UW Bioengineering

Designing a Pseudomonas aeruginosa vaccine antigen Bioen 588, Lab 5

Albert Nguyen

2-7-2017

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INTRODUCTION

Pseudomonas aeruginosa is an opportunistic, Gram-negative bacterium that frequently

causes life-threatening infections in burn, cancer, cystic fibrosis, and immune compromised

patients. It is especially dangerous due to a naturally high innate resistance and high frequency of

acquired anti-microbial resistance and thus there is considerable interest in vaccine development

against this pathogen. [1] Infection begins with bacterial adherence to host mucosal cells via type

IV pilus on the surface of the bacteria. These pili are composed of repeating units of structural

proteins known as pilin which contain a functional receptor binding site near the C-terminus. [1]

The high abundance of surface pilin, however, also makes the protein an ideal vaccine target as it

can be more easily recognized by the immune system. Furthermore, as pilin is crucial to the

initiation of infection, patient antibodies that block the binding site can effectively prevent the

infection.

Given the desperate need for a P. aeruginosa vaccine, the purpose of the following

experiments was to design two potential protein antigens that could be used in such a vaccine. The

first experiment involved using homology modeling software Modeller [2] to create a homology

model of type IV fimbrial precursor pilin protein based off the known structure of truncated PAK

pilin protein. These were chosen as both play important roles in bacterial adhesion and due to a

predicted homology between them. Moreover, the structure of the former is currently unknown

which necessitates the creation of a homology model. The second experiment involved using

Chimera and Foldit software [3, 4] to redesign the truncated PAK pilin protein to be more suitable

for use in a vaccine. This meant mutating various residues to improve stability around the binding

region and/or outright eliminating regions of the protein to reduce size and thus cost to produce.

Five redesigns in total were made and run through the in lucem molecular mechanics simulation,

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ilmm, [5] using the XSEDE Stampede supercomputer [6] to determine how our redesigns would

fare in in-vivo conditions. Data from these simulations were then used to create a final redesign

which was also run through the ilmm simulation to determine the success of the redesign.

METHODS

Homology Model for Type IV Fimbrial Precursor Pilin

FASTA sequence for type IV fimbrial precursor pilin was obtained from the NCBI

database [7] while truncated PAK pilin structure in a PDB file was obtained from the RCSB Protein

Databank (PDB code 1DZO). [8] These were used as inputs for Modeller to generate a homology

model of the Type IV pilin. Modeller does this by comparing the FASTA sequences of the proteins

then predicting a structure of the unknown protein using the assumption that sequence alignment

correlates with structure alignment. [2] We then analyzed the structures of the pilin proteins using

Chimera’s MatchMaker function. [3]

Redesign of Truncated PAK Pilin

Five redesigns of the truncated PAK pilin (PDB code 1DZO) were made using Chimera

and Foldit software and saved as PDB files. [3, 4] While performing each redesign we made sure

to not make any changes to the binding region of the protein which was identified in a previous

study. [9] In the first redesign we used Foldit’s automated mutation and conformation algorithms

to create a more stable protein, calling this design Automated Foldit Design (AFD). In the second

redesign, we used our own intuition to manually redesign the protein while also using Foldit’s

algorithms as guidelines. The structure obtained from this procedure was labeled Manual Foldit

Design (MFD). In the third redesign, we also used our intuition to mutate specific residues and

change their conformations but instead used Chimera software. This design was labeled Intuitive

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Design (ID). For the fourth and fifth designs, we first eliminated all residues in the PAK pilin

protein that we felt were unnecessary for preserving the structure of the binding region, which was

identified in previous studies [10, 11], to create a PAK pilin fragment. In the first of the fragment

designs the residues in this fragment were left unchanged and was labeled Fragment Wild Type

(FWT). In the second we used Foldit to mutate the residues in the fragment as we saw fit along

with the software’s stabilization algorithms and labeled this design Fragment Manual Design

(FMD).

Molecular Simulations and Final Redesign

With the five protein redesigns and the wild type structure as PDB files we were then able

to run them through ilmm molecular dynamics software by accessing the XSEDE Stampede

supercomputer. [5, 6] The ilmm software places the protein in a simulated 10Å solvation cube and

uses molecular dynamics algorithms to replicate protein behavior in solution. We used a simulation

length of 5ns over 5000 steps and, to mimic conditions in the human body, a temperature of 37°C

(310K). The simulations then output various data for analysis including Define Secondary

Structure of Proteins (DSSP) plots, C-α root mean square deviation (RMSD) – deviation with

respect to a minimum energy structure – data, C-α root mean square fluctuation (RMSF) –

deviation with respect to the starting structure – data, and animations of protein dynamics. DSSP

plots assign secondary structure identities to residues in the sequence based on hydrogen bonding

in the protein backbone. We then made a final redesign (RD) of the pilin protein with Foldit using

the outputted data as a guide and performed the ilmm simulation on this redesign to determine the

success of our changes. Ultimately, the process of designing RD consisted of making mutations in

AFD which was identified as the most stable of the initial redesigns.

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RESULTS

Homology Model for Type IV Fimbrial Precursor Pilin

Chimera MatchMaker alignment (Figures 1bc) of the homology model and the reference

PAK pilin revealed that the two structures had an overall C-α RMSD of 0.581Å despite only having

a sequence identity of 56.52%. While the the reference structure had a slightly longer main alpha

helix and, in certain areas, longer beta strands, the C-α RMSDs for most residues in non-loop

regions still fell within 0.2Å (Figure 1b). However, the small alpha helix near the C-terminus of

the reference was absent in the homology model. In addition, the greatest C-α RMSDs, i.e. those

above 0.4Å, between the two structures generally occurred in loop regions where the sequences

didn’t align (Figures 1bc). Finally, residues that play major roles in pilin binding [11] in the

truncated PAK pilin protein were not completely conserved in the analogous region of the

homology model, namely with modifications Q136M and T138I.

Redesign of Truncated PAK Pilin: Simulation Results

The simulation C-α RMSD values for all the initial designs and wild type protein reached

an equilibrium near the end of each simulation (Figure 3). In addition, they all equilibrated to about

the same value of 2Å. On the other hand, this was not the case for either of the fragment designs

or the final redesign (FWD, FMD, and RD); in fact, the RMSD of the mutated fragment (FMD)

was increasing by the end of the simulation. Moreover, of all the untruncated designs the final

redesign in general had the highest RMSD over all time points. In terms of residue fluctuations,

protein animations (not included) and C-α RMSF plots (Figures 4ab) indicated that loop regions

in all designs corresponded to areas with the greatest fluctuations. This was especially apparent at

the loops near the N termini of the designs. Moreover, the fragmented designs had RMSF values

that were generally larger than those of the untruncated proteins (Figure 4b).

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DSSP plots (Figures 5a-c, g) revealed that the untruncated designs and wild type protein

had relatively consistent secondary structures with no significant difference among each design

and the wild type. The only noticeable disappearance was that of the beta bridge in the Manual

Foldit Design and wild type protein in the loop region around residue 68. In addition, the intuitive

design adopted an additional alpha helix in the region around residue 55. We tried to replicate this

helix in the final redesign by matching the sequence in the corresponding region but

unsuccessfully. On the other hand, neither of the fragment designs could maintain their secondary

structures (Figures 5de). In the case of the mutated fragment the beta sheets and alpha helix were

completely lost while in the case of the wild type fragment only the helix was lost.

DISCUSSION

Homology Model for Type IV Fimbrial Precursor Pilin

The data suggest that the homology model successfully recreated the major secondary

structures of the PAK pilin reference. The beta sheets and alpha helices of the reference structure

were replicated in the homology model to within 0.4Å C-α RMSD with only minor differences in

structure size, i.e. being a couple residues longer (Figures 1a-c). Although there was a small helix

near the C-terminus of the reference that was not present in the homology model, the data for the

corresponding region in the homology model show that the C-α RMSD fell within 0.2Å indicating

similarity between the two structures (Figure 1b). It may be possible that this region in the

homology model may in fact be a helix but was unidentified due to some quirk in Modeller. This

is supported by the conformation of the backbone in this region of the homology model which has

some resemblance to a single turn helix. As a result, the homology in backbone structure between

the two in this region indicate the patient’s adaptive immune system may be able to recognize both

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using the same binding ligands. However, residues identified as host cell binding participants in

PAK pilin [9] weren’t present in the corresponding region of the Type IV pilin homology model

(Figures 1ac) suggesting that Type IV pilin cannot completely substitute PAK pilin. It should be

noted that the homology model is only a predictor of the protein structure and future X-ray

crystallography experiments must be performed to determine the success of this study in

establishing the structure of Type IV pilin.

Redesign of Truncated PAK Pilin and Effects on Stability

Trends in C-α RMSD for the initial, untruncated, designs (AFD, ID, and MFD) suggest

that while the untruncated designs could maintain a stable structure, the fragment designs could

not. This is because they were all able to reach an equilibrium within 5ns (Figure 3). By contrast,

the RMSD of both fragment designs and the final redesign (FMD, FWT, and RD) did not

equilibrate and were in general larger signifying that these structures were much less stable than

the untruncated designs. The ability or lack thereof of the designs to maintain their secondary

structures according to DSSP plots (Figures 5a-g) further corroborates this. Surprisingly, the DSSP

plot of the intuitive design also revealed that the structure adopted an alpha helix between residues

53 and 58 but attempts to recreate this in the final redesign via sequence matching were

unsuccessful.

In terms of preserving the binding region, all the untruncated designs had lower or

equivalent RMSF values around the binding region relative to the wild type suggesting that these

designs were successful at stabilizing the binding region (Figures 4ab). The DSSP plots also

indicated no major changes in major secondary structure in this region (Figures 5a-c, g). By

contrast, both fragment designs had much higher C-α RMSF around the binding region relative to

the other designs and the wild type protein and could not maintain their major secondary structures.

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This indicates these designs had a destabilizing effect on the binding region and were thus

unsuccessful designs. As a result, we conclude that the removed regions of the protein were critical

to maintaining structure and recommend future vaccine designs shouldn’t be truncated to the same

degree performed in this study.

CONCLUSIONS

We created a total of six antigen designs for use in a P. aeruginosa vaccine based off Type

IV fibrial precursor pilin and truncated PAK pilin. The first was a homology model of the former

protein while the remainder were modifications of the latter. Though the homology model could

recreate the majority of the secondary structures in the PAK pilin reference, there were slight

differences in sequence around the binding region. Despite this, similarities in backbone structure

around the region mean it may be possible to create a vaccine antigen that allows the immune

system to recognize both antigens on P. aeruginosa. Protein redesigns of PAK pilin had mixed

results in stabilizing the protein around the binding regions; untruncated designs could stabilize

the protein while truncated ones could not. Thus we do not recommend any future redesigns of

PAK pilin to truncate the protein to the same degree we did. Future tests are required to determine

whether our designs in actuality prove to be more stable than the wild type PAK pilin but we are

confident any of our untruncated designs will be helpful in the creation of a future vaccine.

REFERENCES

1. Keizer, D. W. et al. Structure of a pilin monomer from Pseudomonas aeruginosa:

Implications for the assembly of pili. J. Biol. Chem. 276, 24186–24193 (2001).

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2. Sali, Andrej. MODELLER. Computer software. MODELLER, Program for Comparative

Protein Structure Modelling by Satisfaction of Spatial Restraints. Vers. 9.17. Sali Lab,

UCSF, n.d. Web. <https://salilab.org/modeller/tutorial/basic.html>.

3. Chimera. Computer software. Vers. 1.11.2. UCSF, n.d. Web.

<http://www.cgl.ucsf.edu/chimera/>.

4. Foldit. Foldit Solve Puzzles for Science. University of Washington, n.d. Web.

<http://fold.it/portal/>.

5. Beck, DAC, McCully, ME, Alonso, DOV, Daggett, V. in lucem molecular mechanics,

University of Washington, Seattle, 2000-2017.

6. XSEDE User Portal | TACC Stampede. National Science Foundation, n.d. Web. 26 Jan.

2017. <https://portal.xsede.org/#/guest/>.

7. "Type 4 Fimbrial Precursor PilA [Pseudomonas Aeruginosa PAO1] - Protein -

NCBI." National Center for Biotechnology Information. U.S. National Library of

Medicine, n.d. Web. 09 Jan. 2017.

8. RCSB Protein Data Bank, Hazes, B., Sastry, P.A., Hayakawa, K., Read, R.J., Irvin, R.T.

"1DZO." RCSB PDB - 1DZO: Truncated PAK Pilin from Pseudomonas Aeruginosa

Structure Summary Page. N.p., n.d. Web. 09 Jan. 2017.

9. Wong, W. Y. et al. Structure-function analysis of the adherence-binding domain on the

pilin of Pseudomonas aeruginosa strains PAK and KB7. Biochemistry 34, 12963– 12972

(1995).

10. Hodges, Robert S., William Paranchych, Kok K. Lee, Sastry A. Parimi, Randall T. Irvin,

and Peter C. Doig. Synthetic Pseudomonas Aeruginosa Pilin Peptide Vaccine. The

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Governors of the University of Alberta, assignee. Patent US 5612036 A. 18 Mar. 1997.

Print.

11. Wong, W. Y. et al. Structure-function analysis of the adherence-binding domain on the

pilin of Pseudomonas aeruginosa strains PAK and KB7. Biochemistry 34, 12963– 12972

(1995).

FIGURE LEGENDS

Figure 1

A) Reference truncated PAK pilin structure. Residues involved in host cell binding are colored

magenta while residues that participate that don’t participate in binding but are still the target of

our vaccine design are colored cyan. Sequence was identified in previous work. [10, 11]; B) Type

IV Fimbrial Precursor Pilin homology model with color spectrally assigned based on C-α RMSD

with respect to truncated PAK pilin: Blue = 0 Angstroms, White= 0.2 Angstroms, Red = 0.4

Angstroms; C) Homology model with blue indicating sequence alignment and red indicating

where sequence doesn’t align.

Figure 2

A) Fragment Wild Type (FWT); B) Fragment Manual Design (FMD); Mutations shown.

Figure 3: C-α RMSD of designs as a function of time. AFD = Automated Foldit Design; ID =

Intuitive Design; MFD = Manual Foldit Design; WT = Wild Type Protein; FMD = Fragment

Manual Design; FWT = Fragment Wild Type.

Figure 4

C-α RMSF as a function of residue position for untruncated designs (A) or all designs with adjusted

residue numbers (B). Adjusted residue number relates residue position in fragments with the

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analogous position in the untruncated designs. Residues 104-120 are identified as the binding

region. AFD = Automated Foldit Design; ID = Intuitive Design; MFD = Manual Foldit Design;

WT = Wild Type Protein; FMD = Fragment Manual Design; FWT = Fragment Wild Type.

Figure 5a-f

DSSP plots of protein designs and wild type. AFD = Automated Foldit Design; ID = Intuitive

Design; MFD = Manual Foldit Design; WT = Wild Type Protein; FMD = Fragment Manual

Design; FWT = Fragment Wild Type.

Figure 6

FASTA Sequences for proteins and redesigns used in study.

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FIGURES

Figure 1

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Figure 2

Figure 3

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C-α RMSD vs Time

AFD

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RD

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Figure 4

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Residue #

A. C-α RMSF vs Residue #

AFD

ID

MFD

WT

RD

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90 95 100 105 110 115 120

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B. C-α RMSF vs Adjusted Residue #

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Figure 5

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Wild Type Protein Sequences

Type IV Fimbrial Pilin Precursor

ARSEGASALATINPLKTTVEESLSRGIAGSKIKIGTTASTATETYVGVEPDANKLGVIAVAI

EDSGAGDITFTFQTGTSSPKNATKVITLNRTADGVWACKSTQDPMFTPKGCDN

Wild Type PAK Pilin (WT)

GTEFARSEGASALASVNPLKTTVEEALSRGWSVKSGTGTEDATKKEVPLGVAADANKL

GTIALKPDPADGTADITLTFTMGGAGPKNKGKIITLTRTAADGLWKCTSDQDEQFIPKGC

SR

Redesigns of PAK Pilin

Automated Foldit Design (AFD)

RRERAREYGQRALNSVNRLQEVVERALRRGWAVRSGTGREDKREKKVPLGRKDDQN

RLGRIELRSDPADGRRDIRIRFRMEGAGPKNKGKVITLERESKKGRWKCTSDQDEQFIP

KGCSR

Intuitive Design (ID)

RRERARREGESALDKVNPLKTTVEEALSRGWSVKSGTGTEDATKKEVPLGVAADANK

NGTIALKPDPADGTADITLTGTMGGAAPKNKGKIITLTRESKKGLWKCTSDQDEQFIPKG

CSR

Manual Foldit Design (MFD)

GTQFARSEGESALRSVNRLKTTVEEALSRGWSVKSGTGTEDATKKEVPLGVAADANKL

GTIALKPDPADGTADITLTFTMGGAGPKNKGKIITLTRTAADGLWKCTSDQDEQFIPKGC

SR

Fragment Manual Design (FMD)

KITTDTRTAADGLKKCTSDQDEQFIPKGCSR

Fragment Wild Type (FWT)

KIITLTRTAADGLWKCTSDQDEQFIPKGCSR

Final Redesign (RD)

RRERAAEYGQRALNSVNRLQEVVERALRRGWAVRSGTGREDKREKKVPLGRKADAN

RNGRIELRSDPADGRRDIRIRFRMEGAGPKNKGKVITLERESKKGRWKCTSDQDEQFI

PKGCSR

Figure 6