The BIR-Arg Motif of Inhibitor of Apoptosis (IAP) Proteins is Rare in Non-IAP Proteins

Mark Van DykBiochemistry Research

Spring 2014

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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

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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,

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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."

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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.

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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

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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

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# 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)

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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

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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

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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

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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

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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

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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

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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

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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)

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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

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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

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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

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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.

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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

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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.

Van Dyk 27

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AppendixTable A: Table of Residues Involved in BIR-Arg Motifs of IAP Proteins

Van Dyk 29

Table B: Table of Residues Involved in BIR-Arg Motifs of Non-IAP Proteins