A Personal View of Structural Biology

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A Personal View of Structural Biology In South
Africa
Trevor Sewell
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Structural Biology in South Africa is hard to sell
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Structural Biology 1. Determination of the
structure of biological entities ranging from
cells to macromolecules using advanced physical
techniques. 2. The derivation of insight into the
mechanism of operation of biological systems
through an understanding of their structure.
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Why do we need a programme in Structural Biology?

• Structural biology can leverage and enhance
  studies in all branches of biological science.
• "Third generation" biotechnology depends on
  stuctural information - this includes crucial
  aspects of drug / pesticide /herbicide design and
  the design of industrial enzymes.
• Structural Biology is mainstream science.
• National pride!

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Where do protein structures come from?
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Structures give powerful and useful
Structures give powerful and useful
insights easily!
insights easily!
Toyoshima et al, Nature, September 2004, Calcium
ATPase
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In August 2007 Should SA do SB? has ceased to
be an issue.
The issue is now How should SA do SB?
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The creation of a successful Programme in
Structural Biology required three
factors Substantial foreign money International
collaboration Joining of forces by local
universities
The Masters programme provided a focus of
activity and a launch-pad for the discipline. It
was funded by the Carnegie Corporation of New
York (1.2m)
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DWNN spectrum
David Pugh, UWC, 2003
600 MHz, three channel NMR
DWNN structure
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Master of Science in Structural Biology
Registered with Department of Education in 2002
Programme runs over two years. 1 yr coursework,
assessments10 internal and 1 external 1 yr
project dissertation, 2 external, 1 internal
examiner
Arvind Varsani
Muhammed Sayed
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Foreign Participants
Professor Sir Tom Blundell FRS University of
Cambridge (Programme evaluator)
Professor Helen Saibil University of London (3D
electron microscopy)
Dr Lena Orlova University of London (3D electron
microscopy)
Professor Ed Egelman University of
Virginia (Helical Structures)
Dr Alan Roseman, MRC-LMB Cambridge (3D electron
microscopy)
Dr Gwen Nneji University of London (Protein
Crystallization)
Dr Jim Pflugrath Rigaku MSC, Houston (Crystallogra
phic Data Processing)
Dr Mike Lawrence CSIRO, Melbourne (Protein
Crystallography)
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The Electron Microscope Unit at UCT
Jeol 1200EXII cryo, pre-owned by Helen Saibil.
Wellcome Trust
Zeiss 912 cryo, pre-owned by Arvid Maunsbach.
Zeiss, UCT, SMM
Brandon Weber
Mohamed Jaffer
Sean Karriem
Miranda Waldron
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Coming Soon..
FEI Tecnai F20 cryo, pre-owned by Richard
Henderson, MRC-LMB, UCT, National Equipment
Programme (R1.6m)
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The Process
Negative stain or cryo
Image using low dose
Digitize film
Select images
Classify images
Starting model using a common-lines based method
Match images to projections of model
iterate
Reconstruct new model
iterate
Interpret the structure
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Negatively stained native
B. pumilus
nitrilase, pH8
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Multi-reference alignment
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The refinement of the structure of the
nitrilase from
Bacillus pumilus
(11661 images)
video made by Paul Chang
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Final interpreted, published structure of the
cyanide dihydratase from Bacillus pallidus AK67.
Map deposited in EMDB (EBI, Hinxton)
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The First African Structural Biology Conference

• The Wilderness, 24-27 October 2006
• 18 International Plenary Lectures
• 150 South African delegates
• Commercial exhibition

http//sbio.uct.ac.za/conference
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Synthetic chemistry
Drug Discovery Signature Theme
Molecular modelling
Testing of new compounds
X-ray structure determination
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National Structural Biology Strategy
The DST has awarded a grant of R1.1m to the Cape
Biotech Trust to (1) enable the existing UCT/UWC
structural biology programme to continue and (2)
to set up a committee to devise a national
strategy in Structural Biology.
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DST has applied to the treasury for a large
amount of funding for a national programme in
structural biology pending a detailed proposal
from the committee which would contain the
following elements 1. National strategy agreed
to by the role players 2. The possibility of
setting up of Competence Centres with equipment,
scientific and technical expertise (The Centres
will operate within the Innovation Space and
will leverage existing "biotechnology" projects
irrespective of their sponsor - specifically in
the areas of drug/vaccine design,
pesticides/agrochemicals and fine chemical (i.e.
drug and drug precursor) manufacture.) 4. The
programme would guarantee to recruit and train
people from historically disadvantaged
backgrounds.
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What worked at UWC?

• One department (Biotechnology) took responsbility
  and appointed two staff (Pugh and Sayed)
• The industrial enzymes were low hanging fruit
• Symbolism of the x-ray diffractometer
• Masters graduates at every ceremony
• R13500 total course fee (for both years of MSc)
• Science faculty accepted coursework as an honours
  programme

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What good things happened at UCT?

• Spectacular progress in research on ACE,
  nitrilases, GS, chromatin, viruses.
• Drug discovery signature theme
• Well developed bursary infrastructure
• Basis for acquisition of 3 second hand electron
  microscopes

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What went wrong at UCT?

• Although many departments wanted a share of the
  benefits no single department was prepared to
  take responsibility strategic plan, staff
  appointments, budget
• Principal driver was appointed as a manager and
  technician and was never appointed programme
  convener and was not a member of an academic
  department.
• Perceived conflict of interest EMU seen to
  favour biology over materials.

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What went wrong at UCT?

• Rule of ten no course is deemed sustainable
  unless it attracts ten students per year.
• R19500 per annum course fee
• Teaching was not credited as departmental duty
• Everybody changed DVC, deans, HoDs
• Key people were lost for various reasons
• UCT graduates not visible

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Projects
DRUG TARGETS Glutamate dehydrogenase Glutamine
synthetase HIV protease Glutathione
S-transferase Angiotensin converting
enzyme INDUSTRIAL ENZYMES Nitrilases Amidases Nitr
ile Hydratases CHAPERONINS GroEL E461K
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GroEL E461K
Sewell,BT, Best,RB, Chen,S, Roseman,AM, Farr,GW,
Horwich,AL Saibil,HR (2004) Nature Structural
and Molecular Biology.
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Glutathione S-transferase I219A The first African
entry in the PDB
Mutation site
Diane Barnwell, Wits, first successful crystal
structure Kuehnert et al, JMB, 2005
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Angiotensin-converting Enzyme co-crystal
structures
Jean Watermeyer
Itai Chitapi, Muhammed Sayed
Crystallisable glycosylation mutant
Seeding co-crystallisation
Domain-selective Inhibitors
structure-based design
5 Inhibitor datasets
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Domain-selectivity in the S2 pocket
N-ACE-84 C-selective Ki N 196 ?M Ki C 0.8 ?M
4.23A
4.59A
N-ACE-86 not selective Ki N 23.7 ?M Ki C
84.3 ?M
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Alignment of crystal structures of tACE bound to
lisinopril, captopril, N-ACE-84 and N-ACE-86.
Structures were aligned by the zinc-binding
residues of tACE.
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Outcome of ACE work

• Using structure based design principles a
  compound with a 13 fold enhanced differential in
  ACE domain binding capability has been produced
  potentially eliminating the side effects of
  current anti-hypertension drugs.
• If this can be developed as a drug it has the
  potential for market dominance in an annual
  market of 5bn
• Address investment enquiries to Ed Sturrock!

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Investigation of the binding of endozepine to
palmitoyl-CoA
James Onyemata
 A
15N
HN
Overlay 15N-HSQC spectra of 1.2 mM un-liganded
spectra of endozepine (red) and endozepine fully
saturated with 2 mM palmitoyl-CoA (black).
Perturbations to more than half of the resonances
indicate that palmitoyl-CoA interacts with
endozepine. Squares indicate pairs of resonances
used to calculate .
B
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Chemistry
Nitrilase - cyanide dihydratase - CynD - B.
pumilus, P.stutzeri
Cyanide hydratase - G. sorghi
Amidase G. pallidus
Carbamylase 1ERZ Agrobacterium sp.
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Nitrile Hydratase from Bacillus pallidus RAPc8
Tsepo Tsekoa
Samuel Kwofie Ozlem Tastan-Bishop
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Relationship of solved structures to the
bacterial and plant nitrilases, CynD, and fungal
cyanide hydratrases
Plant nitrilases
Solved structures
CynD
Bacterial nitrilases
Fungal cyanide hydratases
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Some of our structures
pumilus
sorghi
Bacillus pumilus CynD pH 6
Bacillus pallidus amidase
B. pumilus CynD pH 5.4
stutzeri
J1
Gloeocercospora sorghi cyanide hydratase
Pseudomonas stutzeri CynD pH 8
Rhodococcus rhodochrous J1 nitrilase pH 8
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Homology model of a dimeric module of P.
stutzeri CynD
D
E1
C
A
E2
E2
C
E1
D
Sewell BT, Thuku RN, Zhang X, Benedik MJ (2005).
Ann N Y Acad Sci.1056 153-9.
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The spiral elongates by associating across the A
and C surfaces In the 14 subunit spiral
termination results from the E surface interaction
Sewell, B.T., Berman, M., Meyers,
P.R.,Jandhyala, D., and Benedik, M.J. (2003).
Structure
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The dimers closest to the two fold axis have
nearly perfect 51 symmetry. The symmetry is
distorted towards the terminal dimers which move
closer to the spiral axis.
-7
-5
-3
E1
D
-1
D
-6
2
E2
-4
4
E2
-2
6
D
1
D
3
E1
5
7
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Contacts a and b result in the terminal dimer
having an inwards tilt of 12 thus preventing the
addition of a further dimer.
-2
6
E1
a
E2
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Is it possible to explain the transition from
spirals to helices which occurs in B. pumilus
CynD at pH 5.4?
The pH 6 structure is possibly a hybrid between a
terminating 14mer and a terminating 18mer
Margot Scheffer
Johann Eicher
E
90o
pH 6.0
pH 5.4
Recall that the histidines are located in the
C-terminal extension on the inside of the spiral
Hypothesis The charged histidines repel one
another disrupting the E surface interaction and
create space for an additional dimer which is
located by the D surface interactions.
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Investigation of the Cyanide Hydratase
fromNeurospora crassa by IHRSR
Kyle Dent
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Bacillus pallidus RAPc8 Amidase
Vinod Agarkar
Serah Kimani
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The Amidase has the extended C-terminal but not
the insertions
Nitrilase
Amidase
Solved structures
Extended C-terminal
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The 66 C-terminal amino acids interlock by
swapping subunits across the 2-fold axis
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Model of D-lactamide acyl intermediate
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The study led to a proposal for a different role
for glu 142
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Vision
Create a network of collaborating sites at which
the structural paradigm is employed to solve
problems related to disease on the one hand and
industrial biotechnology on the other