The Structural Basis of Peptide Channel Formation - American

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13 The Structural Basis of Peptide Channel Formation Garland R. Marshall and Denise D. Beusen Center for Molecular Design, Washington University, St. Louis, MO 63130

The increased interest in peptide ion channels as model systems for larger, physiological channels coincides with improvements in experimental techniques that make peptides more accessible and enable the determination of their structures. This chapter surveys peptides from natural sources of less than 50 residues that are known to function as ion channels and for which three-dimensional structural information is available. Although the helix was initially recognized as a structural motif that made insertion of a peptide into the lipid bilayer energetically feasible, this survey suggests it is not unique. While the magainins, the peptaibols, and the cecropins are examples of helical structures, the role of beta-sheet structures is apparent in tachyplesin and the defensins. Other peptides, such as the lantibiotics, are as yet structurally unclassifiable. A number of structural themes emerge from this analysis that help to explain how such small chemical entities can form pores that enable the transit of ions across the lipid bilayer.

THE

ABILITY O F PEPTIDES T O F O R M C H A N N E L S ( I )

i n m e m b r a n e s has b e e n

an active area o f investigation since the seminal observations o n alamethicin by M u e l l e r a n d R u d i n ( 2 ) i n 1968. I n recent years, the n u m b e r o f peptides k n o w n to act o n membranes has increased dramatically. T h e motivations to study these molecules are diverse a n d i n c l u d e the development o f antibacte rial a n d antiviral agents a n d the possibility that those peptides that f o r m voltage-gated channels c a n act as simple m o d e l systems f o r naturally occur ring i o n channels i n nerve membranes (3, 4). T h e lure o f a small, chemically defined system f o r the study o f i o n selectivity ( 5 ) and the role o f the electric field i n triggering changes i n c o n d u c t i o n have b e e n strong. N u m e r o u s b i o 0065-2393/94/0235-0259$ 14.48/0 © 1994 American Chemical Society

Blank and Vodyanoy; Biomembrane Electrochemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1994.

260

BIOMEMBRANE ELECTROCHEMISTRY

physical techniques have b e e n exploited to p r o b e the c h a n n e l - f o r m i n g activity o f peptides. A l t h o u g h significant progress has b e e n made i n understanding the details o f some systems, i n n o case has a general mechanism evolved to explain a l l the observations. T h e perspectives o f these studies have b e e n many a n d varied: characterization o f the p e p t i d e - m e m b r a n e interaction, the effect o f the peptide o n the l i p i d phase, a n d elucidation o f the structures o f the peptides involved i n i o n channel formation. Because a comprehensive survey o f all o f these efforts f o r every k n o w n peptide i o n channel w o u l d b e enormous a n d because interactions o f peptides w i t h membranes have b e e n reviewed previously ( 6 , 7 ) , w e have focused o u r efforts o n the structural aspects o f c h a n n e l - f o r m i n g peptides that consist o f 50 residues o r less. T h e synergy between the molecular architecture (8) o f p o r e - f o r m i n g peptides ( 9 ) and mechanistic theory has heightened i n recent years as structure-determ ination techniques a n d access to peptides have i m p r o v e d . Information attain able includes the three-dimensional structure o f peptides, b o t h i n solution and b o u n d to the m e m b r a n e , orientation o f peptides i n membranes i n the o p e n o r closed state o f the channel, degree o f aggregation i n solution o r i n the m e m b r a n e , a n d the aggregation n u m b e r a n d tertiary structure o f t h e aggregate. A l t h o u g h the amphipathic helix ( F i g u r e 1) (10) is a r e c u r r i n g m o t i f i n many o f the peptides that show pore formation, it is b y n o means u n i q u e . A n examination o f the structural data o n the w i d e variety o f peptides that f o r m i o n channels i n membranes reveals other themes, many o f w h i c h are sufficiently n e w that their real significance is to provide fertile g r o u n d f o r future investigation.

Peptide Antibiotics N u m e r o u s examples exist i n w h i c h c o m p o u n d s isolated as antibiotics f o r m channels i n l i p i d bilayers ( 7 ) . Certainly, the most highly refined channel structure is that o f g r a m i c i d i n A , a n d m a n y o f the experimental paradigms used to elucidate its structure a n d function are b e i n g a p p l i e d to other systems.

Gramicidin A. G r a m i c i d i n A is a h y d r o p h o b i c , linear peptide o f 15 residues arranged i n alternating chirality (11, 12) a n d toxic to G r a m - p o s i t i v e bacteria. P r o d u c e d b y Bacillus brevis (13), the biological function o f g r a m i c i d i n A i n transcriptional regulation (14, 15) appears to b e unrelated to its most interesting biophysical activity: It acts as a c h a n n e l for monovalent cations i n l i p i d membranes (16, 17). A n impressive arsenal o f c h e m i c a l a n d HCO-L-Val-Gly-L-Ala-D-Leu-L-Ala-D-Val-L-Val-D-Val-LTrp-D-Leu-L-Trp-D-Leu-L-Trp-D-Leu-L-Trp-CONHCH CH OH 2

Gramicidin A


2

13.

MARSHALL AND BEUSEN

Peptide Channel Formation

261

biophysical techniques has b e e n d i r e c t e d to the study o f g r a m i c i d i n A over the last 20 years, a n d the resulting literature is covered i n several review

articles (18-21). Initial structural models o f the g r a m i c i d i n channel (22,

23)

based o n

experimental results that i m p l i c a t e d a d i m e r (24, 25) have persisted ( F i g u r e 2). These models consist o f helical dimers ( F i g u r e 2 A a n d B ) a n d d o u b l e stranded helices ( F i g u r e 2 C a n d D ) , all o f w h i c h are helically w o u n d beta-sheet structures i n w h i c h the peptide dipoles alternate i n orientation a n d result i n no net dipole. T h e absence o f a net dipole a n d any charged amino acids may explain the lack o f voltage dependence i n g r a m i c i d i n . Qualitatively, the two classes o f structural models are similar i n pore size a n d length. B o t h classes have side chains o r i e n t e d to the l i p i d a n d polar carbonyl groups l i n i n g the central pore that presumably act to solvate the i o n . T h e helical dimers characterized b y U r r y et al. (22)

consist o f single-

stranded helices stacked e n d - t o - e n d ( F i g u r e 2 A a n d B ) . O f the theoretically possible forms, the β

6 , 3

d i m e r best fits expectations o f length ( 2 5 - 3 0 Â) a n d

pore size (4 Â) n e e d e d for c h a n n e l activity a n d i o n selectivity. T h e head-toh e a d d i m e r ( F i g u r e 2 A ) is currently favored as the active f o r m based o n ΝMR

(26)

a n d other biophysical techniques

cross-linked d i m e r analogues (22,

27).

as w e l l as the activity o f

Recent solid-state N M R studies

(28)

are consistent w i t h a right-handed helical d i m e r , a conformation also seen i n sodium dodecyl sulfate micelles (29). I o n - b i n d i n g sites o f g r a m i c i d i n i n c o r p o rated into phospholipids have b e e n characterized b y cated at residues 1 1 - 1 4

(30-32),

1 3

C N M R a n d are l o

approximately 20 A apart.

T h e double-stranded helical ( F i g u r e 2 C a n d D ) models o f g r a m i c i d i n were proposed b y V e a t c h et al. (23)

to explain the presence o f four intercon-

verting conformations i n dioxane. These structures are u n l i k e l y to b e the c o n d u c t i n g species, but they have b e e n difficult to dismiss because o f their frequent occurrence i n organic solvents. M a n y o f the possible conformational perturbations due to chain orientation (parallel vs. antiparallel), residues p e r t u r n , helical handedness, a n d chain stagger have b e e n observed i n solution (for a recent

review, see

reference

crystalhzed f r o m ethanol (33,

21). X - r a y analyses o f g r a m i c i d i n A

34) ( F i g u r e 3) a n d f r o m m e t h a n o l - C s C l (35)

reveal structures o f this class. T h e observation o f potential i o n - b i n d i n g sites i n the first case a n d the actual presence o f C s ions i n the pore i n the second study suggest that these types o f structures are capable o f i o n b i n d i n g a n d raise additional questions about their biological significance. Several studies suggest that the conformation (double-stranded helix or helical m o n o m e r ) o f g r a m i c i d i n inserted into the m e m b r a n e is that f o u n d i n solution (36-39). U p o n heating, the m e m b r a n e - b o u n d , antiparallel, d o u b l e stranded helix converts to a helical d i m e r (37).

Subsequent studies have

suggested that the initially inserted double-stranded helical forms are u n l i k e l y to have any conductance capabilities

(40).


262


Figure 1. Amphipathic helices cluster hydrophilic residues on one face, which results in a cylinder that has one hydrophobic and one hydrophilic surface. The first 18 amino acid residues of magainin II are arranged on an α-helical wheel diagram (A) and 3 -helical wheel diagram (B). 10


13.

MARSHALL AND BEUSEN


263

Figure 1.—Continued. In the α-helical conformation, the amphipathicity is evident. In an even more striking comparison, the first 18 residues of the S4 segment of the sodium ion channel are plotted in the same two ways (C and D). Here the 3 -helical conformation (D) clearly segregates the positively charged Arg residues on the same face, whereas an α-helical conformation (C) results in a uniform arrangement of the charged residues. 10


264


Figure 2. Schematic models of gramicidin A dimers: (A) head-to-head helical dimer; (B) tail-to-tail helical dimer; (C) antiparallel double-stranded helix; (D) parallel double-stranded helix. (Reproduced with permission from reference 301. Copyright 1986 Biophysical Society.)

Peptaibols. A l a m e t h i c i n is the best k n o w n m e m b e r o f the class o f fungal antibiotics k n o w n as peptaibols (Table I). T h e name arises f r o m shared structural features: the presence o f several α-methylalanine ( M e A ) or aminoisobutyric a c i d ( A i b ) residues a n d a C - t e r m i n a l a m i n o a c i d alcohol such as phenylalaninol (Phol). T h e voltage-dependent conductance o f peptaibols i n black l i p i d membranes was first demonstrated b y M u e l l e r a n d R u d i n (2), w h o suggested that alamethicin p r o v i d e d a site that b r i d g e d the bilayer through self-aggregation. A w i d e variety o f naturally o c c u r r i n g variants a n d analogues o f the peptaibol antibiotics exhibit voltage dependence a n d f o r m single-channel, m u l t i l e v e l conductance states i n planar l i p i d bilayers ( F i g u r e 4) (41). These are attributed to differing numbers o f helical monomers that aggregate to f o r m the pore (42). A recent study suggests that rather than increase the pore (or " b a r r e l " ) size b y increasing the n u m b e r o f monomers (or "staves"), these conductance states represent clusters o f " p i p e s " i n w h i c h differing numbers o f pipes w i t h different diameters are present (43). Strong aggregation o f alamethicin i n the l i p i d bilayer is observed spectroscopically above a critical concentration, a n d the onset o f pores can b e characterized b y a critical voltage. M o d u l a t i o n o f these two parameters b y the addition o f N a C l to the m e d i u m o r cholesterol to the bilayer are highly correlated, w h i c h suggests that voltage dependence is due to the electric field effect o n the partition between the aqueous a n d membrane phases (44). I o n channels have b e e n observed a n d characterized for alamethicin ( 2 , 41), e m e r i m i c i n (I. Vodyanoy, personal communication), paracelsin (45), trichorzianin (46), a n d the zervamicins (47). O t h e r neutral peptide sequences i n addition to peptaibols are capable o f voltage-dependent conductance changes i n l i p i d bilayers. M o l l e et al. (56)


13.

MARSHALL AND BEUSEN

265


Figure 3. Stereoview (backbone atoms only) of the gramicidin A antiparallel double-stranded fi -helix crystallized from ethanol Narrow lines indicate hydrogen bonds. (Reproduced with permission from reference 33. Copynght 1988 AAAS.) 56

showed that an analogue o f alamethicin i n w h i c h a l l M e A residues were replaced b y L e u a n d C - t e r m i n a l P h e o l was replaced b y P h e - N H

2

retained its

conductance properties, b u t w i t h a higher threshold voltage a n d w i t h m e a n o p e n lifetimes 5 - 1 0 times smaller. Peptides such as oligoAla that have sufficient length ( 1 5 - 2 0 residues d e p e n d i n g o n composition) a n d are c o m posed o f n o r m a l amino acids also show channel formation i n l i p i d bilayers (57).

O n e advantage o f peptaibol study, however, relates to the structural



1

5

Ala

Ac- MeA-Ala-MeA-Ala- MeA -I ™

/

/

ÏPII\

j-Gln-MeA-l MeA ^

\ 1

0

1

5

2

0

I-Gln-Gln- Phol

/ M P A \

- MeA -Gly-Leu-MeA- Pro-Val-MeA-l

1 5 10 15 20 Ac- MeA -Pro-MeA-Ala- MeA-Ala-Gin-M eA-Val- MeA -Gly-Leu-MeA-Pro-Val- MeA-MeA-Glu-Gln- Phol 1 5 10 15 Ac- MeA -Gly-MeA-Leu- MeA -Gln-MeA-MeA-MeA- Ala -Ala-MeA-Pro-Leu- MeA -(R)EtA-Gln-Valol 1 5 10 15 Ac- Phe-MeA-MeA-MeA- Val -Gly-Leu-MeA-MeA- Hyp -Gln-(R)EtA-Hyp-MeA- Phol 1 5 10 15 Ac- Phe-MeA-MeA-MeA-(R) EtA -Gly-Leu-MeA-MeA- Hyp -Gln-(R)EtA-Hyp-MeA- Pro -Phol 1 5 10 15 Ac- Leu -Ile-Gln-Iva- He -Thr-MeA-Leu-MeA- Hyp -Gln-MeA-Hyp-MeA- Pro -Phol 1 5 10 15 20 Ac- MeA -Ala-MeA-Ala- MeA -Ala-Gln-MeA-Val- MeA -Gly-MeA-MeA-Pro- Val -MeA-MeA-Gln-Gln- Phol

Sequence

1 5 10 15 Trichorzianine IIIc (55) Ac- MeA-Ala-Ala-MeA- MeA -Gln-MeA-MeA-MeA- Ser-Leu-MeA-Pro-Val- MeA -Ile-Gln-Gln-Trpol

Suzukacillin A (53, 54)

Paracelsin A (49)

1

[Leu ]Zervamicin (52)

Antiamoebin I (51)

Emerimicin IV (50)

Trichotoxin Α-40 (49)

Alamethicin (48)

Peptaibol

Table I. Amino Acid Sequences of Several Peptaibols

13.

MARSHALL AND BEUSEN

267


200

ms

Figure 4. Multistate conductance shown by alamethicin on a planar bilayer membrane with applied potential of 210 mV (top) and on frog sarcolemmal membrane with — 110-mV resting potential (bottom). Current bursts begin at A and continue until B. The different levels observed are not integral multiples of unit current conductance, which implies different states of the pore. (Upper figure reproduced with permission from reference 41. Copyright 1972 Elsevier. Lowerfigurereproduced with permission from reference 302: Copyright 1979 Macmillan Magazines.)

roles o f the unusual amino acids that characterize their sequences. T h e conformational constraints i m p o s e d b y the α,α-dialkyl a m i n o acids, such as M e A ( A i b ) a n d α-ethylalanine ( E t A or Iva), dramatically l i m i t the conforma tions that must be considered i n any m e c h a n i s m . α,α-Dialkyl a m i n o acids have b e e n shown theoretically b y M a r s h a l l a n d Bosshard (58)

a n d later b y

others ( 5 9 - 6 2 ) to favor Φ , Ψ torsional values associated w i t h either 3 - or 1 0

α-helix ( F i g u r e 5). T h e large n u m b e r o f crystal structures for peptides that contain M e A (63-65)

c o n f i r m the propensity o f this residue to direct the


268


180

!80

ψ 0 ψ 0

-180

Μ,Ι,Ι.Ι,Μ.Ι,Ι,Ι.Ι,Ι,Ι,Ι,Ι,Ι,Ι,Ι,ι

0

Μ.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1,1.

. „

ο

180 -180

φ

180

φ

Figure 5. Experimentally observed values for torsional angles Φ and Ψ observed for alaninyl-type residues (left) and a-methylalanine in high-resolution crystal structures (right). (Reproduced with permission from reference 64. Copyright 1987Wiley-Liss.)

conformation to be helical: either the α-helix, the 3 - h e l i x , o r some c o m b i n a 10

tion o f the two is seen p r e d o m i n a n t l y . I n the case o f A i b ( M e A ) oligomers, Bavoso et al. (66) suggested that the 3 - h e l i x is p r e f e r r e d . K a r l e et a l . (67) 10

suggested that t h e 3 - h e l i x is p r o m o t e d b y M e A i n short peptides o r i n 10

longer peptides ( > 7 residues) i f M e A comprises 5 0 % o r m o r e o f t h e peptide, b u t t h e α-helix is p r e f e r r e d w h e n t h e M e A residues comprise o n e - t h i r d o f the longer peptides. C o m p a r i s o n o f the crystal structure o f e m e r i m i c i n 2 - 9 , w h i c h is a 3 - h e l i x (68), w i t h that o f e m e r i m i c i n 1 - 9 , w h i c h 10

is a n α-helix (69), suggests instead that the energy difference between the two

forms is small a n d selection o f a c o n f o r m e r is dependent o n e n v i r o n m e n

tal

factors. A qualitative explanation o f the balance o f forces that determine

a - o r 3 - h e l i x preference has b e e n presented (69), a n d Z i m m - B r a g g theory 10

has b e e n m o d i f i e d t o explain the c o m p o s i t i o n a n d sequence dependence o f helix preference (70, 71). A l t h o u g h the difference b e t w e e n a - a n d 3 - h e l i c e s 10

stabilized b y t h e multiple M e A residues is m i n i m a l i n terms o f torsional angles ( Φ , Ψ), the consequences i n terms o f relative side-chain position a n d helix length are dramatic. A c o m p a r i s o n o f the characteristic parameters for the two helix types is given i n T a b l e I I a n d s h o w n i n F i g u r e 6. Recent investigation o f the relative stabilities o f these two h e l i c a l forms for M e A a n d Ala

oligomers has refined o u r understanding o f the forces that determine

their occurrence u n d e r a given set o f experimental conditions (72, 73). F u r t h e r m o r e , m o l e c u l a r dynamics simulations have b e e n u s e d to construct an energy surface f o r t h e transition between t h e two h e l i c a l forms a n d to


13.

MARSHALL AND BEUSEN

269


T a b l e I I . C o m p a r i s o n o f Properties o f α-Helix w i t h 3 - H e l i x 10

a-Helix

3 -Helix

Residues per turn Atoms in Η-bonding ring

3.6

3.0

13

10

Rise per residue (Â) Number of H bonds for Ν residues Average torsional angles (73)

1.5

2.0

N-4

N-3

-55

-50

-52

-31

Property

Φ

10

Figure 6. Comparison of structures for the α-helix (upper left and middle) and 3 -helix (lower left and far right). The increased length of the 3 -helix for the same number of residues is evident, as is the difference in hydrogen-bonding pattern: 1

The Structural Basis of Peptide Channel Formation - American

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