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The BIR-Arg Motif of Inhibitor of Apoptosis (IAP) Proteins is Rare in Non-IAP Proteins
Mark Van DykBiochemistry Research
Spring 2014
Van Dyk 2
Abstract
Determining conservation of motifs involves an additional level of complexity
over analysis of protein sequences. While residues may be conserved within a motif,
these residues may come from different regions of each protein sequence. Additionally,
protein sequences cannot be analyzed without additional constraints, as intervening
sequences between desired residues are superfluous to identifying 3-D structures.
Although some difficulty was present in finding a program that best suit the purposes of
this experiment, the Swiss-PdB Viewer molecular graphics program was found to be
most suitable for identifying conservation of motifs. The goal of this experiment was to
determine whether a specific motif known to be present in Inhibitor of Apoptosis (IAP)
proteins was conserved in this protein family, and whether it was also present in other
(non-IAP) protein families. The motif, with naming suggested by myself and Dr.
Matthew Junker, will henceforth in this paper be called the BIR-Arg motif.
Based on previous research, the BIR domain DIAP1_BIR2 (PDB code 1JD4) was
used as a template for the BIR-Arg motif for this experiment. This motif is known to
contain bridging hydrogen bonds, a cation-π interaction between an Arg and an aromatic
residue, and edge-to-face packing of aromatic residues. In order to create the template,
distances from specific atoms in involved residues as well as lower and upper bounds
were established using Swiss-PdB Viewer.
The template of the BIR-Arg motif was searched against a 90-percent non-
redundant subset of proteins containing 14,431 structures from the Protein Data Bank
(RCSB PDB), resulting in a list of matches that matched the specifications of the
template. Results were further analyzed to ensure that each match contained a cation-π
Van Dyk 3
interaction, that residues for each motif came from the same protein chain (this is trivial,
however it is not a feature present in Swiss-PdB Viewer), and that the geometry of
residues in each match was similar to the template. The European Bioinformatics
Institute (EBI) protein database was used to identify characteristics of each protein (such
as protein family) based on the list of PDB names output by Swiss-PdB Viewer. And the
online program CaPTURE (Gallivan, J.P.; et al. Cation-pi Interactions in Structural
Biology. Proceedings of the National Academy of Sciences, 1999, 96, 9459.
http://capture.caltech.edu/) was employed to ensure that each match contained a cation-π
interaction--a necessary component of the BIR-Arg motif. After narrowing down matches
based on the above specifications, the molecular software analysis program MolMol was
used to fit each hit to the model motif (DIAP1_BIR2). Fitting between proteins enabled
both qualitative and quantitative comparisons of differences between motifs.
Results indicate that the BIR-Arg motif is found to be conserved among all
structures of IAP proteins, being present in 9 IAP proteins and 13 out of 14 BIR domains.
The motif is present, but not conserved, among the protein families of the 7 non-IAP
proteins containing the BIR-Arg motif. Results suggest that the BIR-Arg motif is
essential to IAP function and is unique due to the lack of conservation among any non-
IAP protein families.
Introduction
In order to describe the importance of the BIR-Arg motif, it is first necessary to
provide some background information about proteins. Proteins are long polymers made
of amino acid building blocks that, besides water, constitute the second largest
component of a cell. They have varied roles in cells such as having catalytic activity,
Van Dyk 4
serving as structural elements or signal receptors, or transporting specific substances into
and out of cells. Constituent building blocks of proteins—amino acids—are relatively
small molecules that ubiquitously contain amino and carboxyl groups but contain varying
functional groups. Functional groups of these building blocks vary from small to bulky
nonpolar groups, positive or negatively charged moieties, or aromatic cyclic groups or
other unusual moieties.1 It is due to diversity of functional groups in amino acids that
proteins are provided the ability to have a variety of functions.
Due to diversity of functional groups in amino acids, amino acids can interact
with each other through various weak interactions, from hydrophobic, hydrogen bonding,
van der Waals, to electrostatic interactions.2 Various interactions such as these enable
proteins to fold into unique three-dimensional (3-D) structures including secondary α-
helices, β-pleated sheets, and supersecondary motifs. Just as interaction of amino acids
produces proteins with different characteristics and functions, motifs also have varied
characteristics. The cation-π interaction is an important component of some motifs. The
interaction is a strong noncovalent binding force that stabilizes secondary structure of
proteins and that is involved in various drug-receptor interactions.3 In this interaction, an
aromatic residue such as Phe, Tyr, or Trp donates electron density to a positively-charged
residue such as Lys or Arg.
The cation-π interaction is a fundamental component of the Inhibitor of Apoptosis
(IAP) protein family, and the IAP protein family is a vital component of living
organisms. Apoptosis or programmed cell death occurs to eliminate unfit or damaged
cells, helping to maintain homeostasis and to allow proper development of organisms. It
is essential to maintaining a constant cell number of around 1011-1012 cells per day (for a
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healthy adult human).4 Increases in cell death have been reported to occur in AIDS,
neurodegenerative disorders, and ischemic injury, while decreases have been found t o
contribute to cancer, autoimmune diseases, and restenosis.4 On a molecular level,
apoptosis is initiated through proteolytic cascades of hierarchical groups of caspases.
Positive and negative regulation occurs through activation of inhibitors and stimulators.5
IAP proteins negatively regulate the apoptotic pathway by inhibiting caspases to, while
stimulators such as Smac (humans)/DIABLO (mice) provide positive regulation as they
allosterically bind to IAP proteins.5 Since IAP proteins are an essential component in the
regulation of cell growth and proliferation of cells, they are an important area of
investigation in anti-cancer and other subjects of research.
As regulation of apoptosis is necessary to ensure homeostasis, one would surmise
that IAPs must have a highly conserved domain. In fact, IAP proteins contain a highly
conserved baculoviral IAP repeat (BIR) region or domain that is essential for anti-
apoptotic protein.5 The domain contains about 65 amino acids and can be repeated from
one to three times per IAP protein.5 Currently, the PFAM protein family database6 lists
128 BIR sequences that exist from 69 distinct BIR-containing proteins derived from
yeast, nematodes, insects, birds, mammals, and 19 different viruses. Alignment of the 128
sequences from PFAM has yielded a consensus BIR sequence at an 85% threshold level
affirming conservation of the domain.7 In addition, a critical Arg residue appears to be
almost invariant in BIR-containing proteins, being present in >99% of 887 BIR
sequences in the PFAM database.
In Figure 1, a sample of sequence alignments of several BIR domains illustrates
the almost invariant nature of this Arg residue. Previous research has indicated that a
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missense mutation to Ala has resulted in loss of IAP inhibition in O. pseudotsugata,8
while a mutation to the chemically similar residue Lys has resulted in weakening of
binding of Smac and Hid to IAPs from Drosophila as well as the baculovirus Orgyia
pseudotsugata.7 While Lys is chemically and structurally similar to Arg, researchers posit
that Lys cannot substitute for Arg in this motif due to its smaller size--Lys may be less
able to simultaneously form cation-π and hydrogen bonding interactions.7 Furthermore, it
is proposed that the Arg is highly conserved because it is central to a conserved motif that
is essential to apoptotic function of all BIR domains. For clarity, Dr. Matthew Junker and
I propose to call this motif the "BIR-Arg motif."
Van Dyk 7
Figure 1: Conservation of Arginine and Aromatic Residues Involved in BIR-Arg Motif
The BIR-Arg motif has a unique structure further highlighting its importance in
Chemistry. The motif contains backbone carbonyls, an Arg, and two aromatic residues.
Participating residues interact to provide continuity in the motif. Backbone carbonyls
donate electron density to hydrogen atoms of the Arg residue, which in turn forms a
cation-π interaction with an aromatic residue that in turn forms edge-to-face packing with
a second aromatic residue (Fig. 2). However, while the Arg residue is critical to the
Legend:
Green Star = Arginine
Blue Star = 1st Aromatic Residue (F/Y)
Red Star = 2nd Aromatic Residue (F/Y)
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motif, some deviation can occur from substitution in participating aromatic residues (can
be Phe, Tyr, or Trp).
Figure 2: Cation-π Motif in 1JD4 Protein (Schematic Rendered with RasMol9)
While previous research indicates that the motif is essential to apoptotic function
of IAP proteins, there is little research verifying this. This experiment sought to provide
verification by determining whether the BIR-Arg motif is conserved among all IAP
proteins or any non-IAP protein families. It was hypothesized that if the motif was found
in all IAP proteins, it must have an essential role in apoptosis. Additionally, if the motif
was found in other protein families, then it may have a larger biological role than in
providing regulation of apoptosis. However, if the motif was not found in other protein
families, this would solidify the importance of the BIR-Arg motif in apoptosis.
Van Dyk 9
The challenge to identifying the BIR-Arg motif in IAP and non-IAP proteins is
that the motif cannot be searched by standard sequence alignment. In making the spatial
interactions within the BIR-Arg motif, the three critical residues (Arg and two aromatics)
can be separated by any length of amino acid sequence and even be positioned in
different orders within the primary sequence. In addition, the two backbone carbonyls of
the bridging hydrogen bonds can be contributed by any of the 20 primary amino acids
found in proteins. Identifying the BIR-Arg motif necessitates searching in 3-dimensional
space for the correct positioning of the interacting side chains and carbonyl oxygens.
This was achieved by using a software program that enabled creation of a 3-dimensional
template of the BIR-Arg motif that could then be searched against known structures of
proteins.
Conservation of the motif was identified by a three-step process. First, a 3-D
structure search program was used to create a template of the BIR-Arg motif. This
template was then searched against known protein structures (the RCSB PDB database10).
Finally, if the motif was identified in any non-IAP proteins, further analysis was made to
determine if the motif was conserved among corresponding protein families.
Methods
Several software programs and protein databases were used throughout the
experiment. Both PINTS11 and Swiss-PdB Viewer12 enabled searching of structural
motifs, however each program had varying levels of success. Initially in the experiment,
PINTS was used to identify conservation of the BIR-Arg motif. However, it proved to be
unsatisfactory as it did not allow substitution of amino acids of similar structure, resulting
in exclusion of too many matches. Swiss-PdB Viewer was tested next to identify
Van Dyk 10
conservation of the motif. While Swiss-PdB Viewer had some limitations, it ultimately
proved to be successful in identifying the motif.
In order to define motifs, different constraints must be set with Swiss-PdB
Viewer. Constraints include amino acid type, secondary structure, geometry (i.e.
distances), and sequence separation.13 After constraints are defined, they can then be
input into a text file that is searched against a subset of the PdB. This searching is
performed through Swiss-PdB Viewer's servers in Geneva, Switzerland against a 90%
non-redundant subset of the RCSB containing 14,431 PDB structures with resolution of
3.0 Å or better.14 Depending on the complexity of the motif, the search would output a list
of results after a few minutes to an hour. The list would contain names of PDB structures,
as well as participating residues, that fit the constraints as defined within the template.
Through Swiss-PdB Viewer, a template was created from BIR2 of DIAP1 (PDB
Code 1JD4). Parameters were optimized to produce the final template (Fig. 3):
Figure 3: BIR-Arg Motif Template Created from DIAP1_BIR2 (PDB Code 1JD4)
#SEARCH3D# pattern defined from: 1jd4# list of residues# GroupNum allowed_kind allowed_Sec_Struct ; name chain num ss scoreGROUP 0 R * ; 'ARG' 'A' '229 ' 'h'GROUP 1 FY * ; 'PHE' 'A' '264 ' 's'GROUP 2 FY * ; 'PHE' 'A' '292 ' 'h'GROUP 3 RHKDESTNQCUGPAILMFWYV * ; 'ALA' 'A' '224 ' 'c'GROUP 4 RHKDESTNQCUGPAILMFWYV * ; 'ALA' 'A' '248 ' 'h'# distances constraints# (FromGrp FromAtom ToGrp ToAtom minDist optimalDist maxDist)DIST 1 CZ 2 CZ 3.36 4.36 5.36DIST 1 CZ 2 CE1 2.78 3.78 4.78DIST 1 CZ 2 CE2 3.77 4.77 5.77DIST 3 O 0 NH2 1.81 2.81 3.4DIST 0 NH1 4 O 1.63 2.63 3.2DIST 0 CZ 1 CE2 2.74 3.74 4.74DIST 0 CZ 1 CD2 2.82 3.82 4.82DIST 0 CZ 1 CG 3.66 4.66 5.66DIST 0 CZ 1 CD1 4.30 5.30 6.30DIST 0 CZ 1 CE1 4.25 5.25 6.25DIST 0 CZ 1 CZ 3.52 4.52 5.52
Van Dyk 11
# backbone separation# (FromGrp ToGrp min max)# END
In this template, substitution is permissible between aromatic residues (either Phe
or Tyr). Carbonyl oxygens in DIAP1_BIR2 were originally from the backbones of two
Alanine residues (Ala224 and Ala248) in DIAP1_BIR2, however this was relaxed in the
template so that carbonyl oxygens could come from any of the 20 primary amino acids.
In addition, distances characteristic to the motif were defined in the template. Edge-to-
face distances between various carbon atoms of Phe264 and Phe292 (i.e. 1 CZ<=>2 CZ,
1 CZ<=>2 CE1,1 CZ<=>2 CE2) as well as distances between carbonyl oxygens from Ala224
or Ala248 to hydrogen atoms from Arg229 (i.e. 3 O<=>0 NH2, 0 NH1<=>4 O) were
specified. Additionally, distances from Arg229 CZ to various carbon atoms of Phe264
(i.e. 0 CZ<=>1 CE2/CD2/CG/CD1/CE1/CZ) were included to ensure that Arg is parallel with
the aromatic residue it interacts with in forming a cation-π interaction.
For the template, the optimal distance (optimalDist) was defined as established
distances between atoms of DIAP1_BIR2 as involved in the template. Minimum and
maximum bounds for distances (minDist and maxDist respectively) were generally set as
20% less or greater than the optimal distance, however some bounds were set higher or
lower in optimization of parameters. This ensured that all BIR domains would be picked
up as matches for the motif.
A graphical representation of the template is shown in Figure 4. In the figure,
dashed lines represent inter-atom distances that were used in defining the template. Each
match obtained by Swiss-PdB Viewer was required to have all 11 of the defined inter-
atom distances within the minimum and maximum distance constraints.
Figure 4: Pictorial Representation of Template (Rendered with Swiss-PdB Viewer12)
Van Dyk 12
After obtaining a list of results through Swiss-PdB Viewer, the European
Bioinformatics Institute (EBI) protein database15 was used to identify characteristics of
the proteins based on PDB names (such as protein name and protein family). CaPTURE16
was used to determine whether structures obtained from PDB contained cation-π
interactions. As the cation-π interaction is a crucial component of the BIR-Arg motif, hits
that did not contain cation-π interactions were excluded from the list of results. The list
was carefully inspected to ensure that each match contained requisite interactions of the
BIR-Arg motif as well as desired Arg=>aromatic and aromatic=>aromatic geometries.
Finally, after inspecting each PDB file, MolMol17 was used to fit each match to the model
motif (DIAP1_BIR2, PDB code 1JD4). Fitting enabled quantitative and qualitative
comparison to be made between BIR-Arg motifs of IAP and non-IAP proteins.
Results
Much time was spent on refining the template so that it would accurately
represent the BIR-Arg motif (i.e. the final template in Figures 3-4). Initial templates
yielded lists of structures that included false positives, therefore this required time to be
Group 2: Phe292
Group 1: Phe264
Group 0: Arg229
Group 4: Ala248
Group 3: Ala224
Van Dyk 13
spent to increase the stringency of the template. One such was of doing this was by
defining additional distances within the template; this helped ensure that only true
matches were included in the search results.
Using Swiss-PdB Viewer’s built-in search function, the final template (Fig. 3-4)
was searched against a 90 percent non-redundant subset of the PDB14. After analyzing
results, it was determined that the BIR-Arg motif is present in 15 different proteins
(Tables 1-2), including 9 IAP proteins (with 13 of 14 BIR domains containing the motif)
and 7 non-IAP proteins (defined as proteins that came from other protein families). Non-
IAP proteins came from different protein families such as transferases, dioxygenases, and
other proteins involved in metabolic pathways (Table 2 and Fig. 5). Residues that are
involved in the motif of each IAP and non-IAP protein can be found in Tables A and B in
the Appendix.
Table 1: IAP Proteins Containing the BIR-Arg Motif
Van Dyk 14
Number Protein Name PDB Code
1 NIAP_BIR2 2VM5
2 CIAP1_BIR1 3M1D
3 CIAP1_BIR3 3D9T
4 CIAP2_BIR1 3M0A
5 CIAP2_BIR3 2UVL
6 Survivin 2QFA
7 Survivin-mouse 1M4M
8 Livin, ML-IAP 1TW6
9 Testes IAP 1XB0
10 DIAP1_BIR1 -
11 (Model) DIAP1_BIR2 1JD4
12 XIAP_BIR1 2QRA
13 XIAP_BIR2 1I3O
14 XIAP_BIR3 1NW9
Table 2: Non-IAP Proteins Containing the BIR-Arg Motif
Van Dyk 15
Number Protein Name PDB Code
1 Salmonella Typhimurium Cob(I)alamin
adenosyltransferase
1G5T
2 Human phosphotransferase 1RJB
3 Maize 4-hydroxyphenylpyruvate dioxygenase 1SP8
4 Thale cress 4-hydroxyphenylpyruvate dioxygenase 1TFZ
5 Chlorobium tepidum
(Green sulfur bacterium)
RuBisCO-like protein
1YKW
6 Elizabethkingia meningoseptica
Endo-beta-N-acetylglucosoaminidase
2EBN
7 Thale cress hydrolase 2FGE
Figure 5: Pie Chart of Types of Proteins Containing the BIR-Arg Motif
Van Dyk 16
Little variation was present in the BIR-Arg motif of IAP proteins, with 13 of 14
BIR domains containing the motif and residues overlapping almost perfectly (Fig. 6).
Aromatic and Arg residues were found to have great overlap and carbonyl oxygens
involved in hydrogen bonding were all within an acceptable range (as defined in the
template). In contrast to IAP proteins that had high conservation, a higher degree of
deviation was present among non-IAP proteins. Non-IAP proteins contained slightly
different geometry to DIAP1_BIR2 due to different rotation of aromatic residues in
space. However, despite small differences in geometry, all of the non-IAP proteins were
found to contain each requisite residue and interaction unique to the motif (Fig. 7-9).
Figure 6: Alignment of BIR-Arg Motif in 13 out of 14 BIR Domains
Van Dyk 17
Figure 7: Alignment of DIAP1_BIR2 and Maize 4-hydroxyphenylpyruvate dioxygenase
Figure 8: Alignment of DIAP1_BIR2 and Chlorobium tepidum (Green sulfur bacterium)
RuBisCO-like protein
Van Dyk 18
Figure 9: Alignment of DIAP1_BIR2 and Elizabethkingia meningoseptica Endo-beta-N-
acetylglucosoaminidase
Although the BIR-Arg motif was similar in structure among all IAP and several
non-IAP proteins, protein size and intervening structure were not as constant. A visual
illustration is presented between DIAP1_BIR2 and PDB file 1SP8 (4-
hydroxyphenylpyruvate dioxygenase of Zea mays) (Fig. 10). 1JD4 contains 1,586 protein
atoms, 119 hydrogen bonds, 14 alpha helices, 6 strands, and 22 turns while in contrast,
1SP8 contains 11,809 protein atoms, 1,036 hydrogen bonds, 55 alpha-helices, 124 beta
strands, and 128 turns. In the IAP proteins, the Arg and two aromatic residues are spaced
approximately 30 residues apart from each other in primary sequence. However, in 4-
hydroxyphenylpyruvate dioxygenase, the two aromatics are reversed in order in the
primary sequence. Additionally, one aromatic residue is adjacent to the Arg while the
other is more than 250 residues away. Despite such deviation in overall protein structure
between IAP and non-IAP proteins, it should be noted that each match contained all
Van Dyk 19
residues and interactions requisite of the BIR-Arg motif. Furthermore, each match
contained all 11 distances within the bounds established in the template. For further
analysis, Table 3 provides a comparison of inter-atom distances between DIAP1_BIR2
and non-IAP protein corresponding to PDB 1SP8. All distances except two (1 CZ<=>2
CE2 and 0 CZ<=>1 CE2) differ by less than ± 10%.
Figure 10: Comparison of Structure between IAP Protein (DIAP1_BIR2, PDB code
1JD4) and Non-IAP Protein (PDB code 1SP8)
Van Dyk 20
Table 3: Comparison of Inter-Atom Distances between IAP Protein (DIAP1_BIR2, PDB
code 1JD4) and Non-IAP Protein (PDB code 1SP8)
Distances In 1JD4 In 1SP8 Percent Difference
1 CZ<=>2 CZ 4.36 Å 3.99 Å 8.49%
1 CZ<=>2 CE1 3.78 Å 4.07 Å 7.67%
1 CZ<=> 2 C E2 4.77 Å 3.82 Å -19.92%
3 O<=>0 NH2 2.81 Å 3.05 Å 8.54%
0 NH1 4 O 2.63 Å 2.80 Å 6.46%
0 CZ<=> 1 C E2 3.74 Å 5.72 Å 52.94%
0 CZ<=>1 CD2 3.82 Å 3.90 Å 2.09%
0 CZ<=>1 CG 4.66 Å 4.96 Å 6.44%
0 CZ<=>1 CD1 5.30 Å 5.77 Å 8.87%
0 CZ<=>1 CE1 5.25 Å 5.72 Å 8.95%
0 CZ<=>1 CZ 4.52 Å 4.84 Å 7.08%
The uniqueness of the BIR-Arg motif was supported through a small experiment
(Fig. 11). In the experiment, a simplified version of the motif was created containing all
residues except for the second cation-π aromatic residue (Group 4 in the template, Fig. 3).
For the corresponding “simplified BIR-Arg motif” template, all distances were present
except for the removal of edge-to-face distances between edge-to-face aromatic residues
(1 CZ<=>2 CZ, 1 CZ<=>2 CE1, and 1 CZ<=>2 CE2).
Results indicate that the BIR-Arg motif is much more rare than the simplified
motif. While over 50,000 matches were obtained for the simplified motif, only 18
Van Dyk 21
positive matches (including both IAP and non-IAP proteins) were found to contain the
BIR-Arg motif. [Note: The 50,000 hits for the simplified motif contained both “positive”
and “negative” matches. (Negative matches are those that contain the requisite residues,
but whose geometry is too dissimilar to be considered a true match.) Due the sheer
number of results, results were not screened to yield only positive matches.] Since the
simplified motif--containing an Arg residue with bridging hydrogen bonds and a cation-π
interaction--is common, it follows that the BIR-Arg motif is more unique.
Figure 11: Comparison of Structure between BIR-Arg Motif and “Simplified” BIR-Arg
Motif
Van Dyk 22
Discussion
While structural motifs do not allow prediction of biological function within
proteins having dissimilar functions, conservation of a structural motif within a protein
family may indicate the importance of this motif to protein function. BIR-Arg motifs
were found in all structures of IAPs, suggesting that the BIR-Arg motif must be integral
to IAP proteins. Furthermore, conservation of the motif reveals the importance of this
structure to inhibition of apoptosis. The BIR-Arg motif was found to be not essential to
any non-IAP protein family as only sporadic hits of non-IAP proteins were obtained with
the motif template. These results reinforce that the BIR-Arg motif is essential to the
apoptotic pathway.
It was challenging finding a program that satisfied the requirements of this project
—to identify a structural motif while allowing variability of intervening sequences. The
web-based PINTS program11 appeared promising at first; however, it ultimately was not
useful. The lack of ability to substitute amino acids made resulting templates too
restrictive, resulting in exclusion of too many matches. The alternative 3-D structural
search software Swiss-PdB Viewer12 was successful in identifying the BIR-Arg motif.
However, a few limitations were present that without careful planning would have
limited results. Inability to allow equivalency of CD1 and CD2 or CE1 and CE2 atoms of
aromatic residues affected planning of the template. Specific distances had to be selected
that would accurately represent the motif without limiting how many hits were obtained.
For example, the “first” aromatic residue involved in a cation-π interaction with an Arg
residue was represented in the template with 0 CZ (Arginine)<=>1 CE2/CD2/CG/CD1/CE1/CZ
(Aromatic) distances (see Fig. 3 for convention). Lower and upper bounds for these
Van Dyk 23
distances had to be relaxed enough that the aromatic residue could be identified if the CA
atom connecting the backbone of the residue to the ring was oriented in both +x or -x
directions—as long as the aromatic residue was parallel with the Arg. Additionally, while
Swiss-PdB Viewer allows for fitting of 3D structures, its fitting function was too limited.
Swiss-PdB Viewer permitted fitting by Cα, backbone atoms, sidechain atoms, or by all
atoms. However, in this experiment it was important that residues be able to be fit atom
by atom. Residues involved in forming hydrogen bonds (Groups 3 or 4 in Fig. 3) were
desired to be fit solely through their carbonyl oxygens rather than their entire residues.
Similarly, Arg residues were desired to only be fit through atoms of its guanidino
functional group (CZ, NH1, NH2, NE). Finally, aromatic residues were desired to be fit
through all sidechain atoms. This combination of desired specifications for fitting
required the use of software that enabled fitting by atoms. Consequently, the molecular
graphics program MolMol17 was used for fitting in the experiment as it permitted fitting
by atoms.
Non-IAP proteins contained more variation in geometry of participating residues
than BIR domains of IAP proteins. However, all requisite residues and interactions
unique to the BIR-Arg motif were present in the 7 identified non-IAP proteins. In Figure
7, the structure 1SP8 contains hydrogen bonding to hydrogen atoms of the guanidino
functional group of the Arg residue, a cation-π interaction from the Arg to an aromatic
Phe residue, and an edge-to-face aromatic-to-aromatic interaction from the Phe to another
Phe residue. In this protein, if the guanidino group is designated as pointing in the +x
direction, the only difference in the BIR-Arg motif is that the backbone of Phe residue
involved in the cation-π interaction is facing in the +x rather than in the –x direction.
Van Dyk 24
This a trivial distinction since the aromatic group itself is symmetric. Similarly, the face
of the second aromatic packs against a different edge of the first aromatic but again, this
is still an edge-to-face interaction.
The ability for atoms of the cation-π aromatic residue to be parallel or anti-
parallel to the Arg guanidino group was an important consideration in designing the
template. It was important to allow this flexibility as it did not matter where the backbone
was facing as long as the aromatic ring was within range to donate electron density to the
Arg (i.e. by satisfying distance constraints of the template in Figure 3). The only
requirement for this aromatic residue was that the ring must be parallel with the Arg.
Another area of consideration is that the template could be better improved in the
future. While the template in Figure 3 was sufficient for this experiment, several changes
could be made to better represent the motif and to facilitate searching. Limitations in the
software as well as missing or too restrictive distances resulted in several false matches
that had to be excluded by hand. By improving the template, analyzing the motif would
be easier as less time would be needed to exclude matches. Changes could be made to
eliminate false matches, such as those that contain residues from different chains (i.e. an
Arg from chain A, a Phe from chain B, and a Phe from chain C) as well as non-IAP
proteins that contain internal geometries too dissimilar to that of the BIR-Arg motif.
Finally, allowing substitution of Trp to other aromatic residues Phe or Tyr would
provide a stronger analysis of non-IAP proteins containing the BIR-Arg motif. Trp was
found not to be present in BIR-Arg motifs in IAP proteins,6 however it could be possible
that it may be present in BIR-Arg motifs in non-IAP proteins. Additional complexity
would be present in substitution of Trp--a cation-π interaction containing a Trp residue
Van Dyk 25
could have electron density be donated from either ring or from the center of the Trp.
Furthermore, edge-to-face aromatic-to-aromatic interactions would become more
complex with the presence of one or two Trp residues. While not enough time was
present in this experiment to allow substitution of aromatic residues with Trp in the
motif, this could be something that could be expanded upon in the future.
Conclusion
The BIR-Arg motif was successfully identified as conserved among IAP proteins
and present in 7 non-IAP proteins. Furthermore, the motif was found not to be conserved
among any non-IAP protein families. Results from the experiment suggest that the BIR-
Arg motif is integral to IAP function, and lack of conservation among non-IAP protein
families indicates the uniqueness of this motif.
To improve findings, three areas in this experiment could be improved. The
template for the BIR-Arg motif in Figure 3-4 was sufficient to identify the motif in all 9
structures of IAP proteins, however exclusion of false matches could help facilitate
searching in the future. Additionally, while BIR-Arg motifs in IAP proteins do not
contain Trp,6 it is possible that some non-IAP proteins may have been excluded that
contained BIR-Arg motifs with Trp instead of Phe or Tyr. Finally, Swiss-PdB Viewer’s
automatic search feature against a 90 percent non-redundant subset of the PDB did not
identify all 13 of 14 BIR domains so it may also have missed several non-IAP proteins.
To improve results, the search should optimally be performed against the entire PDB
database.
Another area that could be investigated in the future is analyzing protein
sequences of IAP and non-IAP protein families. Although it was weakly inferred that the
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BIR-Arg motif was not conserved among non-IAP families due to matches corresponding
to different protein families, analyzing sequences will make this definitive. (If the BIR-
Arg motif was conserved among non-IAP families, it would be expected that multiple
matches be obtained for each non-IAP protein family.)
Much research has been made analyzing the signal transduction pathway
involving hierarchical groups of caspases and IAP proteins.5 Smac (in humans) and
DIABLO (in mice) have been found to bind to XIAP (and possibly other additional
IAPs), displacing caspases, and negatively regulating IAP activity.5 Findings from this
experiment may guide researchers towards better understanding the process of allosteric
regulation of apoptosis with IAP proteins.
Finally, while all 9 structures of IAP proteins were found to contain the BIR-Arg
motif, only one BIR domain (BIR1 of DIAP1) out of 14 from the 9 IAP proteins was
found not to contain the motif. As the motif was found to be highly conserved within
existing structures of IAP proteins, the lack of the motif might signify that this BIR
domain might have some important role disparate from that of other BIR domains. As
more structures of IAP proteins become available, methods from this experiment may be
able to be used to analyze the structures in order to gain a better understanding of the
function of the BIR-Arg motif. Perhaps, the development of techniques to identify
conservation of the BIR-Arg motif could even be used as a baseline for analysis of other
structural motifs or other protein analysis for other protein families.
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AppendixTable A: Table of Residues Involved in BIR-Arg Motifs of IAP Proteins
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Table B: Table of Residues Involved in BIR-Arg Motifs of Non-IAP Proteins