Queiroz et al.

BMC Bioinformatics (2020) 21:143

https://doi.org/10.1186/s12859-020-3474-1

RESEARCH ARTICLE Open Access

ppiGReMLIN: a graph mining based

detection of conserved structural
arrangements in protein-protein interfaces
Felippe C. Queiroz1* , Adriana M. P. Vargas2 , Maria G. A. Oliveira2,3 , Giovanni V. Comarela4 and Sabrina A.
Silveira1,5

*Correspondence:
[email protected] Abstract
1
Department of Computer Science, Background: Protein-protein interactions (PPIs) are fundamental in many biological
Universidade Federal de Viçosa, Av
Peter Henry Rolfs, Viçosa, MG, Brazil
processes and understanding these interactions is key for a myriad of applications
Full list of author information is including drug development, peptide design and identification of drug targets. The
available at the end of the article biological data deluge demands efficient and scalable methods to characterize and
understand protein-protein interfaces. In this paper, we present ppiGReMLIN, a graph
based strategy to infer interaction patterns in a set of protein-protein complexes. Our
method combines an unsupervised learning strategy with frequent subgraph mining
in order to detect conserved structural arrangements (patterns) based on the
physicochemical properties of atoms on protein interfaces. To assess the ability of
ppiGReMLIN to point out relevant conserved substructures on protein-protein
interfaces, we compared our results to experimentally determined patterns that are key
for protein-protein interactions in 2 datasets of complexes, Serine-protease and BCL-2.
Results: ppiGReMLIN was able to detect, in an automatic fashion, conserved structural
arrangements that represent highly conserved interactions at the specificity binding
pocket of trypsin and trypsin-like proteins from Serine-protease dataset. Also, for the
BCL-2 dataset, our method pointed out conserved arrangements that include critical
residue interactions within the conserved motif LXXXXD, pivotal to the binding
specificity of BH3 domains of pro-apoptotic BCL-2 proteins towards apoptotic
suppressors. Quantitatively, ppiGReMLIN was able to find all of the most relevant
residues described in literature for our datasets, showing precision of at least 69% up to
100% and recall of 100%.
Conclusions: ppiGReMLIN was able to find highly conserved structures on the
interfaces of protein-protein complexes, with minimum support value of 60%, in
datasets of similar proteins. We showed that the patterns automatically detected on
protein interfaces by our method are in agreement with interaction patterns described
in the literature.
Keywords: Protein-protein interaction, Data mining, Interaction patterns

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Queiroz et al. BMC Bioinformatics (2020) 21:143 Page 2 of 25

Background
Protein-protein interactions (PPI) are fundamental in many biological processes, includ-
ing metabolism, information processing, decision making, transport, and structural
organization. They are essential on the understanding of cellular physiology and consti-
tute intricate networks of interactions which produce highly organized, dynamic cellular
systems [1, 2].
There are a variety of online resources, such as databases and functional genomics net-
works, devoted to protein interactions. Some databases, such as HINT [3], BioGRID [4]
and APID [5], build genomic networks that cover protein interactions experimentally
determined. Other resources, such as ConsensusPathDB [6], IMP [7], IID [8], STRING
[9], FunCoup [10] and GeneMANIA [11], include not only experimentally determined
protein interactions but also predicted ones (coupled with quality or confidence scores)
in an attempt to increase coverage.
These resources support the process of knowledge discovery on protein interactions.
In this context, Li and colleagues devised, in [12], a human protein-protein interaction
network that enabled to interpret approximately 4,700 genomes and genes involved in
autism. The interactions between proteins were considered at the molecular level and
used to study the human interactome. This kind of study is relevant because despite
being feasible to experimentally test large sets of proteins for thousands of interac-
tions, only relatively small portions of the interactome of many organisms is elucidated
by recent screens for protein-protein interactions (for instance, 4-22% of the human
interactome [13, 14]). Thus, the computational integration of heterogeneous databases,
involving different organisms and different interaction experiments produce protein
networks that help to increase coverage of interactomes and improve the quality of
annotations.
Another interesting example of knowledge discovery on protein interactions is the focus
on understanding the potential of PPIs as targets, which has been addressed by many
studies as [15–19]. Here we mention a few interesting findings on this topic. Protein–
protein interactions are involved in the regulation of biological systems and can thus be
implicated in a number of diseases, which makes PPIs potential targets of great therapeu-
tic interest. PPIs are attractive yet challenging pharmaceutical targets. The identification
of inhibitors for protein interactions is considered challenging due to the shallow and
extended nature of PPI interfaces [20]. Also, in the interaction between two peptides there
is not a well defined binding site and the peptides involved can be intrinsically disordered
when not in complex [19]. Compared to classical protein-ligand interactions (PLI), that
target one large pocket, PPI inhibitors are smaller in volume and tend to target several
pockets [21]. The development of PPI inhibitors aims to mimic peptides, using mimetics
that are peptide or non-peptide based [22].
In this work, we are particularly interested in protein-protein interactions in a fine
degree of granularity, more specifically, in the residue and even atom level. In this con-
text, Khashan and colleagues [23] proposed SPIDER, a scoring function for docking
experiments based on frequent interaction patterns between residues in protein-protein
interfaces. It uses a graph representation of interactions based on contact information at
the residue level along with Almost-Delaunay tessellation to produce a set of graphs rep-
resenting the interface. Finally, graph mining techniques were employed over the set of
graphs in order to detect frequent patterns of interacting residues.
Queiroz et al. BMC Bioinformatics (2020) 21:143 Page 3 of 25

Morozova and colleagues [24], in contrast, focused on patterns between protein-RNA

interactions. In this case, structural information from protein-RNA complexes was used
to unveil patterns in nucleoside binding pockets. A three-dimensional superimposition
strategy was devised based on the interatomic non-covalent interactions between each
RNA base and protein structures obtained from PDB. The computation of interactions
was performed considering pyshicochemical properties of the atoms and a distance cut-
off. The method was able to single out frequent interactions in nucleoside structure that
are discriminant to the binding of each base.
Melo and colleagues [25], in turn, proposed a method based on contact map matching
to uncover relevant patterns in protein-protein interactions. The algorithmic approach
was based on inter-chain contact maps of protein residues and used image processing
to detect conserved interactions in protein-protein interfaces. The authors were able to
identify important contacts in complexes for a set of protein families.
A variety of methods were devised to characterize, understand and detect patterns
on protein-protein interfaces. Nonetheless, in spite of the relevant contributions of the
majority of the works, strategies that depend on multiple structural superimposition
might be prohibitively expensive for large scale processing. As the amount of biologi-
cal data has been growing in a fast pace, scalable techniques are pivotal to perform such
tasks in a real-world scenario. Furthermore, strategies that work at the residue granular-
ity might not reveal the specific details involved in the molecular recognition process that
takes place on protein-protein interfaces.
To overcome these challenges, this article proposes ppiGReMLIN, a graph based strat-
egy to detect conserved structural arrangements on protein-protein interactions. By
structural arrangements, we consider not only specific interacting residues in PPIs, but
more general biding motifs that may disclose more information on how interactions
occur. Protein-protein interfaces are modeled as graphs in which atoms and non-covalent
interactions represent nodes and edges respectively. Nodes and edges are labeled accord-
ing to their physicochemical properties and distance criteria. The resulting graph dataset
is encoded as feature vectors, which serve as input for a clustering analysis. The result-
ing set of clusters go through a frequent subgraph mining (FSM) to reveal substructures
that are conserved across the whole dataset of protein-protein interactions. ppiGReMLIN
requires a set of protein-protein complexes involving proteins of interests and is derived
from GReMLIN (GRaph Mining strategy to infer proten-Ligand INteraction patterns)
[26], which is a graph-based strategy successfully used to infer patterns in protein-
ligand interactions. Our method does not rely on sequence alignment nor structural
superimposition, and can be used in large-scale datasets of protein interactions.
ppiGReMLIN is based on ideas developed in some of our previous works, concerning
the defense of plants against insects and pathogens. The insect Anticarsia gemmatalis
Hübner is a pest that attacks soybean. The plant, when injured by this insect, produces
the Kunitz trypsin inhibitor (KTI), which impairs the process of proteases degradation in
the caterpillar gut [27, 28]. Inspired by this natural inhibitor produced by soybean, we are
interested in proposing peptides or mimetic peptides to inhibit the proteases of the insect
gut, that is, to carry out the ecological control of this pest insect. We used to perform the
peptide design manually, with the support of some bioinformatics tools. ppiGReMLIN
aims to provide the conserved substructures that should be used in the peptide design
process, so that it is performed in a guided way, resulting in peptides or mimetic peptides
Queiroz et al. BMC Bioinformatics (2020) 21:143 Page 4 of 25

that can potentially inhibit proteases of the Anticarsia gut. It is important to note that our
method is not specific to soybean and its insect pest Anticarsia gemmatalis Hübner and
can be used in other datasets involving protein-protein complexes.

Methods
This section details ppiGReMLIN, our method to infer conserved structural arrange-
ments on protein-protein interface at the atomic level. We describe the datasets, the
problem modeling, the data mining tasks, and the evaluation strategy. Figure 1 presents
the workflow that outlines our method.
Interactions on the protein-protein interface are computed for a set of complexes at
atomic level according to physicochemical properties of atoms and distance criteria in
the Data acquisition and modeling block (Fig. 1a). The Clustering analysis block (Fig. 1b)
takes as input a set of graphs that represent the protein-protein interfaces and segments
them in groups containing similar graphs to mine frequent substructures in the next
block. The resulting clusters serve as input for the Conserved substructure mining block
(Fig. 1c), where a FSM task is performed to search for conserved substructures in each
group.

Datasets
We instantiated our strategy with two main datasets of protein-protein complexes
obtained from PDB. Serine protease dataset is composed of trypsin and trypsin-like pro-
teins coupled with some peptide inhibitor and the BCL-2 dataset is composed of protein
complexes belonging to the BCL-2 family.

Serine protease dataset

Anticarsia Gemmatalis (AG) is one of many defoliating caterpillars affecting crop yields
in agriculture. In north and south America, it infests mainly soybean, causing damage to
plants, and potentially destroying entire crops [29]. Agrochemicals are the most sought
after solution to control the population of these caterpillars; however, its use in agricul-
ture is highly debated nowadays due to various issues concerning its toxicity not only to
the environment, but also to human health. In addition, the employment of these prod-
ucts may cause the surge of other resistant populations of new parasites, which requires
the use of even stronger products or higher dosages. One alternative method to circum-
vent the aforementioned issues consists in the inhibition of the digestive proteases in the
caterpillar, which was shown to reduce its growth rate and survival ratio [30, 31].

Fig. 1 ppiGReMLIN workflow. The workflow is composed of three blocks: Data acquisition and modeling;
Clustering analysis; and Conserved substructure mining. Rectangles indicate processing steps; ellipsoids
denote output files; and hexagons represent input files or parameters
Queiroz et al. BMC Bioinformatics (2020) 21:143 Page 5 of 25

The basis to construct the Serine protease (SP) dataset was a protease sequence from
caterpillar digestive system sequenced in our previous work [32], available at GenBank
[33]. We searched the Protein Data Bank (PDB) [34] for proteins with at least 30%
sequence similarity using our protease sequence as query. Then, these PDBs were filtered
resulting in structures containing a protease chain complexed with some other protein
or peptide. This process resulted in 93 PDB entries, which contain mainly serine pro-
teases in complex with some peptide inhibitor. The similarity between inhibitors was not
taken into account because we are interested in detecting protein-protein patterns that
characterize the caterpillar protease inhibition process.

BCL-2 dataset
The BCL-2 proteins are key regulators of programmed cell death, acting in the regulation
of cytochrome c release on the mitochondrias [35, 36]. They consist in a set of proteins
that share homology with the BCL-2 (BH) domains (1-4), being divided into two main
sub-groups: the anti-apoptotic proteins and the pro-apoptotic proteins. The former ones
are composed by BH domains 1-4, which act preserving the outer mitochondrial mem-
brane integrity by inhibiting its pro-apoptotic counterparts. The pro-apoptotic ones, on
the other hand, may have multiple BH domains (such as BAK, BAX, BOK) or just a single
BH3 domain (such as BIM, PUMA, NOXA, BID, etc.) Ultimately, the cell status depends
on the balance of the levels of these proteins, whose expression can be modulated by
means of cell signaling to tip the equilibrium towards survival or death [37]. Indeed,
apoptosis has been established as a critical tumor suppression mechanism [38]. Also, a
cell’s ability to evade apoptosis alongside with oncogenic mutations that deregulate cell
growth and cell cycling greatly enhance tumorigenesis, therefore characterizing evasion
from apoptosis as one of the ‘Halmarks of Cancer’ [39, 40].
For this dataset, we queried PDB for proteins with a minimum of 30% sequence similar-
ity with the structure of Mcl-1 (extracted from pdb id 2KBW), an anti-apoptotic human
protein of the BCL-2 family. Results were then filtered down to structures containing the
query sequence target protein complexed with some other protein. In this proccess, 72
PDB entries were found, which composes our BCL-2 dataset.

Modeling PPIs as graphs

We computed protein-protein interactions at the atomic level by using a cutoff based
strategy (Fig. 1a). Here we use interactions and contacts as synonyms. For two different
atoms, i and j, in the protein-protein interface, we say that i is in contact with j if j is inside
of a sphere centered in i with radius r, called cutoff [41, 42].
In our modeling, nodes represent atoms and edges represent interactions between
them. We consider only those interactions that connect atoms from different protein
chains, which we name chains A and B. Therefore, we have a bipartite graph G(P, I, E)
whose vertices can be segmented in 2 disjoint sets, P (nodes from A) and I (nodes from
B), such that every edge in E connects a vertex in P to a vertex in I.
Nodes were labeled according to their physicochemical properties as acceptor (ACP),
aromatic (ARM), donor (DON), hydrophobic (HPB), negative (NEG) or positive (POS) as
in [26, 43–46]. Edges were labeled as aromatic, hydrogen bond, hydrophobic, repulsive
and salt bridge, based on the physicochemical properties of their atoms and on a distance
criteria. Table 1 provides the distance criteria considered for each interaction type.
Queiroz et al. BMC Bioinformatics (2020) 21:143 Page 6 of 25

Table 1 Physicochemical properties of atoms and distance criteria (in Å) to compute interactions
Distance
Interaction type Atom types
MIN MAX
Aromatic stacking 2 aromatic atoms 1.5 3.5
Hydrogen bond 1 acceptor and 1 donor atom 2.0 3.0
Hydrophobic 2 hydrophobic atoms 2.0 3.8
Repulsive 2 atoms with the same charge 2.0 6.0
Salt Bridges 2 atoms with opposite charge 2.0 6.0

Some atoms are associated with more than one label due to their ability to interact in
different ways with other atoms. For instance, nitrogen atoms ND1 and NE2 in a histidine
residue can be labeled aromatic, positive, donor and acceptor according to our criteria. As
a consequence of this manifold aspect of node labels, multiple edges with different labels
can also be attributed to a single pair of nodes. Thus, we modeled interaction graphs as
multigraphs in order to capture these physicochemical aspects of protein interactions.
However, its important to notice that not all node labels were attributed to the nodes.
After computing interactions, only those node labels that composed interactions with
other nodes were considered as final labels for the respective nodes in the graph.
Finally, for each complex (PDB entry), we computed the connected components, which
serve as input data for the clustering analysis and FSM of ppiGReMLIN. So, a graph in
our method corresponds to a connected component.

Clustering analysis
A clustering analysis (Fig. 1b) takes as input a set of graphs at the atomic level representing
protein-protein interfaces and organize them in similar clusters, based on physicochemi-
cal properties of its nodes and edges and graph topology, for pattern detection in the next
step.

Graph dataset summary

In order to perform the clustering analysis, we propose a counting matrix, in which graphs
are represented based on the labels of each of their pair of nodes. Our representation
is inspired in some fingerprints that characterize small molecules [47, 48]. Suppose a
graph G1 that has nodes A, B, and C, labeled as donor (DN), acceptor/negative (AC/NG),
donor/positive (DN/PS) respectively, and edges A-B and B-C, representing a hydrogen
bond and a salt bridge respectively, as shown in Fig. 2. To represent such graph, each
pair of its nodes is labeled by joining their corresponding node labels into a single label.
For example, the label DN-DN/PS represents pairs of interacting nodes where one of its
atoms (DN) is a donor atom and the other one (DN/PS) is a donor/positive atom. The
labels produced would be DN-AC/NG, for pair (A,B) and DN/PS-AC/NG for pair (B,C).
In the resulting matrix, each row represents a graph and each column represents one of
the labels produced. The counting matrix for the set of graphs in Fig. 2, which contains
graph G1 , is shown in Table 2.

Dimensionality and noise reduction

Singular Value Decomposition (SVD) was used to perform dimensionality and noise
reduction in ppiGReMLIN, resulting in a compact representation of the input data for
Queiroz et al. BMC Bioinformatics (2020) 21:143 Page 7 of 25

Fig. 2 Graph sample. Graph set example for constructing the proposed counting matrix

clustering analysis (which we named matrix ), that can be manipulated more efficiently
in terms of memory requirement and execution time [49].
SVD decomposes a rank-l r by c matrix X into the product UV T , where: U is an r by l
orthogonal matrix, whose columns are the left singular vectors of X;  is a l by l diagonal
matrix, with elements of the diagonal, denoted singular values, being positive and sorted
in decreasing order; and V is a c by l orthogonal matrix, whose columns are the right
singular vectors of X.
Noise reduction can be achieved by truncated SVD. In this case, a rank-d approxima-
tion of X can be obtained by retaining only: the first d columns of U, resulting in the r by
d matrix Ud ; the first d columns and d rows of , resulting in the d by d matrix d ; and
the first d columns of V, resulting in the c by d matrix Vd . It is possible to show that Xd =
Ud d VdT is the best rank-d approximation of X with regard to the root-mean-square
error [50].
Dimensionality reduction can be performed by calculating the truncated SVD and tak-
ing the product Ud  d , which results in an r by d matrix, that is an approximation of X
with less columns. The rationale behind using Ud  d product to approximate X is that
columns of Ud capture patterns among data objects in Xd [51].
The distribution of singular values was analyzed to select an appropriate d, reducing
matrix dimensionality and keeping relevant information. The attributes of the reduced
matrix are a linear combination of the attributes of the original data matrix. The

Table 2 Counting matrix example

Graph AC-DN/PS AC/NG-DN AC/NG-DN/PS DN/PS-NG DN/PS-DN/PS NG-NG
G1 0 1 1 0 0 0
G2 1 1 1 1 0 0
G3 0 0 0 1 1 1
Queiroz et al. BMC Bioinformatics (2020) 21:143 Page 8 of 25

number of dimensions of the reduced data matrix, d, was chosen considering a mini-
mum threshold of 95% as the relative amount of the variance of the original data to be
preserved [52].

Clustering
A clustering analysis was performed on the reduced counting matrix , in order to group
similar graphs for the subsequent step of FSM. The Spectral Clustering [53] algorithm
was applied in this analysis, as it is able to group data with non-convex shapes.
The Spectral Clustering algorithm uses an internal graph representation of the data as
the basis for clustering. In this graph representation, edges and their weights are con-
veyors of similarity information among its individuals. A similarity matrix of the data
objects is used to build the graph representation. This matrix is calculated based on some
measure of similarity and through different strategies, such as k-nearest neighbors or ε-
neighborhood [54]. The clustering is then defined based on the n smallest eigenvalues and
correspondent eigenvectors associated the with the Laplacian matrix of the graph. Here
we used the scikit-learn [55] (version 0.19.2) implementation of Spectral Clustering and
the “k-nearest neighbors" to build a graph representation of the input data. The similarity
measure adopted was the euclidean distance.
One critical aspect of Spectral Clustering algorithm is the definition of a suitable num-
ber of clusters c and the number of neighbors k. It is especially hard since the selected
clustering algorithm is able to identify non-convex shaped clusters in n-dimensional
space. To circumvent this issue, we use the eigen gap heuristic [54] to support on the selec-
tion of an appropriate number of clusters by identifying the maximal difference between
consecutive eigenvalues associated with the Laplacian.

Evaluation strategy
The appropriate number of clusters n given by the eigen gap heuristic was evaluated for
different values of k (number of neighbors). These values were selected as a percentage p
of the number of entries in each data matrix, starting at 1%, with and increment of 1%,
up to 90%. Then, k was selected as the minimum value for which the respective similarity
graph was fully connected. This is important since disconnected components in spectral
clustering constitute themselves as clusters, which is not an ideal situation unless some
prior information on the data indicates that [54], which is not the case.
Finally, on each dataset, evaluation of n and k was done for all rank-d matrices obtained
from SVD. For each pair (n, k) obtained over each d, we selected the one at which k was
minimal, within the minimal threshold of d, considering 95% of variance in the reduced
matrix .

Conserved substructure mining

This block (Fig. 1c) takes as an input the groups of similar protein-protein interactions to
conduct a FSM experiment which aims to detect conserved substructures in each group.

Subgraph mining
A FSM experiment was conducted to find conserved structural arrangements (frequent
subgraphs) on protein-protein interactions for each group resulting from the clustering
analysis.
Queiroz et al. BMC Bioinformatics (2020) 21:143 Page 9 of 25

In accordance with [56], considering a graph dataset D = G0 , G1 , ..., Gh , support(g)

denotes the number or fraction of graphs in D that have g as a subgraph. The FSM task
consists in finding any subgraph g whose support(g) ≥ minSup (a minimum threshold for
the support). The FSM algorithm adopted was gSpan [57].
Experiments were performed with support varying from 0.1 to 1.0, with step 0.1.
Resulting subgraphs went through a filter in order to extract the maximal frequent sub-
graphs. In a general manner, FSM algorithms are able to detect frequent patterns and
point out in which input graphs such patterns were found. However, it is not possible to
directly map each node/edge of a frequent pattern to a specific node/edge in an input
graph. In this work, being able to perform this mapping is relevant due to the biolog-
ical semantic of patterns, so that the domain specialist knows which are the relevant
atoms and interactions to allow protein-protein complexes to be formed. For details,
see [26, 58].

Subgraph-graph isomorphism
In order to find a mapping between a subgraph H  of a graph H and a graph S, we
employed the VF2 algorithm [59]. However, implementations currently available of VF2
perform the subgraph-graph isomorphism task only for vertex-induced subgraphs. For
these subgraphs, the set of edges between any pair of vertices has to be the same as the
corresponding pair in their supergraphs (Fig. 3). Frequent subgraphs generated by FSM
algorithm are not necessarily vertex-induced subgraphs, so we used line graphs as an
alternative approach to overcome this issue.
A line graph L(H) of a simple graph H (Fig. 3) is obtained by associating a ver-
tex with each edge of the original graph and connecting two vertices with an edge
in the new graph if the corresponding edges of the original graph H have a vertex
in common[60]. As stated in [61], if graphs H and H  have isomorphic line graphs,
then H and H  are also isomorphic, with some exceptions that do not occur in our
application.
Then we can simply provide VF2 with the line graphs of the original input graphs for
which we want to find a mapping. However, in order to be able to use line graphs, graphs
from the datasets and patterns from FSM have to be converted into simple graphs. The
strategy employed at this point was to instantiate the simple graphs based on the same
set of nodes of the original graphs, and connect those that are adjacent in the original
graphs. Labels from multiple edges of multigraphs are joined into single labels in the new
generated graphs. Next, the subgraph-graph isomorphism task is performed using node
match functions so that nodes whose labels are a subset of one another may be proper
evaluated as a match during the process.

Results and discussion

To explore and confirm the ability of ppiGReMLIN to characterize and detect conserved
structural arrangements on protein-protein interfaces at the atomic level we instantiated
our method on two relevant datasets of protein-protein complexes. First, we show that
our method can be applied to detect frequent substructures on protein-protein interfaces
in large-scale. Then we compare the frequent substructures automatically detected by
ppiGReMLIN to relevant residues and interactions that were experimentally determined
and described in the literature.
Queiroz et al. BMC Bioinformatics (2020) 21:143 Page 10 of 25

Fig. 3 Induced subgraphs and linegraphs . Graphs on the left represent a graph G and a set of induced
subgraphs G , G , G of G. Red dashed edges in G and G are the edges missing in each graph for them to
become vertex-induced subgraphs of G. G is the only vertex-induced subgraph of G, as its contains all
edges of its supergraph between its nodes. The linegraphs of each graph are represented on the right. Edges
in a lighter color represent missing edges in L(G ), L(G ) and L(G ), relative to the linegraph L(G). Although
the original graphs are not all vertex-induced subgraphs, their line graphs are
Queiroz et al. BMC Bioinformatics (2020) 21:143 Page 11 of 25

Clustering analysis
As mentioned before, the clustering parameters were defined experimentally based on the
eigen gap heuristic, which was evaluated for every instance of the count matrix regarding
the number of dimensions in its reduced form. The results are summarized in Fig. 4.
For the Serine protease dataset, the number of clusters selected was n = 11, for which
the number of neighbors k = 35 was the minimum for the experiment. For the BCL
dataset, the minimum k for valid instances of the counting matrix (variance greater than
95%) was k = 35, at which point the optimal number of clusters varies between 10 and
13. However, n = 13 was selected as it occurs more frequently over a relatively larger
range of d values (d ∈[ 10, ..., 20]) when compared with n = 10 (d ∈[ 21, ..., 24]). Also, we

Fig. 4 Eigen gap heuristic results. Graphs above show the results of the eigen gap heuristic for both datasets,
for each reduced matrix Xd . The number of neighbors for each experiment is also shown, represented by gray
diamonds
Queiroz et al. BMC Bioinformatics (2020) 21:143 Page 12 of 25

believe the difference of the values in lower dimension instances of the data matrix is a
result from the noise reduction feature offered by SVD decomposition.

Conserved substructures mining

This section presents how proper support values were selected for each dataset and the
conserved substructures detected by ppiGReMLIN in Serine protease and BCL datasets.
As a general guideline, the minimum support value was set to 0.5. By increasing the sup-
port, graphs tend to be smaller in size, but more frequent in groups. Thereupon, the
selection was made as a compromise between frequency of patterns and pattern size.

Serine protease dataset

For the serine protease dataset, at support 0.7, the largest patterns had 6 nodes and
occurred in group 6. For two out of three patterns found in this group, two had real sup-
port equal to 0.83. By reducing the support value to 0.6, the number of patterns in this
group went down to two, while growing in size to 7 nodes (See Supplementary material
Figure 1A). As we are interested in the most frequent substructures, we believe 0.7 to be
a suitable value for minimum support, considering the small increase in pattern size.
From this point on we discuss some of the interesting patterns found by ppiGReMLIN
and summarize other groups for the selected support. Figure 5 shows the patterns we
deemed most interesting and a few sample graphs in which they occur.
The largest pattern from group 6 is depicted as F1 in Fig. 5. The central vertex in this
structure (4-degree vertex in the graph) represents either one of the oxygen atoms (accep-
tor/negative) in the carboxyl group from aspartate residues in the protease chain. The four
atoms with which they interact are the nitrogen and carbon atoms (donor/positive and
positive respectively) in the guanidinium group from arginine residues in the inhibitor
chain. The last vertex in the structure is an oxygen atom (acceptor or acceptor/donor)
from a glycine or serine residue in the protease chain. All interactions between nodes
in this group occur by means of salt bridges and hydrogen bonds, as determined by the
analysis of the input graphs in the group.
Regarding the residue position in the protein chain sequences for pattern F1, the major-
ity of aspartate interacting residues (located in 32 graphs) correspond to the aspartate
present in the specificity pocket (S1 pocket) of trypsin and trypsin-like proteins (See
Supplementary material Figure 2). The only exception occurred in graph 24, where the
interacting aspartate residue from the P1 pocket was displaced in the trypsin from the rat
anionic trypsin complexed with protein ihibitor APPI (PDB id 1BRC). The serine residues
(from the protease) mentioned previously were spotted in 30 of the 33 graphs where F1
was found, located right next to the aspartate P1 in the residue chain sequence. As for
the glycine residues, they are positioned further in the sequence, but spatially close due
the protein folding. Figure 6a shows the pattern in the crystal structure of the complex
formed between bovine beta-trypsin and MCTI-A, a trypsin inhibitor of squash family
(PDB id 1F2S).
Another interesting pattern, F2, is presented in Fig. 5. It was obtained from group 10
and consists of 5 nodes connected by hydrogen bonds. The central atom in the structure
(3-degree vertex in the graph) represents an oxygen atom (acceptor) from the inhibitor
chain interacting with two nitrogen atoms (donor) and one oxygen atom (acceptor/donor)
from the protease chain. In each graph where this pattern was found, two of the atoms
Queiroz et al. BMC Bioinformatics (2020) 21:143 Page 13 of 25

Fig. 5 Protease conserved substructures. F1, F2 and F3 show some interesting patterns and some of their
respective input graphs. Nodes and interactions in patterns might contain only a subset of the labels of its
corresponding nodes in the graphs. For example, red nodes in F3 are labeled negative, meanwhile their
corresponding nodes (in light green) are labeled negative and acceptor. Also, patterns may have multiple
mappings to their corresponding input graphs. For example, the acceptor node from F3 (in blue) may be
mapped separately to the acceptor or acceptor/donor nodes in graph 1

from the protease chain, an oxygen and a nitrogen atom, were part of the same serine
residue, and the other atom, a nitrogen was from a glycine residue. The central atom in
the graph represents a nitrogen atom from the inhibitor chain.
Considering pattern F2, the interacting serine residues are part of the catalytic
triad in serine proteases, commonly referred as SER195, HIS57 and ASP102. (See
Supplementary material Figure 2). The interaction of both the serine and the glycine
residues (usually referred as GLY193) with the carbonyl atom in the substrate in struc-
ture F2 is described as an intermediate state during the process of catalysis. Figure 6b
shows the interaction pattern F2 in the kunitz type trypsin inhibitor complex with porcine
trypsin (PDB id 4AN7).
The last patterns of Fig. 5, represented by F3, similarly to F1, shows both oxygen atoms
(acceptor negative) from the carboxyl group in aspartate residues together with an oxy-
gen (acceptor or acceptor/donor) from a serine residue, all of them interacting with the
Queiroz et al. BMC Bioinformatics (2020) 21:143 Page 14 of 25

Fig. 6 Interaction patterns presented in the context of protein structures. a F1 is represented in the crystal
structure of the complex formed between bovine beta-trypsin and MCTI-A, a trypsin inhibitor of squash
family (PDB id 1F2S). Residues in gray represent protease residues and the ones in orange represent inhibitor
residues. Oxygen and nitrogen atoms are shown in red and blue respectively. b F2 is represented above in the
kunitz type trypsin inhibitor complex with porcine trypsin (PDB id 4AN7). The oxyanion hydrogen bonds are
depicted by red dashed lines, while the yellow ones represent the other hydrogen bonds that compose the F2

nitrogen atoms from lysine residues in the inhibitor. Interactions in this pattern represent
hydrogen bonds and salt bridges. This pattern was obtained from group 8 and occurred
in all graphs in the group.

BCL-2 dataset
For the BCL-2 dataset, the FSM algorithm was performed with support values above 0.6
due to the size of graphs in this dataset being relatively larger (up to 30 vertices) than the
ones in the Serine protease dataset, which generated a large number of substructures, in
the order of 104 to 105 . At support 0.6, the group with the highest number of patterns
Queiroz et al. BMC Bioinformatics (2020) 21:143 Page 15 of 25

had over 50 thousand substructures, which were reduced to approximately 1,500 maximal
patterns, from which only the 10 largest structures were selected for analysis.
The support selected for the BCL-2 dataset was 0.6, since it presented the largest graphs
, with 10 vertices at group 6 and 11 vertices in group 7, as opposed to 8 and 10 ver-
tices in the same groups respectively, at support value 0.7. (See Supplementary material
Figure 1B). Figure 7 shows some of the patterns and groups deemed interesting to
illustrate the ppiGReMLIN results.
Group 2 is composed of hydrophobic and aromatic atoms connected by hydrophobic
and/or aromatic interactions. One of the two patterns found in this group (F4 in Fig. 7)
has five hydrophobic atoms connected by hydrophobic interactions. Graph 0 (structure
of Bcl-xL-Bak peptide complex PDB id 1BXL, chain A), depicted as F4-A in Fig. 7, shows
one input graph in which this pattern is found.
Graphs from groups 6 and 7 are composed of acceptor/negative and donor/positive
atoms connected by salt bridges, hydrogen bonds, and repulsive interactions. Patterns
F5 and F6 in Fig. 7 shows one of the largest patterns from each group. One common
feature in the graphs where these patterns were found is the interaction of atoms from the
carboxyl group in aspartate residues in the pro-apoptotic protein chain with atoms from
the guanidinium group in arginine residues in the pro-survival protein. These interactions
were predominantly spotted as salt bridges. In Fig. 7b the aspartate nodes are highlighted
in graph 39 (structure of the Bcl-XL:Beclin 1 complex, PDB id 2P1L, chain A) and graph
401 (crystal structure of alpha-beta-foldamer 2c in complex with Bcl-xL, PDB id 4A1U,
chain A).
Another common feature in the graphs from these groups regards some interacting
nodes from lysine or arginine residues in the pro-survival chain, highlighted in Fig. 7. In
arginine residues, these nodes represent the atoms from the guanidinium group, while in
lysine they represent the single nitrogen atom from its side chain. These atoms are found
in graphs composing repulsive interactions with the arginine atoms from the pro-survival
protein described before, and salt bridges or hydrogen bonds with other atoms from the
pro-survival chain.

Comparison of ppiGReMLIN with experimental patterns

We searched the literature for conserved interacting residues or structural arrangements
in PPI’s for the BCL-2 and the SP datasets, which are conserved datasets regarding their
sequences and structures. In order to perform a quantitative evaluation of ppiGReM-
LIN we used precision and recall. Borrowing the idea of these metrics from machine
learning: precision determines the fraction of patterns that actually turns out to be rel-
evant among those our strategy declared as relevant; recall measures the fraction of
relevant patterns correctly recovered by our strategy. In this analysis, we consider a
pattern to be relevant if the it contains at least one of the relevant residues reported
by literature.
For the SP dataset, the residues considered relevant for the quantitative analysis were
those experimentally determined already documented in the literature [62–64] (further
details in the “Serine protease experimental patterns” section below):

• I: the reactive serine from the catalytic triad in serine proteases (usually SER195);
• II: the aspartate (usually ASP189) at the bottom of S1 pocket;
Queiroz et al. BMC Bioinformatics (2020) 21:143 Page 16 of 25

Fig. 7 BCL-2 conserved substructures. F4, F5, F6 show some interesting patterns and some of their respective
input graphs. a Input graphs colored according to its node labels. b The same input graphs with its nodes
colored according to their source chain in the protein. In this example, blue nodes represent atoms from the
BCL-2 antiapoptotic part of the protein complex, and green nodes represent atoms from the other protein
chain in the complex. The larger green atoms atoms are part of binding hotspot residues described in
literature relative to the motif LXXXXD

• III: the histidine, usually HIS40, that play an important role in the zymogen activation
in the enzymes;
• IV: a small structure composed of two nitrogen atoms connected to a single oxygen
Queiroz et al. BMC Bioinformatics (2020) 21:143 Page 17 of 25

atom by means of hydrogen bonds was used in order to target a more complex
structure found in serine-proteases know as the oxyanion hole.

The numbering of residues used for the results for the SP dataset above is in agreement
with PDB structure crystal structure of the complex formed between bovine beta-trypsin
and mcti-a (PDB id 1F2S).
For the BCL-2 dataset, five residues in specific positions in the BH3 domain of pro-
apoptotic proteins were considered as relevant for the quantitative analysis, as they
play an important role in the binding of BCL-2/BH3 (further details in the “BCL-2
experimental patterns” section below):
• 1: the leucine (L0), at the beginning of the motif LXXXXD, highly conserved in BH3
domains.
• 2: the aspartate (D+5), at the end of the same motif.
• 3: residue at postion −4 (multiple possible residues)
• 4: residue at position +3 (multiple possible residues)
• 5: residue at position +7 (multiple possible residues)

Results concerning precision and recall are presented in Tables 3 and 4, for the SP and
the BCL-2 datasets respectively. In each table, the results are separated by each of the
groups found in the clustering step, and in the bottom line they are aggregated to give
the overall perspective in the supports considered. The overall precision in each table is
determined by the ratio between the total number of patterns pointing to at least one of
the relevant residues and the total number of patterns found across all groups. The overall
recall, on the other hand, is the ratio between the number of relevant residues mapped by
ppi patterns and the total number of relevant residues.
The analysis of the results shows the overall precision at 69% (SP) and 100% (BCL-2),
while recall is 100% for both datasets. For the SP dataset, the lower precision is due to
the patterns in groups 2,4,5 and 11 as they do not contain residues considered relevant
documented in the literature. However, we were able to find all the relevant structures in
the ppiGReMLIN patterns, as shown by the overall recall (100%). Interestingly, some of
the test structures were identified alone in specific groups, e.g., III in group 1, IV in group
7, and I in groups 6,8 and 9, for the SP dataset; and structure 2 in groups 6,8,11 and 12,

Table 3 Analysis of precision and recall for the SP dataset at minimum support 0.7
Group Patterns Precision Recall Residues
1 1 1.00 0.25 III
2 1 0.00 0.00
3 1 1.00 0.50 II,III
4 1 0.00 0.00
5 1 0.00 0.00
6 3 1.00 0.25 I
7 1 1.00 0.25 IV
8 1 1.00 0.25 I
9 1 1.00 1.00 I
10 1 1.00 0.50 II,III
11 1 0.00 0.00
All 13 0.69 1.00 I,II,III,IV
Queiroz et al. BMC Bioinformatics (2020) 21:143 Page 18 of 25

Table 4 Analysis of precision and recall for the BLC-2 dataset at minimum support 0.6
Group Patterns Precision Recall Residues
1 1 1.00 0.80 1,3,4,5
2 2 1.00 0.60 1,4,5
3 2 1.00 0.60 1,3,5
4 1 1.00 0.80 1,3,4,5
5 1 1.00 0.80 1,3,4,5
6 10 1.00 0.20 2
7 10 1.00 0.40 2,4
8 1 1.00 0.20 2
9 1 1.00 0.80 1,3,4,5
10 1 1.00 0.80 1,3,4,5
11 7 1.00 0.20 2
12 4 1.00 0.20 2
All 41 1.00 1.00 1,2,3,4,5

for the BCL-2 dataset. This shows how ppiGReMLIN not only can identify interaction
patterns, but also is able to single out some relevant interactions with specific residues
in PPI’s, which also demonstrates the importance of the clustering step in the strategy
workflow.
The fact that we did not find patterns documented in the literature in groups 2,4,5 and
11 does not mean that there are no relevant binding patterns in these groups. This only
means that the patterns found are not documented in the literature. There is a possibility
that such patterns are relevant and related to protein-protein interactions. Thinking of
proteins as a network of interaction between residues, there are very few residues at the
binding site and even less residues at the active site. Nonetheless, there are a number of
residues that can be important in this network of residues to help, for example, stabilize
the residues that are at the binding site. We believe that the patterns in the groups 2,4,5
and 11 should be further investigated to understand whether they are relevant.

Serine protease experimental patterns

According to Perona and Craik [64], position 189 located at the base of the S1 pocket
(Fig. 8), is highly conserved as an aspartate in enzymes with trypsin-like specificity
towards substrates that contain arginine and lysine. The role of the negatively charged
ASP189 in binding and catalysis has been addressed in many studies [65–68]. In [64], this
interaction is said to occur by means of hydrogen bonds mediated by water molecules for
substrates with lysine at position P1. For substrates with arginine residues, it is also possi-
ble that these interactions are formed as salt bridges. Our results from patterns in groups
6 and 10, described in the previous section, shows these interactions predominantly as
salt bridges, even for lysine residues. The reason for this lies in the criteria employed
in the contact prospecting phase, in which hydrogen bonds mediated by water were not
considered, only direct hydrogen bonding between atoms were taken in account. Thus,
we would not expect to find such interactions. However, due to the overlapping distance
range of both hydrogen bonds and salt bridges, and physicochemical properties of inter-
acting atoms, the strategy was able to spot the residues as relevant in the protein-protein
interaction interface.
Queiroz et al. BMC Bioinformatics (2020) 21:143 Page 19 of 25

Fig. 8 Interacting residues at trypsin S1 pocket. The S1 pocket is shown above in the kunitz type trypsin
inhibitor complex with porcine trypsin (PDB id 4AN7). The protease surface is depicted in gray with the S1
pocket highlighted in blue. Residue at position S1 from the protease and P1 from the inhibitor are indicated
along with ASP189 and GLY193, which are relevant residues as described in literature. Pattern F2 is
represented above in the interactions of the P1 residue with ASP189, along with other two neighboring
residues. F1 is represented by the interaction of residues in position P1, S1 and GLY193, with the hydrogen
bonds from an oxyanion hole depicted in red dashed lines

Another interaction mentioned in the literature [64] was found in group 10 (described
by F2), and in many graphs in group 3. The structure contains an interaction known as the
oxyanion hole, composed of two amide nitrogen atoms, usually from SER195 and GLY193,
in the protease chain interacting with a carbonyl oxygen atom in the inhibitor chain by
means of hydrogen bonds. The role of the oxyanion hole in the catalysis is to stabilize
the negative charge in the carbonyl group after it becomes a tetrahedral intermediate, i.e,
the double bond between its carbon and oxygen atoms becomes a single-bond [69]. The
structure is depicted in Fig. 6b.
Additionally, the GLY193 residue, which is highly conserved in serine proteases, is
frequent in the patterns described above. According to [70], it makes a crucial con-
tribution to substrate binding in both the enzyme ground and transition states during
catalysis.

BCL-2 experimental patterns

The molecular recognition between pro-apoptotic and anti-apoptotic members of the
BCL-2 family is driven by the ability of the amphipathic BH3 α-helix of regulators to
be accommodated in the hydrophobic groove formed by the four BH domains in the
suppressors [71] (Fig. 9). This interaction paradigm is shared between both BH3-only
(activators) and multi-domain (effectors) proteins of pro-apoptotic BCL-2 members,
which act competitively towards pro-survival members in order to regulate apoptosis.
Natively, effectors are complexed with anti-apoptotic members in healthy cells, being dis-
placed by activators in response to pro-apoptotic signals, which ultimately culminates in
cell death [72].
Queiroz et al. BMC Bioinformatics (2020) 21:143 Page 20 of 25

Fig. 9 Critical interactions for the BCL-2/BH3 binding. Critical residue interactions are shown in the structure
of the Bcl-XL:Beclin 1 complex (PDB id 2P1L). The Beclin 1 chain, a BH3 only protein, is shown in orange in the
hydrophobic groove of Bcl-xL, an anti apoptotic protein. Hydrophobic interactions are depicted by dashed
lines in blue, salt bridges in green, and hydrogen bonds in orange. The five interacting residues from BH3
chain represent those found in patterns and in literature

The BH3 domain of both effectors and activators, typically about 20 amino acids in
length, is characterized by the presence of the motif LXXXXD. While interactions may
occur along the BH3 chain, only a few of its residues have been characterized as critical
to the specificity of binding towards suppressors. Apart from the leucine (L0), flanking
the motif LXXXXD, hydrophobic residues at positions −4, +3 and +7, represent bind-
ing hotspots in the molecular recognition [71]. The hydrophobic interactions promoted
by these residues were found in patterns from groups 1, 2, 3, 4, 5 and also in patterns from
group 9. Graph 0, in Fig. 7b, highlights atoms from the leucine L0 and from isoleucine at
position +3 interacting with other atoms in the suppressor chain by means of hydropho-
bic interactions, and atoms from isoleucine at position +3 . The structure can also be
visualized in Fig. 10a.
The aspartate (D+5) in the motif LXXXXD, is the last of the critical residues men-
tioned in literature, described to interact with atoms from suppressor residues by means
of hydrogen bonds or salt bridges [71]. This interaction was found in patterns from groups
6, 7, 8 and in patterns from groups 10, 11, 12. F5 and F6, in Fig. 7b, highlight atoms from
aspartate D+5 interacting with atoms in the guanidinium group from arginine residues in
the suppressor chain. Figure 10b shows a visualization of input graph 401, one structure
on which F5 was found, with aspartate D+5 (here as ASP121) highlighted.
Interestingly, on the same structures described above, atoms from residues at position
+1 were also found composing interactions. These residues were either arginine or lysine.
F5 and F6 in Fig. 7 highlight these residues, which are shown as LYS117 in graph 39
and ARG95 in graph 401. Although this position is not critical for the binding of BH3
domains and BCL-2 anti-apoptotic proteins, their role in the binding affinity cannot be
overlooked. Boersma and colleagues[73] demonstrated that substitution for glutamine
inhibited binding of pro-apoptotic Bim-BH3 with anti-apoptotic proteins Bcl-xL and Mcl-
1, while remaining unchanged for uncharged residues. Furthermore, Dutta and colleagues
Queiroz et al. BMC Bioinformatics (2020) 21:143 Page 21 of 25

Fig. 10 Interaction patterns presented in the context of protein structures. a Graph 0, containing F4, is
represented in the structure of Bcl-xL-Bak peptide complex (PDB id 1BXL). Residues in gray represent protease
residues and the ones in orange represent inhibitor residues. Oxygen and nitrogen atoms are shown in red
and blue respectively. Residue L0 from motif LXXXXD in BH3 pro-apoptosis regulator Bak is shown here as
LEU578 (b) Graph 39, containing F5, is represented above in the crystal structure of alpha-beta-foldamer 2c in
complex with Bcl-xL (PDB id 4A1U). Salt bridges are depicted in green, hydrogen bonds in orange, and
repulsive interactions in light yellow. Red dashed lines represent edges where interactions are described
both as hydrogen bonds and salt bridges. Residue at position D+5 is highlighted here as ASP121

[72] showed different binding profiles for mutated Bim-BH3 towards Bcl-xL and Mcl-
1. Substitutions with negatively charged residues were tolerated for binding to Mcl-1,
whereas for Bcl-xL, it resulted in low binding affinity.

Conclusions
This work proposed a graph based strategy to detect conserved structural arrange-
ments on protein-protein interface, which we named ppiGReMLIN. In this method,
Queiroz et al. BMC Bioinformatics (2020) 21:143 Page 22 of 25

protein-protein interfaces are modeled as graphs at the atomic level. Atoms and non-
covalent interactions are represented by nodes and edges respectively, which are labeled
according to their physicochemical properties and distance criteria. The resulting set of
graphs is segmented in similar clusters that serve as the input for a FSM, revealing sub-
structures that are conserved across the whole dataset of protein-protein interactions.
Our method does not rely on sequence alignment nor structural superimposition, and
can be used in large-scale datasets of protein-protein interactions.
Results show the method is effective in finding conserved structures described in the
literature in an automatic fashion. At the supports chosen for each of our main datasets,
precision range from 69% up to 100%, with recall of 100%, considering the most relevant
structures found in literature. For instance, from the serine protease dataset, patterns
were found with support higher than 70%. Among these, structures such as the SER195
and GLY193 oxyanion, an important interaction in intermediate states of catalysis, and
the ASP189 interactions in the binding specificity pocket of trypsin, were detected in
more than a single cluster. Relevant interacting residues were also found for the BCL-
2/BH3 dataset with support higher then 60%. Residues at critical positions relative to the
motif LXXXXD, which characterizes the BH3 domain of apoptotic regulators, were found
in many patterns across different groups. Additionally, relevant residues were also found
in non-critical positions in BH3 domains, that influence the binding affinity of BCL-2
members.
As future work, we plan to calculate water mediated interactions by considering struc-
tural water molecules. Also, we intend to make ppiGReMLIN available as a web-server
and as web services, which will allow our method to be easily accessible for users and to
be included in automatic pipelines.

Supplementary information
Supplementary information accompanies this paper at https://doi.org/10.1186/s12859-020-3474-1.

Additional file 1: Supplementary Material.

Abbreviations
AG: Anticarcia gemmatalis; FSM: Frequent subgraph mining; KTI: Kunitz trypsin inhibtor; PDB: Protein data bank; PPI:
Protein-protein interactions; RNA: Ribonucleic acid; SVD: Singular value decomposition

Acknowledgements
Not applicable.

Authors’ contributions
FCQ and SAS conceived the ppiGReMLIN strategy. FCQ and SAS designed and implemented the algorithms and the
visualization prototype. FCQ, APV, MGO, GVC and SAS analyzed data and results. FCQ, GVC and SAS wrote the
manuscript. APV and MGO contributed materials/analysis tools. All authors read and approved the final manuscript.

Funding
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) -
Finance Code 23038.004007/2014-82 - Grant 051/2013; Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq); Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG). The funding agencies had no role in
study design, data collection, analysis and interpretation, decision to publish, or preparation of the manuscript.

Availability of data and materials

The source code and datasets are available at: https://github.com/ppigremlin/ppiGremlin.
The prototype tool that allows visualization of results is available at: https://ppigremlin.github.io/.

Ethics approval and consent to participate

Not applicable.
Queiroz et al. BMC Bioinformatics (2020) 21:143 Page 23 of 25

Consent for publication

Not applicable.

Competing interests
The authors declare that they have no competing interests.

Author details
1 Department of Computer Science, Universidade Federal de Viçosa, Av Peter Henry Rolfs, Viçosa, MG, Brazil. 2 Department

of Biochemistry and Molecular Biology, Universidade Federal de Viçosa, Av Peter Henry Rolfs, Viçosa, MG, Brazil. 3 Instituto
de Biotecnologia aplicada a Agropecuaria, BIOAGRO-UFV, Av Peter Henry Rolfs, Viçosa MG, Brazil. 4 Department of
Computer Science, Universidade Federal do Espírito Santo, Av Fernando Ferrari, Vitória, ES, Brazil. 5 European Molecular
Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), CB10 1SD Hinxton, UK.

Received: 7 August 2019 Accepted: 27 March 2020

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