SER: Technique and Structures

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SER Technique and Structures

• The Derewenda Lab
• University of Virginia
• June 9, 2009

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Specific Aims

• Development of surface engineering methods for
  crystals generation.
• Determination of structures of PSI targets
  recalcitrant to crystallization
• Optimization of crystallization chaperones

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()it is shown here that crystal-packing
interfaces display quite a small area relative to
those among protein subunits. Moreover,
crystal-packing contacts involve protein surface
patches with atom composition indistinguishable
from that of the proteins solvent-accessible
surface.
These results clearly point to protein
crystallization as a nearly stochastic
phenomenon. It would nonetheless be important for
crystal growers to predict to some extent the
packing of a protein crystal to find easily
experimental crystallization conditions and to
make crystals more stable by opportune protein
engineering for better crystallographic resolution
in tertiary structural analyses
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Q. Are crystal contacts of stochastic nature?
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A statistical analysis of crystal contacts in 821
unambiguously monomeric proteins crystallized in
51 different space groups
Q. Is the crystal-contact forming propensity
directly proportional to the solvent accessible
surface area presented by a particular amino
acid?
A. No, the relationship is not linear. The more
buried an amino acid is, the less likely it is to
form a contact.
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Q. Are crystal-contact forming propensities a
function of physicochemical properties of amino
acids?
A. Given the same exposed surface, small and
hydrophobic amino acids have larger propensity to
form crystal contacts than charged residues.
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Contact rim
Contact core
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A. We conclude that crystal contacts are not
stochastic in that there is a preference for
certain amino acids, and that the distribution of
amino acids within contacts is not
random. and others agree.
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A recent statistical analysis of 679 well
expressed proteins, of which 157 yielded crystal
structures
Our statistical analysis of large-scale protein
crystallization results demonstrates that the
mean entropy of exposed side chains and predicted
backbone disorder both anti-correlate strongly
and significantly with successful structure
determination. Combining these results with the
observation that stability is not a significant
determinant of success leads to the conclusion
that the dominant factor determining protein
crystallization outcome is the prevalence of
well-ordered surface epitopes capable of
mediating stereochemically specific interprotein
packing interactions.
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Q. Are crystal contacts uniform in their nature?
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An alternative approach comparative analysis of
crystal contacts formed by the same protein in
different crystal forms.
the globular domain of RhoGDI a paradigm for a
protein recalcitrant to crystallization
The classic
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Mapping of crystal contact forming epitopes in 21
crystallographically independent molecules, in 10
different crystal forms to the sequence of GDI
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203
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Eight out of ten crystal forms contain identical
or similar head-to-head dimers



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Three different crystal forms containing similar
hexamers of RhoGDI dimers
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Very often crystal contacts can easily be
categorized as either primary (larger, often
two-fold related) or secondary, often much
smaller and responsible for the ultimate SG
symmetry and non-crystallographic symmetry
example - 1KMT.
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Target Evaluation
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Tm1679

Predicted to be ß-lactamase. Clustered into
COG1237 Metal-dependent hydrolases of the
beta-lactamase superfamily II
Site 1) K159,E160 Site 2) K78,E79 Site 3) K100,
K101
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Tm1679 K100Y, K101Y (pdb 3H3E)
The SER mutations (K100Y,K101Y) are in a crystal
contact.
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Structure Statistics
Spacegroup P61 a127.2 Å b127.2 Å c41.0
Å Resolution 30 1.9 Å Rwork 15.1 Rfree 20.7
RMSDbonds 0.008Å RMSDangles 1.097
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ß-Lactamases
J. Antimicrobial Chemotherapy 551050-51
Metallo-b-lactamases can degrade all classes of
b-lactams except monobactams and are special for
their constant and efficient carbapenemase
activity. This is a most worrisome characteristic
because carbapenems, which are stable against the
vast majority of serine-b-lactamases produced by
resistant pathogens, are the antibiotics with the
broadest spectrum of activity. Moreover,
metallo-b-lactamases are not susceptible to
therapeutic b-lactamase inhibitors. Carine
Bebrone Biochemical Pharmacology 741686-1701
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Related Structures
Tflp T. tengcongenesis Pdb code 2p4z Proteins
72531-536
Protein Pockets by Global Protein Surface Survey
with BLM ligands from Dali Hits gt 12
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An Interesting active site
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An interesting feature
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An interesting feature
Of the top 100 Blast hits, 19 of them have this
PCHC motif.
They also have another conserved cluster.
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40 proteins contain both
Thermotoga maritima MSB8 Aeromonas hydrophila
subsp. hydrophila ATCC 7966 Pelobacter
propionicus DSM 2379 Chlorobium phaeobacteroides
DSM 266 Thermotoga petrophila RKU-1 Serratia
proteamaculans 568 Salmonella enterica subsp.
enterica serovar Paratyphi B str.
SPB7 Clostridium bartlettii DSM 16795 Salmonella
enterica subsp. enterica serovar Saintpaul str.
SARA29 Salmonella typhimurium LT2 Salmonella
enterica subsp. enterica serovar 4,5,12i- str.
CVM23701 Salmonella enterica subsp. enterica
serovar Kentucky str. CDC 191 Salmonella enterica
subsp. enterica serovar Schwarzengrund str.
SL480 Salmonella enterica subsp. enterica serovar
Heidelberg str. SL486 Salmonella enterica subsp.
enterica serovar Hadar str. RI_05P066 Salmonella
enterica subsp. enterica serovar Newport str.
SL317 Salmonella enterica subsp. enterica serovar
Weltevreden str. HI_N05-537 Anaerofustis
stercorihominis DSM 17244 Chlorobium limicola DSM
245 Chlorobium phaeobacteroides BS1 Chlorobaculum
parvum NCIB 8327 Salmonella enterica subsp.
enterica serovar Agona str. SL483 Salmonella
enterica subsp. enterica serovar Saintpaul str.
SARA23 Proteus mirabilis HI4320 Salmonella
enterica subsp. enterica serovar Dublin str.
CT_02021853 Salmonella enterica subsp. enterica
serovar Javiana str. GA_MM04042433 Thermotogales
bacterium TBF 19.5.1 Salmonella enterica subsp.
enterica serovar Typhi str. 404ty Thermotoga
neapolitana DSM 4359 Blautia hydrogenotrophica
DSM 10507 Proteus penneri ATCC 35198 Citrobacter
youngae ATCC 29220 Proteus mirabilis ATCC
29906 Dethiosulfovibrio peptidovorans DSM
11002 Desulfomicrobium baculatum DSM
4028 Oxalobacter formigenes HOxBLS Oxalobacter
formigenes OXCC13 Citrobacter sp. 30_2 Salmonella
enterica subsp. enterica serovar Choleraesuis
str. SC-B67 Pelobacter carbinolicus DSM 2380
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Comparison w/ COG1237
Residues conserved in COG1237
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Conserved in COG1237 Additionally Conserved
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Spacegroup P414141 ab68.16 c302.0 Resolution
40 2.3 Å Rwork 19.4 Rfree 26.9 RMSDbonds 0.022
Å RMSDangles 2.1
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Yphp and Thioredoxin
(B) a single molecule of YphP with the secondary
structure elements identified (?-helices red, and
?-strands yellow) and the two Cys side chains of
the CXC motif shown as spheres and labeled the
additional N-terminal ?-helix unique to the
DUF1094 family is shown in cyan (C) analogous
view of the human thioredoxin (PDB code 1AUC )
shown for comparison.
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YphP has a low catalytic activity
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The Catalytic Loop and Mechanism
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Tm0439 E118A, K119A, K122A (pdb 3FMS)
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Structure Statistics
Spacegroup C2 a85.2Å b71.7Å c43.3Å ß104.6
Resolution 40 2.2Å Rwork 15.7 Rfree 22.8
RMSDbonds 0.017Å RMSDangles 1.31
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Acta Cryst D65356-365
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Tm0439 and Related Structures
The paper also described three other unpublished
FCD domains
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Tm1382 (pdb 3E57)
K159A, E160APhasing Wild Type Refinement
Pyrophosphohydrolases that act upon Nucleoside
DIphosphate connected to another moiety (X) Such
substrates include (d)NTPs (both canonical and
oxidised derivatives), nucleotide sugars and
alcohols, dinucleoside polyphosphates (NpnN),
dinucleotide coenzymes and capped RNAs. The
substrate diversity requires equally diverse
chemistries. Consensus Nudix Sequence
Gx5Ex5UAxREx2EExGU Tm1382 Sequence Gx4Ex5LxRE
x2EExDV Metals participate in catalysis.
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Structure Statistics
Spacegroup P21 a47.6 b62.9 c73.9
ß98.37 Resolution 40 1.89 Å Rwork 19.9 Rfree
24.0 RMSDbonds 0.010Å RMSDangles 1.26
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NUDIX Hydrolases
AP4AP (2PBT)
MutT (2YYH)
GDPMH (1RYA)
ADPRP (1G0S)
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Tm1821

• Wild Type Tm1821 was found to have an error in
  the stop codon. About 20 extra, junk residues
  were being translated.
• After fixing the construct, the protein yielded
  13/96 hits in the JCSG screen and 18 hits in the
  JCSG screen with 1.5M NaCl reservoirs.

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Tm1821 Diffraction5 seconds _at_ home.
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2qr0

• Structure of VEGF complexed to a Fab containing
  TYR and SER in the CDRs

Surface Engineering of a FAB
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Picking the sites

• Light Chain
• Lys A 107 -- isolated on flat surface.
• Glu A 123 -- salt bridge with K126
• Gln A 124 -- sc faces interior
• Lys A 126 -- salt bridge with E123
• Glu A 143 -- exposed side chain w/ no
  interactions.
• Lys A 145 -- has potential to interact w N147,
  but not in this structure. K-gtR?
• Gln A 147 -- sc in h-bonding network
• Lys A 149 -- sc in h-bonding network K-gtR ?
• Gln A 155 -- mostly buried
• Gln A 160 -- participates in dimerization
• Glu A 161 -- h-bond with other B-sheet
• Glu A 165 -- mostly buried, interacts with VD
• Gln A 166 -- mostly buried, interacts with VD
• Lys A 169 -- primarily exposed, on loop near VD
• Lys A 183 -- tip exposed, but caps N-term of a
  helix. K-gtR
• Glu A 187 -- exposed. no real contacts in this
  structure, but could.
• Lys A 188 -- salt bridge with D185
• Lys A 190 -- disordered side chain, good pick.

• Heavy Chain
• Lys B 117 -- exposed, non-contributing side chain
• Lys B 143 -- buried
• Glu B 148 -- hb with buried Tyr hydroxyl
• Gln B 171 -- mostly buried with hb potential
• Gln B 192 -- Good pick.
• Lys B 201 -- weak interaction with carbonyl on VD
  (old), but potential
• Lys B 206 -- primarily exposed, with few
  interactions.
• Lys B 209 -- at least partially buried, possible
  K-gtR
• Lys B 210 -- disordered, possible salt bridge
  with E219, possible target.
• Glu B 212 -- at C-term, possible target
• Lys B 214 -- at C-term, possible target

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Fab Variants
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Approximate Location of Fab mutations
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Streamlining the protocol

• Re-calculated the CRAP media recipe
• Same final concentrations, easier to deal with
  volumes, and less sterile transfers.
• Re-configured chest incubator
• 8 flasks at a time
• If transformations would work
• Worked out AKTAxpress protocol
• Performs 2 Purifications at a time
• Performs Protein A followed by Cation Exchange
• Long and step learning curve
• Difficult to intervene

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The Xpress protocol

• System Procedures
• Remove ethanol from system
• Fill sample inlets
• Wash Frac Tubing
• 1st Chromatography Step
• rProtein A Column
• Remove ethanol from columns
• Equilibrate column
• Step Elution
• Collect Peak to internal loops
• 2nd Chromatography Step
• Cation Exchange
• Remove Ethanol
• Load Largest Peak from Column 1
• Gradient Elution
• Collect peak in fraction collector
• Re-equilibrate for 2nd sample
• System procedures
• Cleaning in Place of Protein A column

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ÄKTAxpress results
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Tolerated Mutations
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Fab Diffraction (Fab1)
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• UVA
• Zygmunt Derewenda
• David Cooper
• Ulla Derewenda
• Natalya Olekhnovich
• Ankoor Roy
• Darkhan Utepbergenov
• Marcin Cieslik
• WonChan Choi
• Meiying Zheng
• Tomek Boczek
• Kasia Grelewska
• Gosia Pinkowska
• Michal Zawadzki

Los Alamos National Lab Tom Terwilliger Chang
Yub Kim UCLA David Eisenberg Luki
Goldschmidt Tom Holton Lawrence Berkeley Natl
Lab Li-Wei Hung Minmin Yu (Big Thanks) Jeff
Habel And ALL ISFI members!
The ISFI is funded by NIH U54 GM074946.