Journal of Structural Biology 186 (2014) 357–366
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Journal of Structural Biology
journal homepage: www.elsevier.com/locate/yjsbi
A soluble mutant of the transmembrane receptor Af1503 features strong
changes in coiled-coil periodicity
Marcus D. Hartmann, Stanislaw Dunin-Horkawicz 1, Michael Hulko 2, Jörg Martin, Murray Coles ⇑,
Andrei N. Lupas ⇑
Department of Protein Evolution, Max-Planck-Institute for Developmental Biology, 72076 Tübingen, Germany
a r t i c l e
i n f o
Article history:
Available online 22 February 2014
Keywords:
Coiled coil
HAMP domain
Two component signal transduction
Transmembrane signaling
a b s t r a c t
Structures of full-length, membrane-bound proteins are essential for understanding transmembrane
signaling mechanisms. However, in prokaryotic receptors no such structure has been reported, despite
active research for many years. Here we present results of an alternative strategy, whereby a transmembrane receptor is made soluble by selective mutations to the membrane-spanning region, chosen by
analysis of helix geometry in the transmembrane regions of chemotaxis receptors. We thus converted
the receptor Af1503 from Archaeoglobus fulgidus to a soluble form by deleting transmembrane helix 1
and mutating the surface residues of transmembrane helix 2 to hydrophilic amino acids. Crystallization
of this protein resulted in the structure of a tetrameric proteolytic fragment representing the modified
transmembrane helices plus the cytoplasmic HAMP domain, a ubiquitous domain of prokaryotic signal
transducers. The protein forms a tetramer via native parallel dimerization of the HAMP domain and
non-native antiparallel dimerization of the modified transmembrane helices. The latter results in a
four-helical coiled coil, characterized by unusually large changes in helix periodicity. The structure
offers the first view of the junction between the transmembrane region and HAMP and explains the
conservation of a key sequence motif in HAMP domains.
Ó 2014 Published by Elsevier Inc.
1. Introduction
Prokaryotes sense and respond to their environment via an array
of dimeric transmembrane receptors. These couple input at an
extracellular sensor module to regulation of an intracellular
effector. As sensor and effector are separated by the cell membrane,
signaling must take place by conformational changes in the linking
segments, including the transmembrane helices. However, despite
active research for over 20 years, the nature of these changes
remains elusive, largely due to the lack of definitive structural data
on intact, membrane-bound receptors. Here we present an
alternative approach to this problem, aimed at producing soluble
versions of transmembrane receptors. The strategy for achieving
⇑ Corresponding authors. Address: Department of Protein Evolution, Max-PlanckInstitute for Developmental Biology, Spemannstr. 35, D-72076 Tübingen, Germany.
Fax: +49 7071 601 349.
E-mail addresses: [email protected] (M. Coles), andrei.lupas@
tuebingen.mpg.de (A.N. Lupas).
1
Present address: Laboratory of Bioinformatics and Protein Engineering,
International Institute for Molecular and Cell Biology, 02-109 Warsaw, Poland.
2
Present address: Gambro Dialysatoren GmbH, 72379 Hechingen, Germany.
http://dx.doi.org/10.1016/j.jsb.2014.02.008
1047-8477/Ó 2014 Published by Elsevier Inc.
this relies on establishing the geometry of the membrane-spanning
helices and mutating residues pointing towards the membrane to
hydrophilic residues. As a model system we chose Af1503 from
Archaeoglobus fulgidus, an archaeal receptor built around the transmembrane helical hairpin typical of the group (TM1 and TM2), with
a GAF sensor domain in the extracellular loop and an intracellular
HAMP domain at the C-terminus (Hulko et al., 2006). This protein
has proven to be a robust and flexible model for prokaryotic signal
transduction, and we have successfully incorporated its HAMP
domain into functional chimeras with chemoreceptors, histidine
kinases and adenylyl cylclases (Hulko et al., 2006; Ferris et al.,
2011, 2012; Mondejar et al., 2012). Af1503 HAMP has also been
very useful in producing chimeric constructs for structural studies,
including two presented in this special issue (Ferris et al., 2014a,b).
To create a soluble construct of Af1503, we deleted TM1 and
selectively mutated residues on the surface of TM2. The resulting
protein, Af1503-sol, is soluble and the size expected for a fulllength tetramer, but the crystal structure shows only a fragment,
encompassing the modified membrane-spanning region and the
intracellular HAMP domain. This fragment shows how the
protein tetramerizes as a dimer-of-dimers, forming a four-helical,
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antiparallel coiled coil. This mimics the native transmembrane region in several key respects, providing a first glimpse of the junction between the transmembrane segment and HAMP.
Coiled coils are one of the most common structural motifs in
proteins (Lupas and Gruber, 2005). They are a-helical bundles that
adopt superhelical structures in order to present a regular arrangement of sidechains to the bundle core. By far the most common
form is based on a repeating 7-residue sequence motif called the
heptad repeat, where positions are labeled a–g. Each heptad forms
two helical turns with one core residue per turn. This requires a
helical periodicity of 3.5 residues per turn (7 residues over 2 turns,
with 7/2 = 3.5), slightly over-wound with respect to the 3.63 residues per turn of regular, straight a-helices. To compensate, heptad
coiled coils are supercoiled in the opposite sense to the component
helices, forming bundles with a characteristic left-handed supercoiling. Coiled coils can also adopt other periodicities, based on
the combination of sequence elements of three and four residues
(Hicks et al., 2002; Gruber and Lupas, 2003). The most common
are built from hendecads (a–k, 3 + 4 + 4 = 11 and 11/3 = 3.67) or
pentadecads (a–o, 3 + 4 + 4 + 4 = 15 and 15/4 = 3.75), with the helices of hendecad coiled coils being substantially straight and those
of pentadecad coiled coils adopting right-handed supercoiling. The
structure we present is an example of such variation, combining
periodicities of 18/5 (3 + 4 + 4 + 3 + 4 = 18 and 18/5 = 3.60) and
19/5 (3 + 4 + 4 + 4 + 4 = 19 and 19/5 = 3.80).
2. Experimental procedures
2.1. Bioinformatics
Sequence regions between the N-terminus of TM1 and C-terminus of TM2 of Escherichia coli Tsr, Tar, Trg, and Tap proteins were
extracted and used as queries for BLAST searches (Altschul et al.,
1997) on the nr database. After removing redundancy, the resulting sequences were clustered in CLANS (Frickey and Lupas, 2004)
at a p-value cut-off 1e-57 and four clusters corresponding to the
respective homologs were defined. For each cluster a multiple sequence alignment (MSA) was built with MUSCLE (Edgar, 2004)
and manually corrected. Regions corresponding to TM1 and TM2
were extracted from the MSAs and position-specific conservation
was calculated for each helix using AL2CO (Pei and Grishin, 2001).
To predict the periodicity of each of TM helix, each residue was
assigned a vector with a length corresponding to its conservation.
The first vector was set to an angle of 0, and all further vectors
were transformed by a stepwise increment of x degrees, where
97 < x < 104 (at 7/2 the angle between consecutive positions is 103
degrees, whereas at 11/3 it is 98 degrees). The value of x that yields
the greatest resultant vector (conservation moment vector) indicates the optimum angle between positions and can be used to designate the periodicity: 11/3 (97 < x < 99), 18/5 (99.5 < x < 101.5), and
7/2 (102 < x < 104). A coiled-coil register was assigned based on the
angle u between the conservation vectors of individual positions
and the conservation moment vector of the helix; positions where
u reaches the minimum were assumed to form the hydrophobic
core.
2.2. Cloning, expression and purification
Using genomic DNA of A. fulgidus (ATCC 49558) as template,
two PCR fragments were initially generated covering the sequence
of the extracytoplasmic domain (T31-Q253) and the cytoplasmic
C-terminal part of Af1503, including the HAMP domain (T276K338). DNA encoding for the connecting coiled-coil sequence
VKNLLTLAADRAEQIVNDLAST was synthetically generated by PCR
with overlapping primers. The three DNA fragments were
combined into one final construct by overlap extension PCR followed by ligation into pet30b expression vector (Novagen) using
Nde/Hind restriction sites. Protein Af1503-sol was expressed in
BL21-Gold (DE3) cells for 4 h at 37 °C after induction with 1 mM
IPTG (isopropyl-b-D-thiogalactopyranoside) at an OD600 of 0.6.
Purification from soluble cell extract included anion-exchange
chromatography (QHP, 40 ml, GE Healthcare) in 30 mM MOPS/
NaOH pH 7.2 with a salt gradient from 50–600 mM NaCl, followed
by precipitation of pooled fractions with 30% (w/v) ammonium sulfate. Protein was resuspended in 30 mM MOPS/NaOH pH 7.2,
100 mM NaCl, 10 mM EDTA and run on a preparative Superdex
200 26/60 gel filtration column (GE Healthcare) in the same buffer
without EDTA. Purified Af1503-sol was concentrated to 10 mg/ml
for crystallization. To determine the oligomeric state of Af1503sol, protein was run on an analytical Superdex 200 10/300 GL
gel-sizing column (GE Healthcare) calibrated with suitable size
markers.
2.3. Crystallization, data collection and structure solution
Crystallization trials were performed at 297 K via the sittingdrop vapor-diffusion method in 96-well format with a reservoir
volume of 50 ll and drops consisting of 400 nl protein solution
and 400 nl reservoir solution. Best diffracting crystals were obtained with 25% (w/v) PEG 3350 and 100 mM HEPES, pH 6.5. Crystals were loop-mounted and flash-cooled in liquid nitrogen.
Diffraction data were collected at 100 K and a wavelength of
0.85 Å on a MARCCD 225-mm detector at beamline PXII of the
Swiss Light Source (PSI, Villigen, Switzerland). Data were indexed,
integrated and scaled to a resolution of 1.7 Å in space group P21
using XDS (Kabsch, 1993). As the space group and unit cell parameters (Table 1) were not consistent with a folded, full-length protein, we suspected that the crystals contained a degradation
product. In the hope that this fragment contained an intact HAMP
domain, we carried out a molecular replacement search with MOLREP (Vagin and Teplyakov, 2000) using the wild-type structure of
the Af1503 HAMP domain taken from PDB entry 3ZRX (Ferris
et al., 2012). While the rotation search did not reveal clearly outstanding orientations, the translation search returned a convincing
solution with a high contrast. Initial rigid body refinement using
REFMAC5 (Murshudov et al., 1999) yielded traceable electron density of multiple additional a-helical segments. Subsequent automated chain tracing with Buccaneer (Cowtan, 2006) revealed a
second HAMP dimer in the asymmetric unit. This second HAMP dimer was overlooked during molecular replacement as we expected
and searched only for a single dimer. The structure was completed
by cyclic manual modeling with Coot (Emsley and Cowtan, 2004)
and refinement with REFMAC5. The final model comprises four
Table 1
Data collection and refinement statistics.
Data collection
Space group
Unit cell parameters
Resolution range (Å)
Completeness (%)
Redundancy
I/r(I)
Rmerge (%)
Refinement
Resolution range (Å)
Rcryst (%)
Rfree (%)
Residues in the core/allowed region of the
Ramachandran plot (%)
P21
a = 44.07 Å, b = 48.04 Å,
c = 95.19 Å, b = 98.04°
37.7–1.70 (1.80–1.70)
99.2 (97.1)
3.79 (3.75)
11.2 (2.17)
7.7 (68.4)
37.7–1.70 (1.74–1.70)
19.3 (28.6)
24.3 (32.5)
96.3/3.7
Values in parenthesis refer to the highest resolution shell; Ramachandran statistics
were determined by PROCHECK (Laskowski et al., 1993).
M.D. Hartmann et al. / Journal of Structural Biology 186 (2014) 357–366
chains of the fragment N234 to K338. Data collection and refinement statistics are summarized in Table 1. The structure was
deposited in the PDB under accession code 4CQ4.
3. Results
3.1. Construct design
In order to design mutations to solubilize Af1503, it is necessary to identify which residues point towards the membrane and
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are thus not involved in inter-helical contacts. This requires
knowledge of the inter-helical geometries. Crystal structures
show that transmembrane helices in homo-oligomeric bundles
are frequently straight and adopt coiled-coil interactions
(Walters and DeGrado, 2006); indeed, the only membrane-bound
bacterial receptor of known structure, NpHtrII from
Natronomonas pharaonis, shows a hendecad coiled coil (1H2S,
2F93, 2F95: Gordeliy et al., 2002; Moukhametzianov et al.,
2006). The transmembrane helices of Af1503 would therefore
most likely adopt packing interactions based on 11/3 or 18/5
Fig.1. Design of the Af1503-sol construct. (A) Conservation momentum analysis defines the hendecad registers of the chemotaxis receptors Tar, Tsr, Trg and the 18/5 register
of Tap. Low values of the angle u between the overall conservation moment vector and the conservation vectors at individual positions are assumed to correspond to core
positions. The E. coli sequences are shown above the plots as representatives of each group, with core positions in bold face and residues expected in da-geometry in blue.
Exposed c-, f- and j-positions of the hendecad are in red. The resulting core assignment is given below each plot, identifying different registers for Tar and Tsr (top) and Trg
(centre). Positions where efficient cross-linking between cognate cysteines has been shown in singly substituted Tar and Trg are marked with ‘‘c’’ on the relevant plots. The
core assignment derived from the structure of HtrII from Natronomonas pharaonis (2F93: Moukhametzianov et al., 2006) agrees with that for Trg. (B) The Tar/Tsr and Trg
registers can be inter-converted by 33° axial helix rotation. A helical wheel diagram with hendecad (11/3) periodicity is shown with the positions of the two registers labeled
a–k (Tar/Tsr in blue and Trg in green). Core positions are shown in bold face, linked with solid lines. The dashed lines link the helix axis to the respective bundle axes. (C)
Design of surface mutations. The sequence of the Staphylothermus marinus tetrabrachian precursor (1YBK: Ozbek et al., 2005) is shown aligned to Af1503 using the hendecad
register of Trg. Residues in green lie outside the bundle core. These were transferred to equivalent positions in Af1503. One hydrophobic residue from tetrabrachian (red) was
transferred to an exposed j-position of Af1503 (V267) and this was mutated to glutamine. For convenience, residue numbers for Af1503-sol used in the text refer to those of
wild-type Af1503.
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Fig.2. The structure of the Af1503-sol fragment. (A) Sequence of the Af1503-sol construct. The extracellular GAF sensor domain is shown in orange and the intracellular
region including the HAMP domain and helical extension in green. Residues deleted from the N-terminus, i.e. the intracellular loop and TM1 are in grey. The modified TM2
region is shown in blue with residues mutated relative to wild-type Af1503 highlighted in grey (see also Fig. 1C). Residues observed in the crystallized fragment are
underlined. (B) Assembly of the Af1503-sol tetramer. The top view shows the monomer with the modified transmembrane helix and HAMP N-helix in green, the HAMP linker
in grey and the HAMP C-helix in blue. The distinct helical kinks associated with transitions between the body, neck and HAMP regions of the coiled coil are apparent. The
central view shows native parallel dimerization to form the HAMP domain, with monomers distinguished by light and dark colors. The bottom view shows antiparallel
dimerization over the modified transmembrane helices to form the tetramer, with the second dimer shown in yellow and orange. (C) Coiled-coil parameters for Af1503-sol. A
plot of axial helix rotation (effective Crick angle deviation) is shown layer-by-layer for the four-helical regions, as calculated using the program samCC (Dunin-Horkawicz and
Lupas, 2010a). The traces are colored following panel A; residue numbering refers to the green traces. Plots are shown for one HAMP domain only. The rotations states are
relative to an idealized coiled coil with 18-residue periodicity (18/5 = 3.6 residues per turn, i.e. straight a-helices). Straight lines fit to individual segments can be used to
derive average helix periodicities. These are shown for one parallel dimer, illustrating the sharp changes in periodicity between the segments. The HAMP domain is better
described using heptad (7/2 = 3.5 residues per turn) periodicity (see Fig. 5B).
periodicities. Due to the hydrophobic environment, hydrophobic
residues predominate in all positions of transmembrane helices
and hydrophobicity cannot be used as a marker for establishing
helix periodicity and core assignments. We therefore took an
alternative approach based on sequence conservation, under
the assumption that conservation is most pronounced in
M.D. Hartmann et al. / Journal of Structural Biology 186 (2014) 357–366
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Fig.3. Structure of the Af1503-sol ‘‘body’’ region. (A) Side and top views are shown, coloring is as in Fig. 2. Individual coiled-coil layers are shown on the left, illustrating close
to canonical packing geometries. The small helical crossing angle and almost straight helices are commensurate with the average helical periodicity of 3.56. (B) Core
assignments for the antiparallel coiled coil. The sequence of the modified transmembrane helices is shown for parallel and antiparallel orientations (top and bottom,
respectively), with core residues in bold face. The observed core assignment and that expected for hendecad periodicity are shown above and below, with residues in x- and
da-geometry in red and blue, respectively. Residues expected within the core for hendecad packing, but which lie outside the core in the observed structure, are shown in
purple.
residues engaging in packing contacts and less so for residues
pointing towards the membrane.
We first carried out conservation momentum analyses on the
chemotaxis receptors Tar, Tsr, Trg and Tap. These are ideally suited
to this task as many homologs are known that span a broad sequence range and there is a wealth of biochemical data available,
including the results of cysteine cross-linking studies. For TM1
and TM2 of Tar, Tsr and Trg we identified a clear 11/3 pattern in
the conservation of residues (Fig. 1). This pattern can be translated
into a coiled-coil register by defining an angle u between the conservation vector at each position and the conservation momentum
vector for the helix (see Section 2 for details). Low values of u are
assigned to core positions (Fig. 1A). With the aid of cysteine crosslinking data (Pakula and Simon, 1992; Lee et al., 1994; Hughson
et al., 1997), we assigned two distinct registers, related by a 33° axial rotation of the helices (Fig. 1B), one in Tar and Tsr and the other
in Trg. For TM1 and TM2 of Tap, we observed an 18/5 pattern in
residue conservation, but could not confirm this with cross-linking
data, as these are not available.
We next determined the appropriate register for Af1503 by noting a clear hendecad sequence pattern in the Af1503 extracellular
domain, immediately upstream of TM2, which was continuous
with the Trg register. Selecting this register, we chose residues at
surface positions and mutated them to the equivalent residues of
a soluble four-helical, parallel coiled coil with hendecad periodicity; that from the tetrabrachian precursor of Staphylothermus marinus (1YBK: Ozbek et al., 2005). This process resulted in one
hydrophobic residue from tetrabrachian being transferred to a
highly exposed j-position on the surface of the construct, and we
chose to mutate this to glutamine (V267Q) to give the final set of
14 solubilizing mutations (Fig. 1). We also deleted the residues
representing TM1 from the N-terminus. We named this construct
Af1503 solubilized (Af1503-sol). Note that the deletion of TM1
results in an offset of 30 in residue numbers for Af1503-sol with
respect to the wild-type protein. For convenience, the numbering
used in this manuscript refers to the wild type.
3.2. Structure of the Af1503-sol fragment
Expression and purification yielded a soluble, well-folded protein, as judged by tryptophan fluorescence and circular dichroism
(Tm = 90 °C), and bound calcium to the same extent as native
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Fig.4. Side and top views of the ‘‘neck’’ region of Af1503-sol. Coloring is as in Fig. 2. Individual coiled-coil layers are shown on the left, illustrating the packing geometries.
Residues in x-geometry are labeled in red. The large helical crossing angle and strong right-handed supercoiling are commensurate with the under-wound helices (average
periodicity 3.84), in contrast to the adjacent body region (Fig. 3).
Af1503 (data not shown). Based on analytical gel-sizing chromatography, Af1503-sol formed a complex corresponding to a tetramer of
the full construct (4 34.6 kDa). Crystallization trials yielded crystals diffracting to a resolution of 1.7 Å. However, when we obtained
a full dataset, we found the space group and unit cell dimensions
were not compatible with full-length protein in any oligomeric
form. This led us to expect a degradation product of unknown size
and sequence. Fortunately, a molecular replacement search with
the AF1503-wt HAMP domain rewarded us with a high scoring
solution. After initial rigid body refinement, well-defined electron
density for alpha helices was visible throughout the crystal. When
tracing the density it became obvious that there were actually 4
chains in the asymmetric unit, corresponding to the C-terminal
fragment spanning residues N234 to K338. This fragment comprises a small portion of the extracellular domain (N234–Q253),
the modified transmembrane helices (V254–F275) and the cytoplasmic region encompassing the entire HAMP domain (T276–
E331) and a short C-terminal extension (S332–K338). The fragment
forms a tetramer best described as a dimer-of-dimers (Fig. 2). The
monomer dimerizes in parallel to yield the native HAMP domain,
with the modified transmembrane helices as N-terminal extensions. These dimerize further in a non-native, antiparallel manner,
such that the entire fragment forms a four-helix bundle. This bundle can be described as a coiled coil with three distinct regions: an
antiparallel body, a connecting ‘‘neck’’ and the peripheral HAMP.
The body region (A250–I268) is an antiparallel bundle covering
the majority of the modified transmembrane segment. Analysis
of four-helix bundle parameters with the program samCC
(Dunin-Horkawicz and Lupas, 2010a) shows the average helical
periodicity over this region is 3.56 residues per turn (Fig. 2B), i.e.
intermediate between heptad periodicity (7 residues over 2
turns = 3.5) and 18-residue periodicity (18 residues over 5 turns =
3.6). This intermediate nature is reflected in core geometry and helix supercoiling. While the core is best assigned according to the
heptad repeat, the helices are almost straight and have low crossing angles (Fig. 3). Note that our original mutation strategy was
based on the expectation of hendecad periodicity in this region
(11 residues over three turns = 3.7 residues per turn). The coiled
coil consists of two symmetry-related sets of three core layers.
As expected for the antiparallel orientation, each mixes d- and
a-positions of the heptad repeat. The core packing is also close to
the canonical knobs-into-holes geometry expected for a heptad
coiled coil, deviating slightly towards the end of the segment by
axial rotation of up to 10°.
The second segment of the coiled coil is a connecting ‘‘neck
region’’. This consists of the N-terminal region of the fragment
(I239–G249) packed antiparallel to the region bridging the modified transmembrane helices and HAMP (V269–I280). The neck
deviates strongly in helical periodicity from the adjoining regions;
both helices are considerably under-wound, with periodicities
close to 19 residues over 5 turns (19/5 = 3.8). Accordingly these
helices show strong right-handed supercoiling and large helical
crossing angles (Fig. 4). It is notable that the large transitions in
periodicity between the neck and the flanking coiled coil regions
are accompanied by disruptions in canonical helical hydrogen
bonding: at G249 and N270 in the transition to and from the body
region and at P283, a conserved proline residue in the transition to
HAMP. This results in two distinct kinks in the monomer structure
(Fig. 2). The neck consists of three coiled-coil layers. The central
layer is made up of four residues assigned to the l-position of a
M.D. Hartmann et al. / Journal of Structural Biology 186 (2014) 357–366
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Fig.5. The HAMP domain of Af1503–sol. (A) Side and top views of the HAMP domain in Af1503-sol, colored as in Fig. 2. Individual coiled-coil layers are shown on the left,
illustrating the packing geometries. Residues in x- and da-geometries are labeled in red and blue, respectively. In complementary x-da packing, the helices alternate in
contributing one residue in x- and two residues in da-geometry to the bundle core in successive layers. The layers have distinct rhombic cross-sections, leading to deviations
from ideal packing geometry. In Af1503, the HAMP domain is decorated by a short, C-terminal helical extension, which continues the HAMP coiled coil register. (B) A samCC
plot of axial helix rotation (effective Crick angle) relative to an ideal coiled coil with heptad periodicity. The traces are colored following panel A, and show average helix
rotation of 23.8° and +16.4° for the N- and C-helices, respectively. This compares with the theoretical values of ± 26° for ideal complementary x–da packing.
19-residue repeat unit that adopts canonical packing. The two outer layers mix two residues in h-positions in canonical geometry
with two residue in x-geometry, i.e. pointing directly towards each
other across the bundle core (Fig. 4).
The HAMP domain (Fig. 5) is very similar to that previously
determined for Af1503 in a number of settings by NMR (e.g.
2L7H; RMSD 0.9 Å over backbone atoms) and crystallography (e.g.
3ZRX; RMSD 0.6 Å). The domain consists of parallel N- and C- helices connected by a structured loop. These dimerize to form a fourhelical, parallel coiled coil with an unusual core packing. Termed
complementary x–da, it is a rotational variant of canonical knobsinto-holes packing, whereby the N- and C-helices are axially rotated
by 26° in opposite directions. This results in introduction of two distinct packing geometries into the core: x-geometry, where residues
point directly toward each other across the bundle axis and dageometry, where residues form a ring around a central cavity. In
complementary x–da packing each helix alternates in contributing
one residue in x-geometry and two residues in da-geometry in successive layers (Fig. 5). The HAMP N-helices have a periodicity of
3.56 residues per turn, similar to that of the body region, while
the C-helices have the expected heptad periodicity of 3.5 (Figs. 2B
and 5B). Rather than the supercoiling expected for helices with
these periodicities, sidechain packing is accommodated over their
short length by distortions from regular, square bundle shapes.
Af1503 belongs to an unusual class of receptors that contain no
clear effector module. Rather, HAMP is decorated by a short
C-terminal extension, which the current structure shows for the
first time. As expected from its sequence, it forms a two-helical
coiled coil continuous with the hydrophobic register of the HAMP
C-helices. Receptors of this class are very often associated with a
small cytosolic protein of unknown function (e.g. Af1502 in the operon of Af1503) and have gene environments that include SLC-like
membrane transport proteins (Korycinski, M. and Lupas, A.N.,
unpublished data). It is therefore possible that they exploit such
components to form multi-molecular effectors, presumably via
interactions with HAMP.
4. Discussion
4.1. Localized versus delocalized periodicity changes
We had originally designed the solubilizing mutations to Af1503
based on the hendecad register of observed for the transmembrane
helices of Trg (Fig. 1). Accordingly, the average helical periodicity
across the modified transmembrane helices is 3.65, almost exactly
that expected for a hendecad. However, rather than maintaining
this periodicity continuously, requiring almost straight helices,
the region breaks into the longer ‘‘body’’ segment of slightly lefthanded helices and the shorter ‘‘neck’’ segment of strongly underwound helices with right-handed supercoiling. The difference in
core assignment is shown in Fig. 3B; the expected core can be expressed as two hendecads: (3 + 4 + 4) + (3 + 4 + 4) = 22, while the
observed pattern is three heptads with a single residue insertion:
(3 + 4) + (3 + 4) + (4 + 4) = 22. This is equivalent to resolving the
insertion into the heptad repeat locally, rather than delocalizing
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Fig.6. Analogy between Af1503-sol and native transmembrane receptors. The left
view shows the modified transmembrane region of Af1503-sol, colored is as in
Fig. 2. The right view shows the dimeric transmembrane region of TprII of
Natronomonas pharaonis (2F93: Moukhametzianov et al., 2006), with TM1 in green
and TM2 in yellow and monomers distinguished by light and dark colors. The green
pair of helices in the Af1503-sol tetramer packs into the spaces occupied by the
TM1 helices in the native receptor dimer. The positions of the sensor and HAMP
domains expected for Af1503 in this analogy are indicated.
over a wider range. The result is that I268 is involved in the core instead of V269, while four alanine residues expected in d- or e-positions of the hendecad (A240, A250, A262 and A273) are on the
surface.
The sharp changes in helix periodicity observed in the Af1503sol fragment are rare in coiled coils. Indeed, we have previously observed similar behavior only in the trimeric, parallel stalk regions
of auto-transporter adhesins (Alvarez et al., 2010). Here, the largest
periodicity changes are observed in a segment linking righthanded (15/4) and left-handed (heptad) supercoiling. This region
adopts strong right-handed supercoiling, involving a short stretch
of 19/5 periodicity. In line with the current structure, the core of
the adhesin stalks can be assigned to an alternative register, such
that periodicity changes could be delocalized over a wider region,
but again deviations are resolved locally in a short, highly divergent segment (Alvarez et al., 2010).
In the context of native transmembrane receptors, we expect a
transition in periodicity from the hendecad register of the transmembrane helices to the heptad register of HAMP. It is not clear
whether this necessarily involves the strong periodicity changes
observed in the current structure. Indeed, a larger periodicity transition from the neck region to HAMP is localized to the distortion
inherent in incorporating P283 into the helix. It thus seems likely
that the smaller change between a canonical hendecad and heptad
registers could be accommodated in a similar manner. This possibly explains the conservation of this proline in HAMP domains
immediately following transmembrane segments.
4.2. Comparison to NpHtrII
The only structure of a bacterial transmembrane receptor in the
membrane is the sensor rhodopsin II complex from Natronomonas
Fig.7. The transition between the transmembrane region and HAMP in Af1503. (A) Under the analogy presented in Fig. 6, intermolecular contacts between the modified
transmembrane helices and HAMP in Af1503-sol predict those between TM1 and HAMP in the native receptor. Coloring is as in other figures. The inset shows hydrogen
bonding (dashed yellow lines) between E311 and the N-terminal capping residues of an adjacent antiparallel helix. This interaction explains the conservation of E311 as part
of the DExG motif in HAMP domains immediately following transmembrane segments. (B) Justification of the DExG motif in Af1503. A detailed view of the motif (D310–
G313) is shown with coloring as in (A). In addition to the contacts made by E311, D310 makes numerous contacts at the N-cap of the HAMP C-helix. The short distance
between R308 Ca and G313 Ca (dashed green line) explains the conservation of glycine in the motif.
M.D. Hartmann et al. / Journal of Structural Biology 186 (2014) 357–366
pharaonis (Gordeliy et al., 2002; Moukhametzianov et al., 2006).
This complex consists of a dimeric transduction subunit (NpHtrII)
that contains the archetypal TM1–TM2 helical hairpin with short
extracellular loops and a sensor formed by two flanking rhodopsin
subunits (NpSRII) within the membrane. In contrast to the current
structure, the NpHtrII transmembrane helices are substantially
straight and show continuous hendecad packing in the register
identified for Trg in conservation momentum analysis (Fig. 1).
We chose this register in the design of the current construct, as
it continues the register of upstream helices of the Af1503 sensor
domain. Conservation momentum analysis also identifies an alternative hendecad register for Tar and Tsr, likewise supported by
cysteine cross-linking data. Examination of structures for the sensor domains of Tar (1VLS, 1VLT: Yeh et al., 1996) and Tsr (2D4U,
3ATP: Tajima et al., 2011) shows this alternative register also continues that of the upstream helical bundles. Thus both registers appear to be relevant in transmembrane receptors. It is notable that
these two registers are inter-converted by axial rotation of all four
helices by 33° (Fig. 1B). Thus these different chemoreceptors conserve a fixed rotational relationship between the sensor and the
membrane, and therefore between the sensor and HAMP.
4.3. Af1503-sol mimics the TM region of native receptors
Native transmembrane receptors are built around a four-helix
bundle formed by parallel dimerization of the TM1–TM2 hairpin.
In creating Af1503-sol we deleted TM1, meaning this native bundle
cannot form. Rather, in the crystal structure, the space left by deletion of TM1 is effectively filled by two TM2 helices from another
dimer, packed antiparallel to the first. Thus the tetrameric bundle
mimics the transmembrane helices in native receptors (Fig. 6). Given the non-native nature of the antiparallel dimerization, an analogy with native receptors must be made with caution. However, it
does offer an explanation for a key sequence motif in HAMP
domains.
In an extensive bioinformatics study, we found that HAMP domains immediately following transmembrane segments display a
characteristic sequence, DExG, at the N-terminal cap of the C-helix
(Dunin-Horkawicz and Lupas, 2010b). The conservation of the
aspartate and glycine residues in this motif can be rationalized as
forming a compact cap structure, whereas x represents a hydrophobic residue forming part of the HAMP heptad repeat (Fig. 7B).
In contrast, the conservation of glutamate in the second position,
the most conserved position in the motif, is not well explained.
In Af1503-sol, this glutamate (E311) forms intermolecular hydrogen bonds to the N-terminus of a second monomer (Fig. 7). This
suggests that E311 makes analogous contacts within the native
receptor, i.e. capping the N-terminus of TM1. Accordingly, this residue is far less conserved in HAMP domains not directly proximal
to the membrane, such as those following other cytoplasmic domains or within poly-HAMP arrays.
The DExG motif can be extended to include the proline residue
in the N-helix (P283) and the preceding arginine residue, which
show very similar conservation pattern; i.e. RP[. . .]DExG (DuninHorkawicz and Lupas, 2010b). We argue above that the proline acts
as an adaptor between the TM2 and HAMP coiled-coil registers.
The arginine most likely interacts with the polar head groups at
the membrane surface. Thus the motif is involved in structural
organization at the TM–HAMP junction, characterizing the transmembrane and HAMP domains as tightly integrated, four-helical
bundles with highly conserved interactions.
Acknowledgments
We thank Astrid Ursinus for help with protein purification. We
also thank Kerstin Bär and Kornelius Zeth for crystallographic
365
sample preparation and data collection and are grateful to the staff
of beamline X10SA at the SLS for excellent technical support.
Bioinformatics was by S.D.-H. and A.N.L. Structure determination
and analysis was by M.D.H., S.D.-H., A.N.L. and M.C. Molecular
Biology and protein biochemistry was by M.H. and J.M. The manuscript was written by M.C. and A.N.L.
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