Background
Genomic cloning has revealed that most of the enzyme
families essential for maintaining cell growth have been
conserved throughout evolution [ 1 ] . However, mammalian
enzymes with different functional activity may have evolved
by combining elements from several bacterial ancestral
genes. Even small proteins may contain several individual
domains that link them to different superfamilies [ 2 ] .
While many endonucleases share a common active site that is
highly conserved across many subfamilies, identifying
residues that control substrate specificity requires
sophisticated analysis that combines both sequence
conservation and structural data [ 3 4 5 ] .
In this paper we distinguish, using a word-based
"molego" approach, structural elements that control
substrate specificity. We postulate here that elements
conserved in all the members of related protein families
dictate common structures and also common "functions",
i.e., individual steps in a complex reaction. Areas that
affect substrate specificity will be less conserved in the
superfamily than they are in subfamilies of enzymes that
catalyze specific activities. We have chosen to illustrate
this approach using the multifunctional family of DNA
repair proteins, the apurinic/apyrimidinic endonucleases
(APEs), which have a clearly defined bacterial ancestor,
E. coli exonuclease III (ExoIII), and
are distantly related to several enzymes with varying
substrate specificity.
APEs are essential for mammalian cell growth and
bacterial survival in the presence of ionizing radiation
and DNA mutagens [ 6 ] . They initiate repair of an abasic
DNA site by cleaving the phosphodiester backbone 5' of the
phosphodeoxyribose. This generates the necessary 3'
hydroxyl group for DNA polymerases (pol β, δ or ε, in
eukaryotes) to insert the correct nucleotide in later steps
in the base excision repair pathway (BER-pathway) [ 7 8 ] .
Recent crystal structures of huAPE1 complexed with DNA
containing an abasic site [ 9 10 11 ] , combined with
sequence analysis and site-directed mutagenesis, have
defined the residues that participate in metal ion based
cleavage of the phosphate backbone of the DNA [ 12 13 14 15
16 17 ] .
Mutations that greatly diminish the enzymatic activity
of huAPE1 do not affect, and may even increase binding to
damaged DNA, while non-specific DNA binding remains low [
16 18 ] . Further, mutations that have little effect on APE
activity in vitro prevent complementation of DNA repair
deficient
E. coli . As seen with other DNA
repair enzymes [ 19 ] , specificity determining residues,
as yet unidentified for APEs, must be distinct from those
involved in phosphorolysis.
To better assess which residues determine specificity,
we assume that functions unique to APEs will be determined
by motifs that are not conserved in a similar fashion in
families with a different activity spectrum of functions.
Besides cleaving the phosphate backbone, to achieve
specificity APEs must coordinate a series of functions,
including: interaction with target DNA in a series of
small, possibly repetitive steps (scanning), locating
damage sites, establishing the transition state complex,
completing the cleavage, re-adjusting the charge status
within the active site, and regulating release of product
after interaction with the next enzyme in the BER pathway [
20 21 22 23 24 25 ] . A finer breakdown of these functions
can be achieved at the molecular level once all the
residues in the reaction mechanism are known. APEs also
have RNase H, 3'-exonuclease, and 3'-phosphodiester
activities that are particularly high in the bacterial
members of the family [ 26 13 ] .
Our web-based MASIA program [ 27 ] was used to rapidly
decompose the sequences of APEs and related protein
families into motifs, areas of significant conservation in
members of identical function, which could then be
correlated structurally using data from crystal structures.
Having determined that 12 motifs were common to all APE1's,
we compared the structure of the subset of these that
occurred in both DNase 1 and synaptojanin, a member of the
IPP family. These shared motifs had a similar 3D structure
in representatives of these functionally diverse families,
and we therefore called these motifs "molegos" (molecular
legos). We then demonstrated that the shared molegos served
a similar role in substrate binding by comparing the DNA
binding profile of huAPE1 with that for the less specific
enzyme DNase 1. The molegos present in both enzymes
interact with target DNA in a similar fashion, while
residues in molegos distinctive for APE1 control
specificity by binding primarily to the bases around the
apurinic site. Matching of molegos, guided by the degree of
conservation of individual residues across the three
families, allowed a better alignment of the individual
secondary structure elements among the proteins than DALI
achieved. This word based, sequence (motif) to structure
(molego) to function method has clear implications for
genomic analysis and template based homology modeling, as
well as immediate application in recognizing specificity
determinants in proteins that share active sites common to
many enzymes [ 28 ] .
Results
Total sequence decomposition of human Ape1 with
MASIA
MASIA identified 12 motifs as conserved in all members
of the APE family (Figure 1and Table 1). As table 1, last
column, illustrates, these motifs include all the
residues known to be essential for DNA cleavage. Most of
the highly conserved (greater than 90%) residues have
been shown by previous mutagenesis studies to affect
activity. The 12 motifs are also structurally conserved,
as demonstrated by the low RMSD values between segments
in the crystal structures of bacterial ExoIII and of
huAPE1. These two proteins are only 26% identical (based
on a DALI, structure based alignment) and most of the
similar segments are contained in the molegos. As the
third column of the table demonstrates, the backbone
deviation of the segments is overall <1 Å and for 5 of
the motifs, <0.5 Å. We have chosen the name "molegos"
for the structural units associated with motifs, which
are presented pictorially in Figures 2and 3. Most of the
DNA and metal ion binding molegos form individual
β-strands at the core of the protein that orient the
absolutely conserved residues toward the substrate, but
several have a helical or hydrogen bonded coil
structure.
The 12 motifs, which account for about half of the
protein, are bridged by areas that vary in the different
members of the APE family. These connecting regions may
account for the differing activities of the bacterial and
mammalian proteins. The longest molego, 7, was broken
down into two areas, with the contiguous region labeled
7a. The first 7 residues of the 7a area molego are quite
similar in the bacterial and mammalian APE. However, the
end is differently conserved in eukaryotes. The
endonuclease activity of DNase 1 is reduced many fold by
integrating this loop from
E. coli exonuclease III, but the
mutant cleaves at abasic sites in DNA with low efficiency
[ 29 ] . Thus additional residues in the APEs control
specificity while still allowing a reasonable rate of
phosphorolytic cleavage.
Finding APE molegos in the DNase 1
superfamily
In an effort to functionally annotate the molegos of
APE1, we next sought to find them in other proteins that
shared some structural similarity to APE. The APEs,
DNase-1 and inositol 5'-polyphosphate phosphatases (IPP)
have been grouped according to the SCOP database [ 30 ]
as the DNase-1 like superfamily. Although DNase 1 has
only 18% overall sequence identity and the IPP domain of
synaptojanin, 14%, to APE1, we could show that most of
the areas of identity were in molegos common to all three
proteins. Motifs in other protein families were
identified by genomic cross-networking with PSIBLAST (see
methods for details). Our analysis identified 5 molegos
that are common to the DNase 1, IPP and APE families,
which roughly correspond to areas of sequence similarity
identified previously [ 31 32 ] . The structural
similarities of molegos 1,2, 7, 11 and 12 (i.e., the
segmental RMSD's) between APE1 and representatives of the
distantly related DNase 1 and IPP families are comparable
to those found between members of the APE family (Figure
4and Table 1& 2).
Common molegos form a similar active site in two
distant relatives
The 12 conserved molegos form the β-barrel core of
huAPE1. The completely conserved residues of huAPE1
concentrate, for the most part, at one end of this
framework to form the metal ion binding active site
(Figure 4). This core is also common to DNase 1 and
synaptojanin(an IPP family member), which share the
functions of metal ion based cleavage of a phosphate
backbone. The shared molegos define an active site
architecture conserved in all three proteins, including
the orientation of the substrate toward the metal binding
site.
Molegos define functional areas common to DNase 1
and APE1
A contact plot of huAPE1 with the DNA in the 1DE8
crystal structure (Figure 5) shows that motifs 1-3,5-8,
and 10-12 all have residues close to the substrate, an
oligonucleotide containing an abasic site (AP-DNA). The
N-terminal motifs 1-3 and 5 bind primarily 5' to the
apurinic site and to the 3' end of the undamaged strand.
The other motifs bind more to the area 3' of the damage
site. Motifs 10 and 12 span both strands of the DNA.
Although motif 12 contains several highly conserved
residues that, according to mutagenesis results (Table 1)
contribute to APE1 activity, only His309 is very close to
the abasic site in the DNA. Molegos 4 and 9 contain no
residues in contact with the DNA or metal ion. Comparing
the binding of APE in a substrate complex (Figure 6,
left) suggests that APE's binding to the 5' end of the
DNA after cleavage (Fig. 5), especially that mediated by
molego 3, is stronger, while the distance from the
protein to the DNA 3' of the cleavage site increases.
The contact plots of APE1 and DNase1 with their
respective substrates (Figure 6) documents that the
similar molegos in the proteins serve similar functions.
The N-terminal 100 residues of both proteins, including
molegos 1 and 2, bind 5' of the cleavage site and to the
3' end of the opposite DNA strand. Molegos 7, 11 and 12
bind to one base 5' and the next base 3' of the cleavage
site in both proteins. Overall, the pattern of protein
contacts to the cleavage site, the area 5' of the
cleavage site, and the 3' end of the opposite strand are
common to both proteins, suggesting that the functions of
forming the substrate complex and the actual
phosphorolysis are similar in both proteins.
While the length of the DNA in both cases is similar,
DNase 1 clearly has less binding to bases opposite and 3'
of the cleavage site. The extensive contacts that APE1
makes to these positions are mediated by molegos it does
not share with DNase 1. Molegos 6, 7a and 10 all have
residues within hydrogen bonding distance of the three
basepairs 3' of the AP-site. This redundancy of binding
to the 3' side is unique to APE, as is its strong binding
to the DNA opposite the abasic site.
The importance of such bonds for activity was shown in
other work, where huAPE1's binding to the DNA backbone is
only inhibited by ethylation of the phosphates two and
three positions 3' to an abasic site [ 18 ] . Mutation of
R177A, at the end of Molego 6, that binds to this region
and to the bases opposite the AP-site had enhanced
activity [ 11 ] , while mutations at W280 (Molego 11) and
F266 (Molego 10) [ 33 ] reduce activity and, in the
latter case, substrate selectivity.
In work from this group that will be described
separately, we used this analysis to generate mutants of
APE1 with altered activity. An alanine substitution
mutant, N226A, of a conserved residue at the end of
molego 7a that forms a hydrogen bond with the second
phosphate group downstream of the abasic site, had
enhanced APE activity but increased Km and Kd values,
similar to an alanine mutant of R177, which binds to the
same site, reported previously [ 34 ] . A combination of
the two mutants, N226A and R177A, substantially reduced
the ability of APE1 to bind to DNA containing an abasic
site (Izumi et al., in preparation). Thus, molegos can
effectively guide the redesign of enzymes to alter
specificity.
Molegos to improve structural alignment
Using molegos may also help in aligning proteins for
template based modeling, by determining the end points of
secondary structure elements in alignments with many gaps
and insertions. According to MASIA analysis, the residues
K/R and DI at the N-termini of motifs 1 and 2 are
absolutely conserved in the three families, APEs, DNase1s
and IPPs. However, matching these conserved residues
between synaptojanin and DNase1 or APE requires a gapping
that would not be consistent with CLUSTALW or a
structural (DALI [ 35 ] ) alignment of these proteins
(Table 2). If the local alignment with synaptojanin is
gapped to align these residues in the three proteins
(Table 2, gapped), the RMSD for the two sections
separated by the gap is much lower than that if one tries
to align the whole ungapped segment. As Fig.
7illustrates, the local environments of both conserved
residue pairs DI and QE are structurally equivalent in
all three proteins, indicating that a motif based
alignment with a two residue gap is correct. The first
two β-strand molegos in synaptojanin are 2 residues
longer than in APE or DNase 1. By regarding these
elements as simple lego style blocks, and recognizing the
connectivity, molego based alignment correctly defined
the changing length of the secondary structure
elements.
Discussion
Is the specificity of APE determined by binding 3'
to an abasic site?
Crystal structure data, coupled with molego analysis,
outlined the areas of APE1 that distinguish its mode of
DNA binding from the less specific DNase 1. Contact maps
(Figure 6) illustrate how the conserved motifs direct DNA
binding in the distributive (i.e., rapidly releasing
substrate/product), relatively non-specific DNase1 as
opposed to the processive, highly specific huAPE1. Both
enzymes cleave only one DNA strand in a duplex and
bacterial Xth cleaves ssDNA containing an abasic site [
42 ] . The additional contacts huAPE1, compared to DNase
1 (Figure 6), makes 3' to the damage site and to the
opposite strand lower its turnover rate and its potential
to cleave normal DNA. The residues contacting the region
3' to the abasic site come from three different uniquely
conserved areas of APEs (molegos 6,7a, 10) as well as 11,
a molego that is similar to that in DNase-1. These
observations, coupled with DNA ethylation data [ 18 ] ,
indicates that 3' binding is a key element in specific
recognition by APEs.
This is confirmed by site directed mutagenesis
studies. Of the four protein areas that bind to the DNA
3' of the abasic site, mutating F266 (molego 10) or W280
(middle of molego 11) decreases APE activity [ 33 ] . The
F266 mutation is particularly interesting, as the mutants
at this position had reduced substrate specificity and
enhanced 3'-exonuclease activity. However, an R177A
mutant had
enhanced APE activity [ 11 ] ,
as do mutations at N226 (Izumi et al., in preparation).
Combining these mutations however greatly decreases
substrate binding (Izumi et al., in preparation). The 3'
approach to the DNA [ 34 ] and the wide area covered by
the protein on both sides of the abasic site [ 14 ] are
both consistent with the need to hold the product until
the correct polymerase moves in 5' to 3' to complete the
repair [ 25 ] . This implies that the mammalian enzyme
has evolved to be processive, to facilitate more
efficient functioning of the overall BER pathway, and may
not be optimized for simple catalysis. Processivity is an
important facit of the activity of enzymes that function
in complex pathways [ 43 ] . Reduced processivity may
explain, for example, the repair deficits in Xeroderma
pigmentosum (XPA) cells [ 44 ] . Our molego approach
provides a basis for exploring the role of segments of
the protein in its functions, rather than relying only on
data from missense mutations.
Using molegos to detect structural and functional
homologues
We have demonstrated here the derivation and uses of
molegos for analyzing the specificity of enzymes, based
on those derived from the APE family. The methodology can
be used to complement searches with programs such as
PSIBLAST and PROSITE [ 41 ] to detect distantly related
functional or structural homologues in sequences revealed
by genome sequencing. PSIBLAST searches often reveal
areas of local similarity in proteins that have no
significant overall sequence identity. Molego analysis
could be useful to analyze the significance of such
findings. The combined sequence and structure definition
makes molegos more flexible for defining shared protein
elements than methods such as PROSITE that require a
strict one-dimensional definition. An improved motif
definition method, based on physical property similarity
[ 42 ] , which has been incorporated into our MASIA tool]
also promises to enhance the usefulness of the method.
This may eventually lead to a method to find functional
relationships between proteins with even lower overall
sequence similarity.
Another potential area for applying the molego
approach is in homology modeling. Molegos may prove
useful in to check alignments for template based modeling
of homologues with low identity (Table 2and Figure 7), if
the "anchoring" residues are conserved in sequence or
property across the members of both subfamilies. Our
molego approach is closest in principle to that of the
ROSETTA program [ 45 ] whereby the latter seeks only to
connect structure, not function, to a sequence element.
We are currently testing the usefulness of the molego
approach in modeling in the CASP5 competition.
Conclusions
The MASIA program can parse sequences into discrete
blocks of significant conservation. The motifs identified
in the APE family could be structurally annotated using
crystal data to derive molegos, words in the protein
sequence that correlate with structural elements. These
molegos could in turn be functionally annotated by
comparing the DNA binding profile of APE1 with that of the
less specific nuclease DNase 1. This analysis indicated
that residues binding 3' to the site of phosphorolytic
cleavage control the substrate specificity of APE1. These
results indicate that molegos can provide a useful basis
for identifying specificity determining regions in enzymes
with similar active sites but different activity spectra [
46 28 3 ] . Site directed mutagenesis based on these
results can define the function of the unique elements of
the APEs, and aid in the design of enzymes with altered
specificity.
Materials and Methods
Sequence alignment
A BLAST [ 47 ]
http://www.ncbi.nlm.nih.gov/BLAST/search of the
"non-redundant" protein database using the whole sequence
of human APE1 yielded over 100 related sequences. Some
sequence entries represented the same protein, called by
different names or isolated in different screens,
including many entries for huAPE1,
Drosophila Rrp 1 protein (~40%
identical to the mammalian APE1 in the C-terminal third
of the protein), Xth from
E. coli , and counterparts of this
and exodeoxyribonuclease (exo A) sequences from many
bacteria, which are about 25% identical to mammalian
APE1. The mammalian sequences are highly conserved, with
only 6 non-conservative residue variations between the
human and murine sequences, 5 of which occur in the
apparently unstructured N-terminus. Several proteins with
more distant relationship to APE1, such as mammalian and
yeast APEIIs, and the CRC protein from
Pseudomonas , which has no APE
activity [ 48 ] were in the BLAST list, but were not used
for this analysis. To derive functional motifs, the BLAST
list was culled to 37 unique sequences with identity
ranging from 25% to 98%. These were aligned using the
default parameters of CLUSTALW [ 49 50 ]
http://www2.ebi.ac.uk/clustalw/. Sequences were the APE1
protein from human, bovine, monkey, rat murine,
Arabidopsis thaliana (Mouse-ear
cress)
Dictyostelium discoideum (Slime
mold),
Schizosaccharomyces pombe (Fission
yeast),
Caenorhabditis elegans ,
Saccharomyces cerevisiae (Baker's
yeast),
Thermoplasma acidophilum ,
Neisseria meningitidis .
Methanobacterium
thermoautotrophicum ,
Leishmania major ,
Trypanosoma cruzi ,
Coxiella burnetii ; the Rrp1
protein of
Drosophilia ; exonuclease III from
E. coli ,
Bacillus subtilis ,
Mycobacterium tuberculosis ,
Haemophilus influenzae ,
Salmonella typhimurium ,
Helicobacter pylori ,
Rickettsia prowazekii ,
Archaeoglobus fulgidus ,
Actinobacillus
actinomycetemcomitans ,
Streptomyces coelicolor ,
Synechocystis sp. PCC 6803,
Haemophilus influenzae ;
Exonuclease A from
Steptococcus pneumonia ,
Treponema pallidum ,
Borrelia burgdorferi .
Plasmodium falciparum . Different
set conditions and sequence lists were tested in CLUSTALW
for their effect on alignment and subsequent motif
definition with MASIA. Areas peripheral to the
endonuclease domain, such as the 50 amino acid mammalian
and the 428 residue
Drosophila Rrp-1 N-terminal regions
were eliminated to improve the consensus.
Identification of motifs using MASIA
Motifs were identified in the aligned sequences using
the MASIA consensus macro
http://www.scsb.utmb.edu/masia/masia.html. Motifs start
when at least 3 of 4 consecutive positions are more than
40% conserved according to the dominant criterion [ 51 ]
, and extend until at least 2 positions in a row are less
than 40% conserved. To allow for mistakes in the
alignment of all the sequences, essential residues are
those >90% conserved by MASIA criteria over all
sequences in the alignment.
Genomic cross-networking with PSIBLAST
A PSIBLAST search, using huAPE1 as the founder
sequence, with an e-value of 0.1 per iteration, did not
converge after 6 iterations, but few new sequences were
added in the last 2 cycles. Searches with an e-value of
0.01/iteration had similar results, but members of
several families were not included until later cycles.
Members of the DNase 1, LINE-1 repeats, inositol
5'-polyphosphate phosphatase, Nocturnin, CCR4, cytolethal
distending toxin, neutral sphingomyelin
phosphodiesterase, and amino acid methyltransferase
families were found with expectation values of 10 -4or
less to be significantly similar to APE1. To determine
the presence of motifs in these relatives, a CLUSTALW
alignment of at least 5 representatives of a protein
family was prepared and analyzed with MASIA for
significant areas of conservation. In some cases,
alignments taken from literature references (e.g., for
IPPs [ 32 ] ) were used to confirm MASIA results. The
motifs common to these families were compared with the
APE motifs of Table 1. Criteria for inclusion (presence
of motif) included conservation of residues >90%
conserved (side chains shown in blue in the tables) and
patterns of polarity (as determined with a macro included
in the MASIA packet as a user specified feature).
Molego building and comparison
The drawings of "molego blocks" and structures
(Figures 2, 3, 4and 7), contact plots of protein/DNA
(Figures 5, 6), and calculation of the RMSD between
similar segments (Table 2) were done with MOLMOL
http://www.mol.biol.ethz.ch/wuthrich/software/molmol/ [
52 ] . The RMSD values in Table 1were calculated using
SwissPDB viewer and the "fit selected residues"
option.
Authors' contributions
CHS developed the molego concept, performed the sequence
analysis and prepared the manuscript. NO analyzed the
structural properties of the molegos, and prepared figures
2, 3, 4, 5, 6, 7. TI prepared mutants of human APE1 protein
to test the conclusions of this paper, and he provided
background and list of mutations affecting APE function. WB
designed and developed the MASIA program and the modular
analysis approach to enzyme functions.
Additional File 1
Sequence alignment and MASIA printout for
the APE family The original alignment used for the APE
family and the MASIA consensus macro output. Note that the
A. thaliana sequence is consistently
an outlier in the file, due to the CLUSTAL-W misalignment
of the nuclease area of the protein when the N-terminal
sequence is not removed. The real position of the motifs in
the
A. thaliana sequence are
underlined.
Click here for file
