The Application of Surface Entropy Reduction

1
The Application of Surface Entropy Reduction
David R. Cooper, Tomek Boczek, WonChan Choi,
Urszula Derewenda, Natalya Olekhnovitch, Ankoor
Roy, Darkhan Utepbergenov, Meiying Zheng, and
Zygmunt Derewenda University of Virginia,
Department of Molecular Physiology and Biological
Physics. Charlottesville, VA 22908 The Integrated
Center for Structure and Function Innovation (The
ISFI), a PSI-2 Specialized Center.
Abstract A wide array of factors influence
whether or not a protein will crystallize, and
many otherwise well-behaved proteins seem to
harbor an intrinsic resistance to
crystallization. For some proteins, the presence
of highly entropic residues on the surface can
doom crystallization attempts by creating an
entropic shield that prevents the intermolecular
contacts necessary for crystallization. The
Surface Entropy Reduction (SER) approach of
replacing these entropic residues has proven to
be an effective means of producing crystals for
some of these stubborn crystallizers. However,
this approach is not appropriate for every
difficult protein and the likelihood of success
is highly dependent on whether the protein can be
considered a good SER candidate. Herein, we
present the types of analysis that should be
performed before a protein can be called a SER
candidate, and we outline a strategy for the
application of surface entropy reduction. We also
discuss recent successes of our pipeline and
present an indication of the techniques success
rate. Structures that will be shown include a
transcription factor, two metal dependent
hydrolases, and a disulfide isomerase.

• Is SER right for you?
• Have you already tried
• Reductive methylation? Nature Methods 5853-4
  (2008). Structure 141617-22 (2006).
• Quick and easy. Can be performed in parallel
  with native protein already on hand.
• Reductive cyclic pentylation? Acta
  Crystallographica D65462-9 (2009).
• A recently reported alternative to methylation.
• Alternate Reservoir screening? Acta
  Crystallographica D61490-3 (2005).
• This technique involves setting up each
  crystallization drop as normal, but filling the
  reservoir of every well with something other than
  the crystallization solution. We use 1.5M NaCl.
  Not only do we get more crystals (33 more)
  using these screens, but the concentration of
  salt in the reservoir is an easy to optimize
  parameter. The one tricky bit is figuring our a
  good cryo solution, because you cannot assume the
  concentration of the drop is similar to the
  crystallization solution. Sometimes covering the
  drop with oil before harvesting crystals is
  sufficient.
• Absolute minimum SER Candidate requirements
• The protein is soluble and purifies well.
• It is difficult to crystallize or diffracts
  poorly.
• It contains a cluster of highly-entropic
  residues.
• It lacks large regions of predicted disorder or
  coiled coils.
• Its crystallization has not been hampered by a
  missing co-factor or ligand.
• Bioinformatics checks
• Even if you think you know your protein, check
  the following things.
• Check for disorder. One of the first checks
  should be for regions of high disorder.
  Significant regions of disorder can indicate that
  conventional crystallization screening will fail.
  Some proteins may require co-crystallization with
  a folding partner.
• DisMeta (http//www-nmr.cabm.rutgers.edu/bioinform
  atics/disorder/)

APC1446 Q100A,E101A (pdb 3FHK)
Tm1679 K100Y, K101Y (pdb 3H3E)
Apc1446 is encoded by the the B. subtilis yphP
gene. It belongs to DUF1094, which currently
lacks a functional annotation..YphP has a core
domain with high similarity to thioredoxin, but
with a CxC motif instead of the classical CxxC.
Disulfide isomerase assays show this protein to
have a significant isomerase activity.
TM1679 is a metallo-ß-lactamase (MBL) domain
containing protein that has been assigned to
COG1237, a small group with a distinct set of
conserved residues. MBLs are the most troublesome
source of antibiotic resistance. We have
identified a family of 40 proteins that share a
distinct set of conserved residues with TM1679,
with some of these conserved residues overlapping
with a Family Strand Block of RNA-metabolising
metallo-beta-lactatamases (Blocks server).
YphP (Apc1446) Thioredoxin
(1AUC)
APC1446 crystallizes with 4 molecules in the ASU,
which generates a non-crystallographic two-fold.
Three types of crystal contacts are mediated by
the SER mutations.
SER Basics The Surface Entropy Reduction
technique (SER) promotes crystallization by
altering surface features that inhibit
crystallization. Large, flexible residues
(particularly lysines and glutamates) on the
surface of a protein can create an entropy
shield that impedes crystallization. The SER
method involves replacing clusters of
highly-entropic residues with residues that can
facilitate crystallization.
Tm1679 L1 (1SML)
Tflp
(2P4Z) Subclass B3 Top Dali Hit
The catalytic loop of YphP. Arg121 activates
Cys53 allowing intramolecular disulfide
formation. Cys 55 is in turn activated by
Arg121 providing an escape mechanism.
Glutamate Rotamers
An alanine SER variant
Lysine Rotamers
The original and still most popular replacement
residue is alanine, but tyrosine, threonine, and
methionine are also suitable.
Mutations Facilitate Crystallization In most SER
structures, the mutations participate in crystal
contacts. Here the mutated residues are either
labeled or shown in magenta or pink.
The conserved residues of COG 1237 are blue and
additional residues conserved in the Tm1679-like
family are red. The disulfides in the CHCT motif
are shown. In the close up of the active site,
the color of the Ca sphere indicates its
conservation.
Mutations are in crystal contacts.
The structures of known oxidized CxC motifs, and
the reduced CxC motif of YphP

Tm0439 E118A, K119A, K122A (pdb 3FMS)
Tm1382 (pdb 3E57) This
protein was phased with the SeMet SER variant
K159A, E160A (which grew quickly) and refined
against the native wild type (which took 6
months to grow). Both crystals were grown using
alternate reservoir screens (800 mM NaCl and 1.9
M NaCl for WT).
K159A, E160APhasing Wild Type Refinement
Tm0439 is a member of the GntR superfamily of
dimeric transcription factors. It has a
N-terminal winged-helix DNA binding domain and a
C-terminal FCD domain with an internally bound
metal. The FCD domain also serves as a
dimerization domain, yielding disparate
orientations of the DNA binding domains
Hsp33 Structure 121901
The Nudix superfamily is large and catalyzes a
diverse set of reactions, generally a NUcleotide
DIphosphate linked to another moiety, X. Tm1382
contains the conserved Nudix box,
Gx5Ex5UAxREx2EExGU. A number of NUDIX functions
can be ruled out by the lack of other conserved
motifs, but for now the function of this protein
remains a mystery for biochemists to solve.
RhoGDI K99S, Q100S Acta Cryst D63636
RhoGDI K138Y, K141Y Acta Cryst D63636
RhoGDI K135T, K138T, K141T Acta Cryst
D63636
RhoGDI E155H, E157H Acta Cryst D63636

• Our Current SER Strategy
• Target evaluation and selection See Is SER
  right for you? panel.
• Expression of Wild Type taken through to
  crystallization trials.
• Performed on a chromatography system and eluted
  as a gradient to determine optimal washing
  concentration of imidazole.
• We screen the WT crystals for crystallization.
  If we get crystal hits, we will work with them
  for 2 months before undertaking mutagenesis. If
  the structure can be obtained with the WT
  protein, then the protein does not need SER and
  is not a SER candidate.
• Mutation site selection
• We use the SERp server. See If you need SER ?.
• Primer design.
• We order primers to make Ala and Tyr variants for
  the top 3 clusters.
• PrimerX is a nice web tool for designing
  mutagenesis primers. (http//www.bioinformatics.
  org/primerx/)
• QuikChange mutatgenesis
• We make them all at once.
• Mutant expression, purification, crystallization.
• We often purify several SER variants
  simultaneously, with using gravity columns or an
  AKTAxpress. Variants are washed with the
  imidazole concentration determined for the wild
  type protein.

Mutations are in crystal contacts.
DNA binding model based on pdb 1HW2.
The SER Summary Some protein refuse to
crystallize because their surface is teeming with
highly entropic residues that are the result of
an evolutionary pressure to prevent non-specific
interactions ( Physical Biology 19-13 2004).
These proteins are viable candidates for SER and
other surface altering techniques. As PSI has
pursued targets with little of no functional
annotations, ascertaining whether or not a
protein fits into this category has become a task
unto itself. SER has proven itself to be an
effective technique, but its labor intensive
nature warrants a selection process that will
weed out proteins for which SER will not solve
the root of the crystallization problem.
These structures were aligned using the
N-terminal domain of one monomer.
This work was funded as a part of PSI-2. The ISFI
is funded by NIH U54 GM074946. Data were
collected at SER-CAT (APS 22-ID) and ALS 5.0.2