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LUKASZ LEBIODA, PhD
Lovelace
LL, Minor W, Lebioda L. Structure of human thymidylate
synthase under low-salt conditions. Acta Crystallogr D Biol
Crystallogr. 2005 May;61(Pt 5):622-7
Human thymidylate synthase, a target in
cancer chemotherapy, was crystallized from PEG 3350 with 30
mM ammonium sulfate (AS) in the crystallization medium. The
crystals are isomorphous with the high-salt crystals (
approximately 2.0 M AS) and the structure has been solved
and refined (R = 22.6%, R(free) = 24.3%) at 1.8 A
resolution. The high- and low-AS-concentration structures
are quite similar, with loop 181-197 is in the inactive
conformation. Also, residues 95-106 and 129-135 (eukaryotic
inserts region) show high mobility as assessed by poor
electron density and high values of crystallographic
temperature factors (residues 1-25 and 108-129 are
disordered in both structures). The high mobility of this
region may reflect the situation at physiological ionic
strength. Of the four sulfate ions observed bound at 2.0 M
AS, only two are present at 30 mM AS. The inactive
conformation appears to be stabilized by the side chain of
Val3 or a leucine residue from the disordered regions. The
low-salt conditions of these crystals should be much more
suitable for the study of thymidylate synthase inhibitors,
especially those that utilize sulfate-binding sites to
stabilize the inactive conformation of loop 181-197.
Chai G, Brewer JM, Lovelace LL, Aoki
T, Minor W, Lebioda L. Expression, purification and the 1.8
angstroms resolution crystal structure of human neuron
specific enolase. J Mol Biol. 2004 Aug 20;341(4):1015-21
Human neuron-specific enolase (NSE) or
isozyme gamma has been expressed with a C-terminal His-tag
in Escherichia coli. The enzyme has been purified,
crystallized and its crystal structure determined. In the
crystals the enzyme forms the asymmetric complex NSE x Mg2 x
SO4/NSE x Mg x Cl, where "/" separates the dimer subunits.
The subunit that contains the sulfate (or phosphate) ion and
two magnesium ions is in the closed conformation observed in
enolase complexes with the substrate or its analogues; the
other subunit is in the open conformation observed in
enolase subunits without bound substrate or analogues. This
indicates negative cooperativity for ligand binding between
subunits. Electrostatic charge differences between isozymes
alpha and gamma, -19 at physiological pH, are concentrated
in the regions of the molecular surface that are negatively
charged in alpha, i.e. surface areas negatively charged in
alpha are more negatively charged in gamma, while areas that
are neutral or positively charged tend to be
charge-conserved. Copyright 2004 Elsevier Ltd.
Ortlund E, Chai G, Genge B, Wu LNY,
Roy E. Wuthier RE, Lebioda L. Crystal Structures of Chicken
Annexin A5 in Complex with Functional Modifiers Ca2+ and
Zn2+ Reveal Zn2+ Induced Formation of Non-Planar Assemblies.
Annexins 2004 1:183-190
Annexin A5 (anxA5) binds to acid
phospholipids in a Ca2+ dependant manner and can form
continuous 2D crystalline sheets on the surface of
anion-rich phospholipid bilayers. This protein has been
shown in vitro to possess Ca2+ ion channel activity and
appears to be linked to the Ca2+ flux in matrix vesicles,
structures involved in induction of biomineralization during
bone and tooth development. Chicken anxA5 Ca2+ channel
activity, as well as Ca2+ uptake by avian matrix vesicles is
strongly inhibited by micromolar levels of Zn2+. To
investigate this effect, we co-crystallized anxA5 with this
ion and have discovered that Zn2+ coordinates anxA5
molecules in such a fashion that they no longer form the
typical planar triskelion arrays within the crystal, but
rather, form a three dimensional system. This rearrangement
of the trimer-trimer association apparently prohibits planar
binding of anxA5 to the phospholipid bilayer. We also
present evidence that the Zn2+ linked trimers are present in
solution. The structures of Zn2+ and Ca2+ complexes are
reported at 2.9 Å and 1.4 Å resolution, respectively.
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