Topology of the anion-selective porin Omp32 from Camamonas acidovorans

JOURNAL

OF STRUCTURAL

BIOLOGY

Topology SUSANNE

GERBL-RIEGER, Max

14-24 (1992)

108,

of the Anion-Selective Porin Omp32 from Comamonas acidovorans HARALD

Planck

Znstitut

ENGELHARDT, J~~RGEN PETERS, WOLFGANGBAUMEISTER

fiir

Biochemie,

D-8033

FRIEDRICH

LOTTSPEICH,

AND

of Germany

ions particularly efficient at low salt concentrations, a feature which is unusual among bacterial porins. It is, therefore, of some interest to elucidate the structural basis of the anion selectivity of these porins. Steven et al. (1977) were the first to describe, based on electron microscopy and image processing, the trimeric organization of the porin in the outer membrane of Escherichia coli. Later threedimensional reconstructions of reconstituted membrane crystals revealed that the three pores of a functional trimer merge into a common outlet at one side of the membrane (Dorset et al., 1983; Engel et al., 1985). A very similar structure was found in the outer membrane of species with outer membranes in which the porins naturally form two-dimensional regular arrays (Chalcroft et al., 1987; Rachel et al., 1990). Infrared spectroscopy showed that P-sheets are the dominant secondary structure element of porins (Kleffel et al., 1985; Vogel and Jahnig, 1986; Nabedryk et al., 1988). High-resolution electron crystallography of the porins OmpF and PhoE revealed that the p-sheets are arranged as a barrel which creates the pore walls (Sass et al., 1989; Jap et al., 1990). Topological information obtained in phage and antibody binding or partial proteolysis studies, have further contributed to the understanding of the folding of porin polypeptides in the outer membrane (for a recent review see Tommassen, 1988). Recently, X-ray crystallography led to a highresolution three-dimensional structure determination of the Rhodobacter capsulatus porin (Nestel et al., 1989; Weiss et al., 1990, 1991). It confirmed that the P-sheets form the pore as well as the common outlet of the three pores and new features such as the location of amino acid residues supposed to be of functional importance and the distribution of charges were revealed. Although there is little doubt that the bacterial porins are all built on similar structural principles, the overall homology of porins on the primary structure level is relatively weak; it is restricted to eight

Limited proteolysis experiments were performed with outer membranes from Comamonas acidovorans to probe the topology of its major protein component, the anionselective porin Omp32. Proteinase K treatment above a critical temperature of 42°C cleaved the surface-exposed regions of the porin, yielding membrane-embedded fragments which were separated by SDS polyacrylamide gel electrophoresis or reversed phase chromatography. The identification of the proteinase K-sensitive sites was performed by microsequencing. This allowed us to determine six surface-exposed sites of the porin, all located in nonconserved primary structure regions. These results along with the previously determined amino acid sequence and in conjunction with some structural constraints applicable to porins allowed us to propose a chain-folding model of the Omp32 porin. The features of our model are compared with the structure of the Rhodobacter capsulatus porin, recently established by X-ray crystallography (Weiss et al., 1991) and they are used to elucidate the 0 1992 Academic structural basis of the anion selectivity. Press, Inc.

INTRODUCTION

Comamonas acidovorans, a member of the P-subdivision of the Proteobacteria, possesses an outer membrane rather simple in protein composition. It contains two major proteins, the regularly arrayed surface protein which despite its peripheral location is intimately associated with the outer membrane (Chalcroft et al., 1986; Engelhardt et al., 1991) and the transmembrane protein Omp32; moreover two minor proteins, Omp37 and Omp21, are regularly occurring outer membrane components (Engelhardt et al., 1990). The Omp32 has all the functional and structural attributes of a porin (Engelhardt et al., 1991; Gerbl-Rieger et al., 1991). Like the functionally well-characterized Omp34 of the closely related species Acidovorax delafieldii (Brunen et al., 1991) it is strongly anion-selective. The porins Omp32 and Omp34 appear to be capable of accumulating ions in the vicinity of the pore, making the diffusion of an14 1047-8477/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

KEHL,

Federal Republic 28, 1991

Martinsried,

Received November

MARIA

THE

PORIN

OF COMAMONAS

conserved regions (Gerbl-Rieger et al., 1991). In this communication we present a folding model of the porin Omp32 of C. acidovorczns based on its primary structure (Gerbl-Rieger et al., 1991) and the results of microsequencing of proteolytic fragments obtained in controlled proteolysis experiments. Other structural data available to us, such as the p-sheet content, the pattern of regional similarities, and the hydrophilicity profile were used as constraints. MATERIALS a.

strain

&ZCteFid

and

AND Growth

METHODS

K Treatment

of Intact

and

Disrupted

in 1% bacglucose at

Cells

A loo-ml overnight culture was harvested by centrifugation and washed with 20 ml of 30 mM TrisHCl, pH 8.1. The cells were resuspended in 60 ml buffer and half of the cells were broken in a Vibrogen cell mill in the presence of DNase II. The proteinase K treatment was performed with an enzyme-to-protein ratio of approximately 1:20 for 1, 2, 3, and 5 days at 37°C and for 24 hr at 42°C. Intact and disrupted cells were also incubated in the absence of proteinase K to examine whether autoproteolysis or cell lysis occur. The incubation was terminated by harvesting the cells by centrifugation at 5000g. The cells were resuspended in 30 r&f TrisHCl, pH 8.1, 5 mM MgCl,, 2 n&f PMSF, and 50 pgiml DNase II and broken as described above. After incubation at 37°C for 2 hr the glass beads were removed by filtration and the cell envelopes collected by centrifugation at 48 OOOg. c. Proteinase

K Treatment

of Cell Envelopes

Outer membranes were suspended in 30 m&f Tris-HCl, pH 8.1, at a protein content of 10 mgiml. Proteinase K was added until the enzyme-to-protein ratio was 1:50. The samples were incubated for 1, 2, 4, 6, 10, 15, and 30 hr at 37, 42, or 5O”C, respectively. Also outer membranes were incubated in 30 mM Tris HCl, pH 8.1, 5 mM EDTA, or in 30 m&f Tris-HCl, pH 8.1, 10 ti MgCl,, or in 30 m&f Tris-HCl, pH 8.1, with an enzyme-to-protein ratio of 1:20 for 48 hr at 37, 42, 50, and 60°C. The digestion was stopped by the addition of phenylmethanesulfonyl fluoride (PMSF) to a final concentration of 2 n&l. The digested outer membranes were sedimented at 48 OOOg for 20 min. ti

PAGE

and

Ammo

Acid

Sequence

Analysis

Aliquots of untreated and proteinase K-digested outer membranes were dissolved in sample buffer (125 m&f Tris- HC!, pH 6.8, 5% SDS, 10 n&f EDTA, 10 mii! DTT, 10% mcrose, 0.02?{ bromophenol blue) and incubated at 30 or 100°C for 5 min. SDS PAGE was performed according to Laemmli (1970) in a linear gradient gel (7-15% acrylamide) or in a tricine gel (14% acrylamide) according to Schagger and Jagow (19871. The polypeptides were transferred from the tricine gels onto silicon-glass fiber membranes (Eckerskorn et al., 1988) in 50 mM boric acid, pH 9.0, and 20% methanol. Electroblotting was performed in a semidry-apparatus (Sartorius, Gottingenl at 1 mA/ cm2 for 2-5 hr. The blotted protein fragments were stained with 0.1% Coomassie R250 in 40% methanol and 10% acetic acid for 2 min, washed in H,O, and dried. The protein bands were cut out and subjected to N-terminal amino acid sequence determination or amino acid analysis as described elsewhere (Gerbl-Rieger et al., 1991).

15

e. Molecular Sieve Chromatography Phase Chromatography

and

Reversed

The proteolytic fragments in the range of about 2000-6000 Da were isolated by means of chromatography. All the peptides still residing in or attached to the membrane after protease treatment were fractionated on a TSK 3000 column (LKB Pharmacia) in 30% acetonitrile, 0.1% TFA. The fractions containing peptides smaller than about 6000 Da were collected and separated on a Lichrosorb RP 8 reversed phase column (Merck) in a gradient of 060% acetonitrile, 0.1% TFA. The absorbance was measured at 206 nm. The resulting fractions were subjected to amino acid sequence determination. r ATR

Conditions

Comamonas acidovoruns (ATCC 15688) was grown totryptone, 0.5% yeast extract, 1% NaCl, and 0.1% 30°C as described in detail in Chalcroft et al. (1986). b. Proteinase

ACIDOVORANS

-IR Spectroscopy

Outer membranes were analyzed before and after proteinase treatment by means of infrared spectroscopy. Attenuated total reflection infrared spectroscopy (ATR-IR) was performed using a Perkin-Elmer 325 IR spectrometer or a Nicolet FTIR 740 using Ge crystals as supports. Bandshape analysis of the amide I and the amide II regions were performed as described by Kleffel et al. (19851 to quantitatively determine the protein secondary structure. g. Differential

Scanning

Calorimetry

(DSC)

DSC curves of the outer membranes were determined in the same buffer as used for the proteinase K digestion experiments. Two milliliters of a suspension containing membranes at a protein concentration of 50 mgiml were scanned in a Microcalorimeter MC-2 (Microcal, Amherst, MA) over the range from 30 to 70°C and at a heating rate of l”/min. h. Electron The copper stained examined cation

Microscopy

membrane preparations were adsorbed to carbon-coated grids, made hydrophilic by glow discharge, and negatively with 2% uranyl acetate for 30 sec. The membranes were in a Philips EM 420 electron microscope at a magnifiof X 36 000. RESULTS

a. Proteinase K Treatment of Intact Isolated Outer Membranes

Cells and of

When intact cells were exposed to proteolytic digestion with proteinase K (Eberling et al., 1974) under a variety of conditions (37 and 42°C for 24 hr, enzyme-to-protein ratio of 1:20) it turned out that the Omp32 porin was not efficiently cleaved at 37°C even after prolonged incubation for up to 5 days; this applies to intact as well as to broken cells. The incubation of intact cells at temperatures above 42°C led to cell lysis. The outer membranes which were isolated from these lysed cells were free of the “cobblestone” motif characteristic for the surface protein and contained cleaved Omp32. The pattern of the proteolytic fragments found was identical to the pattern obtained with isolated outer membranes after proteinase K treatment at 42°C (Fig. 1, lane h). Time course experiments performed at various temperatures with isolated membranes showed that the proteolytic sensitivity of Omp32 increased significantly at temperatures higher than 42°C (enzyme-to-protein ratio of 1:50). Concomitantly the regularly arrayed surface protein disappeared (Fig.

16

GERBL-RIEGER 37 c kDa

AL.

42~C

c d

5O’C

*a-----Y-

---1--s-.

a 1b

ET

e

f

g

h I I

k Ib

g=c I_-

d’ f’

h‘I

a

i

k/b

cd

e

f

g

hbd’f’h II ’

~uQjjj#jj-

I/

Ib c d

*

e

f

g

hlld

d’

1’ I, ia

I

kOd

k

FIG. 1. Polypeptide patterns of outer membranes from Comamonas acidovoruns after proteolytic digestion with proteinase K (enzymeto-protein ratio 150) at 37, 42, and 50°C over increasing periods of time. Proteins were separated on a 7-15% polyacrylamide gradient gel. Control samples are in lane (a) proteinase K, in (i) membrane digested in the presence of 0.2% SDS, for 30 hr, and in (k) membranes incubated for 30 hr without any proteinase present. Samples were digested with proteinase K for 1 hr (b), 2 hr (c), 4 hr (d), 6 hr (e), 10 hr (f~. I5 hr (a), and 30 hr (h). The samples in lanes a through k were solubilized at 100°C for 5 min; the samples labeled b’, d’, f, and h’ were solub&ed at 30°C.

1). The kinetics were clearly temperature dependent, but the pattern of proteolytic products found in the membrane after incubation for 30 hr at 42 and at 50°C respectively, were identical. It can be ruled out that the peptide pattern obtained is due to proteolysis occurring while the samples are solubilized in 5% SDS at 100°C for 5 min for PAGE; otherwise the polypeptide composition at the earliest stage of the kinetics in lane b (37°C) and lane i in Fig. 1 (and consistently all the other samples) should also show the characteristic pattern of lane h (42 and 50°C). The digestion in the presence of 0.2% SDS led to a large number of small peptides (Fig. 1, lane i). This indicates that the susceptibility of the porin to proteinase K depends on the physicochemical status and the integrity of the outer membrane. The solubilization of the proteolytically cleaved porin with SDS at 30°C resulted in high molecular weight species (90-100 kDa) typical for porins (Lugtenberg et al., 1975; Nakamura and Mizushima, 1976) suggesting that the cleavage products remain physically associated with each other in the membrane. Presumably they still form trimeric complexes. The only low molecular weight band visible under these conditions can be assigned to the uncleaved Omp21 which is resistant to proteinase K treatment even at 50°C and over prolonged incubation periods (Fig. 1, lanes b’, d’, f’, and h’).

multiple cleavage, remain physically associated (Fig. 1, lanes b’, c’, f, and h’). In order to determine whether the Omp32 retained its secondary structure upon treatment with proteinase K, infrared spectra were recorded (Fig. 2). The apparent p-structure content of the isolated porin of C. acidouoruns has been estimated to be 70 et al., 1991). After incubation + 8% (Engelhardt with proteinase K (enzyme-to-protein ratio 1:50) at 42°C for 30 hr the residual membrane, predominantly containing the cleaved Omp32, showed an increased p-structure content of 82 + 10% as indicated by the significant increase of the band at 1632 cm -i in the amide I region of the IR-spectrum (Fig. 2, inset). The high p-structure of the proteinase K-treated membrane along with the results of the solubilization at 30°C (Figs. 1 and 2) is a strong indication that the secondary structure of the membraneembedded portion of Omp32 is affected by neither the incubation at 42°C nor the cleavage of the surface-exposed sites. The relative increase of the p-structure content is consistent with the proposition that the p-strands are located inside the membrane and that surface-exposed loops are removed.

b. Electron

The Omp32 porin remained almost uncleaved when incubations were done at 37°C (Fig. 1). But after 30 hr at a much higher enzyme concentration (enzyme-to-protein ratio 1:20) the surface protein was removed from the membrane (Fig. 3, lane b), yielding two fragments (apparent molecular weight of 27-29 kDa, data not shown) which were found in the supernatant after isolating the membrane by sedimentation. In order to investigate this phenomenon in greater detail we studied the influence of divalent cations on the susceptibility to proteolysis of the

Microscopy

and ATR-IR

Spectroscopy

Electron microscopic investigation confirmed that the regularly arrayed surface protein with its characteristic “cobblestone” pattern completely disappeared upon treatment with proteinase K (enzymeto-protein ratio 1:50) at 42°C for 30 hr (Fig. 2). The resulting membrane sheets appear smooth and featureless, making it unlikely that the Omp32 occurs in the outer membrane of C. acidouoruns regularly arrayed. Such membranes contain the proteolytic products of the Omp32 protein which, despite the

c. Effect of Cations on the Proteolytic the Omp32 in the Outer Membrane

Stability

of

THE

PORIN

OF COMAMONAS

17

ACZDOVORANS

FIG. 2. Electron micrographs of C. acidouoruns outer membranes negatively stained with uranylacetate. (A) Native membranes showing the regularly arrayed surface protein. Inset: IR (infrared) spectrum of the native outer membrane. (B) Proteinase K-treated outer membranes appear smooth and featureless. The regularly arrayed surface protein has disappeared. Inset: IR spectrum of proteinase K-treated outer membranes. The bar represents 200 nm. Quantitative bandshape analysis of the IR spectra (for details see Kleffel et al., 1985) revealed the contribution of the component characteristic for p-strands to the amide I band at 1632 cm i. X-axis (wavenumber cm ‘1, Y-axis (absorbance).

porin. In membranes incubated at 37°C for 30 hr with proteinase K (enzyme-to-protein ratio 1:20) in the presence of 5 mM EDTA, the Omp32 protein was degraded, whereas the addition of 10 miU MgCl, protected the porin at incubation temperatures as high as 42 and 50°C (Fig. 3). At 60°C the presence of MgCl, had no longer a protective effect. It is noteworthy that all proteolytic products obtained under these conditions are identical with those shown in Fig. 1 (lane h). The susceptibility of the Omp32 protein to proteolytic attack correlated with the phase behavior of the outer membrane which was determined by means of differential scanning calorimetry, using the same buffer conditions as with the digestion experiments (Figs. 3 and 4). The DSC curves show broad peaks, characteristic for natural membranes containing various lipids and proteins (McElhaney, 1982). The onset of the uptake of heat was about 39”C, just beyond the temperature where no or almost no proteolysis was observed (37”C, Fig. 1; Fig. 3, lane b). DSC curves of outer membranes mea-

kDa 95 68 45

-

29

-

21

-

12.5-

-

IlW-.w -

6.5 --

abcdefghi FIG. 3. Polypeptide patterns of outer membrane from C. acidouorans after proteolytic digestion with proteinase K (enzymeto-protein ratio 1:20) for 30 hr at various temperatures in the presence of EDTA and MgCl,. Peptides were separated on a 14% polyacrylamide gel (Schigger and Jagow, 1987). All samples were incubated in 30 mM TrisHCl, pH 8.1, for 30 hr. Lane a, outer membrane incubated at 50°C without any enzyme present; lane b, outer membrane incubated at 37°C; lane c, 37°C + 5 n-u%4 EDTA; lane d, 42°C; lane e, 42°C + 10 m&f MgCl,; lane f, 50°C; lane g, 50°C + 10 mkf MgCl,; lane h, 60°C; lane i, 60°C + 10 mM M&I,.

18

GERBL-RIEGER

/

A

F--fJfl

35

45

I 55

I

65

75

OC

75

oc

I

I

I

35

I

45

I

55

I

65

FIG. 4. Differential scanning calorimetry (DSC) curves of outer membrane of C. acidot~oruns in the temperature range between 30 and 70°C. (A) Phase behavior of the outer membrane in 30 mM Tris-HCl, pH 8.1. (B) Phase behavior of the outer membrane in 30 mkf Tris-HCl, pH 8.1, and 5 mM EDTA added. (0 Phase behavior of the outer membrane in 30 n-&f Tris-HCl, pH 8.1, and 10 mkf MgCl, added. Arrows indicate the temperatures 37, 42, and 5O”C, respectively.

sured in the presence of 5 mM EDTA showed a significant shift toward lower temperatures, the beginning of heat uptake already being at ~30°C while 10 mM MgCl, shifted the onset of phase transition to about 53°C. This is in good agreement with the observation that proteolysis occurred at 60 but not below 50°C. Obviously, divalent cations have an effect on the mobility of lipid, lipopolysaccharide, and protein by binding to and cross-linking negatively charged groups. Preincubation of the membranes at 60°C for 4 hr and subsequent digestion at 37 and 42°C with or without EDTA and MgCl, gave identical results (data not shown). This means that incubation at temperatures up to 60°C does not cause denaturation; but it appears to reversibly change the physicochemical state of the outer membrane. d. N-Terminal Sequence Determination of Fragments Obtained by Proteinase K Digestion

In order to identify the proteinase K-sensitive regions in the amino acid sequence of Omp32, the pep-

ET AL.

tides produced by limited proteolysis were separated by PAGE, electroblotted, and sequenced. Five N-terminal sequences of proteinase K-sensitive sites were obtained via this approach, i.e., the sites al, cl, dl, d2, and el (Table I). Four of the proteolytic products had staggered ends (Fig. 5 and Table I, peptides 2,4, 5, and 6). Only peptide 3 (Fig. 5) gave a discrete N-terminal sequence (Table I). The peptides 8, 9, and 10 (Fig. 5) did not give any sequence information. These proteolytic fragments probably contained the blocked N-terminus of Omp32 (GerblRieger et al., 1991) and were possibly truncated at the C-terminal ends. The N-terminal sequence of peptide 7 is identical with that of the proteinase K. The N-terminal sequence of peptide 1, a prominent component of 5- to 6-kDa size, could not be related to the Omp32 at all and possibly originated from the regularly arrayed surface protein. We are currently investigating whether this peptide represents the membrane anchoring domain of the surface protein. The apparent molecular weights of the identified peptides varied between 6.5 and 32 kDa. For the isolation of peptides smaller than 6 kDa, molecular sieve fractionation combined with reversed phase chromatography were used. This allowed to identify additional proteinase K-sensitive sites (Table I, sites bl, fl) by means of the N-terminal amino acid sequence determination. Other sites (~2, e2) were confirmed by the existence of the fragments RP 16 and 17 and RP 21 (Table I) obtained by reversed phase chromatography. Figure 6 shows a scheme with the digestion products aligned to the complete primary structure; the positions of the C-terminal ends were deduced from the apparent molecular weights as determined by SDS-PAGE. In summary, six surfaceexposed sites were detected in the sequence of the Omp32. DISCUSSION

Porins have some structural attributes which greatly facilitate attempts to model the folding of their polypeptide chains. Already prior to the availability of high-resolution structures from X-ray crystallography (Weiss et al., 1990, 1991) and electron crystallography (Jap et al., 19911, the high p-structure content of about 60% has stimulated proposals for their basic structural organization and polypeptide chain folding (Rosenbusch et al., 1982; Kleffel et al., 1985; Paul and Rosenbusch, 1985; Nabedryk et al., 1988). Vogel and Jahnig (1986) presented a refined model taking into account a motif of alternating hydrophobic and hydrophilic residues in the p-strands. As in bacteriorhodopsin (Fimmel et al., 1989) information about surface-exposed sites played an important role in refining and verifying the models. Topographic information was derived from antibody and phage binding studies, limited

THE

PORIN

OF COMAMONAS

19

ACIDOVORANS

TABLE1 N-Terminal

Origin of peptides and numbers PAGE RP PAGE

Acid Sequences of the Proteinase K Digestion Products of the Omp32

Amino

Approximate K

N-terminal

10 000

Arg

(151

~6000

Gly

(6)

20000

Ala

~6000

Gly

(2)

v - Gly v -- Asn v - Gln

- Leu v - Phe v Gly

v -

- Asp v - Ser

v - Asp

v -

-

PAGE

(5)

16800

Thr

- Ala v - Ala

PAGE

(4)

15 300

Thr

v -

PAGE RP RP

(3) (21) (3)

11700 ~6000 1500

Ala Thr Ala

- Thr - Asp - Gly

RP

(16,171

- Tyr v - Phe v - Gly

Ala

Asp Asp

Asp Arg Glu

v v -

- Ser

- Gly

- Ala

- Ser

Lys

- Arg

- Arg

- Ser

- Thr

- Val

Ala

Asp

- Asp

Asn

Gly

-

Asn

- Gly

- Ile

- Arg

- Ala

-

Val

- Gly

- Arg

- Tyr

Ser

v -

Val

- Gly

- Arg

- Tyr

- Ile

- Arg - Glu - Ser

-

Glu Ile Gln

~ Ile - Thr - Thr

- Thr - Leu - Gly

- Leu - Gly - Val

- Gly - Ala - Gln

RP: Peptides

kDa

‘0. ;z 6543-

2- .’ - 6.5 b

sequences

- Thr

proteolysis of surface-exposed protein portions, as well as from experiments involving insertion mutagenesis (Agterberg et al., 1987; Bosch and Tommassen, 1987; Morona et al., 1985; Schenkman et al., 1984; Korteland et al., 1985; Van der Ley et al., 1985,1987). The recently published X-ray structure determination of the R. capsulatus porin (Weiss et that 16 antiparallel al., 1990, 1991) confirmed P-strands form a barrel creating the pore in the

a

acid

Gly

Note. PAGE: Peptides isolated from polyacrylamide gels; alternative cleavage sites obtained in proteolysis experiments.

l-

amino

Cleavage sites (see Fig. 6) and sequence positions

m

FIG. 5. Silicon-glass fiber (SGF) Western blot of an electropherogram of proteinase K digested outer membranes. Outer membranes were incubated with proteinase K (enzyme-to-protein ratio of 150) for 30 hr at 42°C (lane b) and separated on a 12% polyacrylamide gel (Schagger and Jagow, 19871, transferred onto SGF-membranes, and stained with Coomassie brilliant blue. The fragments l-10 were subjected to N-terminal amino acid sequence determination. Lane a, outer membrane proteins; lane m, molecular weight markers.

isolated

- Asn

al (2&30) - Ser

bl

(70-743

- Asn

cl (123126) c2 (126) dl(16P 1671

by reversed

- Ser - Val

- Tyr

- Asn

phase

chromatography.

- Phe

Arrows

d2 (164 168) el (200) e2 (201) fl (317) indicate

outer membrane. The high-resolution electron crystallography studies of the porins OmpF (Sass et al., 1989) and PhoE (Jap et al., 1990) make it likely that a p-barrel forming the pore wall is a common feature of the various porins from E. coli. We used the approach of limited proteolysis to identify peptide regions located at the membrane surface or sequence portions which are embedded in the membrane interior. The proteinase-treated membranes remained intact as judged by electron microscopy. This observation justifies the assumption that the cleavage sites are indeed located at the surface of the membrane, probably very close to the interface where further degradation is sterically hindered. The fact that the relative p-structure content actually increased indicates that the membrane-embedded portions of the Omp32 mainly represents p-sheet elements. Although there appears to be little primary structure homology among porins from different organisms, we have shown recently that eight sequence regions do possess significant local similarities even among porins from distantly related species (GerblRieger et al., 1991). The highest similarity that we found with the recently published primary structure is the strongly anion-selective porin from Bordetella pertussis (Li et al., 19911, which is also a member of the P-subdivision of Proteobacteria (data not shown). The eight regions of homology alternate with the hypervariable peptide regions as identified in the E. coli porins (Mizuno et al., 1983) which are likely to represent surface-exposed regions. As discussed ear-

20

GERBL-RIEGER

A

I 26-30

, B

I

70-74 1

I 123-120

I

ET

I 164-107 i

AL.

I

I

200 I

317 I

NH

COOH

dl d2

r\ al

el e2--

bl

fl-

c2-FIG, 6. Schematic representation of the proteinase K cleavage products and the proteinase K-sensitive sites of the Omp32 in comparison with regional porin similarities. (A) Motif of regional sequence similarities obtained by a comparison of porin primary structures (from Gerbl-Rieger et al., 1991). (B) Proteinase K-sensitive sites in the Omp32. Numbers give the N-terminal amino acids obtained by microsequencing of the proteolytic products of the Omp32. (Cl Approximate size and position of the proteolytic peptides in the primary structure of the Omp32. Fragments al, cl, dl, d2, and el correspond to the peptides 2, 3, 4, 5, and 6 isolated by PAGE. Fragments bl, c2, e2, and fl are peptides smaller than 6000 Da and were isolated by reversed phase chromatography.

lier (Gerbl-Rieger et al., 1991) we assume that the eight conserved regions are located inside the membrane and represent the stretches of the polypeptide chain forming the p-barrel. This is consistent with the assumption that the six cleavage sites (Figs. 6 and 7, sites a-f) we have been able to identify in the C. acidouoruns Omp32 are exclusively located in the nonconserved sequence regions. It is noteworthy that they are all close to the

hydrophilic regions predicted by the hydrophilicity profile of the amino acid sequence. Studies performed by Bosch and Tommassen (1987) and Van der Ley et al. (1986) similarly provided evidence that the surface exposed regions of the E. coli porins are located in hypervariable regions. The surface exposed regions of PhoE are located in maxima of the hydrophilicity profile which are all separated by approximately 40 amino acid residues (Tommassen,

LO

FIG. 7. Folding model of the Omp32 based on the primary structure, secondary structure data from IR spectroscopy, limited proteolysis, and hydrophilicity profile: the pattern of porin regional similarities (see Discussion) were also taken into account. The black arrows represent the six (a through f) proteinase K cleavage sites. The white columns indicate the hydrophilic maxima of the amino acid sequence of Omp32 according to Kyte and Doolittle (1982). The dotted columns represent the p-strands with hydrophobic amino acids supposed to be oriented toward the lipid phase printed in bold letters. The superimposed bars (1 through 8) mark the pattern of regional sequence similarities obtained by comparison with various porins from E. coli and Neisseriu (Gerbl-Rieger et al., 1991).

THE

PORIN

OF COMAMONAS

1988). In the sequence of Omp32 the proteinase K cleavage sites a-b, c-d, and d-e are separated by approximately 40 amino acids from each other. The distance from e to f is 117 residues, i.e., approximately 3 x 40 residues. Assuming that two neighboring surface loops are intervened by ca. 40 amino acid residues forming two p-strands, we obtain at least 12 p-strands. In addition there is one N-terminal (amino acid residue 1 to site a) and one C-terminal (site f to residue 332) p-strand. The region between the sites b and c, consisting of approximately 50 amino acid residues, could make two further p-strands as well as a large loop. The R. cupsuZatu.s porin indeed possesses a large loop in this particular sequence region (Weiss et al., 1991). Hence there is good evidence for at least 16 antiparallel p-strands in the Omp32. This provides the basis for the following consideration and the folding model for the porin Omp32 of C. acidouoruns (Fig. 7). In order to estimate the extent of the individual p-strands we used the occurrence of alternating hydrophilic and hydrophobic amino acid residues as observed by Vogel and Jahnig (1986) in the OmpF and OmpA proteins of E. coli; another criterion we took into account is the location of the proteinasesensitive sites. The resulting model predicts an overall p-structure content of about 58% and an average length of 12 amino acid residues per p-strand. These values are in reasonably good agreement with estimates from IR spectra (70 + 8% and 11-12 amino acids average length per P-strand (Engelhardt et al., 1990). In our model the lower rim of the P-sheet consists of hydrophobic amino acids, most of them probably facing the lipid (Fig. 7). It is striking that 8 of the 16 p-strands do possess Tyr or Phe in the first or second position. These aromatic residues apparently form one of the rings which were first described for the R. capsulatus porin (Weiss et al., 1991; Schiltz et al., 1991; W. Welte, personal communications). A second (upper) ring of aromatic amino acid residues appears also to exist in the Omp32. Again 8 of the 16 P-strands have 1 or even 2 aromatic amino acid residues near the upper end. Interestingly, most of the p-strands appear to possess an aromatic amino acid either at the lower or the upper end. These residues are not exactly horizontally aligned in our model, but one has to take into account that the p-strands might be variously tilted with respect to a normal to the membrane plane as shown by Nabedryk et al. (19881, Jap et al. (19911, Walian and Jap (19901, and Weiss et al. (1990, 1991). The seven loops or turns at the lower end of the P-sheet are much shorter than the loops on the upper end. Five of these turns coincide with local maxima of the hydrophilicity profile. Most of the proposed turns consist of amino acids allowing or even

ACIDOVORANS

21

promoting p-turns (Chou and Fasman, 1978; Milner-White and Poet, 1987). The two outermost loops on the lower side (Figs. 7 and 8) are the longest on this side and flank the five small turns. This part of the folding pattern is again in good agreement with that of the R. capsulatus porin. Weiss et al. (1991) reported that the larger terminal loops form the protein-protein interface of the trimer, whereas the five small p-turns are exposed to the lipid phase. The loops on the other side of the P-sheet are larger. Seven of the eight loops on this side coincide with maxima of the hydrophilicity profile (Fig. 7), while two hydrophilic maxima are located within the putative p-strands 13 and 14. Possibly these regions contribute to the polar interface mediating the protein-protein contacts in the trimeric complex as found in the R. capsulatus porin (Weiss et al., 1991). Since we did not obtain proteinase K cleavage sites in these loops, the suggested folding of the sequence region from positions 202 to 295 is the least certain one in our model. It is interesting to note that particular amino acids or sites found in other porins to be of functional importance are conserved in the Omp32 polypeptide and appear in appropriate regions of our folding model. Examples are the amino acids Args,, ArgT4, and Asp,,, of OmpC which contribute to its functional properties; point mutations changing one of these charged residues to uncharged amino acids increased the size of the OmpC porin channel (Misra

FIG. 8. Folding model of Omp32 displaying the distribution of the charged amino acids and the location of aromatic amino acid residues. Diamonds represent the negatively charged amino acids D (Asp) and E (Glu). Filled circles represent the positively charged amino acids R (Arg) and K (Lys), hatched circles represent H (His). Crosses represent the aromatic amino acids F (Phe), Y (Tyr), and W (Trp) which possibly form a lower and an upper ring of aromatic amino acids anchoring the porin in the membrane. The horizontal lines enclose the region which possibly forms the channel portion not merging into the common outlet of the porin trimer.

22

GERBL-RIEGER

and Benson, 1988; Lakey et al., 1991). The Omp32 polypeptide has Arg residues at the positions 38, 75, and 76 (and Lys at position 74) and Asp at position 105. These residues are located in the lower part of the P-sheet, supposed to form the pore, and in the large loop (Asp,,,) which contributes to the formation of the pore in the R. capsulatus porin (Weiss et al., 1991). The amino acid residues Arg15s, Argaol, Gl~ms, and Gly,,, and the amino acid at the position 315 of the porin PhoE from E. coli are cell surfaceexposed sites as shown by antibody and phage binding studies (Korteland et al., 1985; Van der Ley et al., 1986; Bosch et al., 1989). Interestingly, the corresponding positions in the Omp32 folding model, i.e., J-JYs~~~,Arg2037 G~Y~B, Gly2s4, Gly277y and Vals15, are all situated in loops at the upper side (Fig. 7). In view of the anion selectivity of the Omp32 the distribution of charged amino acids in the model is of particular interest. One may distinguish three characteristic regions. The loops and turns at the lower side apparently contain four positively and four negatively charged residues and thus a net charge equal to zero. The sequence portions in the loops at the upper side, which form the part of the pore merging into a large outlet in the trimeric complex (Weiss et al., 1990,1991), apparently contain 20 negatively charged and 16 positively charged amino acid residues and, in addition, two His. The net charge, therefore, is again close to zero; at least, there is no clear evidence for an electric potential. However, the positive and negative charges are not evenly distributed over the loops. The loops 2 to 5 contain 14 negative and only 7 positive charges, while the loops 1 and 6 to 8 together carry 6 negatively and 9 positively charged amino acids and, in addition, 2 His residues (Fig. 8). The third region which we suppose to represent the portion of the pore which does not contribute to the common outlet in a porin trimer (indicated in Fig. 8) is clearly charged. Eleven positive charges (and one His in addition) dominate the 4 negatively charged residues. If we only take into account residues of the P-sheet orientated toward the interior of the pore according to our model (Fig. 8) there is still a surplus of 5 positive charges inside the channel. Further investigations will establish whether the Lys and Arg residues in this part of the channel create a surface potential capable of accumulating negatively charged ions as it was postulated for Omp32 (Engelhardt et al., 1990) and for the Omp34, the anionselective porin of the closely related species Acidouorax delufieldii (Brunen et al., 1991). The number of surface charges expected according to the functional model of Brunen et al. (19911, i.e., the potential equivalent to l-2 elementary charges near both channel entrances, is in good agreement with the

ET

AL.

net number of positive charges suggested by this structural model. Whether the clustering of positively and negatively charged amino acids in the loops and in the channel contributes to the formation of charged “semicircles” described by Weiss et al. (1991) for the R. capsulatus porin remains to be established. We cannot yet unambiguously decide on the sidedness of the porin. There is some evidence, however, that the side with the larger loops, which is presumably the side forming the common outlet of the three pores in the assembled trimer-provided the structure is similar to the R. capsulatus porinrepresents the outer surface. First, the proteolytic attack of the porin was always correlated with the disappearance of the regular surface protein, and according to our model all cleavage sites are located on one side. Second, the proteolysis was inhibited by Mg2+ over a wide temperature range but promoted by EDTA. These temperature-dependent effects are correlated with the phase behavior of the outer membrane. Since LPS, which is located in the outer leaflet of the outer membrane, binds divalent cations quite effectively and alters its phase behavior accordingly (Brandenburg et al., 19901, it is tempting to speculate that LPS is responsible for providing the protection against proteolysis. Two facts corroborate this view: (i) LPS is indeed associated with the porin of Comamonas acidouorans (Engelhardt et al., 1990) as well as with other porins (Reinhart et al., 1977; Hoenger et al., 1990); and (ii) the long carbohydrate side chain and the core region of the LPS may shield the porin. The latter was elegantly demonstrated in antibody binding studies (Bentley and Klebba, 1988; Hackstadt, 1988) and phage binding experiments (Van der Ley et al., 1986). If the common outlet of the pore of the Omp32 is indeed orientated toward the cell surface and not toward the periplasmic space, the orientation would be the same as found for the Thermotoga maritima porin by deep etching in conjunction with surface relief reconstruction (Rachel et al., 1990). Surface relief reconstruction experiments of crystalline patches of the porin in native outer membranes could help to establish the sidedness in a definitive way. We thank Wolfram Welte for helpful discussions and ther thank Bing Jap for critical reading the manuscript. work was supported by a grant of the SFB 266.

we furThis

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