THESIS - DOCTOR OF PHILOSOPHY - JAMES Amy Midori - 2017

146 Pages·2017·6.87 MB·English

by  Joanne Edmondston

Evolutionary insights into two plant protein families: Bowman-Birk inhibitors
and asparaginyl endopeptidases
Amy Midori James
Bachelor of Science (Honours), University of Victoria, BC, Canada
Master of Science, University of Victoria, BC, Canada

This thesis is presented for the degree of Doctor of Philosophy of The University of Western Australia
School of Molecular Sciences
2017

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ABSTRACT
This thesis is divided into two parts and is presented as a series of manuscripts that are published or are
formatted for publication. Part I of the thesis describes the investigation of a class of plant protease inhibitors,
the Bowman-Birk inhibitors (BBIs). Part II of the thesis investigates the mechanism of a class of proteases that
possesses a second function as cyclizing enzymes, asparaginyl endopeptidases (AEPs). The initial interest in
these two distinct and unrelated protein families, the BBIs and the AEPs, was their common association with a
small, cyclic protease inhibitor, Sunflower Trypsin Inhibitor-1 (SFTI-1). SFTI-1 is processed into its mature form
by AEPs and shares structural homology with BBIs.
Part I of the thesis investigates the evolution of Bowman-Birk Inhibitors and includes one research paper which
is described briefly here. Despite sharing structural, functional and sequence similarity with BBIs, SFTI-1 has
distinct evolutionary origins to BBIs and has evolved by convergent evolution. BBIs have previously only been
described in the legume and cereal families. This distant phylogenetic distribution implies BBIs evolved
independently by convergence yet their high structural and sequence similarity suggests they share a common
ancestor. Using targeted bioinformatic searches for the highly conserved BBI inhibitory loop, we discovered
BBI-like sequences exist in many plant species, including the basal angiosperm Amborella trichopoda and in the
lycopod Selaginella moellendorffii. Lycopods diverged ~200-230 million years before the common ancestor of
angiosperms. We demonstrated that protein encoded by one of the S. moellendorffii BBI-like sequences is
capable of inhibiting trypsin. Identification of functional BBIs in Selaginella and BBI-like sequences in the basal
angiosperm A. trichopoda implies legume and cereal BBIs share a common ancestor and did not evolve by
convergent evolution.
Part II of the thesis investigates the mechanism of AEPs and includes three research chapters which are
described briefly in the following paragraphs. SFTI-1 is processed from a precursor protein, PawS1
(Preproalbumin with SFTI-1), which is also precursor to a seed storage albumin. PawS1 is processed by AEPs,
which release the albumin subunits as well as SFTI-1. To gain a greater understanding of the processing of the
dual-destiny precursor protein, we determined the structure based on NMR spectroscopy of PawS1. The
structure showed two well-defined domains, consisting of SFTI-1 and the albumin subunits, separated by a
flexible linker peptide. To gain further insight into maturation of albumin from PawS1, PawS1 was incubated
with sunflower extracts as well as with recombinant sunflower AEP1 (HaAEP1). Both studies provided evidence
for multiple cleavage sites in PawS1 for HaAEP1.
AEPs have been recruited for the synthesis of cyclic peptides multiple times through plant evolution. In
addition to SFTI-1, the unrelated cyclic peptides, cyclic knottins and kalata-type cyclic peptides, also require
AEP for their synthesis. To gain an understanding of the mechanism of cyclization by AEP, we determined the
crystal structure of a sunflower AEP capable of performing a cyclization reaction. To further probe which
residues of the active site influence activity, a series of mutants were generated and tested for cyclizing ability.
These results, along with the structure of the sunflower AEP, enabled the modelling of SFTI-1 maturation and
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defined structural characteristics of AEP that have enabled the recruitment of AEPs for synthesis of cyclic
peptides compared to other highly similar proteases.
One example of an AEP has been suggested to have specialized as a cyclizing enzyme and has apparently lost
its protease activity. Butelase 1, an AEP from Clitoria ternatea, is a very efficient cyclase apparently lacking the
ability to cleave yet with no discernible differences in primary sequence to explain this lack of cleavage activity.
We expressed Butelase 1 in Arabidopsis lacking expression of any endogenous AEPs and produced
recombinant Butelase 1 to test its cleavage ability. Both approaches suggested that Butelase 1 maintains its
cleavage activity, although with less efficiency compared to other AEPs. Possibly AEPs are evolutionarily
constrained to maintain some cleavage activity as AEPs are known to self-process. AEPs are expressed in an
inactive form and require the removal of a cap domain by AEP to become active.
This thesis investigates two interesting plant protein families using bioinformatics, biochemistry and structural
biology. The major findings presented are: identification of ancient origins for BBIs; structural elucidation of
the processing of the SFTI-1 precursor; determination of structural features influencing bifunctionality of AEP;
and determination of a predicted evolutionary constraint on complete specialization of AEPs.

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TABLE OF CONTENTS
Thesis declaration ................................................................................................................................................... ii
Abstract ................................................................................................................................................................. iii
Table of contents .................................................................................................................................................... v
Acknowledgements ............................................................................................................................................... vi
Authorship (cid:282)eclaration: Co-authored publications .............................................................................................. vii
List of abbreviations ............................................................................................................................................... 1
Preface and outline of thesis .................................................................................................................................. 3
Part I: Evolution of Bowman-Birk inhibitors ........................................................................................................... 4
Chapter 1: General introduction......................................................................................................................... 5
1.1 Biological function of Bowman-Birk inhibitors ........................................................................................ 5
1.2 Distribution and molecular evolution of Bowman-Birk inhibitors ........................................................... 5
1.3 Structural characteristics of Bowman-Birk inhibitors .............................................................................. 6
1.4 Scope of study presented in Chapter 2 .................................................................................................... 7
1.5 Literature cited......................................................................................................................................... 7
Chapter 2: Evidence for ancient origins of Bowman-Birk inhibitors from Selaginella moellendorffii (James et
al., 2017; The Plant Cell) ................................................................................................................................... 10
Chapter 3: General Discussion .......................................................................................................................... 50
3.1 Summary and major outcomes .............................................................................................................. 50
3.2 Internal tandem duplication of BBI inhibitory motifs ............................................................................ 50
3.3 BBIs as a model for gene family loss and gene family expansion .......................................................... 55
3.4 Literature cited....................................................................................................................................... 55
Part II: Structural and biochemical investigations of asparaginyl endopeptidases .............................................. 62
Chapter 4: General introduction: Macrocyclization by asparaginyl endopeptidases (James et al., 2017; New
Phytologist) ....................................................................................................................................................... 63
Chapter 5: Two proteins for the price of one: Structural studies of the dual-destiny preproalbumin with
Sunflower Trypsin Inhibitor-1 (Franke et al., 2017; Journal of Biological Chemistry) ...................................... 70
Chapter 6: Structural basis of ribosomal peptide macrocyclization in plants (Haywood et al., 2018; eLife) ... 85
Chapter 7: Auto-catalytic cleavage is an evolutionary constraint for macrocyclizing endopeptidases (James et
al., in prep)...................................................................................................................................................... 108
Chapter 8: General discussion ........................................................................................................................ 133
8.1 Summary of key findings ...................................................................................................................... 133
8.2 Future directions: Understanding the (cid:349)(cid:374)(cid:3)(cid:448)(cid:349)(cid:448)(cid:381) mechanism of maturation of SFTI-1 from PawS1 ........ 134
8.3 Literature cited..................................................................................................................................... 137
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ACKNOWLEDGEMENTS
This thesis comprises the work of many people to whom I am extremely grateful for generously providing their
time and expertise. As this thesis is prepared as a series of papers, the work presented is the result of
collaboration between many great scientists. I benefitted greatly from the diversity of expertise represented
by my co-authors. All papers presented were led by my supervisor, Dr Joshua S. Mylne. I am thankful for his
creativity and curiosity that initiated all the work presented in this thesis. I would also like to thank my co-
supervisor, Prof. Charles S. Bond, who offered essential scientific and technical advice to support the work
presented. I also thank the examiners for taking the time to critique my thesis.
I would like to thank all the members of the Mylne group, the Bond group and other members of the school of
Molecular Biosciences and Plant Energy Biology who kindly provided advice and support on countless
occasions. Specifically, I would like to thank Dr Kalia Bernath-Levin for teaching me about protein expression
and purification; Dr Gavin Knott for teaching me techniques in protein purification, protein crystallization, and
isothermal calorimetry; Maxime Corral and Julie Leroux for teaching me techniques in working with
Arabidopsis; Dr Ricarda Fenske, Mark Fisher and Jingjing Zhang for assistance with mass spectrometry; Dr
Achala Jayasena for assistance with bioinformatics; and Dr Joel Haywood and Dr Jason Schmidberger for advice
on optimization of protein crystallization.
In addition to my scientific education during my studies at the University of Western Australia, I am grateful for
many other opportunities provided during my degree including opportunities to be part of the undergraduate
lab practical teaching program. I would like to thank Assoc. Prof. Martha Ludwig, Dr Thomas Martin and Dr
Heng Choii for the opportunity to be a demonstrator for several undergraduate laboratory courses. I am also
grateful to Karina Price, the science communications officer for Plant Energy Biology, for many opportunities to
participate in science communication activities. I am grateful to have taken part in the Scientific Mentorship
program at UWA and to Dr Laura Boykin for her valuable mentorship.
Finally, I would like to thank my loving husband, Alec James, for travelling across the world to support me in
my pursuit of knowledge. I am forever thankful to my family for supporting us in our decision to come to
Australia. The experience of living and studying in a different country has been invaluable and I am indebted to
my supervisor, Dr Joshua S. Mylne, for providing an enriching and scientifically exciting environment.

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AUTHORSHIP DECLARATION: CO-AUTHORED PUBLICATIONS
This thesis contains work that has been published and/or prepared for publication.
Details of the work:
James, Jayasena, Zhang, Berkowitz, Secco, Knott, Whelan, Bond, Mylne (2017) Evidence for
ancient origins of Bowman-Birk inhibitors from Selaginella moellendorffii. The Plant Cell 29:461-
473
Location in thesis:
Part I: Chapter 2
Student contribution to work (Overall contribution: 90%):
Amy James along with Joshua S. Mylne wrote the manuscript. Amy James performed all
experimental work with the exception of library preparation and sequencing (Oliver Berkowitz,
David Secco, James Whelan), RNA extractions (Joshua S. Mylne) and transcriptome assembly
(Achala Jayasena, Jingjing Zhang). Gavin J. Knott and Charles S. Bond provided advice on
optimization of protein purification.

Details of the work:
James, Haywood, Mylne (2017) Macrocyclization by asparaginyl endopeptidases. New
Phytologist. Accepted 24 Jan 2017. DOI: 10.1111/nph.14511
Location in thesis:
Part II: Chapter 4
Student contribution to work (Overall contribution: 70%):
Amy James along with Joshua S. Mylne and Joel Haywood wrote the manuscript. Amy James
produced Figure 1 and Joel Haywood produced Figure 2.

Details of the work:
Franke, James, Mobli, Colgrave, Mylne, Rosengren. (2017) Two proteins for the price of one:
structural studies of the dual-destiny preproalbumin with Sunflower Trypsin Inhibitor-1. Journal
of Biological Chemistry 292:12398-12411
Location in thesis:
Part II: Chapter 5
Student contribution to work (Overall contribution: 10%):
Amy James generated recombinant AEP and contributed to the editing of the final manuscript.
The paper was written by Bastian Franke, Josh Mylne and K. Johan Rosengren. Bastian Franke
performed all additional experimental work with assistance with NMR data recording,
processing and analysis (Mehdi Mobli), with structure calculations and analysis (K. Johan
Rosengren), and with mass spectrometry recording and analysis (Michelle L. Colgrave).

Details of the work:
Haywood, Schmidberger, James, Nonis, Sukhoverkov, Elias, Bond, Tawfik, Mylne. Structural
basis of ribosomal peptide macrocyclization in plants. Manuscript submitted for review.
Location in thesis:
Part II: Chapter 6
Student contribution to work (Overall contribution 30%):
Amy James performed site directed mutagenesis to generate Cys AEP mutant, assisted in
generation of activate recombinant AEP protein, assayed AEP and AEP mutants for activity and
contributed to editing of the manuscript. Joel Haywood wrote the manuscript and performed
all experimental work with assistance from Jason Schmidberger and Charles S. Bond in
production of the HaAEP1 protein crystal and solving the structure, from Amy James, Samuel G.
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Nonis and Kirill Sukhoverkov in producing recombinant protein, from Amy James and Samuel G.
Nonis in performing enzyme assays and from Kirill Sukhoverkov in circular dichroism assays,
Details of the work:
James, Haywood, Leroux, Elliott, Fisher, Ignasiak, Nonis, Fenske, Mylne. Auto-catalytic cleavage
is an evolutionary constraint for macrocyclizing endopeptidases. Manuscript prepared for
submission.
Location in thesis:
Part II: Chapter 7
Student contribution to work (Overall contribution 70%):
Amy James wrote the manuscript with assistance from Katarzyna Ignasiak and Joshua S. Mylne.
Amy James performed all experiments with the following exceptions: Joel Haywood solved the
crystal structure of Butelase 1, Amy James, Julie Leroux and Alysha Elliott generated the
transgenic Arabidopsis lines, Mark Fisher performed LC/MS analysis, Katarzyna Ignasiak and
Samuel G. Nonis generated recombinant protein for and performed activity based probe
analysis, and Ricarda Fenske and Amy James performed MRM analysis of seed extracts.
Student signature:
Date: (cid:68)(cid:258)(cid:396)(cid:272)(cid:346)(cid:3)(cid:1013)(cid:853)(cid:3)(cid:1006)(cid:1004)(cid:1005)(cid:1005)
I, Joshua S. Mylne certify that the student statements regarding their contribution to each of
the works listed above are correct
Coordinating supervisor signature
Date:
(cid:26)(cid:14)(cid:46)(cid:66)(cid:83)(cid:14)(cid:19)(cid:17)(cid:18)(cid:25)
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LIST OF ABBREVIATIONS
AEP asparaginyl endopeptidase
BBI Bowman Birk inhibitor
BLAST Basic Local Alignment Search Tool
cDNA complementary DNA
CHCA α-cyano-4-hydroxycinnamic acid
D O heavy water or deuterium oxide
2
DNA deoxyribonucleic acid
DTT dithiothreitol
EDTA ethylenediaminetetraacetic acid
ER endoplasmic reticulum
FPLC fast protein liquid chromatography
hAEP human AEP
HSQC heteronuclear single quantum coherence
kD or kDa kilodalton
LB Luria-Bertani medium
LC/MS liquid chromatography mass spectrometry
LSU large subunit
m/z mass to charge ratio
mAEP mouse AEP
MALDI matrix-assisted laser desorption/ionization
MES 2-(N-morpholino)ethanesulfonic Acid
MRM Multiple reaction monitoring
mRNA messenger RNA
MS mass spectrometer
NCBI National Center for Biotechnology Information
NEC no enzyme control
NMR nuclear magnetic resonance
1
ORF open reading frame
PAGE polyacrylamide gel electrophoresis
PawS1 Preproalbumin with SFTI-1
PCR polymerase chain reaction
PDB Protein Data Bank
PI protease inhibitor
RNA ribonucleic acid
RP-HPLC reverse phase-high-performance liquid chromatography
SDS sodium dodecyl sulfate
SESA seed storage albumin
SFTI-1 Sunflower Trypsin Inhibitor 1
Snn succinimide moiety
SSP seed storage protein
SSU small subunit
TEV Tobacco Etch Virus
TOF time of flight
Tris tris(hydroxymethyl)aminomethane
WT wild-type

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