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How Translocons Select
Transmembrane Helices
Stephen H. White1,2 and Gunnar von Heijne3,4
1
Department of Physiology and Biophysics and 2 Center for Biomembrane Systems,
University of California, Irvine, California 92697-4560; email: [email protected]
3
Department of Biochemistry and Biophysics and 4 Center for Biomembrane Research,
Stockholm University, SE-106 91 Stockholm, Sweden; email: [email protected]
Annu. Rev. Biophys. 2008. 37:23–42
Key Words
First published online as a Review in Advance on
February 7, 2008
membrane proteins, membrane protein folding, membrane protein
assembly, membrane protein stability, lipid-protein interactions,
hydrophobicity scales
The Annual Review of Biophysics is online at
biophys.annualreviews.org
This article’s doi:
10.1146/annurev.biophys.37.032807.125904
c 2008 by Annual Reviews.
Copyright All rights reserved
1936-122X/08/0609-0023$20.00
Abstract
Like all cellular proteins, membrane proteins are synthesized by ribosomes. But unlike their soluble counterparts, highly hydrophobic
membrane proteins require auxiliary machineries to prevent aggregation in aqueous cellular compartments. The principal machine
is the translocon, which works in concert with ribosomes to manage the orderly insertion of α-helical membrane proteins directly
into the endoplasmic reticulum membrane of eukaryotes or into
the plasma membrane of bacteria. In the course of insertion, membrane proteins come into thermodynamic equilibrium with the lipid
membrane, where physicochemical interactions determine the final
three-dimensional structure. Much progress has been made during
the past several years toward understanding the physical chemistry
of membrane protein stability, the structure of the translocon machine, and the mechanisms by which the translocon selects and inserts transmembrane helices. We review this progress and consider
the connection between the physical principles of membrane protein
stability and translocon selection of transmembrane helices.
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Contents
INTRODUCTION . . . . . . . . . . . . . . . . .
MEMBRANE PROTEIN
STABILITY AND ASSEMBLY . . .
Dynamic Nature of Fluid
Lipid Bilayers . . . . . . . . . . . . . . . . .
Membrane Protein Intrinsic
Interactions . . . . . . . . . . . . . . . . . . .
Membrane Protein Formative
Interactions . . . . . . . . . . . . . . . . . . .
STRUCTURE OF THE
TRANSLOCON . . . . . . . . . . . . . . . .
TRANSLOCON RECOGNITION
OF TRANSMEMBRANE
HELICES . . . . . . . . . . . . . . . . . . . . . . .
A Biological Hydrophobicity
Scale . . . . . . . . . . . . . . . . . . . . . . . . . .
Position Dependence
of Free Energies . . . . . . . . . . . . . . .
Prediction of Transmembrane
Helices . . . . . . . . . . . . . . . . . . . . . . .
Membrane Insertion of
Multi-Spanning Proteins . . . . . .
Helix-Helix Interactions . . . . . . . . . .
THE BIOLOGY-PHYSICS
NEXUS . . . . . . . . . . . . . . . . . . . . . . . . .
24
24
24
26
27
29
30
30
32
32
33
33
36
INTRODUCTION
MP: membrane
protein
Translocon: a
heterotrimeric
membrane protein
found in all
organisms that is
directly responsible
for inserting
α-helical proteins
into membranes
TM:
transmembrane
MD: molecular
dynamics
24
Prediction of the three-dimensional structure
of α-helical membrane proteins (MPs) from
amino acid sequences is a challenging and
important problem. One key to solving this
problem is to understand quantitatively the
formative interactions in biological assembly,
which is initiated by the concerted action of
ribosomes and translocons. Another key is to
understand quantitatively the intrinsic interactions of MPs with the lipid bilayer of the
membrane. The formative and intrinsic interactions must share some common principles, because the ribosome-translocon machinery delivers MPs to bilayers in a manner
that allows MPs to come quickly into equilibrium with the bilayer after assembly (44, 80).
White
·
von Heijne
We attempt in this review to describe what is
presently known about these principles.
We begin with an overview of the properties of lipid bilayers and the general principles
of MP stability and assembly, with an emphasis on the latter. More extensive discussions
of MP stability are available in several comprehensive reviews (47, 61, 67, 80). In two
succeeding sections, we first review progress
toward understanding the structure and function of the translocon machinery, and then
review progress toward understanding the
code the machinery uses to recognize transmembrane (TM) segments of MPs. Finally,
we discuss the inherent physicochemical connection between the formative and intrinsic
interactions.
MEMBRANE PROTEIN
STABILITY AND ASSEMBLY
Dynamic Nature of Fluid
Lipid Bilayers
The lipid bilayers of cell membranes must be
in a fluid state for normal cell function. The
characteristically high thermal disorder of this
state precludes direct three-dimensional determinations of bilayer structure. But the onedimensional crystallinity of multilamellar bilayers dispersed in water or deposited on
surfaces permits the distribution of matter
along the bilayer normal to be determined by
diffraction methods. For example, the combined use of X-ray and neutron diffraction
measurements (76, 77) results in a structure
consisting of a collection of time-averaged
TM probability distribution curves of water and lipid component groups (e.g., carbonyls and phosphates), representing projections of their three-dimensional thermal
motions onto the bilayer normal. Figure 1a
shows the liquid-crystallographic structure
of an Lα -phase dioleoylphosphatidylcholine
(DOPC) bilayer (81) and Figure 1b is a snapshot from a molecular dynamics (MD) simulation of the same system.
Three features of this fluid bilayer structure are important. First, the widths of the
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probability densities reveal the great thermal
motion. Second, the combined thermal thicknesses of the interfaces (defined by the distribution of the waters of hydration) are approximately equal to the 30 Å thickness of the
hydrocarbon core (HC) of the bilayer. The
thermal thickness of a single interface (∼15 Å)
can easily accommodate an α-helix parallel to
the membrane plane. The common lollipop
cartoons of bilayers that assign diminutive
thicknesses to the interfaces are thus misleading. Third, the interfaces are highly heterogeneous chemically. A polypeptide chain in
an interface must experience dramatic variations in environmental polarity over a short
distance because of the steep changes in chemical composition (79) (Figure 1c).
MD simulations of bilayers, which provide dynamic three-dimensional information
at the atomic level, are rapidly becoming an
essential structural tool for examining lipidprotein interactions at atomic scales (3, 22, 57,
59, 68). The future offers the prospect of
combining one-dimensional bilayer diffrac-
tion data with MD simulations in order to
arrive at experimentally validated dynamic,
three-dimensional structures of fluid lipid
bilayers (Figure 1b) with embedded proteins
and peptides (6, 7). A movie constructed
from an MD simulation of a fluid DOPC
a
Interface
Hydrocarbon
core
HC: hydrocarbon
core
Interface
Bulk water
Probability
ANRV343-BB37-02
Waters
of
hydration
-40
0
-20
Methyls
Methylenes
Double-bonds
l
Carbonyls
arbo
Glycerol
l cer
Phosphate
hosp
Choline
h lin
40
20
Distance from bilayer center (Å)
b
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→
Figure 1
c
0.05
Charge density
The liquid-crystallographic structure of a fluid
dioleoylphosphatidylcholine (DOPC) bilayer.
(a) The structure of a fluid DOPC lipid bilayer
(81) consists of a collection of transbilayer
Gaussian probability distribution functions
representing the lipid component groups of the
bilayer unit cell. The areas under the curves
correspond to the number of constituent groups
per lipid represented by the distributions (e.g., one
phosphate, two carbonyls, four methyls).
(b) Molecular graphics image of DOPC taken
from a molecular dynamics simulation by Ryan
Benz. The color scheme is given in panel a. The
image was prepared by S. White using Visual
Molecular Dynamics (36). Two of the lipids are
shown in space-filling representation. (c) Polarity
profile ( yellow curve) of the DOPC bilayer (above)
computed from the absolute values of atomic
partial charges (79). The end-on view in panel c of
an α-helix with diameter ∼10 Å, which is typical
for membrane protein helices (9), shows the
approximate location of the helical axis of the
amphipathic helix peptide melittin (35). The
figures in panels a and c have been adapted from
reviews by White & Wimley (78–80).
0.04
0.03
α-helix
0.02
0.01
0.00
-40
-20
0
20
40
Distance from bilayer center (Å)
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bilayer can be viewed at http://blanco.
biomol.uci.edu/Bilayer Struc.html.
Membrane Protein Intrinsic
Interactions
Because MPs are equilibrium structures, their
intrinsic interactions can be described by any
a
convenient set of experimentally accessible
thermodynamic pathways, irrespective of the
biological synthetic pathway. One particularly
useful set of pathways is the so-called fourstep model (80) (Figure 2a), which is a logical
combination of the early three-step scheme of
Jacobs & White (37) and the two-stage model
of Popot & Engelman (60). Although these
b
4
Whole-residue interfacial scale
Partitioning
Folding
Insertion
ΔGaaIF (kcal mol-1)
Interface
HC core
Association
Coupled
c
ΔGaaWW (kcal mol-1)
2
1
0
I L F V C M AW T Y G S N H P Q R E K D
-1
Peptide bond
-2
d
4
3
3
Wimley-White
whole-residue n-octanol scale
TM α-helix
Side chains
Backbone
ΔG = -10
ΔGsc = -36
ΔGbb = +26
2
1
0
I L F V C M AW T Y G S N H P Q R E K D
-1
Peptide bond
-2
(kcal mol-1)
Figure 2
Energetics of peptide interactions with lipid bilayers. (a) Schematic representation of the four-step
thermodynamic cycle of White & Wimley (80) used to examine the energetics of membrane protein
stability through studies of small, water-soluble peptides (43, 85–87). The association of transmembrane
(TM) helices is probably driven by van der Waals interactions, giving rise to knobs-into-holes packing
(23, 47, 48, 63). (b) The Wimley-White (WW) interfacial hydrophobicity scale determined from
measurements of the partitioning of short peptides into phosphatidylcholine vesicles (86). (c) The WW
octanol hydrophobicity scale determined from the partitioning of short peptides into n-octanol (83). The
free-energy values along the abscissa in panels b and c are ordered in the same manner as in Figure 4e.
(d ) The energetics of TM helix insertion (38, 87) of glycophorin A estimated from the free-energy
contributions of the side chains (Gsc ) and backbone (Gbb ). The net side chain contribution (relative to
glycine) was computed using the n-octanol hydrophobicity scale (83). The per-residue cost of
partitioning a polyglycine α-helix is +1.15 kcal mol−1 (38). Figures adapted from reviews by White and
colleagues (72, 74).
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·
von Heijne
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pathways do not necessarily mirror the actual
biological assembly process of MPs, they are
nonetheless useful for guiding biological experiments, because they provide a thermodynamic context for biological processes. The
objective of describing the stability of MPs
by means of any of these schemes is to determine experimentally the thermodynamic constraints on MP structure formation.
Each of the steps in the four-step model
has been intensively studied by several laboratories during the past 15 years (1, 14, 47,
61, 67, 74, 80). Here we focus primarily on
the energetics of helix stability as established
from measurements of water-to-bilayer and
water-to-octanol partitioning free energies of
model peptides, summarized in Figure 2b,c
(83, 84, 86). The most fundamental conclusion from these studies is that the unfavorable thermodynamic cost (GCONH ) of
partitioning peptide bonds into membranes
can be dramatically reduced by the formation of secondary structures (43, 85), because the partitioning free-energy GHbond of
hydrogen-bonded peptide bonds is considerably lower than GCONH . For example, computational studies (4, 5) of peptides in bulk
alkanes suggest that GCONH for water-toalkane transfer is +6.4 kcal mol−1 , compared
to only +2.1 kcal mol−1 for GHbond . The
per-residue free-energy cost of disrupting hydrogen bonds in an alkane is therefore about
4 kcal mol−1 , meaning that a 20-amino-acid
TM helix would cost 80 kcal mol−1 to unfold
within the membrane hydrocarbon (ignoring
the interfacial gradients). This explains why
unfolded polypeptide chains cannot exist in a
TM configuration.
As discussed in detail elsewhere (38, 74,
80), GHbond sets the threshold for TM helix stability (Figure 2d ). The free energy of
transfer of nonpolar side chains dramatically
favors helix insertion, whereas the transfer
cost of the helical backbone dramatically disfavors insertion. What is the most likely estimate of GHbond ? The practical number is the
cost Ghelix
glycyl transferring a single glycyl unit
of a polyglycine α-helix into the bilayer HC.
The best estimate is +1.15 kcal mol−1 (38),
which fortuitously corresponds to the cost of
transferring a random-coil glycyl unit into noctanol (83). This finding led to a systematic
evaluation of the Wimley-White (WW) noctanol as a hydrophobicity scale for predicting TM helices (38). The prediction accuracy
exceeded 95%.
Membrane Protein Formative
Interactions
Proteins destined for TM export (translocation) or insertion are generally managed by
the concerted action of translating ribosomes
in the cytoplasm and translocon complexes located in the endoplasmic reticulum (ER) of
eukaryotes or in the plasma membrane of bacteria. The operating principles for the machinery of MP assembly (8, 15, 19, 40, 58,
75) are summarized in Figure 3a. The critical MP component of the translocon complex is heterotrimeric Sec61 in eukaryotes or
the highly homologous SecYEG in bacteria.
Cryo-EM image reconstructions (Figure 3b)
of native ribosome-translocon complexes (52)
suggest that the complex is likely composed of
two dimers of the Sec61 heterotrimer and two
copies of the tetrameric translocon-associated
protein (TRAP). At least three other proteins
associate closely with the translocon complex
but do not seem to be part of the ribosometranslocon complex seen in the image reconstructions. These are the translocating chainassociated membrane protein (TRAM) (16,
25); the signal peptidase complex (SPC) (21),
which cleaves signal sequences; and oligosaccharyl transferase (OST) (11), which Nglycosylates -Asn-X-Ser/Thr-sites on membrane and secreted proteins.
The translocon complex acts as a switching station: Secretory proteins are allowed to
pass straight through into the ER lumen or
the bacterial periplasm (secretion), whereas
TM segments of MPs are shunted sideways
into the membrane bilayer. Deciphering the
code that the translocon uses for selecting
elongating segments for TM insertion is of
www.annualreviews.org • Translocons and Transmembrane Helices
WW:
Wimley-White
Translocon
complex: an
assembly of
membrane proteins,
including one or
more translocons,
upon which the
ribosome docks
SecYEG:
translocon in the
plasma membrane of
eubacteria. Y, E, and
G correspond,
respectively, to the
α, γ, and β subunits
of Sec61
Sec61 or
Sec61αβγ:
translocon in the
endoplasmic
reticulum of
eukaryotes
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a
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Ribosome
1
b
SRP
mRNA
Rib
Ribosome
Alu
dom
domain
Exit
tunnel
RNA
Polypeptide
S
S
dom
domain
Emerging signal
L
2
Membrane
Elongation
arrest
Translocon
complex
3
Docking
Cytoplasm
SR
Translocon
Exoplasm
4
Signal transfer
and elongation
N
C
Translocon
N
C
N
C
c
d
Hydrophobic
Hyd
drophobic
collar
co
colla
ar
Ribosom
Ribosome
(bac
(back)
Secβ
Sec
cβ
β
SecY
Se
ecY
TM7
T
M7
M
TM7
7
TM2
TM
TM2B
2B
SecE
SecE
E
Se
E
Exit
28
White
·
von Heijne
(fro
(front)
ont)
TM
TM2B
M2B
T
TM2
TM2A
2A Plu
Plug
ug
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fundamental importance for understanding
the folding of MPs (see below). But the selection of TM segments is only the first step
in the complex process of gathering the TM
segments together to form the native protein
structure (12, 65, 66).
Comparisons of the SecYEβ crystallographic structure with cryo-EM reconstructions (52) suggested that the heterotrimers
form a tetramer arranged as a dimer-ofdimers ordered in a back-to-back configuration (“back” is defined in Figure 3c). No
nascent peptide was observed in the crystallographic structure, which is thus assumed to be
STRUCTURE OF THE
in a closed state. Disulfide cross-linking experTRANSLOCON
iments (10), however, revealed that elongating
The key protein of the eukaryotic translo- chains pass through the so-called hydrophocon complex—the one that acts as the switch- bic collar in the middle of SecY (Figure 3d ),
ing station—is heterotrimeric Sec61αβγ suggesting that translocon-mediated protein
(SecYEG in eubacteria, SecYEβ in archaea) export and membrane insertion involve at any
(56). The Sec61 α-subunit has 10 TM he- particular time only one of the SecY/Sec61
lices, whereas β and γ typically have 1 TM heterotrimers in the translocon complex. The
helix (eubacterial SecE has 3 helices and SecG broad purpose of the posited tetrameric assohas 2 TM helices). Van den Berg et al. (70) ciation of SecY/Sec61 may be to provide an
have determined the crystallographic struc- assembly platform that enables the ribosome
ture of SecYEβ from Methanococcus jannaschii and other members of the Sec family to seat a resolution of 3.8 Å. It is shown embed- crete or insert nascent chains (55).
Figure 3c shows SecYEβ from the viewded in a lipid bilayer in Figure 3c,d. The images are snapshots from an MD simulation point of the ribosome and Figure 3d shows
of the heterotrimer embedded in a palmitoy- a view parallel to the membrane. The 10
TM helices of SecY are arranged to form an
loleoylphosphatidylcholine bilayer (73).
←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
SecYEβ: translocon
in the plasma
membrane of archaea
Figure 3
Membrane protein assembly. (a) The machinery of membrane protein assembly. (Step 1) A ribosome
translating the mRNA of a protein targeted for secretion across or insertion into membranes and a signal
of a recognition particle (SRP), which is a GTPase. The structures of ribosomes are reviewed in
References 18 and 62, and the structure of SRP is reviewed in Reference 54. (Step 2) The ribosome and
SRP recognize a signal peptide as it emerges from the ribosome exit tunnel, bind together, and cause
arrest of elongation in eukaryotes (27, 28, 82). (Step 3) The ribosome-SRP complex binds to the
membrane-bound SRP receptor (SR), another GTPase that associates dynamically with the translocon.
Prokaryotes use a simplified SRP (Ffh) and SR (FtsY) that associate to form a quasi-twofold symmetrical
dimer (20). The binding of SRP to SR causes reciprocal stimulation of their GTPase activities, causing
transfer of the signal peptide to the translocon and resumption of elongation (2). (Step 4) Proteins
targeted for translocation are secreted into the periplasm (bacteria) or endoplasmic reticulum lumen
(eukaryotes), whereas the stop-transfer signals of MPs are transferred to the membrane bilayer. (b)
Cryo-EM image of the canine ribosome-translocon (Sec61) complex. The small and large ribosome
subunits are indicated by S and L, respectively. Modified with permission from figure 4 of Reference 52.
(c) Structure of a single SecYEβ closed-state translocon heterotrimer from Methanococcus jannaschii (70)
that has been embedded in a palmitoyloleoylphosphatidylcholine lipid bilayer (red, headgroups; white,
acyl chains) using molecular dynamics (MD) methods. SecY is viewed from the ribosome along the
bilayer normal. The front and back of the protein are indicated. Sec Y is composed of 10 transmembrane
(TM) helices. Helices 1-5 are colored dark purple, except for TM2B, which is red. Helices 6–10 are
colored orange except for TM7, which is colored red. The presumed lateral exit from the TM7/TM2B
lateral gate is indicated. (d ) SecYEβ viewed along the bilayer plane toward the lateral gate through which
nascent TM helices are believed to move into the bilayer. The TM2A plug helix apparently seals the
translocon in the absence of nascent peptide. The images in panels c and d were prepared from an MD
simulation, courtesy of A. Freites. Both molecular graphics images were produced using Visual Molecular
Dynamics (36).
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inverted “U” (Figure 3c), with TM helices 1–
5 forming one leg and helices 6–10 forming
the other leg. The two sets of helices have a
pseudosymmetric twofold rotation axis in the
plane of the membrane and are connected at
the back by an external loop. This loop and
the single TM helix of SecE prevent lipids
from contacting the interior of SecY from the
backside. The only possible opening from the
interior into the lipid bilayer is through the
so-called lateral gate formed by TM2B and
TM7 (Figure 3c,d ), which is hypothesized to
control passage of nascent TM helices into
the bilayer from the hourglass-shaped waterfilled interior of SecY (Figure 3d ). The two
halves of the hourglass are separated by a ring
of hydrophobic residues (hydrophobic collar)
that are believed to act as a seal around the
elongating chain.
Sitting just below the hydrophobic collar
is a short helix (TM2A) that apparently acts
as a plug to block passage of small molecules
through the translocon in the closed state. Van
den Berg et al. (70) hypothesized that the plug
is displaced by nascent protein translocation.
But the necessity for the TM2A plug for sealing the hourglass in the absence of a translocating nascent chain was discounted in a study
of a so-called plugless Sec61/SecY mutant (41,
49), because excision of TM2A had no effect on the viability of yeast cells. Remarkably,
however, a crystallographic study of plugless
SecY (45) showed that SecY restructures itself
in the absence of TM2A to form a new plug!
The image of SecYEβ in a lipid bilayer
(Figure 3c,d ) is entirely consistent with the
idea that TM helices move into the lipid
membrane from the water-filled, proteinconducting channel by a simple partitioning
process, as suggested by cross-linking studies
of nascent chains (29, 50). In such a scheme,
sufficiently hydrophobic helices prefer the bilayer, whereas more-polar helices favor the
translocon and ultimately the aqueous phase.
That is, the translocon and the lipid bilayer
work in concert to decipher the code for TM
helices embedded in the amino acid sequence.
If this view is correct, then the big question
concerns the code for deciphering the process. Answers to this question should lead to
major improvements in the prediction of the
membrane protein structure.
TRANSLOCON RECOGNITION
OF TRANSMEMBRANE HELICES
A Biological Hydrophobicity Scale
Insights into the process of TM helix insertion have been obtained by Hessa et al.
(32), who used an in vitro expression system (64) that permits quantitative assessment of the membrane insertion efficiency of
model TM segments (Figure 4). Specifically,
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→
Figure 4
Integration of designed transmembrane (TM) segments (H-segments) into the endoplasmic reticulum
(ER) using dog pancreas microsomal membranes. This system was used to explore systematically the
hydrophobicity requirements for TM helix integration via the Sec61 translocon (32). (a) Wild-type
leader peptidase (Lep) from E. coli has two N-terminal TM segments (TM1, TM2) and a large lumenal
domain (P2). H-segments, flanked by glycosylation sites (G1, G2), were inserted between residues 226
and 253 in the P2 domain. For H-segments that integrate into the membrane, only the G1 site is
glycosylated (left), whereas both the G1 and G2 sites are glycosylated for H-segments that do not
integrate into the membrane (right). Redrawn from Reference 32. (b) An example of sodium dodecyl
sulfate gels used in the in vitro determination of the extent of glycosylation of Lep/H-segment constructs
in the absence (−RM) and presence (+RM) of dog pancreas rough microsomes. (c) Equations used by
Hessa et al. (32) for the analysis of gels of the type shown in panel b. (d ) Mean probability of insertion, p,
for H-segments with n = 0 −7 Leu residues. The curve is the best-fit Boltzmann distribution, which
suggests equilibrium between the inserted and translocated states of the H-segments. (e) Biological
Gaa
app scale derived by Hessa et al. (32) from H-segments, with the indicated amino acid placed in the
middle of the 19-residue hydrophobic stretch. ( f ) Correlation between Gaa
app and the WW octanol
)
(Figure
2c).
Data
in
panels
b–e
are
replotted
from
Reference 32.
free-energy scale (Gaa
WW
30
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·
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(G1 and G2) (Figure 4a) can occur only in
the lumen of the RMs, H-segment TM insertion could be distinguished from secretion by simple gel assays (Figure 4b). The
relative fractions of singly (1g) and doubly
(2g) glycosylated molecules allow quantitative assessment of insertion versus secretion
they examined the integration into membranes of dog pancreas rough microsomes
(RMs) of designed polypeptide segments (Hsegments) engineered into the lumenal P2
domain of the integral MP leader peptidase
(Lep) (Figure 4a). Because glycosylation of
the engineered Asn-X-Ser glycosylation sites
a
b
P2
G2
Translocated
G1
66 kDa
H
G1
46 kDa
G1 + G2: translocated, 2g
G1 only: inserted, 1g
Background
Inserted
ER Lumen
H
TM2
TM1
30 kDa
Cytoplasm
P1
G2
MW -RM +RM
standards
P1
P2
c
d
1.0
Probability of insertion:
Probability (p)
P = f1g/(f1g + f2g )
Apparent equilibrium constant:
Kapp = f1g /f2g
Apparent free energy of insertion:
∆Gaaapp = -RTlnKapp
0.8
0.6
0.4
0.2
H: GGPG-LnA19-n-GPGG
0
0
1
2
3
4
5
6
7
8
Number of leucine residues (n)
e
f
4
ΔGaaapp (kcal mol-1)
4
3
E
GGPG
Xaa
GGPG
2
1
0
-1
I L F V CM AWT Y G S N H P Q R E K D
Amino acid
ΔGaaww (kcal mol-1)
ANRV343-BB37-02
3
H
2
R
G
1
A
0
C
V
-1
-2
-3
-1
D
K
I
L
N Q
T S
M
P
Y
F
W
0
1
2
3
ΔGaaapp (kcal mol-1)
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Apparent free
energy (Gapp ): an
operational term for
quantitating the
favorability of
transferring a
polypeptide segment
from the translocon
into the membrane
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(Figure 4c). The first experiments, carried
out using H-segments of the form GGPG(Ln A19−n )-GPGG with n = 0 to 7, revealed
that the probability of insertion, p(n), conformed accurately to a Boltzmann distribution
(Figure 4d ). This showed that transloconmediated insertion has the appearance of an
equilibrium process. Given this key observation, the insertion of H-segments was quantitated using the apparent free energy of insertion (Gapp ) (Figure 4c ).
A biological hydrophobicity scale (Gaa
app )
(Figure 4e) could be derived from studies
in which each of the 20 naturally occurring
amino acids were placed in the middle position of H-segments containing various numbers of Leu and Ala residues chosen to maintain p ≈ 0.5 (Gapp ≈ 0), which is the region of
maximum sensitivity of the assay (Figure 4e).
Considering the complexity of the biological system, the scale correlated surprisingly
well (Figure 4f ) with the WW octanol scale
(Figure 2c). Their overall high correspondence implies that the recognition of TM segments by the translocon likely involves direct
interaction between the segment and the surrounding lipid (29), which seems reasonable
in light of Figure 3d.
Position Dependence
of Free Energies
Does Gaa
app vary with position within the Hsegment? To answer this question, Hessa et al.
(32) performed position scans of two types:
single- and pair-scans. In the simpler singlescan, an amino acid of interest was placed at
different positions in the H-segment sequence
and Gapp was determined. The dramatic results from an Arg scan are shown in Figure 5a
(34). Similar results were found for Lys, Asp,
and Glu scans. The strong dependence on position must be related to the relative ease of
snorkeling of the charge group to the wet bilayer interface (46)—the farther the charge
is from the interface, the greater the energetic cost. The strong position dependence
of Arg explains why it is possible for Sec61
32
White
·
von Heijne
to insert the KvAP S4 voltage-sensing helix,
which contains four Arg residues, across the
ER membrane with Gapp ≈ 0 (34). An MD
simulation of S4 across a lipid bilayer (24)
showed that the arginines snorkel to the bilayer interface to form salt-bridges with the
phospholipid phosphates and hydrogen-bond
networks with water (Figure 5b).
In pair-scans, a pair of residues of a given
kind was moved symmetrically from the center of the H-segment toward its N and C
termini to preclude the possibility of a shift
in helix position across the membrane. Pairscans of charged residues were consistent
with single-scans, suggesting that helix shifts
were not significant. Pair-scans of the aromatic residues, which have preferential interactions with the bilayer interface (42, 86,
88), gave another insight into TM helix insertion. The behaviors of Trp and Tyr were
dramatic (Figure 5c). When placed centrally,
they strongly reduced membrane insertion,
but they became much less unfavorable as they
were moved apart. Indeed, Trp was as favorable as Leu when placed in the outermost positions (Figure 5c). The position dependence
of Phe was different from that of Trp and
Tyr (Figure 5c), because Phe does not have a
strong interfacial preference in MPs (69, 71).
The wave-like pattern observed for the Phe
pair-scan is a result of variations in the hydrophobic moment (amphiphilicity) of the helices (32). These results provided further evidence supporting the idea that protein-lipid
interactions are central to the recognition of
TM helices by the translocon.
Prediction of Transmembrane
Helices
The strong position dependence of Gaa
app
meant that the base biological hydrophobicity
scale would be of limited value for predicting
TM helices by simple hydropathy plot methods; accurate predictions require accounting
for the position dependence of Gaa
app . In a
recent study, Hessa et al. (33) carried out a
comprehensive examination of the position
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dependence of Gaa
app . In addition, they determined how the overall length of the Hsegment affected Gapp . The data enabled
them to derive a simple expression for calculating the expected Gapp for H-segments
given the amino acid sequence and overall
length:
pred
Gapp =
l
i=1
2
Gaa(i)
app + c 0 μ + c 1 + c 2 l + c 3 l ,
helices by the translocon, and support models
based on a partitioning of the TM helices between the Sec61 translocon and the surrounding lipid. The details of the partitioning process remain to be determined, but presumably
the open state of the translocon is a highly dynamic one that permits rapid sampling of the
translocon-bilayer interface by the translocating polypeptide.
1.
aa(i)
Gapp
where l is the length of the segment,
is the matrix element giving the contribution
from amino acid aa in position i, μ is the hydrophobic moment, c0 is the weight parameter
for the hydrophobic moment, and the terms
c1 +c2 l+c3 l 2 account for the dependence of
Gapp on segment length. The optimized matrix was derived by minimizing the sum of the
squared difference. A web server for calculating Gapp profiles across a protein sequence is
available at http://www.cbr.su.se/DGpred/.
pred
Distributions of Gapp values obtained
for mammalian secreted proteins as well as
single- and multi-spanning MPs are shown in
pred
Figure 5d. The overlap between the Gapp
distributions for the single-spanning TM proteins and the secreted proteins is small, and
the two distributions cross close to the zeropoint on the scale defined by the experimental analysis of the designed H-segments. A
surprisingly large fraction (25%) of the TM
helices in the multi-spanning MPs of known
pred
three-dimensional structure have Gapp >
−1
0 kcal mol . Such segments would presumably be only inefficiently recognized as TM
helices by the translocon if they were the only
hydrophobic segment in a protein. This observation suggests that TM helices in multispanning MPs may depend on interactions
with neighboring TM helices for proper partitioning into the membrane. Indeed, a number of such cases have been described in the
literature (66), though their overall incidence
has been unclear.
The results of these studies by Hessa et al.
(32–34) suggest that direct protein-lipid interactions are essential for the recognition of TM
Membrane Insertion of
Multi-Spanning Proteins
How does the Sec61 translocon handle proteins with multiple TM helices? The most revealing study published so far focused on the
6TM protein aquaporin 4 (65). By an extensive analysis using site-specific cross-linkers
introduced into each of the TM helices, the
authors arrived at a detailed picture of when
during biosynthesis each TM helix exits the
translocon and enters into the lipid bilayer. In
general, the helices were observed to follow
each other into the membrane in a strict N- to
C-terminal succession. Certain helices, however, would first completely exit the translocon only to revisit it at a later stage when a
downstream helix had just entered the translocon channel. One is thus left with a picture
of a dynamic translocon that allows multiple
TM helices to interact with each other at early
stages of membrane integration. In this way,
one may envision a mechanism whereby TM
helices that would not by themselves be sufficiently hydrophobic to integrate efficiently
into the membrane become embedded in the
progressively folding protein.
Helix-Helix Interactions
What kinds of interactions might underlie
helix-helix association during transloconmediated membrane insertion into the lipid
bilayer? It is well established that hydrogen
bonding between polar residues such as Asn or
Asp can drive helix-helix interactions in both
detergent micelles and biological membranes
(13, 26, 89, 90) and can facilitate the
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formation of helical hairpins during
translocon-mediated insertion (31). MeindlBeinker et al. recently examined (51)
whether and to what extent interhelix hydrogen bonding could drive the process of
translocon-mediated TM helix insertion
a
GGPG Arg
itself, and whether the separation between
the two helices within the sequence may
influence any such interaction. To address
these questions in a quantitative way, they
extended the systematic approach established
by Hessa et al. (32) to study the effects
b
GGPG
0.5
S4 helix
ΔGapp (kcal mol-1)
Arginine
0.0
4
6
8
10
12
14
16
18
20
22 24
-0.5
10 Å
-1.0
-1.5
= Arg positions in S4 helix
-2.0
Position
Xaa
ΔGapp (kcal mol-1)
GGPG
Xaa
d
GGPG
1.2
Tyr (2Y, 3L, 14A)
1.0
0.8
Phe (2F, 1L, 16A)
0.6 Trp (2W, 2L, 15A)
0.4
0.2
0.0
2
4
6
8
10 12 14
-0.2 0
-0.4
4L, 15A
-0.6
-0.8
0.3
16
18
Frequency
c
Separation
Single-span TM
Multi-span TM
Cytoplasmic
Secreted
0.2
0.1
0.0
-10 -8
-6
-4
-2
pred
ΔGapp
0
2
(kcal
4
mol-1)
P2
G2
e
Translocated
G1
G1
Inserted
ER Lumen
H1
f
H
H2' H
I
II
ER Lumen
H1
H2'
H2'
D
D
H
H2'
D D
D D
Cytoplasm
Cytoplasm
G2
P1
P1
34
White
(II)
ΔGaaapp >>0
·
2D/17L
(I)
ΔΔGapp = ΔΔGapp -ΔΔGapp
P2
ΔGaaapp <<0
19L
von Heijne
H
6
8
10
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of mutual helix-helix interactions on the
efficiency of membrane insertion, using the
scheme shown in Figure 5e.
The experiments (51) yielded several important results (Figure 5f ). First, different
Asn- or Asp-containing H2 sequences did
not affect the insertion of a purely hydrophobic H-segment. Furthermore, little effect was
seen when a signal peptidase cleavage site
was introduced in H2 , or even when the entire H1-H2 region was replaced by the signal
peptide from preprolactin. The H2 sequence
thus had little influence on Gapp when the
H-segment was composed only of hydrophobic residues (cf. Figure 5f, I).
Second, by analyzing model protein constructs in which zero, one, or two Asn or
Asp residues were placed in two neighboring hydrophobic segments (H2 and H), it
was found that Gapp of a marginally hydrophobic H-segment was significantly reduced only if both the H2 -segment and the
H-segment contained two Asn or two Asp
residues (Figure 5f, II) with a spacing of
three, but not one or five, residues (i.e., when
they are spaced one helical turn apart in
both H2 - and H-segments). These results
suggest that interhelix hydrogen bonds can
form during Sec61 translocon-assisted insertion, and that the H2 -segment remains in
←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
Figure 5
Position dependence of Gaa
app and helix-helix interactions in membrane protein assembly. (a) Scan of a
single Arg residue across H-segments of composition 1R/6L/12A. The position of the Arg in the
19-residue hydrophobic stretch is shown on the x-axis. Orange circles indicate the locations of the Arg in
the KvAP S4 helix. Data are replotted from Reference 34. This plot reveals the strong position
Arg
dependence of Gapp . Lys, Asp, and Glu residues show a similar position dependence. (b) Molecular
dynamics simulation of a model S4 voltage-sensor peptide (GGPG-LGLFRLVRLLRFLRILLIIGPGG) in a palmitoyloleoylphosphatidylcholine bilayer. (Left) Cut-away view of the simulation system,
showing bilayer distortion around the peptide and the contacts between phosphate groups, water
molecules, and Arg guanidinium groups. (Right) This space-filling representation of the hydrophilic
neighborhood of the S4 helix, represented as Connolly surfaces, reveals a 10 Å gap that is never occupied
by the Arg guanidinium groups because of snorkeling to the bilayer interface. Red, water; yellow,
phosphocholine headgroups; teal, acyl chains; white, GGPG. . .GPGG flanks; silver, non-Arg S4
residues; dark blue, guanidinium groups. Images modified from Reference 24 with permission.
(c) H-segment pair-scans for Phe, Trp, and Tyr residues in which pairs of residues are moved
symmetrically toward the N and C termini of the sequence. The compositions of the H-segments are
indicated. The dashed line indicates the Gapp value for the 4L/15A H-segment. This shows that the Trp
residues have the same apparent free energy as Leu when placed near the ends of the H-segment. Data
pred
are replotted from Reference 32. (d ) Distributions of Gapp values in cytoplasmic, secreted, and
transmembrane (TM) proteins were computed from Equation 1. The 17- to 33-residue segment with
pred
lowest Gapp was identified in 670 cytoplasmic ( green), 1012 secreted (gray), and 349 single-spanning
pred
TM proteins (excluding signal peptides; blue), and the 17- to 33-residue segment with lowest Gapp
within each annotated helix (plus 10 residues on either side) was identified in 508 TM helices from
multi-spanning TM proteins of known three-dimensional structure ( purple ). Data points show the
pred
relative frequency of proteins with Gapp within ± 0.5 kcal mol−1 of the value given by the x-axis. Data
are replotted from Reference 33. (e) In order to examine helix-helix interactions driven by hydrogen
bonding, the native Lep H2-segment was replaced by an H2 -segment of the general composition
L19-n Nn or L19-n Dn (n = 0, 1, or 2), and a 19-residue H-segment containing one or two Asn or Asp
residues was inserted into the P2 domain. Redrawn from Reference 51. ( f ) To measure the effect of Aspor Asn-mediated interactions between the H2 - and H-segments, two constructs were compared for each
H-segment: one with a uniformly hydrophobic 19-Leu H2 -segment (I) and one with an H2 -segment in
which one or two of the Leu residues were replaced by Asp or Asn residues (II). The interaction free
energy is expressed as the difference (Gapp ) in the apparent free energy of insertion of the H-segment
between the two constructs. In the example shown, the H-segment contains two Asp residues and the
(I )
appropriate number of Leu and Ala residues to make Gapp ≈ 0 kcal mol−1 . Redrawn from Reference 51.
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close proximity to the translocon to offer its
hydrogen-bond donor and acceptor sites to
the incoming H-segment even when the intervening loop is 150 residues long (30, 53,
65).
THE BIOLOGY-PHYSICS NEXUS
The Gapp measurements of Hessa et al.
(32–34) are fully consistent with the simplest
model one can propose for how TM helices
are recognized by the ribosome-translocon
machinery: Helices are somehow allowed to
partition into the surrounding lipid bilayer
because of the free energy of interaction between the TM segment and the lipid. This
would explain the correspondence between
the biological hydrophobicity scale and biophysical scales like that of WW, and it would
explain why the positional variations in Gapp
for residues such as Arg, Trp, Tyr, Phe, and
Gly (32, 34) match the statistical distribution of these residues across the membrane
in the high-resolution X-ray structures (69).
The data at hand thus speak strongly in favor of direct protein-lipid interactions as the
main driving force for the integration of single
TM helices, although the translocon may affect the ability of pairs or higher-order assemblages of TM helices to interact among themselves before partitioning into the bilayer (33,
51).
Although much remains to be done in order to fully understand the results obtained
with the Sec61 translocon system, the H1
and H2 TM helices present in the model
protein (Figure 5e) do not seem to affect
the results in any significant way, as they
can be replaced by a cleavable signal peptide with little effect on the measured Gapp
values (51). Moreover, position-specific contributions to Gapp obtained by single-scans
of a charged or polar residue along an Hsegment predict Gapp values for H-segments
using symmetrical pair-scans, or even natural
TM helices with multiple charged residues
36
White
·
von Heijne
within ∼1 kcal mol−1 (32, 34). This finding suggests that vertical sliding of the Hsegments used in the derivation of the biological hydrophobicity scale is not a serious
problem.
This is probably not the whole story, however. Many polar and charged residues, Arg
included, have rather long and flexible side
chains, making it possible for them to snorkel
toward the lipid-water interface region. At the
same time, lipid molecules located close to a
TM helix can adapt to the presence of polar
residues, and water molecules can help solvate polar groups located well within the bilayer plane (17, 24, 39). One upshot of this
dynamic picture of protein-lipid interactions
is that Gapp profiles, such as the one shown in
Figure 5a, most likely do not provide an accurate representation of the free-energy profile for moving a charged residue all the way
across a membrane (as opposed to inserting it
sideways from the translocon as part of a TM
helix). Presumably, if a helical peptide is pulled
across a lipid bilayer, there is a substantial freeenergy barrier (not seen in the Gapp profile)
when a charged residue has to flip its direction
of snorkeling across the 10 Å hydrophobic gap
(Figure 5b) from one membrane surface toward the other (17). Seen from this perspective, one may regard the translocon as a device designed to lower the activation barrier
for translocation of polar and charged residues
across the membrane. It does so by providing an aqueous channel while making it possible for consecutive segments of the nascent
polypeptide to make lateral excursions from
the channel in order to test whether the free
energy of membrane insertion is favorable.
Despite these caveats, it seems likely that
the biological hydrophobicity scale is a good
measure of the energetics of protein-lipid
interactions in the true biological context,
and as such will help us define the sequence
determinants of membrane-protein assembly
much more precisely than has been possible
so far.
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SUMMARY POINTS
1. MPs are in thermodynamic equilibrium with the cell membrane’s lipid bilayer, which
means that the stability and three-dimensional structure of MPs are ultimately determined by lipid-protein physical chemistry.
2. α-Helical MPs are identified during translation on the ribosome by the signal recognition particle that initiates docking of the ribosome to the membrane-embedded
multi-protein translocon complex.
3. Elongating polypeptides from the ribosome pass through a translocon TM channel
within the translocon complex.
4. The translocon’s U-shaped structure allows diversion of TM helices sideways into the
lipid bilayer.
5. The diversion of the helices into the bilayer appears fundamentally to be a physicochemical partitioning process between translocon and bilayer.
6. The partitioning process can be described quantitatively by apparent free energies
that serve as a code for the selection of TM helices by the translocon working in
concert with the lipid bilayer.
FUTURE ISSUES
1. Much more structural information about translocons and translocon complexes is
needed, especially an atomic-resolution structure of a translocon engaged in polypeptide secretion.
2. Although there is a clear connection between the physical chemistry of lipid-protein
interactions and selection of TM helices by the translocon, a quantitative molecular
description of the empirical apparent free energies of the translocon’s selection code
is needed.
3. The molecular basis for the translocon-assisted assembly of multi-spanning MPs
needs to be established.
DISCLOSURE STATEMENT
The authors are not aware of any biases that might be perceived as affecting the objectivity of
this review.
ACKNOWLEDGMENTS
This research was supported by grants from the Swedish Foundation for Strategic Research, the
Marianne and Marcus Wallenberg Foundation, the Swedish Cancer Foundation, the Swedish
Research Council, and the European Commission (BioSapiens) to GvH, and by grants from
the National Institute of General Medical Sciences to SHW. We thank Michael Myers for
editorial assistance and the TEMPO group at U.C. Irvine for useful discussions.
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experimental
method for
quantitating
translocon
selection of TM
helices and
describes the
determination of a
biological
hydrophobicity
scale.
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that hydrogen
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www.annualreviews.org • Translocons and Transmembrane Helices
69. Derives implicit
MP potential
functions that
mirror the
biological
hydrophobicity
scale of Hessa et al.
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70. Presents the
first
atomic-resolution
structure of a
translocon.
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