1 AN ABSTRACT OF THE DISSERTATION OF Arden Perkins for the degree of Doctor of ...

289 Pages·2015·7.81 MB·English

1

AN ABSTRACT OF THE DISSERTATION OF

Arden Perkins for the degree of Doctor of Philosophy in Biochemistry & Biophysics
presented on March 13, 2015.
Title: Model Systems for Structural Investigations into Peroxiredoxin Catalysis,
Conformation Change, and Inactivation.

Abstract approved: .
P. Andrew Karplus

Peroxiredoxin (Prx) enzymes catalyze the reduction of hydrogen peroxide,
peroxynitrous acid, and organic peroxides, and are extremely efficient peroxidases,
with k /K on the order of 107 – 108 M-1 s-1. Besides their role in oxidative stress
cat M
defense, evidence has accumulated that some eukaryotes, including humans, use Prxs
as switches to regulate peroxide levels for the purpose of signaling events triggered by
hormones and growth factors. Their significance and relevance to human health is
underscored by the occurrence of cancer in some Prx knockout mice; the
overexpression of Prxs in certain cancers, and knockout/knockdown studies that show
Prxs in pathogens can be important, and even essential, for pathogen viability and
infectivity. Further, their ubiquity in all the kingdoms of life implies Prxs provide
indispensible functions.
This thesis reports on work aimed at characterizing aspects of Prx function and
catalysis using model systems that behave well for experimentation, specifically
focusing on detangling the multifaceted roles of conserved residues, catalytic
conformation change, and hyperoxidative inactivation. Five chapters of original work
include two review articles and three primary research reports. One review provides a
relatively broad overview of Prx structure-function (Chapter 2) and the other focuses
on observations related to understanding the physiological role(s) of Prxs especially
summarizing the results of knockout/knockdown studies and assessing the natural
distribution of an enzyme, sulfiredoxin, that is able to reactivate hyperoxidized Prxs
(Chapter 3). The latter shows that many virulent bacterial and eukaryotic pathogens
lack sulfiredoxin, implying that they are unable to rescue hyperoxidatively inactivated
Prxs.
Of the primary research reports, two studies using the model Prx Salmonella
typhimurium alkyl hydroperoxide reductase C (StAhpC) assess the impact of
modifications on structure and dynamics (Chapter 4) and define the roles of highly
conserved residues as they pertain to catalysis, conformation change, and
oligomerization (Chapter 5). The studies with StAhpC were enabled by the discovery
that a previously studied crystal form of the locally-unfolded (LU) conformation of
StAhpC is also able to accommodate the fully-folded (FF) conformation. The work
includes presentations of the first crystal structure of the wild type enzyme in its
substrate-ready form and also the structures of eight mutants of residues that are well-
conserved in the Prx1 subfamily of Prxs. The work led to an awareness of how small
shifts in the relative stabilities of the FF versus LU conformations could strongly
influence Prx function, and this in turn led to the proposal of a novel idea for the
design of selective inhibitors of Prxs as potential drug leads: to target regions involved
in the catalytic conformation change to trap them in inactive states. The third primary
research report (Chapter 6) presents an analysis of three crystal structures of the PrxQ
subfamily that had been solved and deposited in the Protein Data Bank by structural
genomics groups, but not described in publications. These three structures provided
views of the only remaining undescribed type of Prx conformation change – that of the
PrxQ group with a resolving Cys in helix 2. In addition to describing the conformation
change, we also define roles for conserved residues from a structural perspective for
this entire PrxQ subgroup.
Finally, in a forward looking part of the thesis (Chapter 7) I describe initial
work toward developing Xanthomonas campestris PrxQ (XcPrxQ) as a new model
system for study. This enzyme has the advantage of being a monomer, unusual for
Prxs, and this makes it more ideal for probing questions related to dynamics. The
preliminary work with this system includes crystal structures solved at 1 Å resolution,
the highest for any Prx, trapping all relevant active site oxidation states and a ligand-
bound form, NMR backbone assignments for the reduced and oxidized forms, and in
silico docking analyses aimed at discovering a conformation-stabilizing inhibitor.
Furthermore, results showing that the protein in the crystal has strongly enhanced
sensitivity to hyperoxidation validates my proposal that inhibitors stabilizing the FF
conformation of Prxs will lead to hyperoxidative inactivation. Together, these results
establish XcPrxQ as a promising model system for future study.
© Copyright by Arden Perkins
All Rights Reserved March 13, 2015.
Model Systems for Structural Investigations into Peroxiredoxin Catalysis,
Conformation Change, and Inactivation
by
Arden Perkins

A DISSERTATION
submitted to
Oregon State University

in partial fulfillment of
the requirements for the
degree of

Doctor of Philosophy

Presented March 13, 2015
Commencement June 2015
Doctor of Philosophy dissertation of Arden Perkins presented on March 13, 2015.

APPROVED:

.
Major Professor, representing Biochemistry & Biophysics

.
Chair of the Department of Biochemistry & Biophysics

.
Dean of the Graduate School

I understand that my dissertation will become part of the permanent collection of
Oregon State University libraries. My signature below authorizes release of my
dissertation to any reader upon request.

.
Arden Perkins, Author
ACKNOWLEDGEMENTS

There are many people who have contributed to my success and who have
supported me while at Oregon State. First, I would like to thank my parents Lynn and
Gina Perkins, who didn’t mind that their son was more interested in paleontology,
astronomy, and dead garter snakes than working on the family farm. I would like to
thank my Grandma Gloria for all her love and support, and always being willing to
listen to my complex ideas and ambitious plans. I thank my siblings Kelli, Hayden,
and Evan for all their love and encouragement and many hours of competitive video
gaming. I also thank family friends Gene and Coral Rose, Marge-Anne and Helen
Leonnig, Doug Shorey, and Randy and Mary Jane Guyer for their belief in me, and
helping me so much as I was getting ready for the next steps in life. I would like to
thank my former professors at Eastern Oregon University, especially Dr. Joe Corsini
and Dr. Ron Kelley who were excellent teachers and helped convert my passion for
science into an ability to explore and answer questions through experimentation. I
thank all the tough and sometimes crummy jobs I had throughout college as they both
taught me valuable life skills and also encouraged me to strive higher to do what I
really loved.
I thank the faculty of the Oregon State Biochemistry department for giving me
the opportunity to come to OSU. If not for you, I might still be (unhappily) welding
and pulling metal slag out of my toes. I thank the Biochemistry department office
staff, especially Dina Stoneman, for all the helpful advice and guidance on ordering,
reserving, scheduling, and putting a smile on my face every morning. I thank my
committee members for your guidance in the completion of my PhD, in particular Dr.
William Bisson, who allowed me to take part in a study to learn more about
computational biology and drug design. I thank my fellow graduate student friends
who supported me through classes and the many trials and tribulations that come with
the challenge of PhD work. I appreciate the many conversations, both science-related
and not, that helped make my experience at OSU a time of personal and scientific
growth for me. I would especially like to acknowledge and thank Chelsea and Ben
Wolk, who very graciously allowed me to stay with them during periodic trips back to
Corvallis to complete my research projects. I thank the members of the Karplus lab—
Camden Driggers, Kelsey Kean, and Andrew Brereton—for being such a great and
supportive team that created a positive and productive atmosphere. I wish you all the
best in your professional and personal lives.
I thank the many collaborators I’ve worked with: Dr. Kwanho Nam (Harvard
University), Dr. Garry Buchko (Pacific Northwest National Labs), Jared Williams
(Oregon State), and especially Dr. Kim Nelson, Dr. Derek Parsonage, and Dr. Leslie
Poole of Wake Forest University school of medicine who took me in to accommodate
a visit to Wake Forest and who have been such great teammates for the works
presented here.
I want to thank Dr. Dale Tronrud for the countless things he has done to help
me and teach me about the field of protein crystallography, protein structure, and how
to make computers do your work for you. I sincerely appreciate his help and expertise,
and his massive knowledge base, which is very humbling, and that he was never too
busy to answer a question. Thank you for allowing me into your life, helping me in
any way you could, inviting me on hikes, pointing me in the right direction when it
came to a great sci-fi book, and having spirited and enlightening conversations on
politics and ethics.
Central to my success at Oregon State has been my mentor, Dr. Andy Karplus.
Andy, I first want to thank you for meeting me on that Saturday morning when I
couldn’t get away from work during the week. Your thoughtfulness that day left an
impression on me that only deepened as I came to know you better. It was quite a
challenge for me to convert from a knowledge and experience base that was focused
on ecology and biology to the microscopic interactions occurring within individual
cells. But despite that I had little background in biochemistry, your support and faith
in me helped me succeed in the difficult series of first-year courses and inspired a
fascination with these tiny machines called proteins. I’ve never once doubted that you
had my best interests in mind, and I have very much appreciated your honesty, even
when the truth was that I needed to do better. I feel very lucky to have had a PI who
encouraged me to attend conferences, make connections, publish papers, and prepare
myself for a career in science. At the same time I feel extremely privileged to learn
from someone who is a true expert in the field of protein structure. If that wasn’t
enough, you have been so generous to me on a personal level—allowing me to start
the PhD program early, hosting lab get-togethers, funding trips to conferences and to
Wake Forest to foster my career, writing me letters of recommendations and
nominating me for awards, providing advice on difficult issues, and being incredibly
flexible to allow me to work off-site from Washington—I can’t thank you enough for
all you’ve done for me. Andy, I have your guidance and enthusiasm to thank for where
I am now and where I am going.
CONTRIBUTION OF AUTHORS

Kim Nelson contributed to the expression, purification, and kinetic analyses of
proteins used in Chapters 3-7, performed sequence conservation analysis for Chapter 5
and 6, and contributed to writing Chapter 2. Derek Parsonage contributed to writing
and also protein expression, protein purification, and kinetic analyses in Chapters 2-7.
Jared Williams performed the mass spectrometric analyses in Chapter 4. Andrew
Brereton and Steven Hartman contributed to the crystallographic work for Chapter 5.
Leslie Poole and Andy Karplus provided critical review and guidance for experiments
for Chapters 2-7; Andy Karplus also contributed to editing and reviewing the full
thesis text. Michael Gretes reprocessed data for the structures reported in Chapter 6.
Garry Buchko performed the NMR analyses described in Chapter 7 and the appendix
chapter.