 17 ., H %-I IfI !I I5 21 21 I 16 %3

Total

% I!)

torsion

3 %

9

1 13 2 1 8

3 15 3 1 6

60

-

8 11 65

91

5

1 (F240) -180 L

1

Asp7 ODl Asp7 OD% SW22 0 Ala47 0 Ala78 S (:ly84 0 Asp85 0 Thr92 OGI Arg97 NE I&U105 0 Lysl38 NZ Vail39 0 (:lu168 OEI I+169 NZ Lys169 NZ SW172 OG Tyr200 ( )H Arg208 NE Arg208 NH2 Arg212 NE Asp255 0 Asp283 ()D:! Asn284 OD1

Atom

Arg26 NEt Arg26 NH2t Tyr91 OH Ser50 OG (conf’.-I Ser 133 OG Tyr104 OH Lysl98 N% Asp294 OD2 -4sp290 0 Argl 10 NE Asp145 ODlt Thr142 OGI GIu185 OEl Asplil ODli Asp171 onzt Met I78 0 (Znd.50 OE 1 Asp%09 or>zt li.srm)9 It Thr218 0 Thr2.56 OGl Asn284 N Ser289 OG

or)

Distance

)

0

(A)

2.96 3.01 274 2.68 2.80 2.75 P78 2.70 266 275 299 2.67 2.50 330 3.16 2.74 2.64 279 23i 266 2.59 280 2.80

Only hydrogen bonds with D.. A distances less than 2.80 A are given. t For salt-bridges. t,he criterion was relieved to 1). A dist,anvrs less than 3.5 ‘%.

60

120

(deg.)

Figure 7. Dihedral angles of reverse turns represented as arrows with (i+ 1) positions as circles (labeled) and (i+B) positions as triangles. The (i+ 1) positions of the 5 unlabeled reverse turns around (-60”. -30”) start with residues36. 47, 66, 91 and 96.

reminiscent of the protease inhibitor eglin-C (Bode et al., 1987). where an arginine side-chain backed up by the C-terminal carboxylate group stabilizes a loop of about ten residues that arches over a P-sheet and contains the inhibitor binding site. Furthermore, this globule is involved in the crystal packing contact (seeTable 9). (d)

orbe subunit 2

-60 4

Table 4 polar side-chain interactions within

Atom

-120

I (V251)

x, is the dihedral angle around the CA-CB bond as defined relative to the N atom eclipsing with CG (CGl for Ile of Val), OG or SG (IITPAC-IUH convention, 1970). t g+ and g- refer to values of 60” and 300”, as suggested by the IUPA(‘-JUB convention (1983). This is different from earlier reports, e.g. Janin et a.Z. (1978), James & Sielecki (1983) and liarplus $ Schulz (1987). $ A residue assumes an eclipsed conformation when x1 is within 20” of the eclipsed positions at O”, 120” or 240”. 9: For residues assuming more than 1 side-chain conformation. only the major conformation was considered in this analysis (see also Table 5).

Strong

1

1 (N288)

1 (1281) 1 (1,265)

6 %

114

Eclipsedf

7 13 5 3 7 6 10 1 2 9

9 % 4 2 3

58

~~--__

t

1 3 % 5 12 11

255

120

of residues .-I_

3 6 1

‘“r-

angles x 1

flexibility

Chain

The overall average isotropic temperature fact,or (R-factor) of the final model is 32.0 ‘4’. If one considers onlv the main-chain atoms, the N-factor is 26 A2, which agrees with the value derived from the Wilson plot (Fig. I). The R-factor plot of Figure 9 shows that strands jl through fll6 form the most rigid parts of the molecule. The long loop /A?-~6 (largest intermission bet’ween P-st’rands in Fig. 9) Table 5 Residues

Residue

Atoms

Met 13

CG. (k cu. (CN). CGl, (:Gl. (‘(~1

Ser50 Vail52

t Partial adjusted to 1: x1. ,r2, Table 3 for

with

SD. CF SD). GE OG OG CG2 CG2 / (‘(‘2 /T

multiple

Label

conformations

Orcupanry (?,P

I I % I % 3

occupancies were refined add up to 100%. x3 are the dihedral angles the definition of x,.

Torsion angles ((b.) ~-~ -~ -~ x1: x2 13

51 49 60 40

175 180 309 56

ii3

297

26 21

178 63

‘80 249

using

XPLOK

and

along

the side-chain.

29i 153

then See

Figure 8. Stereo view of all atoms and hydrogen bonds of the small globule formed by chain segments 204 to 229 and 252 to 260. Chain cuts are marked by dots. A remarkable hydrogen bond network is created around the side-chain of Arg208, stabilizing the long loop arching over the cc-helix. Water molecules are not included. This globule protrudes from porin in the membrane toward the external medium.

defining the pore eyelet is also very rigid. The most flexible parts are loops /J&/39 and fllO-fill at the periplasmic end of the barrel, and loop p15-fi16 at the external end. Still, there is continuous density for all main-chain atoms at the la level in the final BFOFC-map. The three highest maxima of the B-factor plot correspond to those of the real space K-factor plot of Figure 3(c). (e) Calcium-binding

sites

In the initial preparation and crystallization procedure, EDTA was added to dissociate the trimeric porin into subunits (Nestel et al., 1989). Later on: better diffracting crystals could be grown from EDTA-free preparations (Kreusch ef al.. 1991). During the structural refinement of the EDTA-free crystal form B, three calcium ions were detected. The Ca2+ assignment was confirmed by the H-factors of the ions being close to tfhose of t’he contacting atoms (Table 6). Among the three ions, Ca-I and Ca-II are located in the large loop /?5-fl6 forming the eyelet of the

---

mm

--

--

--

m-w1

1 0

50

100

150 Residue

200

250

I 300

number

Figure 9. Mobility of the polypeptide chain. The average B-factor of the main-chain atoms of each residue is plotted wersus the residue number. P-Strands are indicated by bars.

pore, where they compensate to some extent the negative charge excess of this loop. &III connect,s t’wo subunits and thus stabilizes the trimer. ,Most likely, it is the factor differentiating between crystal forms A and B; its removal with EDTA causes trimer dissociation, as observed with other porins from phototrophic bact’eria (Woitzik et al., 1990). All three calcium ions are in pentagonal bipyramidal ligand arrangements (Table 6). As shown in Figure 10(a). Ca-I is liganded by three protein atoms and four water molecules. The central plane of the bipyramid is formed by both carhoxylatr oxygen atoms of Glu80 (bidentate ligand), Wat305. Wat307 and Wat339. while Asp108 ODl and Wat312 are the apical ligands. Every water molecule in the primary hydration shell is fixed h> additional protein ligands. The apical angle defined by Asp108 ODl, Cit.1 and Wat312 is 175”. In contrast to Ca-J, there are five protein atoms and only two water molecules contacting Cla-11 (Fig. 10(b)). The carboxylate oxygen atoms of Asp93 form a bidentate ligand in the central plane. Other ligands in this plane are AsnlOO OD1, Asp101 OD2 and Wat327. Apical ligands are Asp95 OD 1 and Wat331, giving rise to an apical angle of 159”. With Asp95 OD2 somewhat further away from Ca-IT but st’ill close enough for binding (Table 6). the geometry of this site could be considered as a, pentagonal bipyramid with an apical bidentatr ligand. Ca-I and Ca-II are interconnect’ed by the carboxylate group of Asp101 and two water molecules. Asp101 OD2 is a direct ligand of Ca-IT. whereas Asp101 ODl is hydrogen bonded to Wat307 and Wat339, which in turn are direct Iigands of Ca-T (Table 6). Ca-III has the carboxylate oxygen atoms ot Asp136 as the bidentate ligand in the central plane. This plane also contains Lys138 0, Gly140 0 and Wat314, which is fixed by Asp115 0 and Phe21’ 0 from the neighboring subunit. As shown in Figure 10(c), the apical ligands are Asnll6 ODl from the same subunit and Asn20’ ODl from the neighboring

Porin Structure subunit. The very short ligand distances (Table 6) are remarkable. At 175”, the apical angle is almost ideal. It has been predicted from electrostatic calculations and verified by analyzing a crystal grown in the presence of 20 mM-CaCl, (Weiss et al., 1991b), that the analyzed porin contains another calcium site at the pore eyelet, Ca-IV. A structural refinement based on a 2.2 A resolution data set of the cocrystal with 20 mM-CaCl, showed that the ligand arrangement at Ca-IV is octahedral (Fig. 10(d)). Here the B-factor of the ion does not agree with those of the ligands (Table 6), pointing to less than full occupancy.

Table 6 G’alcium-binding

IOIl

Distance (A)

B-factor m

Ligand

2.52 2.48 224 2.43

Glu80 OEl Glu80 OE2 Asp108 ODlt Wat305

21 23 17 21 21

2.33

Wat307

21

2.28 237

Wat312t Wat339

23

2.44 933 2.40 263 231 225 224 239

Asp93 Asp93 Asp95 Asp95 AsnlOO Asp101 Wat327 Wat331

Ca-I

19

sites Secondary of water

residues ligands

Wat324 (TyrlOQ 0, ThrlO6 0, Glyl07 0), Wat346 (TyrlO4 0) Asp101 ODl, Asp85 ODl Asp85 OD2, Ile102 0 Asp101 ODl

19

(h-11

ODl OD2 ODlt OD2 ODl OD2 t

Ca-111

20 25 22 22 25 23 27 20

Asp101 ODl Glu88 OE2, AsnlOO ND2, Wat349 (Asp85

OD2)

19 2.16

Asnll6

2.35 2.39 255 218 2.35 2.18

A.?~136

ODlt ODl OD2 0 0

212 2.2 1

Asp58 ODl Asp74 ODl Glu109 OEl Wat67@ Wat675$ W&717$

Asp136 Lys138 Gly140 Wat314 Asn20’ ODl t

ca- rvt.

2.21 2.17 2.18 2.21

22 24 21 21 25 16

Asp1 15 0, Phe21’ 0

19 35 16 9 26 56 21 58

Glu49 Glu57 Wat67@

OEI 0 (Asp58

OD2)

t Apical positions of the pentagonal bipyramids. j’ Obtained in a separate 2.2 A X-ray structure analysis of a crystal grown and kept in the presence of 20 mM-CaCl,. This structure has been refined to an R-factor of 19.1% for all data between 10 and %2 A. $ The numbering of the water molecules in the structure obtained in presence of CaCl, starts with number 606 in order to distinguish between the 2 structures. Wat675, however, is close to Wet416 in the structure without CaCl,.

501

(f) Sohent structure The final model contains 274 water molecules subdivided into two groups. The first group consists of 240 usual water molecules, whereas the second contains 34 water models at the non-polar surface of porin. Most probably, these models represent methylene groups of detergents that bind preferentially at the given site. Density distributions and B-factors of all models are given in Figure 11. As expected, the non-polar models concentrate at low densities and have high B-factors. Transferring the detergent content observed with porin crystals of OmpF from E. coli (Garavito et al., 1983) to our crystals, we estimate that our crystals contain 40 y0 (v/v) detergent besides 30% (v/v) buffer and 30% (v/v) polypeptide. This is in general agreement with data from the photoreaction centers (Roth et al., 1989, 1991). Accordingly, the 240 water molecules defined in the model constit,ute 20% of the total of 1200 water molecules in the asymmetric unit, which is in the normal range observed with crystals from water-soluble proteins. Among these 240 water molecules, seven are calcium ligands, 181 are in the first hydration shell at a distance of less than 3.5 A from a protein atom, and the rest is in the second shell; 38 water molecules can be defined as an integral part of the protein if one uses the average main-chain B-factor of 26 A2 as the limiting crit,erion. (g) Detergent positions In the course of the refinement, additional densities were routinely assigned as water molecules. When some of these densities became continuous, they were replaced by C&E4 chains, which is the detergent used for crystallization. Four such detergent molecules were included in the model. Three of them are located at the outer non-polar surface of porin. Since they form no strong contact to the protein, they are unlikely to be of functional importance. Moreover, these models may represent parts of more than one detergent molecule, a clear distinction was not possible. The contacts of three detergent molecules are listed in Table 7; their locations at the non-polar surface are indicated below (see Fig. 13). The occupancy of the detergent molecules was refined yielding values of @83, @69 and 066 for Det546, Det547 and Det548, respectively. The average B-factor of the three detergent molecules is 61 A2, which is higher than the average of 27 A2 for the one complete detergent molecule found in the crystal structure of the bacterial photoreaction center (Deisenhofer 81,Michel, 1989). (h) Ligand-binding

site

The extra density modeled as the fourth detergent molecule is located in a small, mostly non-polar cleft between loop fi5-fiS and helix a3 at the inside of the barrel below the external protrusion

&I. S. Weiss and G. E. Schulz

.502

(a)

95 :

,kI’

.

1W

I

&A$

4-

09

(d)

Fig. 10.

r-i \loo

Pork

Structure

503

Table 7 (continued) Detergent atom?

Electron density

Figure 11. Statistics modeled as water, with

defined

(~1

of the 274 solvent

which have hydrogen bonds

been subdivided (“real” water,

molecules into white

those bars)

and those at the non-polar surface without such bonds (striated bars). In the final model, the numbering of the real water molecules starts at 305, the non-polar models start at 549. Within each group the numbers follow the densities of the final BFOFC-map. The average B-factors corresponding to the 2 groups of water models are given as continuous lines (thin line, “real” water; thick line, non-polar models).

(Fig. 12(a)). As shown has a length of about

in Figure 12(b), this density 12 to 15 a and contains a

relatively thick head, which is not fully accounted for by the fitted linear chain. This binding site is illustrated in Figure 12(c), it contains a few polar atoms at the left-hand side. Table 7 Bound detergent molecules Detergent atom? Det546 C-l c-2 c:-3 C-4 C-6 C-6 C7 (xi o-9 C-10 c-11 o-12 C-13 c-14 o-15

B-factor (A7

51 52 51 51 53 56 60 64 66 66 66 65 65 65 64

Number of contacts1

2 6 5 8 2 4 1 5 3 2 3 2 7 5 1

chest distance

421 374 3.99 359 393 390 4.17 3.86 3.80 394 420 449 353 3.99 443

(A)

Closest atom9

Len265 Gly279 Gly280 Gly280 Asp294 Ile281 Trpl9 I&u295 Leu295 Trpl9 Trpl9 Trpl9 Trpl9 Trpl9 Trpl9

0 0 N N 0 CD1 CH2 CB CD2 CH2 CH2 CE2 CE2 CD1 CG

(‘-16 (‘-17 O-18 (‘-19 (‘-%O O-21 Det547 (‘-1 (‘-% (‘-3 (‘-4 (‘-5 (‘-6 (‘-i (‘-8 O-9 (‘-10 C-11 O-12 (‘-13 c-14 O-15 Cl6 (‘-17 O-18 c-19 C-%0 O-%1 Det548 C-l c-2 C-3 C-4 c:-.r c:-ti (‘-7 (‘-8 O-9 (‘- 10 (‘-11 o-12 c-13 (‘-14 O-15 C-16 C-17 o- 18 C-19 c-20 o-21

B-factor (AZ)

Number of contacts$

Closest distance (A)

62 60 61 63 66 69

3 3 3 6 2 3

3.67 427 3.94 3.99 40; 326

Trpl9 Phe21 Phe21 Phe21 Asp136’ Met134’



2 1 3

408 4.36

Phe21 CEl Va112 GCl Leull9 CD2

3 6

384 33i

Met72’ C‘E Glyl 1 CA

6 1 8 1 4 3 4 1

4.00 425 3.95 4.37 3.96 404 3.77 3.70

Va1297 Gly296 Gly296 lie277 11~277 Val297 lle277 1~~63’

1 1 1

439 394 4.40

Ile277 CD1 Leu39’ CD1 hu.39 CD1

4 8 3 4 1 3 3 1 3 5 5 2 1 2

3.77 417 424 3.67 4.46 422 3.88 42x 356 3.81 3.57 4.30 438

Len186 Tyr200 TyrPOl Tyr200 Tyr2OO Tyr232 Gly233 Len234 Tyr332 Va1%49 Va1249 Va1251 Va1249

(‘B C CB 0 0 CB 0 CB CD% CGl CGl CGl CGI

4.20

Thr261

(‘GZ ,

1 2 2 2 3

4.14 3.61 4.24 4.27 4.36

Thr261 Thr261 Thr263 Tyr263 Tyr263

CG2 CG2 CD2 CG CD1

38 40 43 46 4x 50 51 53 67 61 65 68 71 74 76 71 78 7x 78 78 80

-

-

4li

Closest atoms CB CEl CEl CEl 0 CE

CGl CA CA CG2 CD1 CGl CD1 CD2

The ligand inside the barrel modeled as CsE, (Det545) is not specified here but depicted in Fig. 12, because it may be the wrong compound. t The numbering of the 21 non-hydrogen atoms of CsE, starts at the non-polar side with C-l and runs through O-21. $ Number of protein atoms forming a contact within a distance of 45 A. $ Closest among all protein atoms that rontact ( ~4.5 A) a given detergent atom.

Figure 10. The calcium-binding sites in porin; depicted are all residues that have at least 1 atom closer than is A to the calcium ion and all directly co-ordinated water molecules. The ion-ligand interactions are marked by broken lines, chain cuts by dots. The groups backing up the Ca 2+-liganded water molecules are given in Table 6. (a) Site Ca-I at the pore eyelet. (b) Site Ca-II at the pore eyelet. (c) Site Ca-III at the subunit interface. Residues from the other subunit are given with thick lines. (d) Site Ca-IV at the pore eyelet occupied at 20 mlur-C&l, was taken from a separately refined structure of porin (see the legend to Table 6). The only significant conformational change on Ca2+ binding is a rotation of the carboxyl group of Glu109 (x3) by about 50”. Moreover, 2 of the 3 liganded water molecules are not present in the final model.

504

M. S. Weiss and G. E. Schulz

(b)

Figure 12. The ligand-binding site inside the pore. (a) The chain fold of porin from R. capsulatus given as a C” backbone model with some labeled positions. The view is approximately from the a-fold axis. The location of the binding site is depicted by the electron density of a 2FOFC-map at the la level. The model is a CsE, detergent molecule (Det545). (b) Magnified representationof the final 2FOFC-map (cutoff level la) around the CsE, model fitted to the density. (c) Stereo view of all side-chains with an atom closer than 45 a to the CsE, model, and Wat346, Wat418. Wat424, Wat473 and Wat482 (+).

In a recent report, Bollivar & Bauer (1992) showed that porin of R. capsulatus binds tetrapyrrol

intermediates of the biosynthetic pathway of bacteriochlorophyll a. Tetrapyrrol is not consistent with the observed density, however, leaving the nature of the ligand unexplained. (i) The membrane-facing outer surface The outer surface of porin can be described in terms of four zones depicted in Figure 13. Starting from the smooth periplasmic end of the barrel (bottom of Figs 12(a) and 13), there is a zone of aromatic residues including Phe67, Tyrl21, Tyr123, Phe128, Tyr156, Phe158, Tyrl61, Phe192, Phe240 and Tyr269. It is remarkable that all phenylalanine

point upward to the non-polar part of the surface, while all tyrosine residues point downward. Above this aromatic girdle follows a completely non-polar zone, presenting the side-chains of mostly leucine, valine and alanine residues to the outside. The only polar atom is Tyr121 OH, which is, however, hydrogen-bonded to Phe67 0 and close to the interface. Above this non-polar zone there is a second less well developed aromatic girdle containing Tyrl4, Trpl9, Tyr167, Tyr201, Tyr232 and Tyr263. It contains no phenylalanine residue, but three methionine residues. Again, all tyrosine residues point away from the non-polar part of the surface. The regular arrangements of these two aromatic girdles suggestsa functional role. As indicated in Figure 13, the total height of non-polar surface residues

Porin Structure

Figure 13. Projection of the outer surface of the /l-barrel onto a cylinder. Polar atoms are marked by dots and ionogenic groups by open quadrangles.Detergent moleculesDet546, Det547 and Det548 are labeled close to their C-l atoms. Det546 and Det547 form also contacts to a neighboring subunit in the trimer (see Table 7). The non-polar surface exposed to the membrane is boxed. The vertical height of the box is 24 A. The border lines at the left and right-hand sides separate the membrane-exposed side-chains from those buried by other subunits. In addition to the /I-barrel residues, we added all residues in the loops at the bottom end of the barrel, as well as residues 16, 17,47. 135 to 137, 180, 255, 290 and 291 at the top end for clarity including the aromatic girdles is about 24 A, which is in agreement with the thickness of the non-polar interior of membranes (Zaccai et al., 1979; Lewis & Engelman, 1983; Pastor et al., 1991). Above this second aromatic girdle, there is a very polar zone with numerous negatively charged residues pointing to the outside. These carboxylate groups are likely to participate in the strong and tight Ca2+ . . carboxylate network within the layer of lipopolysaccharide core units such that the interface between these core units and porin becomes as tight as the lipopolysaccharide layer itself, and porin becomes an integral part of the protecting coat. It should be mentioned that this observation

corroborates the orientation of porin as derived from hydropathy plots (Welte et al.. 1991) and accessibility experiments (Tommassen, 1988) with the rough and smooth barrel ends facing the external and the periplasmic space. respectively. (j) The pore Diffusion through porin is mainlv determined by the structure of the eyelet, which ls formed by the long loop between B-strands 85 and /IS narrowing the inner diameter of the p-barrel considerably. The structure of the eyelet is obviously tightened by electrostatic interactions between negatively and

Figure 14. Stereo view onto the pore eyelet. The a-fold axis at the same height is indicated. Depicted are all residues and water molecules (+) of the final model within a distance of 10 A from the center of the eyelet at model co-ordinates (PO, - 17.0 8, +230 A), i.e. Asp7, Arg9, Arg24, Arg26, Lys46, His48, Glu49, Glu54’ (thick line), Asp58, Asp74, Leu76, Glu80, Asp85, Glu88, Thr92, Asp93, Asp95, AsnlOO, AsplOl, Asp108 and Glu109. The calcium positions are highlighted by dots. Clockwise, starting from the lower left-hand side, these are Ca-IV, Ca-I and Ca-II. Ca-IV and its direct water ligands were taken from the separate structure at 20 mM-CaC1, and added to this Figure. The displayed Glu109 conformation is that without bound Ca-TV.

M. A’. Weiss and G. E. Schulz

506

Figure 15. Projection of the outer surface of a b-barrel onto aa cylinder as in Fig. 13, but shifted such that now the interfaces are at the Figure center. The vertical broken line separates the 2 interfaces; it coincides approximately with the projection projection of of the the 3-fold 3-fold axis. axis. Only Only residues residues Leu4 and Phe29 participate in both interfaces. All residues involved in the interfaces (distances I 45 A) are are marked. General interactions are indicated by dots and hydrogen bonds (Table 9) by open quadrangles. Residues involved in binding of Ca-III are emphasized by arrows (see Table 6 and Fig. IO(c)). positively charged residues juxtaposed across the channel (Fig. 14). This arrangement rigidifies the side-chains pointing into the pore center. There is a line of four positively charged residues at van der Waals’ distance, three guanidinium groups (Arg9,

Arg24 and Arg26) and one amino group (Lys46). The central part of this line can be visualized in Figure 2, where two charged guanidinium groups are side by side at van der Waals’ distance in welldeveloped density. These adjacent positive charges require a strong electric field, which has been confirmed in electrostatic calculations (Weiss et al., 1991b). The rigid side-chains pointing into the eyelet’ narrow its diameter considerably. Further narrowing is caused by fixed water molecules. Accounting for their van der Waals’ envelopes, the remaining opening is merely 3 A by 6 A, restricting diffusion without water displacement drastically. Table 8 Polar Atom

interactions

between

1

Atom

2

Glul s Asp36 N

Phe3Ol’ Leu271’

Asnl5 Asn15

ODl ND2

Asp18 Asn20

OD2 ODl

Asn20 Phe21 Ser22 Ser23 Ser25 Ala47

ND2 N OG 0 S 0

Serl43” OG Glu141” 0 Ser143” OG Serl43” O(‘ 1 (k-111” Asp136” ODI Asp136” OD2 Lys138” O$ Gly140” O$ Thr142” 0 Asp136” ODZ Glu141” OE2 Gly56” N Ala53” 0 8er50” OG (conf.-2)

OT2t 0

subunits Distance

(A)

(k) Subunit

The accessiblesurface areas of an isolated subunit and of trimeric

respectively Accordingly, trimer

The distance criterion is 35 A. Interactions through solvent molecules are neglected. t Salt-bridge. f This repulsive interaction may enhance the Ca2+ affinity of these 2 ligands of Ca-III.

porin

are 15,370

A2 and 37,990

A2,

solvent molecules or ions included). an area of about 8120 A2 is buried on

(no

formation,

and the area of one subunit/sub-

unit interface is 1350 A2. A cylindrical projection of the interface region is given in Figure 15. The uper rim and, to some extent, the lower rim of this interface are polar and connected by one salt-bridge between

the N and C termini

as well as by at least

12 hydrogen bonds (Table 8). Between the rims there is a non-polar region, containing four clusters of phenylalanine residues. The largest cluster consists of Phe29, Phe29’ and Phe 29”, which meet at the triad, as well as Phe45 and its equivalents backing them up. Three other clusters consist of (Phe41, Phe299’ and Phe301’) and its two equivalents and are thus shared between subunits. Moreover, Phe21 interdigitizes well with a neighboring subunit, its side-chain contacts as many as seven residues (72”, 117”-119”, 134”-136”). Table 9

2.76 3.14 344 3.36 3.08 2-87 2.18 329 2.99 349 280 298 293 2.77 3.40 3.14 3.11

interface

Crystal Atom

1

Ser37 Ser37 Gly38 Gly38 Glu40 Glu40 Ser124 Ser124 Ser124

0 0 0 0 OE2 OE2 N 0 0

Atom ThrP18* Thr218* Arg212* Thr218* Arg212* Arg212* Ala22 1 * Ala221 * Ala221*

contact 2 N OGI NH1 OGl NHlt NH2t 0 (! 0

interactions Distance

(A)

2.91 309 2.73 341 316 3.16 3.47 330 3.45

The distance criterion is 3.5 AL. Slightly above this limit is the hydrogen bond between Ala66 N and Pro219* 0 with 353 A. Residues involved in general interactions (distance limit 4.5 A) are Asp36, Ser37, Gly38, Leu39, Glu40, Gly65, Ala66, Gly68, Tyr123, Ser124, Gly126 and Ala127 from one side and Ala176, Leu177, Arg212, Bsp216, Leu217, Thr218, Pro219, Va1220, Ala221, Ala222 and Ala223 from the other. t Salt-bridge.

Pork

Stmcturr

507

(a)

(b)

Figure

16. Molecular

packing

in the

The c-axis (b) Stereo

corresponds to the drawing of the same

array illustrated as a cubic closest, packing

crystalline

space group RX which can be described membrane arrangement

normal. viewed

Five trimers approximately

Due to the strong polar and the intertwined nonpolar contacts between the subunits, the threepronged central interface region forms a rigid central core of trimeric porin. This is reflected also in the low B-factors of this core; the average mainchain B-factor of all interface residues marked in Figure 15 is 21 A2. The less rigid p-barrels are built as three arches over this core. These P-barrels are supported by the eyelet-forming inner loop /E-j?6 approximately at the height of the membrane center. A striking feature of the interface is the contact of chain segments 53 to 55, 115 and 141 to 143 with the neighboring subunit. These segments lie right on top of the wall of the neighboring barrel (near /?a). (21~54 even points into the pore of a neighboring subunit as shown in Figure 14, its OEl atom approaches Arg24 NH1 to a distance of 3.78 A.

(I) Crystal packing Space group K3 requires only one type of crystal contact for constructing a three-dimensional lattice. The interface area of the only contact type in our crysteals is about 370 A2. Accordingly, the buried solvent-accessible area on crystallization is about 2220 A2 per trimer, which is around 676 of the total solvent-accessible area. This value is comparatively small. Despite its small area, however, the contact is well defined, including one salt-bridge and six direct hydrogen bonds between protein atoms (Table 9). Chain segments 36 to 40, 65 to 68 and 123 to 127 of one subunit interact with segments 176 to 177 and 212 to 223 of a subunit, from a neighboring trimer.

by (‘” backbone models in the hexagonal unit czell of of porin trimers. (a) Stereo view with vertical c-axis. in 3 layers are shown. All cmntarts are head-to-tail. along

the

c-axis.

The former segments are loops p2-fi3, p-1-85 and BS-87 at the smooth end of the barrel. They contact’ the protrusion and loop fl9-j?lO at the rough barrel end of the neighboring molecule forming three headt,o-tail contacts per trimer (Fig. 16). As can be visualized in Figure 9, the B-factors of the contact’ing segments are in the normal range for the and salt-bridged hydrogen-bonded residues (Table 9), but they are relatively high for segments 65 to 68, 123 to 127 and 176 to 177 t’hat. form only weaker contacts. These strong polar contacts explain the high degree of order in the crystals. There is no lateral non-polar contact between the trimers. The secret of high quality integral membrane protein crystals seems to lie in the exclusive formation of polar contacts. The obtained diffraction limit of 1.8 A or better (Kreusch et al.. 1991) is so far unique for this protein group). This work gemeinschaft wissenschaften. are available

under

the

was supported by the Deutsche Forschungsand by the Graduiertenkolleg PolymerThe co-ordinates and the structure factors from the Brookhaven Protein Data Bank accession numbers ZPOR, and R2PORSF,

respect,ively.

References Benz,

R. & Bauer, K. (1988). Permeation of hydrophilic through the outer molecules membrane of Gram-negative bacteria. Eur. J. Biochem. 176, l-19. Bhat, T. 1L. & Cohen, G. H. (1984). OMITMAP: an electron density map suitable for the examination of errors in a macromolecular model. J. AppE. Crystallogr.

17,

244.-248.

508 Bode,

~zil.

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W.. Papamokos, E. & Musil, D. (1987). The highresolution X-ray crystal structure of the complex formed between subtilisin Carlsberg and eglin-C, an elastase inhibitor from the leech Hirudo medicinalis. Eur. J. Biochem. 166, 673-692. Bollivar, D. W. & Bauer, C. E. (1992). Association of tetrapyrrol intermediates in the bacteriochlorophyll a, biosynthetic pathway with the major outermembrane porin protein of Rhodobacter capsulatus. Biochem. J. 282, 471-476. Briinger, A. T.. Kuriyan, J. & Karplus, M. (1987). Crystallographic R-factor refinement by molecular dynamics. Science, 235, 458-460. Engel, A., Massalski, A.. Schindler, H., Dorset, D. 1,. & Rosenbusch, J. P. (1985). Porin channel triplets merge into single outlets in Escherichia coli outer membranes. Nature (London), 317, 643-645. Evans, P. R. (1987). In Proceedings of the Daresbury Study Weekend on Protein Crystal Data Analysis, Daresbury Laboratory. U.K. French, S. & Wilson. K. S. (1978). On the treatment of negative intensity observations. Acta Crystal&r. sect.A, 34, 517-525. Deisenhofer, ,J. & Michel, H. (1989). The photosynthetic center from the purple bacterium reaction Rhodopseudomonas viridis. Science, 245, 1463-1473. Garavito, R. M. & Rosenbusch, ,I. P. (1980). Threedimensional crystals of an integral membrane protein: an initial X-ray analysis. J. C’ell Biol. 86, 327-329. (iaravito. R. M.. *Jenkins. J., Jansonius, ,J. N.. Karlxson. R. & Rosenbusch, J. P. (1983). X-ray diffraction analysis of matrix porin, an integral membrane protein from Escherichia coli outer membranes. J. Mol. Niol. 164, 313-327. TUPAC-IUB Commission on Biochemical Nomenclature (1970). Abbreviations and symbols for the description of the conformation of polypeptide chains. J. Mol. Riot 52, l-17. Biochemical ICPAC-TUB Joint Commision on Nomenclature (1983). Abbreviations and symbols for the description of conformations of polynucleotidr chains. Eur. J. Biochem. 131, 9Sl5. James, M. N. G. & Sielecki, A. R. (1983). Structure and refinement. of penicillopepsin at 1.8 a resolution .I. Mol. Biol. 163. 299-361. ,Janin, J., Wodak, S., Levitt, M. Cy: Maigret, B. (1978). Conformation of amino acid side-chains in proteins. .I. Mol. Biol. 125, 357-386. ?Jap, B. K. & Walian, P. J. (1990). Biophysics of the structure and fun&ion of porins. Quart. Rev. Biophys. 23. 367-403. Jap. B. K.; Walian. P. .J. & Gehring, K. (1991). Structural archit’ecture of an outer membrane channel as determined by electron crystallography. Nature (London,), 350. 1677170. ,Jones. T. A.. Zou, J-Y., Cowan, 8. W. & Kjeldgaard. M. (1991). Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. sect. A, 47. 110-119. Kabsch, W. & Sander, C. (1983). Dictionary of protein secondary structures: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers, 22, 2577-2637. Karplus, P. A. & Schulz, G. E. (1987). Refined structure of glutathione reductase at I.54 A resolution. J. Mol. Biol. 195: 701-729. Kreusch, A.. Weiss, M. S.. Welte, W., Weckesser, J. &

and

(2. E.

Schulz

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