11 Evolutionary Use Of Domain Recombination A Distinction Between

11 evolutionary use of domain recombination: a distinction between membrane and soluble proteins ======================================

11
Evolutionary Use of Domain Recombination: A Distinction Between
Membrane and Soluble Proteins
===============================================================
Yang Liu, Mark Gerstein, Donald M. Engelman
Department of Molecular Biophysics and Biochemistry, Yale University,
P.O. Box 208114, New Haven, CT, 06520-8114, USA
To whom reprint requests and page proofs should be addressed:
Donald M. Engelman
Department of Molecular Biophysics and Biochemistry, Yale University,
P.O. Box 208114, New Haven, CT, 06520-8114, USA
Phone: 203 432 5601; Fax: 203 432 6381;
E-mail: [email protected]
Abstract
Soluble proteins often contain multiple structural domains, which are
shuffled or recombined to gain new functions in the course of
evolution. We examined integral membrane proteins for evidence of this
mechanism using a classification of polytopic transmembrane domains.
Surprisingly, in contrast to the situation in aqueous solution, we
found that recombination of structural domains is not common inside
membranes: the majority of integral membrane proteins contain only a
single polytopic membrane domain. We suggest that non-covalent
oligomeric associations, which are common in membrane proteins, may
provide an alternative source of evolutionary diversity in this class
of proteins.
Introduction
Protein domains are often mixed to facilitate evolution, usually by
recombination events that place them in single polypeptides (1-4).
Proteomes from archae, prokarya, and eukarya were studied using a
structure-based classification (SCOP) (5), and it was found that a
large majority of domains (approximately 65% in prokarya and
approximately 80% in eukarya) are combined with other domains (6).
Thus, evolution appears to use recombination of domains to generate
new protein structures and functions. However, the structural database
is overwhelmingly biased in favor of soluble proteins, raising the
question of whether the process of domain recombination is also used
inside membranes. Using our classification of polytopic trans-membrane
domains into ~650 families (7), we examined 26 proteomes, and found
that mixed domain proteins are much less abundant inside membranes
than in the aqueous regions of a cell. We argue that the constraints
of the membrane environment, which have been previously noted (8,
9), favor oligomerization, so that covalent links may not be required
for domains to recombine to gain new functions during evolution.
Results
Using sequence data from 26 genomes (8 in archaea, 14 in prokarya, and
4 in eukarya), membrane domains with two or more putative
transmembrane helices (10) were classified into families. Here, we
use the word “domain” to designate a protein with more than one
transmembrane helix, in distinction from the occasional use of the
word to note the independent stability of single helices (9, 11).
Popot (personal communication) has suggested that a useful distinction
can be made between folding domains, which might be single helices,
and functional domains, which would usually require multiple helices.
Our classification was based in part on the Pfam assignments (12)
and in part on clustering by sequence similarities (7). Most (95%)
polytopic membrane domains defined in the families have relatively
short loops (<80 residues) between transmembrane helices. To be
counted as a polytopic domain family, at least four members must be
present. Approximately 650 families were identified, corresponding to
approximately 61% of all predicted integral membrane protein domains.
Because they are the best defined, we chose to examine the cases in
the Pfam-A classified families (see Fig. 1A), and found that most
integral membrane proteins (~78% for archaea and prokarya; ~67% for
eukarya) contain only a single classified membrane domain. It follows
that the level of transmembrane domain recombination in membrane
proteins is less than 33%. Thus, membrane proteins do not exploit
domain recombination to such a large extent as soluble proteins do.
The relative paucity of domain combinations within integral membrane
proteins might be understood as arising from the two-dimensional
structure of the phospholipid bilayer, which facilitates domain
interaction without covalent linkages. Membranes restrict volume,
translational freedom, and rotational freedom of proteins so that the
entropic penalty for oligomerization is reduced. It is notable that
the known membrane protein structures overwhelmingly consist of homo-
and hetero-oligomeric associations (13). Figure 2 shows a
cross-section of cytochrome C oxidases (from bovine heart
mitochondria) (14), photosynthetic reaction center (from
Rhodopseudomonas virdis) (15), and cytochrome bc1 complexes (in
bovine heart mitochondria) (16) at the midplane of the bilayer,
revealing that the identity of individual subunits cannot be seen in
the structure: inspection of the gray representation does not lead to
the identities color-coded in the other view.
Further support for the idea that oligomers emerge as a consequence of
the membrane environment can be found in “split protein” experiments,
where polytopic membrane proteins expressed as fragments are observed
to associate and function (see, e.g., (17); for review see (13)).
The same kind of behavior has been documented in vitro, using
fragments of proteins (18, 19) (20, 21). Separate evolution of
subunits that associate has also been observed (22). That fragments
can re-associate and function argues that the covalent linkage between
them, while perhaps adding stability and/or control of expression, is
not essential.
Of the membrane proteins containing more than one domain, many appear
to have resulted from domain duplication, containing two or more
identical Pfam-A domains (Fig. 1B) (see, e.g., (23)). Eukaryotes
have a higher incidence (~16% on average) of integral membrane
proteins with two or more duplicated domains than do prokarya or
archaea (~9%). Figure 1B lists the most commonly duplicated domains in
integral membrane proteins in the genomes. An interesting observation
is that the 7-TM chemoreceptors and 7-TM rhodopsin families have high
occurrences (48 and 46) and most of them occur in C. elegans (48 and
32). Knowing that C. elegans has an exceptionally large number of 7-TM
receptors and rhodopsin-like membrane proteins (7, 24), it may be
that the duplications imply possible functional relations between
homologous 7-TM domains. This observation is also supported by the
idea that dimerization of G-protein-coupled receptors may be important
for their functions (25).
By contrast with the paucity of covalent combinations of transmembrane
domains, combinations between soluble domains and membrane domains are
frequently observed. We analyzed the membrane proteins having only one
membrane domain to see how many had flanking soluble domains (Fig.
1C). We found that archaea and prokarya have a much larger proportion
(~34% and 24%, respectively) of single-domain membrane proteins
without flanking soluble domains than eukarya (~7%). Consistent with a
previous study of soluble proteins (6), this observation indicates
that genetic recombination can happen for membrane protein genes in a
similar fashion to soluble ones. That the membrane portions do not
show such recombination with each other must then reflect different
constraints.
Another similarity shared by membrane and soluble proteins is the
distribution pattern of protein domain families in the three kingdoms
(Figure 3). Based on previous analysis (7), using just the Pfam-A
families, we found that the 26 proteomes used in this study consist of
1922 soluble domain families and 214 polytopic membrane domain
families. The soluble proteins have almost 10 times more families than
integral membrane proteins, suggesting a higher diversity of structure
for proteins when the membrane constraints are absent. On the other
hand, the proportions of the common and unique families in the three
kingdoms are similar between membrane and soluble proteins, implying
that a similar evolutionary process is shared by these two kinds of
proteins.
Discussion
Our survey of domain combinations in the helical, transmembrane parts
of membrane proteins reveals that most have only one membrane domain.
Either the required functional diversity is much less for membrane
proteins or they may exploit a different strategy to attain diversity
in evolution. The latter possibility is supported by the observation
of a widespread occurrence of membrane protein oligomers, by studies
of split membrane proteins, and by the argument that oligomer
formation is facilitated by the constraints of the membrane bilayer.
Since the same constraint would not apply if one of the domains were a
soluble domain, it is reasonable to find that covalent links are
frequently used between soluble protein domains and membrane domains.
A challenge for future work will be to document the extent to which
alternative oligomerization (the degree to which a given domain may
participate in different oligomeric complexes) may provide an
evolutionary mechanism.
Acknowledgement
MG thanks an NIH grant (GMS4160-07) for financial support. YL was
supported by an NLM postdoctoral fellowship (T15 LM07056).
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Figures
Figure 1. Domain combination of polytopic membrane domains in genomes
(A) The green bars represent the percentage of classified membrane
proteins by Pfam-A that consist of only one polytopic membrane domain,
and the light green bars indicate the percentage of classified
membrane proteins that consist of duplicated polytopic membrane
domains. The archaea group includes Archaeoglobus fulgidus, Aeropyrum
pernix K1, Halobacterium sp., Methanococcus jannaschii,
Methanobacterium thermoautotrophicum, Pyrococcus abyssi, Pyrococcus
horikoshii, and Thermoplasma acidophilum; the prokarya group includes
Aquifex aeolicus, Borrelia burgdorferi, Bacillus subtilis, Chlamydia
pneumoniae strain AR39, Chlamydia trachomatis, Escherichia coli strain
K12, Haemophilus influenzae, Helicobacter pylori strain 26695,
Mycobacterium tuberculosis, Mycoplasma genitalium, Mycoplasma
pneumoniae, Rickettsia prowazekii, Synechocystis sp, and Treponema
pallidum; and the eukarya group includes Saccharomyces cerevisiae,
Drosophila melanogaster, Caenorhabditis elegans, and Arabidopsis
thaliana. Notes on the assignment strategy: ~65% of the assigned
membrane proteins had Pfam-A matches. Pfam-B and the clustered
families were excluded, as they are not as carefully classified as
Pfam-A families. Integral membrane proteins that contain only one
classified membrane domain with no more than one extra TM-helix were
considered to be single membrane domain proteins; otherwise, they were
considered to be multiple membrane domain proteins. (The Pfam
classification does not always consider TM-helix regions). The orange
bars indicate the percentage of single domain soluble proteins based
on the classification of Pfam-A, which can have up to 30 residues next
to their Pfam-A domains.
(B) The table shows the Pfam-A membrane-protein families that occur
most often in tandem duplicated fashion. It ranks the families by the
number of sequences where they are found more than once in a given
gene.
(C) The plot shows the percentage of classified single domain membrane
proteins lacking a soluble domain. The single domains proteins have no
more than 30 residues flanking regions next to the membrane domains.
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Figure 2. Helix interactions in the membrane midplane
A 5-residue section is defined at the apparent center of the membrane
lipid bilayer (inferred from the hydrophobic exterior) and helix
positions are indicated. The grayscale image emphasizes that the
subunits shown in the colored image cannot be inferred from helix
relationships.
Figure 3. Protein domain families shared between the archaea,
prokarya, and eukarya kingdoms
This figure shows the distributions of Pfam-A families in soluble and
membrane proteins among the three kingdoms. The common families shared
by the three kingdoms represent 24% for soluble proteins and 28% for
membrane proteins; while the unique families represent 7%, 24%, and
41% for soluble proteins and 7%, 22% and 33% for membrane proteins in
archaea, prokarya, and eukarya respectively.