Evolutionary mix-and-match with MFS transporters II
M. Gregor Madejaand H. Ronald Kabacka,b,c,1
aDepartment of Physiology,bDepartment of Microbiology, Immunology and Molecular Genetics, andcMolecular Biology Institute, David Geffen School
of Medicine, University of California, Los Angeles, CA 90095
Contributed by H. Ronald Kaback, October 22, 2013 (sent for review October 14, 2013)
One fundamentally important problem for understanding the
mechanism of coupling between substrate and H+translocation
with secondary active transport proteins is the identification and
physical localization of residues involved in substrate and H+bind-
ing. This information is exceptionally difficult to obtain with the
Major Facilitator Superfamily (MFS) because of the broad sequence
diversity of the members. The MFS is the largest and most diverse
group of transporters, many of which are clinically important, and
includes members from all kingdoms of life. A wide range of sub-
strates is transported, in many instances against a concentration
gradient by transduction of the energy stored in an H+electro-
chemical gradient using symport mechanisms, which are discussed
herein. Crystallographic structures of MFS members indicate that
a deep central hydrophilic cavity surrounded by 12 mostly irregu-
lar transmembrane helices represents a common structural fea-
ture. An inverted triple-helix structural symmetry motif within
the N- and C-terminal six-helix bundles suggests that the proteins
may have arisen by intragenic multiplication. In the work pre-
sented here, the triple-helix motifs are aligned in combinatorial
fashion so as to detect functionally homologous positions with
known atomic structures of MFS members. Substrate and H+-bind-
ing sites in symporters that transport substrates, ranging from
simple ions like phosphate to more complex peptides or disacchar-
ides, are found to be in similar locations. It also appears likely
that there is a homologous ordered kinetic mechanism for the
H+-coupled MFS symporters.
membrane transport|sequence alignment|bioenergetics
74 families with members from Archaea to Homo sapiens (1–3).
MFS proteins catalyze transport of a wide range of substrates,
including amines, acids, amino acids, sugars, peptides, and anti-
biotics, in many instances by transducing the energy stored in an
H+electrochemical gradient (Δμ~H+, interior negative and/or al-
kaline) into a concentration gradient of substrate by substrate/H+
symport mechanisms (4).
The lactose permease from Escherichia coli (LacY), a galac-
toside/H+symporter, arguably the most intensively studied sec-
ondary transporter known at present (5, 6), is the paradigm of
the MFS. LacY is comprised of 417 amino acid residues orga-
nized into two pseudosymmetrical six-helix bundles with the N
and C termini on the cytoplasmic face of the membrane (Fig. 1).
To determine which residues play an obligatory role in the
mechanism and to create a library of mutants with a single-Cys
residue at each position of the molecule for structure/function
studies, each residue was replaced individually with Cys in a
functional mutant devoid of native Cys residues (7). The great
majority of the single-Cys mutants are expressed normally in the
membrane and catalyze accumulation of lactose against a signif-
icant concentration gradient, thereby demonstrating that Cys
replacement at most positions does not induce severe pertur-
bations in the structure of LacY or in the symport mechanism. It
is striking that fewer than 10 residues are irreplaceable for active
lactose transport: Glu126 (helix IV) and Arg144 (helix V), which
are critical for substrate binding, as well as Trp151 (helix V)
where an aromatic side chain is essential; Glu269 (helix VIII),
His322 (helix X), and Tyr236 (helix VII), which are likely in-
he exceptionally diverse Major Facilitator Superfamily (MFS)
includes over 10,000 sequenced members and is comprised of
volved in coupling between protonation and sugar binding; and
Arg302 (helix IX) and Glu325 (helix X), which are exclusively
involved in H+translocation (5). As shown in the inward-facing
crystal structures of LacY (8–11), these residues are located at
the apex of a deep central hydrophilic cavity that is open to the
cytoplasm only and tightly sealed on the periplasmic side (Fig.1
A and B). Although very few residues are absolutely irreplace-
able, Cys substitution of 82 additional residues has a significant
effect on activity, inhibiting the steady-state level of accumula-
tion by 50–80% (7, 12).
X-ray structures give the impression that the symporters are
rigid, but various biochemical and biophysical approaches pro-
vide converging evidence that LacY is in a highly dynamic state
(13–15). Furthermore, binding of galactosides induces wide-
spread conformational transitions, increasing the open probability
of a hydrophilic cleft on the periplasmic side of the molecule, with
closure of the cytoplasmic cavity in reciprocal fashion (6, 16).
These coordinated conformational transitions are fundamental to
secondary transport and represent the basis for the alternating
access mechanism. Accordingly, the catalytic cycle of a transporter
does not involve significant movement of sugar- and H+-binding
sites relative to the membrane. Rather, the protein essentially
moves around the substrate, reciprocally exposing the binding sites
to either side of the membrane (i.e., alternating access in ref. 17).
A wealth of biochemical and spectroscopic data demonstrates that
the alternating access mechanism is operative in LacY (reviewed
in refs. 6 and 16). Furthermore, X-ray structures (18–21), as well
as spectroscopic findings (22), indicate that other MFS members
probably function in similar fashion.
LacY is highly dynamic. On one hand, although ∼65% of the
side chains are hydrophobic and buried, ∼85% of the backbone
amide protons exchange with deuterium in 10–15 min at room
temperature (13, 14), with 100% exchange at elevated, non-
denaturing temperatures (15). On the other hand, flipping the
six-helix bundles over and superposing them in structure models
The Major Facilitator Superfamily (MFS), the largest family of
secondary transport proteins, catalyzes transport of a wide
range of substrates. Difficulty discerning underlying mecha-
nistic principles is due to low sequence conservation. However,
a common structural feature of MFS members, suggesting that
they may have arisen by intragenic multiplication, is a repeat of
four three-helix bundles organized in two pseudosymmetrical
domains. An alignment of these triple-helix motifs in combi-
natorial fashion allows detection of functionally homologous
positions. Thus, substrate and H+-binding sites in distantly re-
lated symporters are located at the same relative positions. The
structural organization also suggests that an ordered kinetic
mechanism similar to that determined for lactose permease
may be operative in other MFS symporters.
Author contributions: M.G.M. and H.R.K. designed research, performed research, analyzed
data, and wrote the paper.
The authors declare no conflict of interest.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
www.pnas.org/cgi/doi/10.1073/pnas.1319754110PNAS Early Edition
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representing inward- or outward-open conformations yields an
rmsd of about 1.2 Å. Thus, the two conformations are symmet-
rical, suggesting that the two domains move in rigid-body fashion
during alternating access. However, the conclusion regarding
rigid-body movement is incompatible with the H/D exchange data
and observations that transport is blocked when helices within the
N- or C-terminal helix bundles are cross-linked (23–25). Further-
more, crystal structures have been reported for other MFS trans-
porters in an occluded conformation (20, 26) where the central
cavity is inaccessible from either side of the membrane, which
argues against a simple rocker-switch mechanism (27).
A principal difficulty in comparing MFS proteins is their low
sequence conservation. Despite the conserved fold motif and
in some cases overlapping function, sequence identity ranges
around 12–18% (12). However, sequence similarity is much
higher because most MFS members have a hydrophobic amino
acid content of 60–70%. Moreover, the inherent symmetry of the
proteins with regard to helix kinks and bends provides further
overlapping of residues. In addition, regions of functional simi-
larity (e.g., substrate- or H+-binding sites) align for only very
closely related MFS transporters (28, 29). In less closely related
symporters, residues with similar functional roles are often at
disparate locations in the sequences (12).
The X-ray structures of various MFS symporters published in
the past 2 y (19–21, 26, 30–33) have increased the total number
of unique sets of coordinates from three (8, 27, 34) to nine, not
including coordinates for alternative conformations or mutants
of the same transporter (9–11, 18, 35). With a growing number
of crystallographic structures of MFS symporters, we can begin to
identify functionally related residues, which are uncorrelated in
the primary structures but are correlated in the tertiary structures.
It has been suggested that the MFS transporters may have
arisen by intragenic multiplication of the triple-helix motif to two
pseudosymmetrical six-helix bundles (36, 37), the most common
topological feature of MFS transporters. The recently presented
crystallographic model of L-fucose permease (FucP) was obtained
with the central cavity open to the periplasm, an outward-facing
conformation opposite to that of LacY (21). It was observed that
the transformation between the inward- and outward-facing con-
formations is obtained by an interconversion between conforma-
tions of neighboring inverted triple-helix repeats in the N- and
C-terminal six-helix bundles (38). This observation prompted
a comparative sequence analysis of the triple-helix motifs, screening
for conservation of functional residues. An example of flexibility in
design was observed within LacY and FucP, two MFS symporters
(12). We permuted the order of the triplets in FucP from their
natural order relative to LacY to obtain a higher order of sequence
conservation (Fig. 2). The alignment was tested for conservation by
comparing the 92 LacY mutants that impair function with 34
analogous functional mutations in FucP (12). In contrast to the
conventional, linear sequence alignment, much stronger alignments
between the sugar- and H+-binding sites in the two proteins are
observed with rearrangement of the FucP triplets. We suggested
that LacY and FucP might have evolved from primordial non-
covalently fused helix-triplets that formed symporters. Modern
symporters formed as functional mutations were being introduced
and the functional segments assembled in a varying order. An al-
ternative although less dynamic scenario is that a multispanning
protein evolved first, possibly for stability reasons, and functional
mutations were introduced later. In any case, using the notion of
triple-helix evolution in the MFS, a more useful annotation of se-
quence motifs than a conventional, linear sequence alignment may
be appropriate; we can test this possibility by alignment of func-
tionally significant residues.
Results and Discussion
Although it is generally believed that all MFS symporters oper-
ate by an alternating access mechanism, extensive biochemical
and spectroscopic evidence is available for LacY only (reviewed
in refs. 6 and 16). Furthermore, as a result of intensive study over
three decades, an overall mechanism for coupling in LacY has
been worked out.
An Overall Mechanism for Coupling in LacY.
i) Lactose/H+symport in the uphill or downhill energetic
modes is precisely the same reaction. The difference is in
the rate-limiting step. For downhill symport, deprotonation
is rate-limiting; for uphill transport, deprotonation is no lon-
ger limiting, and either dissociation of sugar or a conforma-
tional change that leads to deprotonation becomes limiting.
ii) Sugar binding and dissociation—not Δμ~H+—are the driving
force for alternating access (Δμ~H+has no effect whatsoever
on equilibrium exchange or counterflow).
iii) LacY must be protonated to bind sugar (the pKafor sugar
binding is ∼10.5).
iv) Galactoside binds by an induced-fit mechanism, which powers
a transition to an occluded state.
v) Sugar dissociates first.
LacY (PDB ID code: 2V8N). Helices are shown as rods; the numbers of helices in
the N-terminal six-helix bundle are colored green, and the numbers of C-ter-
minal six-helix bundle are colored orange. Critical residues are indicated (green,
residues involved in sugar binding exclusively; orange, residues involved in
sugar affinity and H+ translocation; magenta, residues involved in H+ trans-
location exclusively; yellow, weakly salt-bridged residues; details are provided
in the main text). The water-accessible surface of the cavity is shown as a light
blue surface [calculated using the Computed Atlas of Surface Topography of
proteins (CASTp) Web tool with a probe size of 1.4 Å]. (B) Cytoplasmic view; the
color-coding is the same as in A.
LacY structure. (A) Side view of the inward-facing conformation of
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vi) Upon sugar dissociation, there is a conformational change
that causes Arg302 (helix IX) to approximate Glu325, lead-
ing to deprotonation of LacY.
As an initial step to investigate whether a similar overall mech-
anism might apply to other MFS symporters, we have searched for
functional homologies between individual residues in additional
structurally resolved symporters.
Detection of Functionally Homologous Positions. MFS transporters
consist of four symmetrically disposed triple-helix units that can
be aligned individually and examined for conservation of func-
tionally significant residues (Table 1; Fig. 2). The combinatorial
alignment of the symmetry motifs allows detection of function-
ally homologous positions in different MFS transporters. The
procedure relies on mapping known functional markers in the
helix triplets followed by inferential mapping of unknown func-
Arrangement of helix-triplets in XylE and PiPT. The recently solved
structures for XylE and PiPT (20, 26) in the substrate-bound
state allow identification of residues involved in substrate bind-
ing (Fig. 3). Although many substrate-binding residues in LacY
are located in the N-terminal six-helix bundle (e.g., Arg144,
Trp151, and Glu126), in XylE and PiPT, residues from the C-
terminal six-helix bundle predominate. The previous study (12)
comparing LacY with FucP revealed that due to the different
order of the symmetry motifs, functionally equivalent residues
can be located at different positions in the protein. Superposition
of the C-terminal six-helix bundle from XylE or PiPT on the N-
terminal bundle of LacY results in spatial alignment of substrate-
binding residues in these three symporters (Fig. 3 A and B).
However, an alignment of XylE and PiPT with FucP can be
achieved only if symmetry motif D is superposed on motif C in
FucP and, respectively, symmetry motif C is superposed on motif
D in FucP (Fig. 2; Table 1). The N-terminal six-helix bundles
from XylE, PiPT, and FucP superpose on each other without
changing the order of symmetry motifs A and B. However, su-
perposition on LacY succeeds only when symmetry motif C from
LacY is superposed on symmetry motifs B from XylE, PiPT, and
FucP, and symmetry motif D from LacY with the symmetry
motifs A from the other three symporters (Fig. 2; Table 1).
Altered orientation of helix-triplets. In FucP, the orientation of all
four motifs is inverted with respect to LacY; all cytoplasmic
loops superpose on periplasmic loops and periplasmic loops on
their cytoplasmic counterparts in these two symporters (12).
Unlike FucP, in XylE and PiPT, only motifs A and B are inverted
with respect to motifs C and D in LacY, thereby placing the C
terminus of the first six-helix bundle (motifs A and B) and the N
terminus of the following six-helix bundle (motifs C and D) on
opposite sides of the protein in the alignment of the structure
motifs (Fig. 2). This is not the case in the native XylE or PiPT;
rather, in these proteins, the N-terminal six-helix bundle is ro-
tated 180° with respect to the membrane plane relative to LacY.
This is compatible with the hypothesis that the symporters
evolved by intragenic duplication and fusion of the helix-triplets
(12, 36, 38). With single substrate and H+binding-sites (5), the
primordial MFS symporters may have exhibited substantial
flexibility with regard to the orientation of the helix bundles
to each other. This is not surprising considering that substrate-
binding sites are generally located at the approximate middle of
these symporters. Therefore, inversion of triple-helix motifs may
be well tolerated. In this context, although the antiporter EmrE
is not an MFS protein, it is a dimer with the binding site placed at
the dimer interface in the middle of the complex. Moreover, it
has been demonstrated (39) that the EmrE dimer is functional in
both parallel and antiparallel configurations.
Arrangement and orientation of helix-triplets in PepT. The oligopeptide/
H+symporter (PepT) (19, 30, 31, 35) provides another variation
to the order of the triple-helix units because the symmetry motifs
quence. (A) Helix-triplets (represented by colored boxes) from FucP, PiPT, XylE,
and PepT are aligned with LacY (helices 1–3, blue; helices 4–6, green; helices 7–9,
orange; helices 10–12, yellow). The flags indicate the loops within symmetry
motifs. Helix-triplets from LacY are aligned with FucP (B), PiPT (C), and PepT (D)
(helices 1–3, blue; helices 4–6, green; helices 7–9, orange; helices 10–12, yellow).
The alignments are oriented with the LacY cytoplasmic side to the top. The flags
indicate the loops within symmetry motifs (white, cytoplasmic loop; gray, peri-
by the respective loop. See SI Appendix, Fig. S1 for a schematic representation.
Schematic alignment of the helix-triplets in consecutive order in the se-
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in PepT and LacY align in the consecutive order D-B-C-A to LacY
A-B-C-D by using known substrate-binding residues as functional
markers (Figs. 2 and 4C; Table 1). Clearly, the orientations of
motifs A and D are inverted with respect to the membrane relative
to LacY (Figs. 2 and 4C). Consequently, only residues at the center
of PepT superimpose without inversion (Fig. 4C) (Glu310 in PepT
and Asp240 in LacY). In contrast, functional positions displaced
from the center are observed on the cytoplasmic side of the sub-
strate-binding site [i.e., at a symmetry related location on the helix
(Fig. 4C): Glu32 in PepT and Glu325 in LacY].
Alignment of substrate-binding side chains. In the X-ray structure of
the inward-facing conformer of PepTGkfrom Geobacillus kaus-
tophilus with the bound inhibitor alafosfalin (19), contacts to the
inhibitor are provided primarily from motifs A and C. The posi-
tions of most side-chain interactions observed in the crystal
structures of other MFS symporters are also observed in PepTGk
although the side chains themselves vary, as expected. Residue
Gln339 (C-terminal, helix VIII) in symmetry-motif C in PepT and
corresponding to Glu269 in LacY provides contact between the
N- and C-terminal halves of PepTGkby interacting with substrate
ligand Arg43 and with the substrate (Figs. 4C) (N-terminal, helix I).
Gln339 is located on the same face of helix VIII as the substrate
ligand Asn342. The corresponding two residues, which appear to be
involved in substrate binding, are observed in homologous symmetry
motifs in XylE (Glu168, Gln175), PiPT (Gln177, Ser185) (Fig. 4B),
FucP (Gln159, Asn162) (12), and in LacY (Glu269, Asn272) (Fig.
4). In all crystallographic structures with a bound substrate, the
position corresponding to Glu269 in LacY couples the two six-helix
In addition to polar substrate ligands, aromatic side chains
also play an important role in substrate binding. For example,
Trp151 in LacY (8, 40) and Phe308, its counterpart in FucP (41),
are essential for sugar binding (22), and they align with Trp416 in
XylE, Lys459 in PiPT, and Phe170 in PepTGk. Irreplaceable
Arg144 in LacY (42, 43) is in close proximity and on the cyto-
plasmic side of Trp151, and, in FucP, Arg312 (21) is functionally
important and lies in a similar position relative to Phe308 (12).
Similarly, in XylE, Trp416 and Asn415 align with Trp151 and
Arg144 in LacY. Asn415 is involved in a complex H-bound
network with Gln289 and Asn294 that interacts with xylose (20).
In the open-inward, inhibitor-bound structure of PepTGk, the
position of Asn166 (Fig. 6A) corresponds to Asn415 in XylE,
Arg312 in FucP, and Arg144 in LacY (Fig. 4). Although muta-
tion of Asn166 impairs active transport and counterflow (19), in
this conformation, Asn166 in PepTGkis too far from the bound
inhibitor to be involved in an H-bond network analogous to
XylE. However, it is possible that tilting of PepT helix V against
helix I would move Asn166 into proximity with substrate and
close the cavity. Such conformational movement was suggested
for LacY, where helix V and I are thought to undergo a sub-
strate-dependent, scissors-like movement at position Gly24
(helix I) and Cys154 (helix V) (10, 24, 44), and indeed these
positions correspond to residues Gly41 and Ala172 in PepTGk.
Alignment of H+-binding side chains. Helix X in LacY (symmetry-
motif D) corresponds to helix I in PepT, XylE, PiPT (Figs. 2 and
4), and FucP (12) in symmetry motif A. Helix I carries the car-
boxyl side chains (Glu32 in PepT, Asp27 in XylE, Asp45 in PiPT,
and Asp46 in FucP) that align with the well-characterized
Glu325 in LacY. Strikingly, mutants with neutral replacements
for Glu325 in LacY do not catalyze lactose/H+symport, but
counterflow and equilibrium exchange are unaffected (45). This
and additional evidence (5, 46–48) indicate that Glu325 is irre-
placeable and is directly involved in H+symport. Similar findings
have been reported for Asp46 in FucP (22), Glu22 in PepTSt
(31), and Asp27 in XylE, supporting the conclusion that these
residues are functionally homologous to Glu325 in LacY.
Residues on the same face of helix X as Glu325 play a critical
role in the mechanism of LacY (5). In particular, His322, which
is located one helix-turn toward the periplasm from Glu325, is
another functionally irreplaceable residue (49–53). In contrast
to Glu325 mutants, exchange and counterflow are blocked in
His322 mutants, and replacement with Asn or Gln results in a
major decrease in affinity for galactosides (52, 54). However,
mutant H322R catalyzes lactose influx down a concentration
Table 1.Combinations of symmetry motifs in MFS
Helix-triplets A–D from PiPT, XylE, PepT, and, for comparison, FucP are aligned to LacY. The triplets are
color-coded as in Fig. 2. The values represent the similarity score and the functional similarity score
according to SI Appendix, Eqs. S1 and S2 (Q/QAct). For comparison, the respective scores are also stated
for the superposition of the N-terminal six helices (Nter) and the C-terminal six helices (Cter) with LacY for
the respective model.
LacY. (A) C-terminal six-helix bundle of XylE (colored in light olive) is super-
posed on the N-teminal six-helix bundle of LacY (side chains shown colored in
light blue). The ligand of XylE, xylose, is shown as a gold ball-and-stick model.
(B) C-terminal six-helix bundle of PiPT (colored in light olive) is superposed on
the N-teminal six-helix bundle of LacY (side chains shown colored in light blue).
The ligand of PiPT, phosphate, is shown as an orange ball-and-stick model.
Oxygen and nitrogen atoms are colored red and blue respectively. The su-
perposition of XylE and PiPT is provided in SI Appendix, Fig. S2.
Overall architecture of the substrate binding sites compared with
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gradient at a slow rate without H+translocation (52). Although
His322 is clearly involved in affinity for galactosides, it has been
suggested that it is also involved in H+translocation (54).
Moreover, the notion has been put forward that structural water
coordinated within a triad between His322, Tyr236, and Glu269
(two additional irreplaceable residues) may act as a cofactor for
binding and galactoside/H+symport by forming a hydronium ion
intermediate during turnover (54, 55). In this regard, an analogous
situation is apparent in PepTGkwhere Glu35, which corresponds
to His322 in LacY, is not a direct substrate ligand. However,
Glu35 is involved in substrate binding by positioning the guani-
dinium group of Arg36 with substrate (Figs. 4C and 6A) (19).
Functional Correlations. Homology of primary amino acid sequen-
ces reflects evolution and therefore provides a guide to structure,
mechanism, and function. Proteins that are related by common
descent are expected to exhibit homologous structures and func-
tions proportional to the degree of their sequence similarity. This
principle provides the motivation to define protein phylogenetic
relationships and correlate specific residues with function.
A kinetic mechanism for LacY. Varied experimental efforts have been
undertaken with LacY to develop a kinetic scheme for lactose/
H+symport (5, 6, 46), and Fig. 5 depicts a simplified scheme.
The sequence is initiated from the inward-facing conformer
(HEi) in which the carboxyl group of Glu325 is protonated due
and PepT (C) to LacY helix-triplets (stereo-view). The
Cα atom trace is show as wire and colored according
to the helix-triplet (Fig. 2 and Table 1) except for
PiPT in B. Significantly equivalent residue pairs are
shown as sticks in the same color. Labels of LacY
residues are indicated in blue.
Functional alignment of XylE (A), PiPT (B),
Madej and Kaback PNAS Early Edition
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to the low dielectric of the local environment, as evidenced from
the X-ray structures. Because the apparent pKaof Glu325 (helix
X) is ∼10.5 (54), deprotonation is likely due to the transient
proximity of Arg302 (helix IX) (56) (step 1). Subsequently, the
cytoplasmic cavity closes (step 2), resulting in the formation of
the apo-intermediate (En). This conformer can relax back to the
inward-facing conformation or open to the periplasmic side
where it is reprotonated, which may involve Tyr236 (helix VII),
His322 (helix X), and Glu269 (helix VIII) (55) (step 3). The
protein then assumes a conformation symmetrical to the inward-
facing conformer to reorient the helices for transfer of the H+to
negatively charged Glu325 and binding of sugar from the peri-
plasmic side (HEo, steps 4 and 5). All of the specificity of LacY
for substrate is directed toward the galactopyranosyl end of the
substrate, and binding begins with a nonspecific hydrophobic
interaction between the bottom of the galactopyranosyl ring with
the hydrophobic surface of the indolering ofTrp151 (40, 57). Once
the galactoside is oriented properly, formation of the binding site
occurs. Arg144 (helix V) and Glu126 (helix IV), as well as Glu269
(helix VIII), interact with specific OH groups on the galactopyr-
anosyl ring. Thus, galactopyranoside binding probably involves in-
duced-fit (58). As the protonated galactoside-bound LacY ternary
complex forms, the protein closes around the sugar to form an
tothe outward-facingconformationor thecytoplasmiccavity opens
with release of sugar to the cytoplasm (step 7). With release of
sugar, the initial inward-facing conformation is restored (step 8).
By reorientation of helices, Tyr236 (helix VII) is displaced from a
position between Arg302 and protonated Glu325, thereby causing
deprotonation of Glu325 (Ei+H+) with reinitiation of the cycle
(step 1) (41). This ordered scheme was proposed originally in 1979
(59) and has been continuously refined since.
The ordered kinetic mechanism. There is an abundance of evidence
for the ordered mechanism as depicted in Fig. 5 (reviewed in
refs. 5 and 46). As stated above, the pKafor galactoside binding
is ∼10.5. It is apparent that LacY is protonated at physiologic pH
before substrate binding, and recent experiments supporting this
conclusion more directly have been presented (60). Critically,
neutral replacements for Glu325 yield mutants that are totally
unable to carry out any reaction that involves net H+transport but
catalyze equilibrium exchange and counterflow as well as or better
than the WT. As indicated in Fig. 5, a satisfying explanation for
the behavior of Glu325 neutral replacement mutants that provides
very strong evidence for the ordered mechanism shown is that
the mutants can oscillate within the shaded portion of the scheme,
but cannot deprotonate, and therefore cannot form the apo-
intermediate and complete the cycle. In other words, the order of
release in the WT must be dissociation of the galactoside first,
followed by deprotonation. Because FucP, XylE, and PepT all
have a carboxyl group corresponding to Glu325 in LacY that
displays a similar phenotype when neutralized by mutagenesis, it is
apparent that these symporters and perhaps many other MFS
symporters likely operate by a similar ordered kinetic mechanism.
A common mechanistic pattern. As discussed, the initial step in the
cycle for LacY involves deprotonation of Glu325, which is
thought to cause closure of the cytoplasmic cavity (61). As a re-
sult, the central cavity becomes closed on both sides. Although
this conformation has not been observed crystallographically
with LacY, such a conformation was observed with PepTSo(30),
and a homology model for the LacY apo-intermediate is also
available (41). The apo-intermediate can relax to the outside-
open conformation, and the symporter reprotonates from the
outside. An open-outward crystallographic model of FucP (21)
and theoretical models for LacY (38) have been presented.
Binding of substrate to the protonated open-outward conformer
causes the protein to close around the substrate to form the
occluded state. This conformation has been described in XylE
(20) and PiPT (26). Interestingly, in both structures, the sub-
strate-liganding side chains are located in the N- and C-terminal
transport model. Inward-facing (blue) and outward-facing (green) confor-
mations are separated by the apo-intermediate conformational cluster
(gray) or by the occluded-intermediate conformational cluster (orange).
Substrate (S) and H+are indicated. Steps are numbered consecutively: Sub-
strate translocating transitions are indicated by blue arrows (steps 5–8) and
transitions recycling the outward-open cavity are indicated by red arrows
(steps 1–4). All steps are reversible (indicated by double-headed arrows).
The blue-shaded area demarcates the equilibrium-exchange reaction.
Examples of experimental coordinates (transporter and PDBID) associated
with respective conformations are indicated.
Transport cycle of LacY. Overview of the postulated steps in the
substrate-bound state of PepTGk(PDB ID code: 4IKZ). (B) Inward-open, substrate-free state of PepTGk(PDB ID code: 4IKV). The position of the substrate detected
in the substrate-bound state is indicated as white profile. (C) Inward-occluded conformation of PepTSo(PDB ID code: 2XUT). The colors of the bars at the
bottom indicate the mechanistic affiliation of the respective states with regard to the mechanistic model shown in Fig. 5.
Conformational changes in the substrate binding site of PepT (see SI Appendix, Fig. S3 for stero-view version of this figure). (A) Inward-open,
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| www.pnas.org/cgi/doi/10.1073/pnas.1319754110 Madej and Kaback
halves of the symporters. The occluded conformation can either
reopen to the outside or open to the inside with release of
substrate. In LacY, this global conformational change is postu-
lated to be initiated by a localized scissors-like movement be-
tween helices V and I that is induced by galactoside binding (24).
High-resolution X-ray structures of the inward-facing confor-
mation of PepTGk(19), with bound inhibitor (pdbID: 4IKZ),
show helix V with Asn166 displaced from its corresponding po-
sition in XylE (Asn415 on helix XI). It is likely that Asn166
ligates substrate in the fully occluded state and is no longer in
contact with alafosfalin in the inward-facing conformation of
PepTGk(Fig. 6A). Asn166 is presumably important for substrate
binding in PepTGk(19), like Arg144 in LacY (43). Therefore,
movement of helix V may initiate release of substrate on the
cytoplasmic side. As discussed above with respect to LacY, he-
lices V and I cross in the approximate middle of the membrane
where Cys154 (helix V) and Gly24 (helix I) are in close proximity
(8, 44). The C154G mutant binds ligand with high affinity but
catalyzes almost no transport (44, 62). The X-ray structures of
PepT (18, 19, 30) demonstrate that helix V crosses helix I in the
approximate middle of the membrane in such a manner that
Ala171 (helix V in PepTGk) lies close to Gly42 (helix I in PepTGk),
thereby emphasizing the homology to this position in LacY. In the
inward-facing conformation of PepTGkwithout bound substrate
(pdbID: 4IKV) (Fig. 6B), further distortion of the substrate-binding
site is observed. An induced-fit mechanism has already been sug-
gested for LacY (9). Galactoside is thought to induce the formation
of its binding site, primarily through side-chain movements without
a global conformation change. Such a scenario is consistent with
crystallographic structures observed with PepTGk(19).
It is obvious that the formation of the open-outward cavity to
initiate another round of transport requires a conformation in
which substrate is released without rebinding. The structure of
the open-inward conformation of PepTSt(pdbID: 4APS) (31)
provides structural insight in this regard. Here, the substrate-
capping Tyr residue is rotated back toward the center of the
cavity, blocking access of substrate. Further stabilization of this
conformation is represented in the crystal structure of PepTSo(30)
where the Tyr residue is stabilized in a conformation blocking
access to substrate by H-bonding to an Arg32 (Fig. 6C).
Taken together, the striking similarities within the MFS
clearly suggest that a common mechanistic pattern may be
used for catalysis of symport by additional transporters in this
Materials and Methods
Structure and Sequence Alignments. The structural symmetry motifs accord-
ing to Radestock and Forrest (38) were generated from the crystallographic
coordinates of LacY (A, Thr7-Asn102; B, Leu104-Phe187; C, Lys220-Ser309; D,
Ala311-Leu400) (10), FucP (A, Arg22-Met115; B, Asn116-Thr229; C, Arg258-
Ala345; D, Gly347-Phe431) (21), XylE (A, Tyr5-Ile112; B, Tyr125-Pro221; C,
Gly276-Thr365; D, Gly369-Glu465) (21), PiPT (A, Pro30-His125; B, Trp126-
Arg229; C, Thr306-Ile410; D, Gly411-Arg518) (26), and PepT (A, His21-Ile114;
B, Gly117-Leu211; C,Ala286-Ser392; D, His398-Met492) (19), and the super-
imposition of the helix-triplets was carried out as described in Madej et al.
(12). In brief, computer programs COOT v0.7 (63) and UCSF-Chimera (64)
were used for the structure-guided sequence alignment. No positional
restraints were applied to functionally significant residues in the sequence
or structure alignments. The superposition was visually inspected for the
conservation of LacY functional markers. As functional markers, 22 polar
residues in LacY were used where mutations to Cys cause greater than 50%
(SI Appendix, Eq. S2: sAA25–50%) or 75% (SI Appendix, Eq. S2: sAA0–25%) in-
hibition of the transport rate with Cys-less LacY (7, 12).
Discrimination of Defective Alignments. For discrimination of defective
alignments, a similarity score, Q, was defined. A reduction matrix (SI
Appendix, Table S1) of similar side chains was defined based on clus-
tering of reduction groups in similar folds, omitting interlacing for dif-
ferent levels of reduction (65). This reduction matrix was modified with
respect to the clustering for packing values of helix–helix contacts in the
membrane, their natural occurrence in α-helical membrane proteins (66),
and hydrophobicity (67) (SI Appendix, Fig. S4 and Table S1). The main
difference is that His was grouped with positively charged residues Arg
and Lys instead of with Asn in the first reduction level. Additionally,
groups introducing helix irregularities and small side chains (Gly, Pro) are
grouped with Cys at the last reduction level based on similar helix
packing and hydrophobicity to Ser and Thr. Due to high abundance in
helical membrane proteins, the hydrophobic side chains I, F, V, and L
were ignored. The following value scoring of sequence conservation was
used: conserved residue = 20, first reduction level = 10, second reduction
level = 7, and third reduction level = 4. The sum of the scoring values (red
Value) was divided by the total number of analyzed positions multiplied
by the highest score (v = 20) for a conserved residue (SI Appendix, Eq. S1).
For 100% conservation, Q should equal 1. The quantification of func-
tionally overlapping positions was performed by weighting the scoring of
functionally critical positions (AA0–25%, activity inhibited by at least 75%
upon mutation to Cys) and functionally relevant positions (AA25–50%,
activity inhibited by at least 50% upon mutation to Cys) using the re-
duction matrix (SI Appendix, Table S1) and an arbitrary weighting factor
of 5 for functionally critical positions (SI Appendix, Eq. S2). For 100%
conserved transporters with 100% conserved functional positions, this
value, QAct, should equal 1. The best scoring structure alignments were
inspected visually for conservation of the geometry in the functional
site, i.e., if functional groups face each other in the alignment.
ACKNOWLEDGMENTS. We are deeply indebted to Arthur Karlin for editorial
contributions. This work was supported by National Institutes of Health
Grants DK51131, DK069463, and GM073210, as well as National Science
Foundation Grant MCB-1129551 (to H.R.K.).
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| www.pnas.org/cgi/doi/10.1073/pnas.1319754110Madej and Kaback
Mix-and-Match Evolution II
M. Gregor Madej1 and H. Ronald Kaback1,2,3*
1Department of Physiology, 2Department of Microbiology, Immunology &
Molecular Genetics, 3Molecular Biology Institute, David Geffen School of
Medicine, University of California Los Angeles, Los Angeles, CA 90095
Running title: Structure-Function Correlations in the MFS
Keywords: membrane transport | symport | MFS | sequence alignment |
*Corresponding author: H. Ronald Kaback, email@example.com
Figure S1: Clustering of amino acid properties (three-dimensional representation). The
coordinates are computed from the relative occurrence [%] in alpha-helical membrane proteins
for x-coordinate (1), the packing value [val.] for y-coordinate (1) and ∆ hydrophobicity [kcal/mol]
for z-coordinate (2 and http://blanco.biomol.uci.edu/hydrophobicity_scales.html). The amino acid
identity is indicated by single letter code and the positions are colored according to ∆
hydrophobicity (from violet to red). Hydrophobic side chains I, F, V and L display similar helical
packing and hydrophobicity properties paired with high abundance in in alpha-helical membrane
Figure S2: Structure superposition of PiPT and XylE. (A) Schematic representation of
crystallographic structures of PiPT (PDBid 4J05; gray) and XylE (PDBid 4GBY; rainbow). A
satisfying structure alignment is generated (rmsd achieved: 2.6 Å, number of residues reference
(4GBY): 475, number of residues moving (4J05): 422, number of aligned residues: 346,
sequence identity: 19.9%) where the putative H+-binding-sites (B, blue square) and the substrate
binding-sites (C, red square) superpose. In the detailed representations residue labels of XylE are
shown in green color and residue labels of PiPT are shown in magenta color. Xylose is shown a
transparent shape for better visually.
Figure S3: Conformational changes in the substrate binding site of PepT. (A) Inward-open,
substrate-bound state of PepTGk (pdbID: 4IKZ). (B) Inward-open, substrate-free state of PepTGk
(pdbID: 4IKV). The position of the substrate detected in the substrate-bound state is indicated as
white profile. (C) Inward-occluded conformation of PepTSo (pdbID: 2XUT). The colors of the bars
on the right side indicate the mechanistic affiliation of the respective states with regard to the
mechanistic model shown in Fig. 5.
Figure S4: Orientation of helix-triplets. Helix-triplets from FucP , PiPT, XylE and PepT are
aligned with LacY (helices 1–3, blue; helices 4–6, green; helices 7–9, orange; helices 10–12,
yellow) according to Fig. 2, the order of the helices (roman numerals) is shown according to the
alignment to LacY. The N- and the C-termini of the helix-triplets are indicated by red and blue
rectangles respectively. A red dot marks the N-terminus of the protein and a blue dot marks the
C-terminus. The central middle loop is shown as a green line. Although the orientation of
homologous helix-triplets may be flipped relatively to LacY, The N-, the C-termini and the middle
loop are always on the cytoplasmic side.
Table S1: Reduction matrix for sequence simplification. Grouping of residues based on
reasonable simplification of protein sequence with no interlacing for different levels of reduction
(modified from ref. 2). Letter grouped without spaces represent a reduction clade and are treated
as similar in scoring of sequence alignments.
level score reduction alphabet symbol
0. 20 L I V F Y W M G P A T S C N Q E D H R K *
1. 10 LIV F YW M GP ATS C NQED H RK !
2. 7 LIVF YW M GP ATSC NQED HRK :
3. 4 LIVF YWM GPATSC NQEDHRK .
Equation S1: Similarity score.
Q: similarity score, redValue: score assigned from the reduction matrix (SI Appendix, Table S1),
AAall: number of all residues in the alignment, AALIVF: number of not compared side-chains. For
100% conservation, Q should equal 1.
Equation S2: Functional similarity score.
QAct: functional similarity score, sAA0-25%: number of aligned critical positions in the alignment, AA0-
25%: number of all critical positions in the alignment, sAA25-50%: number of aligned functionally
relevant positions in the alignment, AA25-50%: number of all functionally relevant positions in the
alignment. Critical positions are positions where mutations to Cys cause greater than 50% and
functionally relevant positions are positions where mutations to Cys cause 75% inhibition of the
transport rate with Cys-less LacY (3, 4). For 100% conserved transporters with 100% conserved
functional positions this value, QAct, should equal 1.
LacY / PiPT
^ ^ ^ $ # #$
LacY-A.pdb,_chain_B/1-97 NTNFWMFGLFFFFYFFIMGAYFPFF....PIWLHDINHIS.......... 36
PiPT-c.pdb,_chain_A/306-410 TWNHFRNLLGSMLGWFLVDIAFYGINLNQSVVLAQIGFAGKTGDVYDKLF 50
* . ! .
^ ^ #$ $# ^ ^
LacY-A.pdb,_chain_B/1-97 .KSDTGIIFAAISLFSLLFQPLFGLLSDKLGLRKYLLWIITGMLVMFAPF 85
PiPT-c.pdb,_chain_A/306-410 QLATGNIIVTALGFLPGYYFTLF..LIDIVG.....RKKLQFMGFIMSGL 93
! . !* . . . * * * !.
LacY-A.pdb,_chain_B/1-97 FIFIFGPLLQYN 97
PiPT-c.pdb,_chain_A/306-410 FLAILAGEIDHI 105
$^ $ # $# # # ## $$ $ #^## #$
- -= = = - == =-
LacY-B.pdb,_chain_B/1-83 LVGSIVGGI.YLGFCFNAGAPAVEAFI..EKVSRRSNFEFGRARM..FGCV 46
PiPT-D.pdb,_chain_A/411-499 GKGPLLACFTFMQFFFNFGANTTTFIVAAELFPTRIRAS.AHGISAAAGKC 50
*. . * ** . * . * . .. ! *.
## # # # ^^
LacY-B.pdb,_chain_B/1-83 GWALCASIVGIMFTI.NNQFVFWLGSGCALILAVLLF.F 83
PiPT-D.pdb,_chain_A/411-499 GAILSSLVFNQLKAKIGTSAVLWIFFSTCILGFISTFLI 89
* :! * .
#^ $ $# # ^$ ^ ^ $ $
LacY-C.pdb,_chain_B/1-104 LFRQPKL.WFLSLYVIGVSCT.YDVFDQQFANFFTSFFATGEQGTRVFGYVT 50
PiPT-B.pdb,_chain_A/126-229 WDGNRVLTWITICRVFLGIGIGGDYPMSATVVSDR.ANI.HRRGTLLCFIFA 50
! * . * ..** !
$# $ # $ #$# $^ # # $
- = = - = -
LacY-C.pdb,_chain_B/1-104 TMGE.LLNASIMFFAPLIINRIGGKN.ALLLAGTIMSVRIIGSSFATSALEV 100
PiPT-B.pdb,_chain_A/126-229 NQGWGSFVGSLVTIVTISGFKH..RLKSGHTHDVDKAWRILIGLSLIPAFGT 100
* . . ! ! ! ! ** . .*
LacY-C.pdb,_chain_B/1-104 VILK 104
PiPT-B.pdb,_chain_A/126-229 LYQR 104
# # ^# $^ $ ^# $# # ^ #
= = = = =
LacY-D.pdb,_chain_B/1-89 SALEVVILKTLHMFEVPFLLVGCFKYITSQFEVRFSATIY.LVCFCFFKQ 49
PiPT-A.pdb,_chain_A/30-116 LVLLAGVGFFLDAYDL.FII...NQVAPMLAQVYFPKTGLPAQRQDLMKA 46
. ! . . ! . * *
$ ^ ^
LacY-D.pdb,_chain_B/1-89 LAMIFMSVLAGNMYESIG......FQGAYLVLGLVALGFTLISVFT 89
PiPT-A.pdb,_chain_A/30-116 AANI.GCVVGQVMFGVLGDSFGRKFVYGKELILIIVATIFQM.... 87
* . . * * .
LacY / XylE
^ ^ ^ $ # #$ ^
LacY-A.pdb,_chain_B/1-96 TNFWMFGLFFFFYFFIMGAYFPFF.PIWLHDINHISKSDTGII.FAAISLFS 50
XylE-C.pdb,_chain_A/276-365 GVIVIGVMLSIFQQFVGINVVLYYAPEVFKTLG.AS.TDIALLQTIIVGVIN 50
. * : * !* . .
^ #$ $# ^ ^ $
LacY-A.pdb,_chain_B/1-96 LLFQPLFGLLSDKLGLRKYLLWIITGMLVMFAPFFIFIFGPLLQYN 96
XylE-C.pdb,_chain_A/276-365 LTFTVLAIMTVDKFG.RKPL.QII.GALGMAIGMFSLGTAFYT... 90
** * ** * * ! .
$^ $ # $# # # ## $$ $ #^## #$ #
= = = = - == =
LacY-B.pdb,_chain_B/1-88 ILVGSIVGGIYLGFCF.NAGAPAVEAFIEKVSRRSNFEFGRARMFGCV.G 48
XylE-D.pdb,_chain_A/369-465 GIVALLSMLFYVAAFAMSWG.PVCWVLLSEIF..PNAIRGKALAIAVAAQ 47
. * . * * . .* *!* .
# # # # ^^
= = =
LacY-B.pdb,_chain_B/1-88 WALGASIVGIMFTIN...........NQFVFWL.GSGCALILAVLLFFAKTD 88
XylE-D.pdb,_chain_A/369-465 WL.ANYFVSWTFPMMDKNSWLVAHFHNGFSYWIYGCMGVLAALFMWKFV.PE 97
* . . . * * * .!
#^ $ $# # ^$ ^ ^ $ $ $
= - -
LacY-C.pdb,_chain_B/1-91 KLWFLSLYVIGVSCTYDVFDQQFANFFTSFFAT..GEQGTRVFGYVTTMG 48
XylE-B-coot-0.pdb,_chain_A/125-221 YVPEFVIYRIIGGIGVGLASMLSPMYIAELAPAHIRGKLVSFNQFAIIFG 50
* . . . ! .! . *
# $ # $ #$# $^ # # $
= = - = =
LacY-C.pdb,_chain_B/1-91 ELLNASIMFFAPLIINRI.....GGKNALLLAGTIMSVRIIGSSFATS 91
XylE-B-coot-0.pdb,_chain_A/125-221 QLLVYCVNYFIARSGDASWLNTDGWRYM.FASECIPALLFLMLLYTVP 97
! . ! * ! ! ! ! .
# # ^# $^ $ ^# $# #
= - - = -=
LacY-D.pdb,_chain_B/1-101 GSSFATSALEVVILKTLHM.FEVPFLLVG.C.FKYIT.SQFEVRFSATIY 46
XylE-A-coot-0.pdb,_chain_A/5-110 NSSYIFSITLVATLGGLLFGYDTAVISGTVESLNTVFVAPQNLSESAANS 50
** * . ! . . . . ! ! **!
^ # $ ^
LacY-D.pdb,_chain_B/1-101 LVCFCFF.KQLAMIFMSVLAGNMYESIGFQGAYLVLGLVALGFTLISVFT 95
XylE-A-coot-0.pdb,_chain_A/5-110 LLGFCVASALIGCIIGGALGGYCSNRFGRRDSLKIAAVLFFISGVGSAWP 100
* . . .* ! * . ! . . * .
LacY-D.pdb,_chain_B/1-101 LSGPGP 101
XylE-A-coot-0.pdb,_chain_A/5-110 ELGFTS 106
LacY / PepT
^ ^ ^ $ # #$ ^
LacY-A.pdb,_chain_B/1-96 TNFWMFGLFFFFYFFIMGAYFPFFPIWLHDINHIS.KSDTGIIFAAISLF 49
PepT_4IKZ_D.pdb,_chain_A/1-101 GGLVHPIWLVLSYFIVVLGELCLSPVGLSATTKLAPAAFSAQTMSLWFLS 50
* . * . ! ! !. !
^ #$ $# ^ ^ $
=- = -
LacY-A.pdb,_chain_B/1-96 SLLFQPLFGLLSD.KLGLRKYLLWIITGMLVMFAPFFIFIFGPLLQ...Y 95
PepT_4IKZ_D.pdb,_chain_A/1-101 NAAAQAINAQLVRFYTPENETAYFGTIGGAALVLGLILLAIAPRIGRLMK 100
*. . . ! .. * ! .*
LacY-A.pdb,_chain_B/1-96 N 96
PepT_4IKZ_D.pdb,_chain_A/1-101 G 101
$^ $ # $# # # ## $$ $ #
- = = = - =
LacY-B.pdb,_chain_B/1-83 LVGSIVGGIYLG...FCFNAGAPAVEAFIEKVSRRS...N.FE....... 36
PepT_4IKZ_B.pdb,_chain_A/1-100 MAGHIALAIPGGVAALFVSMALIVLGTGLLKPNVSSIVGDMYKPGDDRRD 50
* . * . ! * * ! .
^## #$ ## # # # ^^
= - = = = =
LacY-B.pdb,_chain_B/1-83 ..FGRARMFGCVGWALGASIVGIMFTINNQFVFWLGSGCALILAVLLF.. 82
PepT_4IKZ_B.pdb,_chain_A/1-100 AGFSIFYMGINLGAFLAPLVVGTAGMKYNFHLGFGLAAVGMFLGLVVFVA 100
. * . * .. * .* .. . .
#^ $ $# # ^$ ^ ^ $ $ $#
= - =
LacY-C.pdb,_chain_B/1-88 LWFLSLYVIGVSCTYDVFDQQFANFFTSF.FATGEQGTRVFGYVTTMGEL 49
PepT_4IKZ_C.pdb,_chain_A/1-102 RVIAYIPLFVASAMFWAIQQQGSTILANYADKRTQLDVAGIHLSPAWFQS 50
* !** . ! ! .! !
$ # $ # $ # $ ^ # # $
- = - = - = -
LacY-C.pdb,_chain_B/1-88 LNASIMFF.APLIINRIG.........G.KNA..LLLAGTIMSVRIIGSS 86
PepT_4IKZ_C.pdb,_chain_A/1-102 LNPLFIIILAPVFAWMWVKLGKRQPTIPQKFALGLLFAGLSFIVILVPGH 100
*. ** ! * * ** !.
LacY-C.pdb,_chain_B/1-88 FA 88
PepT_4IKZ_C.pdb,_chain_A/1-102 LS 102
# # ^# $^ $ ^# $# #
= = - = =
LacY-D.pdb,_chain_B/1-89 SALEVVILKTLHMFEVPFLLVGCFKYITSQ............FEVRFSATIY 40
PepT_4IKZ_A.pdb,_chain_A/1-99 HPKGLFTLFFTEFWERFSYY.GM.RAILVYYMYYEVSKGGLGLDEHLALAIM 50
. . * * ! ! : ! !
^ # $ ^
LacY-D.pdb,_chain_B/1-89 LVCFCFFKQLAMIFMSVLAGNMYESIGFQGAYLVLGLVALGFTLISVFT 89
PepT_4IKZ_A.pdb,_chain_A/1-99 SIYGALVYMSGIIGGWLADRVFGTSRAVFYGGLLIMAGHIALAIPGGVA 99
. * . . . ! . !
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